Advances in Environmental Microbiology 7

Christon J. Hurst Editor

The Structure
and Function of
Aquatic Microbial
Communities
Advances in Environmental Microbiology

Volume 7

Series Editor
Christon J. Hurst
Cincinnati, Ohio
USA

and

Universidad del Valle

Santiago de Cali, Valle
Colombia
Periodic spring located adjacent to the Miller-Leuser Log House in Anderson Township, Hamilton
County, Ohio, United States of America. Photo by Christon J. Hurst, used with permission

More information about this series at http://www.springer.com/series/11961

Christon J. Hurst
Editor

The Structure and Function

of Aquatic Microbial
Communities
Editor
Christon J. Hurst
Cincinnati, Ohio, USA
Universidad del Valle
Santiago de Cali, Valle, Colombia

ISSN 2366-3324 ISSN 2366-3332 (electronic)

Advances in Environmental Microbiology
ISBN 978-3-030-16773-8 ISBN 978-3-030-16775-2 (eBook)
https://doi.org/10.1007/978-3-030-16775-2

© Springer Nature Switzerland AG 2019

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Cover illustration: “Volvox reimagined” is a montage image created in 2015 and is being used with
permission of the artist, Christon J. Hurst. Those images incorporated into this montage were: center image
“Aspergillus flavus” (author: Hossein Mirhendi, image supplied by and used with author’s permission);
clockwise from upper right “Micrasterias” (author: Rogelio Moreno Gill, image supplied by and used with
author’s permission), “BrownGiantKelp3600ppx” (author: FASTILY, Creative Commons Attribution
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Can there be a combination of place and time
which leaves a perfect memory, a place to
which you do not return because you know
that it will have changed? I believe that to be
true. During the months of July, August and
September of 1998 I was a Fulbright Senior
Scholar in Cali, Colombia, staying in what
was then named the Hotel Pacifico Royal.
Because I was to be living there for three
months, the hotel gave me a free upgrade to a
two room suite complete with a small dining
table. The university was in fiscal crisis, and
so I agreed ahead of time to use my salary
from the Fulbright Commission to pay for my
own lodging and meals. The financial
arrangement meant that my meals had to be
inexpensive. My evening meal usually was
only what I could heat in my rooms’
microwave oven, and generally all that I could
afford were the corn meal cakes called arepas
plus some marmalade spread on top of them.
Therefore, I initially had little need for a
dining table in my hotel room.
The hotel employees were very kind to me and
quickly became my friends. Breakfast
fortunately was provided by the hotel and
cooked by the breakfast buffet chef, Rocío. She
smilingly reminded me that she could prepare
whatever kind of omelet I might want. But,
Rocío knew that for breakfast I only ate
croissants with of course marmalade, and that
I drank hot chocolate. On days when I seemed
to arrive late for breakfast, Rocío would have
saved for me a plateful of fresh croissants on a
shelf under the buffet table and hot chocolate
would be awaiting me. Once each two weeks I
washed my laundry in the hotel room bathtub
and then hung up the wet laundry all around
my room for drying. The chambermaid,
Blanca, told me that the hotel offered laundry
service, but I said that I had no money to
afford it. Blanca eventually told me that the
hotel would dry all of my clothes for free! That
was very kind, but I felt too guilty to ask the
assistance. Two weeks later, when my suite
was again filled with wet laundry, Blanca
once more offered to have the clothes dried for
me. I thought about it, and asked if they could
dry just my jeans because those took three
days to air dry in the summer humidity.
Following that, each morning after I had done
my laundry the wet jeans would disappear
only to magically reappear a few hours later
dried and carefully folded. The hotel men’s
soccer team adopted me as their unofficial
mascot and made sure that I got to each of
their practice sessions and games. I eventually
learned that one time their team even had
hired the car that transported me to the game
and back. I remember José the doorman, who
always was kind and never would accept a tip
from me because I was his friend. Plus, of
course, there was the concierge María
 Fernanda whose kindness made everyone
around her feel touched by her presence.
I eventually did find a good usage for that
table. Each workday I walked through a
shopping mall during trips between my hotel
and the university. After a couple of weeks,
I noticed that a stationery store in the
shopping mall sold wet clay. My hobbies
included pottery and ceramic sculpture, and
with that I found a solution for occupying my
spare time at the hotel. I covered the top of my
dining table with plastic, paper, and on top of
that I placed some heavy canvas purchased
from a fabric store in the shopping mall. The
clay contained sticks and pebbles that needed
to be removed before the clay could be
worked, and with a few simple tools plus a
ceramic ashtray used as a rolling pin I made
presents for the hotel staff. Almost all of those
things that I made at the dining table can be
seen in this picture.

Sculpture made in Cali Rm 902 Hotel Pacifico Royal 1998

I had no means of firing those objects, and so

they were given away air dried. When my
 birthday came around, María Fernanda
quietly collected enough money as donations
from the hotel staff to buy a glass of wine for
me from the hotel’s bar. At her request, the
hotel kitchen volunteered a slice a cake. I was
out to a museum on that day, but everyone
who could wait did stay until I returned to the
hotel. Together, my friends at the hotel then
presented to me the cake and wine, and they
sang Happy Birthday to me. I again spent
some time at the hotel in 2000, and although
staying there was similar and very nice,
something of what had seemed magical
during that summer in 1998 was a bit
changed. I have not returned to that hotel
because, although I would imagine it
currently is a very nice place, I want my
memories to remain as perfect as it all seemed
in 1998. To whom among those people would
I dedicate my work on this book? Most
certainly it should be María Fernanda, for
that kind specialness in her soul which I am
sure leaves everyone believing the world is a
better place.

María Fernanda Gutiérrez Herrera in Cali 2000

Series Preface

The light of natural philosophy illuminates many subject areas including an under-
standing that microorganisms represent the foundation stone of our biosphere by
having been the origin of life on Earth. Microbes therefore comprise the basis of our
biological legacy. Comprehending the role of microbes in this world which together
all species must share, studying not only the survival of microorganisms but as well
their involvement in environmental processes, and defining their role in the ecology
of other species, does represent for many of us the Mount Everest of science.
Research in this area of biology dates to the original discovery of microorganisms
by Antonie van Leeuwenhoek, when in 1675 and 1676 he used a microscope of his
own creation to view what he termed “animalcula,” or the “little animals” which
lived and replicated in environmental samples of rainwater, well water, seawater,
and water from snow melt. van Leeuwenhoek maintained those environmental
samples in his house and observed that the types and relative concentrations of
organisms present in his samples changed and fluctuated with respect to time.
During the intervening centuries we have expanded our collective knowledge of
these subjects which we now term to be environmental microbiology, but easily still
recognize that many of the individual topics we have come to better understand and
characterize initially were described by van Leeuwenhoek. van Leeuwenhoek was a
draper by profession and fortunately for us his academic interests as a hobbyist went
far beyond his professional challenges.
It is the goal of this series to present a broadly encompassing perspective
regarding the principles of environmental microbiology and general microbial ecol-
ogy. I am not sure whether Antonie van Leeuwenhoek could have foreseen where his
discoveries have led, to the diversity of environmental microbiology subjects that we
now study and the wealth of knowledge that we have accumulated. However, just as
I always have enjoyed reading his account of environmental microorganisms, I feel
that he would enjoy our efforts through this series to summarize what we have
learned. I wonder, too, what the microbiologists of still future centuries would think
of our efforts in comparison with those now unimaginable discoveries which they
will have achieved. While we study the many wonders of microbiology, we also

ix
x Series Preface

further our recognition that the microbes are our biological critics, and in the end
they undoubtedly will have the final word regarding life on this planet.

Christon J. Hurst in Heidelberg

Indebted with gratitude, I wish to thank the numerous scientists whose collabo-
rative efforts will be creating this series and those giants in microbiology upon
whose shoulders we have stood, for we could not accomplish this goal without the
advantage that those giants have afforded us. The confidence and very positive
encouragement of the editorial staff at Springer DE has been appreciated tremen-
dously and it is through their help that my colleagues and I are able to present this
book series to you, our audience.

Cincinnati, OH Christon J. Hurst

Volume Preface

Microbial communities exist in all potential aquatic habitats including surface water,
aquifers, and discarded materials on which precipitation collects. Some members of
those microbial communities will naturally be floating on the surface, many others
will live suspended at particular depths in the water column, a broad range of
microorganisms make their existence in sediments, and still there are other commu-
nities that attach to solid matrices and contribute to the formation of biofilms.
The authors of this book describe how aquatic microbial communities are struc-
tured in ways that optimize the community’s functioning and the authors further
explain that community structuration often includes functional stratification. Struc-
turation can be visibly obvious in biofilms including the presence of layers that
typically extend from a community’s surface of attachment outward into the aqueous
surroundings. Vertical stratification often occurs within photic zones, and the pho-
tosynthetic pigments produced by microbes in photic biofilms may be evident as
color banding, with the banding representing a community structured on the basis of
optimal wavelength usage. In areas of low disturbance by metazoans, that vertical
banding of photic communities can include seasonal laminations. The authors also
explain that aquatic utilization of organic detritus as an energy source often begins
with fungal colonization which starts a food chain. Further processing of sedimented
organic material occurs by microbial communities that may be stratified on the basis
of reduction potential, and their activity can include methanogenesis.
Preparing microbiologically safe drinking water requires eliminating hazardous
components of the aquatic microbial community and often the processed water then
needs to be safely distributed. Vibrio cholerae represents one example of pathogenic
aquatic microorganisms for which consideration must be given whenever microbial
ecology favors presence of that species in water that would be used for drinking.
This book explains the techniques used for accomplishing microbial removal from
water and also addresses the fact that drinking water distribution relies upon
understanding and controlling aquatic environments which are designed to be
isolated from their surroundings. The isolation is intended both to contain the
product water and to reduce contamination caused by external impacts. Most

xi
xii Volume Preface

often, municipal communities will have organized utility services which supply
drinking water via enclosed piping distribution systems. Some human communities,
particularly those which may be remote and isolated, are not serviced by piping
networks and instead reply upon tanker trucks for the transportation of drinking
water. Even with those options, many individuals choose to purchase bottled
drinking water and very often emergency situations temporarily may necessitate
the supplying of bottled water. It must be understood that drinking water provided by
any of those means will not be sterile. The authors provide an understanding of those
microbial communities that are suspended in drinking water and also explain the
microbial biofilms that develop on the inner walls of water distribution systems. We
need to understand those microbial communities and control the health risk which
they may present, both because some of the microbes in drinking water are patho-
genic and because drinking water biofilms can interfere with water disinfection
practices.
Studying aquatic microorganisms often entails identifying them, and for that
reason this book additionally provides knowledge of the techniques that have been
developed for successful isolation and cultivation of bacteria.
I am tremendously grateful to Andrea Schlitzberger, Markus Späth, and Isabel
Ullmann at Springer DE, for their help and constant encouragement which has
enabled myself and the other authors to achieve publication of this collaborative
project.

Cincinnati, OH Christon J. Hurst

Contents

1 Understanding Aquatic Microbial Communities . . . . . . . . . . . . . . . 1

Christon J. Hurst
2 Relationship Between Lifestyle and Structure of Bacterial
Communities and Their Functionality in Aquatic Systems . . . . . . . 13
Luca Zoccarato and Hans Peter Grossart
3 Biofilms: Besieged Cities or Thriving Ports? . . . . . . . . . . . . . . . . . . 53
Otini Kroukamp, Elanna Bester, and Gideon M. Wolfaardt
4 Complex Structure but Simple Function in Microbial Mats
from Antarctic Lakes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Ian Hawes, Dawn Sumner, and Anne D. Jungblut
5 Fungal Decomposers in Freshwater Environments . . . . . . . . . . . . . 121
Vladislav Gulis, Rong Su, and Kevin A. Kuehn
6 The Ecology of Methanogenic Archaea in a Nutrient-Impacted
Wetland . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Andrew Ogram, Hee-Sung Bae, and Ashvini Chauhan
7 Briefly Summarizing Our Understanding of Vibrio cholerae and
the Disease Cholera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
Christon J. Hurst
8 Options for Providing Microbiologically Safe Drinking Water . . . . 185
Christon J. Hurst
9 Microbiome of Drinking Water Distribution Systems . . . . . . . . . . . 261
Laurence Mathieu, Tony Paris, and Jean-Claude Block
10 Isolation and Cultivation of Bacteria . . . . . . . . . . . . . . . . . . . . . . . . 313
Martin W. Hahn, Ulrike Koll, and Johanna Schmidt

xiii
Chapter 1
Understanding Aquatic Microbial
Communities

Christon J. Hurst

Abstract Aquatic environments are divided both physically and functionally into
ecosystems whose community organizations include competitions as well as coop-
erations. Bodies of water that are small and shallow are more likely to be energet-
ically dependent upon allochthonous nutrient inputs from the land. And, at the same
time, small and shallow bodies of water will have less buffering capacity against the
potential abruptness of fluctuations in allochthonous inputs. Larger bodies of water
will by nature of their size have more buffering capacity against allochthonous
impacts, and larger bodies of water also will be more reliant upon their autochtho-
nous energy resources. All of the surfaces within aquatic systems contain biofilms,
and in a sense it often takes a biofilm to nurture a microbe. Being part of a biofilm has
both its blessings and curses, its benefits as well as limitations. Our task of under-
standing the nature of aquatic microbial communities requires recognizing interre-
lationships between the good, the bad, and the ugly, with slimy and smelly being par
for the course.

1.1 Introduction

The life that we know on this planet has a suggested starting point of perhaps
4.5 billion years ago. That beginning likely occurred sometime after the presumed
collision of earth with either a very large asteroid or a small planet, and that object
has since been named Theia. The collision eventually resulted in creation of our
moon. It is understood that the heat arising from such an impact event almost
certainly would have sterilized the earth, eliminating any previously existing life.
Following that impact the earth’s surface eventually would have cooled sufficiently
for liquid water to appear. We presume that the life now recognized on this planet

C. J. Hurst (*)
1814 Woodpine Lane, Cincinnati, OH 45255, USA
Universidad del Valle, Cali, Colombia

© Springer Nature Switzerland AG 2019 1

C. J. Hurst (ed.), The Structure and Function of Aquatic Microbial Communities,
Advances in Environmental Microbiology 7,
https://doi.org/10.1007/978-3-030-16775-2_1
2 C. J. Hurst

began shortly after liquid water recollected, with microorganisms initially evolving
and growing in that water. And so, in a sense, aquatic microbial life must be
considered to represent the start of our biological heritage. Aquatic microbial life
now exists from the very surface in all fresh, estuarine and oceanic waters, to the
abyssal depths of the oceans. It has been suggested that we might even give a name
to the common ancestor of all these life forms, with Glansdorff et al. (2008) having
proposed that to be “Luca”.

1.2 The Water Has Depth and Divides

In ponds and lakes, we describe vertical stratification of the water column from the
surface to the bottom, according naming its three main layers the epilimnion,
metalimnion and hypolimnion. The epilimnion is the uppermost layer and usually
it will be the warmest because of sunlight. The epilimnion layer also will be oxic
because water circulation within the epilimnion distributes atmospheric oxygen
throughout that zone. The hypolimnion is the lowermost and generally coldest
area of the water column, which also has a relatively low dissolved oxygen level
because water circulation within that layer usually is limited to occurring between
the bottom of the waterbody and the position of the thermocline. The metalimnion
marks the thermocline, which is intermediate both with respect to its vertical position
as well as its temperature and level of dissolved oxygen. Each of those zones has its
microbial communities and their ecological activities. The thermocline can rise
during the daylight hours and fall during the nighttime hours on a daily cycle during
much of the year, depending upon the extent to which the surface water is heated by
the sun. Very shallow water bodies may not have a thermocline at all. Moderately
deep waterbodies, of perhaps a meter of so in depth, may have their thermocline sink
to the bottom and disappear during the night and then the thermocline can subse-
quently reform and rise with the next days sunlight. Disappearance of the thermo-
cline is important because it allows more oxygen to reach the otherwise relatively
oxygen deprived bottom of the water column and sediments. I once received a
teaching grant from El Instituto para el Control y la Conservación de la Cuenca
Hidrográfica del Lago de Maracaibo (ICLAM), in Venezuela, where they interest-
ingly designed the depth for a set of wastewater stabilization canals such that a
thermocline would develop in those canals during the day, then the thermocline
would sink and disappear each night. That daily thermocline pattern meant the
sedimented solids could undergo alternating cycles of aerobic and anaerobic micro-
bial activity. Moderately shallow lakes of perhaps a few meters may have their
thermoclines disappear not daily but seasonally, completely dissipating during the
winter and reforming during the spring. Quickly flowing bodies of water, such as
streams, may experience sufficient mixing that thermoclines do not establish. The
oceanic water column generally is divided into five layers called pelagic zones, and
from top to bottom those are the epipelagic, mesopelagic, bathypelagic,
abyssopelagic, and hadopelagic. Beneath the water column is the sediment, which
has a low oxygen level at its surface and becomes anoxic not far beneath the
sediment surface.
1 Understanding Aquatic Microbial Communities 3

In a shallow lake with low turbidity, all of the water column may be photic zone
meaning that sunlight penetrates even to the bottom such that photosynthesis can
occur as a primary energy source throughout the system. In deeper lakes, and in the
ocean, there will be a vast aphotic zone which underlies the photic zone. The amount
of sunlight penetration into the aphotic zone is either too limited, or indeed nonex-
istent, such that other energy sources sometimes drive the deeper water lifecycles.
However, with exception of locations such as deep sea hydrothermal vent plumes,
most life in the column water and sediment is affected by what happens energetically
higher in the water column, and most aquatic life utilizes food chains that are based
upon photosynthesis (Cavan et al. 2018; Salter et al. 2012; Wolff et al. 2011).
There are aquatic microbial mechanisms, including those occurring among the
communities in aquifers, which metabolize metals (Legg et al. 2012). Microbial
mechanisms also help to cycle other natural elements including carbon (Painter et al.
2017; Poulton et al. 2017; Sanders et al. 2016), nitrogen (Painter et al. 2017),
phosphorus (Davis et al. 2014; Painter et al. 2017; Poulton et al. 2017) and sulfur
(Wasmund et al. 2017). Microbial life often converts those from elemental forms
into organically useable forms, and sometimes back into elemental forms, done as
enzymatic processes for deriving operating energy. Microbial processing of the
minerals emitted from hydrothermal sources such as vent plumes have produced
some amazing lifestyles (Dick et al. 2013). Aquatic microbial communities also have
some capacity for degrading compounds such as spilled natural gas and oil (Red-
mond and Valentine 2012).

1.3 And Its Ecosystems Are Filled with Amazing Life Forms

Aquatic environments are divided into ecosystems. When we view from a large
perspective some small body of water, such as a pond or stream which is narrow and
shallow, we typically see that the water has an upper fluid surface exposed to the
atmosphere plus there will be inanimate solid surfaces including the bottom and
sides. Some of those solid surfaces are completely and permanently submerged, but
other surfaces may not be constantly covered with water. We visibly may notice
some living aquatic plants and macroalgae, plus some vertebrates and invertebrates,
all of which also represent aquatic surfaces contained within that body of water.
There additionally will be debris including detritus that once was living. When we
view the ocean from a boat that is distant from shore, we mostly see only the surface
of the water and floating debris, but we presume that the other types of solid surfaces
are present within that ocean even though we cannot see them. When we touch any
of those solid surfaces, even the debris, we will notice that none of it feels clean, and
instead all of it feels slimy because in fact something is growing on it. Even some of
the smallest aquatic organisms have other life forms growing on them (Huq et al.
1983).
Chapter 2 of this book, “Relationship Between Lifestyle and Structure of Bacte-
rial Communities and Their Functionality in Aquatic Systems” by Luca Zoccarato
4 C. J. Hurst

and Hans-Peter Grossart, pp. 13–52, helps us to understand that when we view the
same body of water from a more narrow perspective we can perceive it consists of
numerous microhabitats filled with microscopic life. Broadly grouped, those micro-
habitats include: the diffusion controlled water phase; a colloidal phase of nanogels
and microgels; particles which may be either exudates, or carcasses, or aggregates;
and the living biosphere which has among its components such things as algae,
zooplankton, and fish. Each of those microhabitats represents a different range of
chemical and physical characteristics. Among those characteristics are the associated
abiotic factors such as light, temperature, and oxygen. There also are differences in
the concentration and composition of organic matter. Some of the organic material
will be particulate in nature, and some will be dissolved, although there often is not
an absolute distinction between those two broad categories of organic matter. Much
of the organic matter still is living! A major goal always is to eat and not be eaten.
The variables of a water body’s size and land proximity, or volume and distance
from the center of the waterbody to the shoreline, can be grouped and described as a
factor which contributes to an aquatic ecosystems buffering capacity, with the areas
in smaller bodies of water experiencing less buffering against terrestrial inputs. The
ecosystems within smaller bodies of water, and the near shore areas of larger bodies
of water, will be more subject to short term disruptions effected by allochthonous
materials that arrive from terrestrial environments and also will be more susceptible
to the short term changes in terrestrial inputs caused by climate fluctuations associ-
ated with local and regional weather patterns (Davis et al. 2018) including precip-
itation. It is important to remember that precipitation drives both land surface runoff
and groundwater discharge. Loading received from terrestrial inputs will change
both the microbial community composition and the community functions. Those
allochthonous materials include plant and animal debris which may drive the aquatic
carbon cycle, along with both natural and anthropogenic chemicals that wash in from
the land surfaces.
The flow of air also brings both nutrients and often contaminants to the water.
Windborne solids can travel huge distances, although their impact may be most
noticed near to the shore (Griffin and Kellogg 2004). Those inputs are factors which
can vary daily, they will include larger scale seasonality patterns, and also encom-
pass very long scale climate change patterns. There even are vast swathes of the earth
that have cycled between aquatic environments and dry land in relationship to water
and land usage patterns, with examples being the Mesopotamian Marshes in Iraq,
Iran and Kuwait. Kenya’s Lake Turkana Basin additionally has experienced very
long scale climate change patterns (Bloszies et al. 2015; Goldstein et al. 2017).
Larger bodies of water and also the distantly offshore areas of oceans will have
their ecologies and carbon cycles more driven by sunlight that directly reaches the
water’s surface. In some places oceanic deep sea thermal vents additionally will
make their contribution as a local basic energy source. And thus, the carbon cycles in
those aquatic areas will have an autochthonous base.
Oceanic currents bring changes including a supply of nutrients to aquatic areas
that are far distant from the shore, acting in much the same way as do rivers carrying
inputs from the land surface to the near shore areas. Some of those nutrient changes
1 Understanding Aquatic Microbial Communities 5

which are due to oceanic currents indeed occur very far distant from land while other
changes due to currents can occur relatively near to the shore. Those oceanic flow
patterns often carry contaminants in addition to bringing inputs of nutrients. Fluc-
tuations associated with oceanic currents generally are less erratic than are fluctua-
tions associated with terrestrial rivers, although the fluctuations associated with
oceanic currents can be quite dramatic as in cycles of the El Niño Southern
Oscillation. Oceanic currents which bring upwellings of cold, nutrient laden water
(Hosegood et al. 2017) often result in plankton blooms that feed and increase fish
populations. When those water flow patterns change they can disrupt planktonic
growth, and have an opposite effect by causing fish populations to decline.

1.4 It Often Takes a Biofilm to Nurture a Microbe

Living organisms interact with their surrounding environment, being both affected
by the environment and in return modifying it. Those actions involve the organisms
collecting and taking in what they need from their surroundings and nearly simul-
taneously leaving behind what is, for them, unneeded refuse. The selection processes
as to what is taken in versus left out are not random, they are in fact quite purposeful
and for each species those processes both have driven evolution and been modified
by evolution. And, generally, an organism never exists by itself. It is important to
recognize the fact that life typically is a communal process and we must understand
the key significance of microbial existence within a structured community.
When microorganisms exist in communities, the metabolic functions of individ-
ual organisms become interdependent and often competitive, and the community
develops its communal characteristics. Interdependence within a community can
mean selectively sharing metabolic products with other microorganisms and
macroorganisms in ways that are mutually beneficial. One organism’s requirements
often are different from those of another, and what one microbe either intentionally
overlooks or leaves as waste can represent the essential needs of another organism.
That type of specialization leads to a community organization which efficiently
allows one microbes trash to become another microbes treasure. The resulting
community structuration may include metabolic zonation and also metabolic cou-
pling. Competition often is only unilaterally beneficial and includes cheating, which
can extend to the point of stealing needed resources from your competitors so as to
energetically starve those competitors out of existence, or even just simply and
outright eating your competitors.
There is an African proverb “It takes a village to raise a child”, and by analogy it
often takes a biofilm to nurture a microbe. Microbial communities very often
develop on surfaces which form collection points. The resulting biofilms have
both temporal and spatial heterogeneity as a consequence of the groups organiza-
tional development. That development is a process which strives to best utilize
available nutrient and energy sources. Sometimes, the surface on which the biofilm
exists has been chosen because the surface provides a basic energy source, such as
6 C. J. Hurst

the wall of a metal drinking water distribution pipe, a piece of organic detritus, or a
living being that may become detritus due to actions of the accumulating biofilm. At
other times, the surface may provide an anchorage point which facilitates communal
exposure to an energy source such as sunlight. Energetically, there also may be
organizational aspects which rely upon the availability of electron donors and
acceptors, represented by reduction potential gradients within a biofilm. We humans
are indeed a part of the biofilm which has formed on this rocky planet that we call our
home. Our evolutionary path has been directed in part by the environment imposing
requirements, and we undoubtedly have modified the environment by leaving a tale
of changes in our wake. Through it all we must acknowledge and remember that
microbes were our biological origin, biologically the microbes sustain us, and
biologically the microbes will survive our lifetimes endpoint.
The characteristics and composition of microbial biofilms differ based upon where
they form, the conditions under which they form, and the conditions under which they
either continue to exist healthily or senesce. Chapter 3 of this book, “Biofilms:
Besieged Cities or Thriving Ports?” by Otini Kroukamp, Elanna Bester and Gideon
M. Wolfaardt, pp. 53–90 asks the question of whether biofilms which exist in flowing
conditions are either more like thriving ports where there is organization and good to
be found associated with everything that comes and goes, or if biofilms like besieged
cities are potentially challenged by everything that comes near.

1.5 When Life Hits the Mat

Photosynthetic mats have a biofilm structure that is uniquely interwoven. Chapter 4

of this book, “Complex Structure but Simple Function in Microbial Mats from
Antarctic Lakes” by Ian Hawes, Dawn Sumner and Anne Jungblut, pp. 91–120,
describes and discusses the physical characteristics and cooperation found within the
photosynthetic microbial mats which grow as benthic communities under the con-
tinuous ice cover of Antarctic lakes. Those authors explain that growth in such
microbial communities shows a very high level of evolved structural organization.
The community structure can include seasonal lamination because there is limited
environmental disturbance and also because the habitats lack large metazoans that
might disrupt or potentially even consume portions of the microbial community.
Zonation in those communities is energetically driven and the authors examine the
interacting dynamic forces which produce those mat structures.

1.6 The Fungi Will Get You If You Land in the Water

Fungi play a large role in aquatic recycling of organic carbon through their decom-
position of detritus from plants and animals. That detritus represents a major source
of nitrogen and phosphorus in addition to the obviousness of its carbon content. In
1 Understanding Aquatic Microbial Communities 7

chapter 5 of this book, “Fungal Decomposers in Freshwater Environments” by

Vladislav Gulis, Rong Su and Kevin A. Kuehn, pp. 121–155, its authors explain
that in aquatic environments the fungal biomass tends to have a dominate association
with submerged leaf litter and wood. There, fungi valuably act by enzymatically
breaking down the large plant polymers such as cellulose, hemicelluloses, lignins,
and pectin. The kinetics of those enzymatic activities are affected by the chemical
characteristics and oxygen levels of the surrounding water. Some of the fungal
activity represents a direct recycling of nutrients to produce not only fungal elements
which will be contained in the detritus but also fungal spores that will be released
into the water. Much of the nutrients in that detritus, including the fungal elements
themselves, will then be consumed by other aquatic microorganisms and
macroorganisms. Photosynthetically active algae also play a role in the degradation
of detritus, as do bacteria. Some algae are surface associated while others are
suspended in the water column. The predominance of aquatic bacterial biomass is
associated with fine particulate material.

1.7 Researching Microbiology Even When You Are Up

to Your Waist in Alligators While Studying Respiration
Without Oxygen, in a Swamp

Wetlands are a connecting point between aquatic and terrestrial life. They contain
both aquatic and terrestrial characteristics and in a sense have their own combination
of microbial activities. Seasonal wetlands can be aquatic environments during some
time periods of the year, and terrestrial environments during other times, depending
upon whether the water level is above versus below the ground surface. And,
wetlands merge with one another and do as well merge with stream environments
(Vanderhoof et al. 2016).
The Everglades is a wide and generally quite shallow river in Southern Florida of
the United States, it now is a patterned peat land and serves as an example of a
complex wetland with interdependent ecosystems. Peat is a product of partial plant
material decomposition which occurs in wetlands (Hohner and Dreschel 2015) and
represents long term carbon storage. Conversion of that plant material into coal
would represent longer, geologic scale storage of its carbon. Chapter 6 of this book,
“The Ecology of Methanogenic Archaea in a Nutrient Impacted Wetland” by
Andrew Ogram, Hee-Sung Bae, and Ashvini Chauhan, pp. 157–172, explains that
methane production is a key microbial characteristic of wetlands. The anoxic
environment of peat systems results in slow decomposition of organic carbon.
Therein, the methanogenesis that occurs is a form of anaerobic respiration which
uses as its terminal electron acceptor any available atoms of carbon contained in
either low molecular weight organic compounds or carbon dioxide, and the result is
production of methane. Sulfate reduction also occurs in wetlands, where it is done by
microorganisms that perform anaerobic respiration using sulfate as a terminal
8 C. J. Hurst

electron receptor and reduce that to hydrogen sulfide. Although it often is thought
that methanogenesis generally occurs only when sulfates are depleted, both pro-
cesses can occur in the same zonal area (Sela-Adler et al. 2017).
In a sense, methane production is an indicator of the microbial health of wetlands.
Methane production is an anaerobic activity associated with archaea and that activity
also takes place in the intestines of ruminants (Patra et al. 2017) and many other
mammals. Methane production also occurs in anaerobic wastewater treatment pro-
cesses (Qiao et al. 2015; Świątczak et al. 2017). Methanogenesis additionally occurs
in landfills (Staley et al. 2012). The microbes which generate methane by those
process are termed methanogens.

1.8 And Microbes Are even in the Water that We Would

Want to Drink

Some of the microorganisms present in water cause disease in humans (Hurst 2018)
including the notorious Vibrio cholerae (Ali et al. 2015; Azman et al. 2013) which is
a commensal to crustaceans including those which are considered zooplankton,
notably copepods (de Magny et al. 2011). Vibrio cholerae causes the disease cholera
as explained in chapter 7 of this book, “Briefly Summarizing our Understanding of
Vibrio cholerae and the Disease Cholera” by Hurst, pp. 173–184. One of our public
health goals is removing hazardous microorganisms from drinking water, and for
that purpose we use methods which vary in complexity and effectiveness. Water
treatment processes are designed differently with regard to the volume of water that
can be treated simultaneously (Huq et al. 2010; Hurst 2018) and the microbial
community changes as a result of drinking water treatment processes (Liao et al.
2015). The procedures used for processing water to either remove hazardous micro-
organisms or destroy their infectiousness and thereby render the water safe for
ingestion will differ depending upon the volume of water being treated. Sometimes
the general treatment methodology is similar at large and small size scales but
performed in different ways. The subject of supplying microbially safe drinking
water is presented in chapter 8 of this book, “Options for Providing Microbiologi-
cally Safe Drinking Water” by Hurst, pp. 185–260.
Water treatment for large communities often is followed by efforts to safely
distribute the treated water using community drinking water distribution networks
that involving piping. Those drinking water distribution systems are enclosed man
made aquatic environments colonized by a wide range of both microorganisms and
small macroorganisms that arrive from several sources, as explained in chapter 9 of
this book, “Microbiome of Drinking Water Distribution Systems” by Laurence
Mathieu, Tony Paris and Jean-Claude Block, pp. 261–311. Organisms invariably
will enter into the distribution system as constituents of the source water that is being
distributed, even if that source water was processed by physical and chemical
treatment processes. Those organisms will be joined by other biological
1 Understanding Aquatic Microbial Communities 9

contaminants which represent accidental arrivals that have entered the distribution
system in association with infiltrations related to piping engineering failures. Such
failures include inadequate sealing of the pipe connections, as well as breakage of
piping and pipe connections. Accidental cross contamination of the drinking water
and septage collection systems represents yet another source of microorganisms.
Consequently, the plumbing of drinking water networks are aquatic ecosystems
which contain archaea, bacteria, fungi, viruses, protozoa, and small invertebrate
metazoa, many of which certainly can cause disease in humans who ingest the water
from distribution systems. That accumulation of biomass and its ecosystem structure
must be understood and controlled, because the biomass in drinking water interferes
with chemical disinfectant processes upon which we rely for delivering safe product
water to consumers.
Normally we think of removing phosphorus as something done for treating
domestic wastewater to help protect the natural ecosystem from eutrophication
(Fig. 1.1). However, there also is some consideration that removing phosphorus
during drinking water treatment beneficially may reduce the microbial growth that
occurs within water distribution systems (Wang et al. 2014).

1.9 They Are more than Just Nameless Faces: There Are
Ways to Culture and Identify even the Seemingly
Ungrowable

Aquatic microbial communities contain a vast range of organisms, each with its own
activities and necessary requirements. Often, all but some viruses may be culturable
if the appropriate conditions can be met. The subject of “Isolation and Cultivation of
Bacteria” is addressed in chapter 10 of this book by Martin W. Hahn, Ulrike Koll and
Johanna Schmidt, pp. 313–351. The information which they summarize helps us to
focus upon and understand an important objective. Its successful achievement often
requires complicated technical processes because those microorganisms which rep-
resent the study goal have evolved to naturally exist and thrive in their own
environmental milieu, under conditions that perhaps require very specific physical
and chemical parameter ranges.
Selective physical isolation procedures, including particle size class separations,
often can be developed as a helpful early step to aid with identifying the microbes
present in an environmental sample. The next task is a requirement to replicate the
conditions under which the microorganisms would have been growing in their
natural environment. If existing cultivation media formulations prove inadequate,
then it may be necessary to design new cultivation media and include some provision
of appropriate energy sources such as light of particular wavelength ranges. Flow
cytometry is one of the techniques that can be used to assess cultivation efficiency.
Gene sequencing is the most accurate testing approach for determining if the
resultingly isolated and cultivated organisms either are newly discovered or
10 C. J. Hurst

Fig. 1.1 The image shows eutrophication at a waste water outlet in the Potomac River,
Washington, D.C. It is titled “Potomac green water” by Alexandr Trubetskoy and used under the
Creative Commons Attribution-Share Alike 3.0 license

previously have been described, and for learning if perhaps those organisms have
previously even been named by other researchers.

Compliance with Ethical Standards

Conflict of Interest Christon J. Hurst declares that he has no conflict of interest.

Ethical Approval This article does not contain any studies with human participants or animals.
1 Understanding Aquatic Microbial Communities 11

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Chapter 2
Relationship Between Lifestyle
and Structure of Bacterial Communities
and Their Functionality in Aquatic Systems

Luca Zoccarato and Hans Peter Grossart

Abstract Aquatic bacteria dwell in an unexpectedly heterogeneous world defined

by multiple specific microhabitats of differing complexity, size, and temporal-spatial
dynamics. The diversity in microhabitats is proposed as a potential explanation for
the “plankton paradox” wherein high levels of species and functional richness are
maintained under limited resources. Microhabitats can be classified into broad
groups including the diffusion-controlled water phase (DifP), colloidal phase
(nano- and micro-gels; ColP), particles (exudates, carcasses, and aggregates; Par),
and the living biosphere (algae, zooplankton, and fish; Bio). For each microhabitat,
this chapter examined the various physiochemical properties and principal dynamics
that define these environments and then linked these with major lifestyle strategies
adopted by the bacteria inhabiting these systems. Within this context emphasis was
placed on the revision of the concept of free-living bacteria.
Expectedly it was found that each microhabitat, in fact, selects for distinct
functional guilds. The DifP-associated community is regulated by the dynamics of
dissolved organic matter quality and quantity (e.g., enzymatic dissolution of poly-
meric materials). The communities associated with the ColP component were driven
by the rapid ephemeral nature of gel structures assembled from physical polymer
interactions. The Par microhabitats sustain dense bacterial communities with
remarkable metabolic activity that can result in the complete or partial dissolution
of the particle. Interestingly, Par-associated bacteria are in a state of constant
attachment and detachment in response to physiochemical evolution of the micro-
niches. Moreover, Bio-associated epi- and endobionts present additional levels of
complexity due to the influence of the host-bacteria interfaces, which experience
continued change in response to environmental and biological stressors. Understand-
ing these interactions at local scale is critical for the comprehension of the impact of
anthropogenic activities on microhabitat complexity and is necessary for enabling us
to understand how microhabitat features scale up to major aquatic biomes.

L. Zoccarato (*) · H. P. Grossart (*)

Department of Limnology of Stratified Lakes, Leibniz Institute of Freshwater Ecology and
Inland Fisheries, Stechlin, Germany
e-mail: zoccarato@igb-berlin.de; hgrossart@igb-berlin.de

© Springer Nature Switzerland AG 2019 13

C. J. Hurst (ed.), The Structure and Function of Aquatic Microbial Communities,
Advances in Environmental Microbiology 7,
https://doi.org/10.1007/978-3-030-16775-2_2
14 L. Zoccarato and H. P. Grossart

Table 2.1 Overview on the proposed microhabitat definitions

Microhabitats Size range Major dynamics
Diffusion-con- DifP
0.1 μm to
6  Temporal-spatial heterogeneity in autochthonous
trolled water μm and allochthonous dissolved compounds
phase
Colloidal phase ColP  Highly dynamic conditions at short time scales
(nano- and micro-gel formation)
 As matrices providing ephemeral nutrient
hotspots leading to abrupt fluctuations of the bac-
terial community composition
 Increased nutrient and polymer turnover com-
pared to DifP
Freshwater and Par >0.8 μm to  Particle and bacterial community composition
marine particles several cm and mutually affect each other
m  High temporal-spatial dynamics in particle com-
position leading to different degradation processes
and time scales
 Vertical bacterial dispersion due to particle
sinking
 Specific food web interactions
Living biosphere Bio  Host physiology changes in response to biolog-
ical and environmental factors
 Unspecific and specific interactions between
bacteria and their host (symbioses, parasitism,
commensalism, etc.)
 Changes in bacterial adaptations and behavior
shifts the association with the host, e.g., from
commensalism to parasitism (may also kill the
host)

2.1 Conceptual Idea

Bacteria—called “the unseen majority” less than 20 years ago (Whitman et al.
1998)—nowadays are known to represent the richest life domain in terms of species
diversity and metabolic functionality. Dwelling in almost all ecosystems of the
planet, bacteria show a nonrandom spatial community distribution; however, at
this coarse observation scale, most factors causing the observed patchiness are still
unclear. In this chapter, we try to tackle this issue by embracing the bacterial
perspective in order to shed light on their microscale behavior taking into account
the multitude of different temporal and spatial scales as well as different levels of
environmental and organizational complexity.
The close surroundings of a bacterial cell in aquatic environments consist of
several specific microhabitats extending from μm to several mm and cm (Table 2.1):
the diffusion-controlled water phase, the colloidal phase (nano- and micro-gels), the
marine and freshwater particles (exudates, carcasses, aggregates), and the living
biosphere (algae, zooplankton, fish, etc.) (Grossart 2010). Each of these
2 Relationship Between Lifestyle and Structure of Bacterial Communities. . . 15

microhabitats is characterized by a different range of chemical and physical gradi-

ents and, therefore, harbors a certain level of heterogeneity and complexity provid-
ing multiple conditions for microbial micro-niche formation. These micro-niches
form the basis for microbial diversity and functionality and thus may explain the
so-called plankton paradox (Hutchinson 1961). Hutchinson was the first researcher
wondering about “how it is possible for a number of species to coexist in a relatively
isotropic or unstructured environment” since they were “all competing for the same
sorts of material”? Nowadays, modern investigative technologies provide evidence
of the remarked heterogeneity of chemical and physical properties associated with
each of the microhabitats given in Table 2.1 and how aquatic microbial communities
are adapted to exploit this diversity (e.g., Salcher et al. 2013) and how they interact
(Garcia et al. 2015). The view of aquatic environments as “relatively isotropic or
unstructured environments” has drastically changed during the past decades by
recognizing different levels of complexity when moving from the diffusion-
controlled water phase to the colloidal and particulate phase and lastly to the living
biosphere.
Major abiotic factors influencing microbial microhabitats are light/UV penetra-
tion, temperature, oxygen, pH, pollutants, organic matter (OM) concentration and
composition, chemical gradients, etc. (e.g., Church 2008) but also numerous biotic
factors such as host physiology, symbiosis, commensalism (auxotrophy), parasitism,
predation, competition (including antibiotics and toxins), and viral lysis (e.g.,
Burghardt and Oswald 2015). Moreover, abiotic and biotic factors dynamically
interact and hence can mutually affect each other (Kirchman 2012). As a result,
this microscale diversity affects bacterial community composition by selecting for
those organisms which can rapidly adapt to the existing microhabitat conditions, and
in turn the bacterial activity of the resulting microbial communities will modify and
shape the microbial microhabitat. Interacting with the environment (exoenzymatic
activity, uptake, and secretion), bacteria modify the chemical composition of their
microhabitats by such processes as consuming and transforming available substrates,
as well as secreting new compounds (metabolic by-products, antibiotics, toxins). In
addition, the highly variable and heterogenic composition of micro-niches may
result in different patterns of microbial presence and microbial activities which
form as characteristic features of aquatic environments, e.g., eutrophic coastal waters
with extremely high particle concentrations vs. the oligotrophic, open ocean with a
much more uniform microbial microenvironment. This temporal and spatial vari-
ability of microhabitats at different temporal and spatial scales can further increase
diversity, functionality, and complexity of aquatic microbial communities evolving
in time and space.
Since microbial microhabitats and micro-niches are not constant in time and
space, we propose certain features of the major, above defined microbial
microhabitats:
The diffusion-controlled water phase (DifP) (Fig. 2.1a) represents microhabitats
(for truly free-living microorganisms) that are controlled by the temporal-spatial
dynamics of dissolved molecules, which, e.g., are generated by organismic exuda-
tion and enzymatic dissolution of polymeric materials such as autochthonous and
16 L. Zoccarato and H. P. Grossart

Fig. 2.1 Schematic

representation of the
principal aquatic bacterial
microhabitats such as the
diffusion-controlled water
phase, colloidal phase (a),
freshwater and marine
particles (b), and living
biosphere (c). The
microhabitats are spatially
organized according to the
increase in size, the micro-
niche’s number and
complexity, and the time
scale of the dynamics which
pervade the microhabitat.
The blue halos indicate the
gene richness of the
bacterial genomes present in
the microhabitats. The
dotted lines show the
possible attachment and
detachment of the
chemotactic and generalist
bacteria that switch from
free-living to particle-
attached lifestyle or vice
versa

allochthonous particulate OM. Bacteria adapted to these microenvironments are

directly dependent upon the physical-chemical changes of the surrounding water
phase, with any chemotactic bacteria being able to follow the spatial and temporal
distribution of the dissolved compounds and the non-motile bacteria which remain
randomly distributed consuming those compounds diffusing toward them (Stocker
2012).
The colloidal phase (ColP) (Fig. 2.1a) microhabitats encompass highly dynamic
conditions at short time scales and, therefore, harbor the most ephemeral and
dynamic micro-niches. The fast, spontaneous assembly and subsequent dispersion
of the gel structures deeply affect the chemical and physical conditions of the
microhabitats (Verdugo 2012) leading to variable kinetics of the uptake processes,
2 Relationship Between Lifestyle and Structure of Bacterial Communities. . . 17

different availabilities (e.g., labile vs. refractory) of the bacterial substrates and
nutrients (Simon et al. 2002; Stocker et al. 2008), and abrupt changes in environ-
mental conditions, and additionally the rapid exploitation of nutrient patches within
a gel structure can induce spontaneous fluctuation of the bacterial population that
was occupying that micro-niche.
Freshwater and marine particle (Par) (Fig. 2.1b) microhabitats can be completely
or partially consumed by the associated bacteria, and the remaining refractory
fraction can eventually sink controlling the efficiency of the oceanic carbon pump
(i.e., the abiotic and biological processes of atmospheric carbon dioxide sequestra-
tion in the deep ocean and sediment) (Volk and Hoffert 1985; Jiao et al. 2010);
different sinking rates can also influence the colonization process of the particles
(Turner 2002) and favor bacterial dispersion. The bacterial community evolves over
time in response to modifications of the physiochemical properties; e.g., a sinking
particle enriched in organic substrates is colonized faster by motile bacteria as
compared to a particle with low substrate concentrations since many bacteria can
detect the OM plume surrounding OM-rich particles. Moreover, it has been
suggested that the mean residence time of motile bacteria on a particle is about 3 h
pointing to a continuous exchange between free-living and particle-attached bacte-
ria. The dynamics of the colonization process is a feedback of the changing chemical
environment in the Par microhabitats (Kiørboe and Thygesen 2001; Kiørboe et al.
2002). Shifts in community composition are correlated with alterations of the
metabolic capability of the bacterial community (e.g., Fontanez et al. 2015), which
in turn differently affects and modifies the composition and properties of the particle
microhabitat. Par microhabitats also represent foraging hotspots for bacterivores and
have relevant implications for the trophic food webs since the high prey density leads
to a higher predation efficiency (e.g., limiting energy consumption for prey
retrieval); thus the attached bacterial populations are top-down controlled by grazing
activities which decrease the interspecies competition and increase the biodiversity
of the microhabitats (Kiørboe et al. 2004; Corno et al. 2013).
The living biosphere (Bio) (Fig. 2.1c) microhabitats share part of the temporal-
spatial dynamics of Par microhabitats. In addition, these microhabitats are influenced
by changes in the host-bacteria interface such as modifications of the host physiol-
ogy in response to environmental and biological stressors (Grossart 1999; Grossart
et al. 2005; Amin et al. 2012; Glasl et al. 2016; Seymour et al. 2017). The diatom
Thalassiosira weissflogii, for example, produces transparent exopolymer particles
(TEP), which are responsible for aggregation dynamics, albeit that production occurs
only in the presence of specific associated bacteria. This production process is
controlled by the photosynthetic activity of the diatom, has relevant consequences
on algal sinking, and therefore influences the efficiency of the carbon export (Gärdes
et al. 2011). Bacteria similarly might change their behavior in response to environ-
mental stressors with remarkable implications for the host (Vezzulli et al. 2012), and
such is the case for the Flavobacterium Kordia algicida, which lives associated with
several diatom species. When the population of K. algicida reaches a certain
threshold, an algicidal protease is excreted through a quorum sensing-triggered
mechanism, which renders the commensalistic relationship with the algal host to a
18 L. Zoccarato and H. P. Grossart

parasitic one (Paul and Pohnert 2011). Croceibacter atlanticus was shown to prevent
the algal host to divide and leading it to increase in size (i.e., colonizable surface)
(van Tol et al. 2016).
To date, despite the outstanding heterogeneity of these microhabitats, some of
them are largely overlooked in ecosystem-based investigations (in particular, bacte-
ria associated either with particles or organisms) leading to inaccurate estimations of
the measured microbial processes on a larger scale. Nowadays, new technologies
provide novel and unexpected tools to investigate the genomic diversity and meta-
bolic potential of aquatic bacteria offering the opportunity to tackle with unprece-
dented precision the task of understanding each single type of microhabitat.
Techniques such as next-generation sequencing (also called high-throughput
sequencing) and the related “omics” approaches help to uncover the composition
of the prokaryotic communities as well as their manifold functionalities and phys-
iological responses (Shendure and Ji 2008; Moran et al. 2013). Furthermore,
advances in sequencing and chemistry methodologies enable single amplified
genome (SAG)-based studies linking individual cell traits with potential functions
in the ecosystem. The SAG studies, in combination with new mass spectrometric
(MS) techniques such as MS-MS, and desorption electrospray ionization combined
with MS (DESI-MS), allow the metabolome characterization of single organisms
and thus facilitate deep investigations of both cell-to-cell and cell-to-particle inter-
actions. These approaches help to shed light on microbial dynamics in the Par and
Bio microhabitats.

2.2 How Much Free-Living Is Free-Living?

The differentiation between free-living (FL) vs. particle-attached (PA) bacteria is

based on a conceptual distinction that often lacks a real ecological meaning. Bacteria
are considered FL if they can pass through a filter of a certain pore size, whereas PA
bacteria will be retained on the same filter. This distinction has been earlier intro-
duced for separating organic matter fractions, i.e., dissolved OM (DOM) and
particulate OM (POM). Historically, the threshold in size of the mesh or filter used
to separate the different bacterial fractions was chosen arbitrary and still often varies
among disciplines and between individual studies. Today, there is no general
consensus, and the threshold can vary from 0.8 μm (e.g., Smith et al. 2013) to
5–10 μm (e.g., Bižić-Ionescu et al. 2014).
This methodological inconsistency might be responsible for the partly contradic-
tory conclusions regarding the phylogenetic patterns in community composition of
FL vs. PA bacteria. Some authors have tried to correlate the occurrence of typically
copiotrophic (those found in environments rich in nutrients) and generalist bacterial
groups such as Gammaproteobacteria and Flavobacterium with sources of POM,
while oligotrophic-adapted bacteria (those which typically survive in environmental
conditions that have much lower carbon concentrations) tend to primarily occur in
the dissolved fraction (Biers et al. 2009; Ortega-Retuerta et al. 2013; Rieck et al.
2 Relationship Between Lifestyle and Structure of Bacterial Communities. . . 19

2015). Contrariwise, other researchers have pointed to a more random bacterial

colonization of the POM and, therefore, to the absence of a consistent pattern in
FL vs. PA bacteria communities (Hollibaugh et al. 2000; Ghiglione et al. 2007).
Although we agree with the “lottery” theory for the very initial colonization, which
increases a certain degree of randomness (e.g., seasonality of FL bacteria which
represent the bulk for the “lottery drawings”; Rösel and Grossart 2012), an increas-
ing number of studies highlight marked genomic differences among copiotrophic
and oligotrophic bacteria, which strongly support the existence of a consistent
functional pattern in microbial distribution and community structure among the
various microhabitats (Grossart 2010; Burke et al. 2011; Tang et al. 2014;
Giovannoni et al. 2014).
However, in nature the distinction between DOM and POM is anything but sharp.
In the literature it is described that OM in the environment forms a size continuum
(Azam 1998; Verdugo et al. 2004). Molecules and single polymers in solution
undergo a passive diffusion and can spontaneously coagulate or aggregate to form
marine micro- and macro-gels. These tridimensional structures are formed continu-
ously by purely physical and chemical processes. A physical gel is formed by
polymer interconnections through tangles and cross-links (electrostatic interactions
based on the polymers’ charge density and interactions among hydrophilic and
hydrophobic domains), which are characterized by low activation energy
(<50 kJ mol1). Thus the formation of these gels is a fully reversible process with
a high dynamic in the dispersion and self-assembly equilibrium that stretches from
the polymers’ dispersion phase to polymer aggregation in nanogels (0.1–0.2 μm) and
eventually micro-gels (3–6 μm) (e.g., Verdugo 2012).
The dichotomic distinction between FL-PA bacteria and DOM-POM might not
be enough as the natural distribution of the realistic size structure (both organisms
and OM) within an ecosystem cannot be reduced and just simplified into two size
classes. Therefore, a possible solution is to consider more precise size fractions,
establishing the size threshold based on the true size of particles present within each
specific environment. A more precise distinction of size classes (Mestre et al. 2017)
or particle types with a defined sinking behavior (i.e., specific residence time in the
water column) is necessary to achieve a meaningful ecological simplification of the
real world. It also must be understood that particle size and type distribution within
eutrophic environments are significantly different from that of ultra-oligotrophic
environments (e.g., upwelling areas will have more and larger particles than the open
ocean but presumably less and smaller particles than in estuarine areas).
In order to avoid confusion with the cited literature, in the following sections, we
will keep the distinction between FL and PA bacteria based on the threshold adopted
in the cited paper. However, we will consider the colloidal phase not as a
homogenously diluted environment but rather as a heterogeneous puzzle in which
some pieces are represented by micro-gel structures.
20 L. Zoccarato and H. P. Grossart

2.3 Microhabitat Descriptions

2.3.1 Diffusion-Controlled Water Phase (DifP) and Colloidal

Phase (ColP)

2.3.1.1 Physiochemical Characterization and Major Dynamics

The aquatic environment surrounding the cell is an extreme diluted environment

(Fig. 2.1a) where nutrients and substrates are relatively dispersed (McCarthy et al.
1996). The foraging distance might play a crucial role since at the cell scale, the
aquatic medium is highly viscous (characterized by a low Reynolds number) which
can involve diffusion limitations. Free-living bacteria can be roughly categorized as
either immotile waiting for the food to come to them or choosing to hunt by
swimming and thereby increase their encounter rate with the food particles. Being
motile means that their food intake would increase only with the square root of the
bacteria’s velocity (Purcell 1977). As an alternative intermediate option, the bacteria
can move toward patches with a higher nutrient concentration than the surrounding
water, e.g., micro- and macro-gels, particles, aggregates, etc. It is worth pointing out
that some cells and colonies due to their sizes or shapes with a Reynolds number > 1
can escape the diffusion limitation and experience the advantage of advection
processes (such as catabolite dispersion) (KarpBoss et al. 1996).
Autotrophic bacteria mainly rely on the availability of inorganic nutrients
(Agawin et al. 2000), and their suitable niches are constrained by the need for
light. Inorganic nutrient concentrations are in turn affected by the metabolism of
heterotrophic bacteria, which rely on the availability and composition of the OM
(Hansell and Carlson 2002). However, a large fraction of bacteria in global super-
ficial seawater have shown the capability to utilize both sunlight and OM
circumventing conditions of energy and carbon limitation; these photo-heterotrophic
groups are known as proteorhodopsin-containing bacteria (Béjà et al. 2000) and
aerobic anoxygenic phototrophic (AAP) bacteria (Yurkov and Csotonyi 2009;
Koblížek 2015). The consumption of OM by these organisms through enzymatic
cleavage is coupled with the release of inorganic compounds (e.g., nitrogen and
phosphate), in a process called mineralization of the OM. Thus the distribution and
bioavailability of the OM have dramatic impacts on bacterial community structure
and function. The DOM (which comprises the major fraction of compounds forming
marine gels) represents the largest OM pool, being roughly ten times more abundant
than POM in most aquatic systems, although the DOM present in the open ocean is
predominantly recalcitrant and poorly bioavailable (Williams et al. 1969).
As argued in the previous section (Sect. 2.2), the distinction between DOM and
POM is entirely dependent on methodological constraints since the polymers which
make up the DOM undergo continuous processes of nano- and eventually micro-gel
formation. The marine gels are hotspot of nutrients and OM of the ColP microhab-
itats, since they generally contain a solid/solvent ratio of
1% and thus higher OM
concentrations than DifP microhabitats (i.e.,
10 g L1 against
103 g L1 of the
2 Relationship Between Lifestyle and Structure of Bacterial Communities. . . 21

sea water). The gel formation process is greatly influenced by properties of the water
mass (dielectric properties, osmolarity, pH, and ionic composition) and local envi-
ronmental factors (temperature and pressure) but also by the quality of the polymers
(length and chemical properties) and by their concentrations, which can also influ-
ence the dimension and the stability of the gels (Verdugo 2012 and references
therein).
Input/output and transformation of the DOM have strong implications for the
three-dimensional structure of the ColP microhabitats. The principal DOM input
stems from algal exudates, sloppy feeding associated with zooplankton grazing, and
cell lysis mediated by viral infection and by bacterial lysis (on phytoplankton or
other bacteria) (Carlson 2002 and references therein). Moreover, DOM plumes
created by bacterial POM degradation provide a rich, although ephemeral hotspot
of polymers and nutrients which might be rapidly consumed by FL bacteria (Smith
and Azam 1992; Kiørboe and Jackson 2001) or undergo DifP and ColP microhabitat
formation. Biodegradation represents one of the major processes that affect the
concentration and composition of the present DOM pool. Bacteria can rapidly
degrade carbohydrates, proteins, lipids, and organic acids which represent the labile
fraction of the DOM (although the composition varies according to DOM origin) but
in turn also secrete new compounds and leave the more refractory fractions behind
(Kirchman 2003). Similar to biodegradation, photolysis can greatly modify the
DOM pool by breaking apart polymers and large molecules into small compounds
(e.g., phenol, acetate, and propionate) which can in turn be degraded by the micro-
bial community stimulating their growth and metabolism (Moran and Zepp 1997;
Hutalle-Schmelzer et al. 2010). Refractory, high molecular weight compounds can
also be produced by photooxidation processes devoting a fraction of DOM to
biodegradation (Obernosterer et al. 1999). This physical process can also produce
harmful compounds such as hydrogen peroxide which can induce modifications in
the composition of the associated bacterial community selecting for species resistant
to reactive oxygen species (Scully et al. 2003; Glaeser et al. 2010; Glaeser et al.
2014).
Gel formation has a critical importance for nutrient and metabolite uptake of
bacterial cells since it reduces the dilution of the DOM pool in water. DOM
represents a huge biomass (
700 gigatons, where 1 Gt ¼ 1015 g), but it can be
evenly distributed with final molar concentrations in the order of 106 mol L1. In
contrast, within a micro-gel a particle of 600 Da (upper threshold for microbial
uptake) can reach OM concentrations of 10 g L1. As a result, the matrix of the gel
limits the DOM dispersion of the enzymatically cleaved compounds and thus
decreases the necessary foraging distance allowing bacteria to save energy when
obtaining their nutrient supply (Verdugo et al. 2008) and to increase their growth
yield (Fernandez et al. 2019).
22 L. Zoccarato and H. P. Grossart

2.3.1.2 Bacterial Adaptations: To Eat and Not Be Eaten

The two major concerns of each living organism are to eat and not to be eaten.
Therefore, the FL bacteria have developed several strategies to exploit the available
micro-niches paying attention to these constrains. As a consequence, FL bacteria
tend to be streamlined both in cell and genome size to reduce their expenditures for
maintenance energy and their vulnerability to grazing.
A small cell size offers several advantages to the organism such as a high surface-
to-volume ratio, which maximizes their capabilities for nutrient/substrate uptake and
thus provides a physiological advantage against larger cells in nutrient-limited
environments (Kirchman 2012). Moreover, a reduced cell dimension is a useful
strategy to avoid grazing losses. The freshwater Actinobacteria, for example, have
cell dimensions of 0.1 μm in diameter (ultramicrobacteria), and their populations
reach maximum abundances in the presence of heterotrophic nanoflagellates. This
clearly points to a negative grazing selection on the basis of prey size (Jezbera et al.
2006; Grujčić et al. 2015). Other abundant clades of ultramicrobacteria include the
marine Alphaproteobacteria SAR11 and the freshwater sister group LD12. The
Alphaproteobacteria SAR11 has been defined as cryptic fugitive, being almost
“invisible” to grazers due to its reduced biomass and slow growth rate (Yooseph
et al. 2010). Other strategies adopted from FL bacteria to avoid predation are the
formation of large colonies. This cellular organization confers additional buoyancy
and circumvents the handling capability of smaller grazers (e.g., heterotrophic
nanoflagellates). Filament formation is a common but diffuse trait among different
phylogenetic groups such as Cyanobacteria, Proteobacteria, and Bacteroidetes
(Hahn et al. 1999). Nevertheless, these bacterial colonies can be efficiently ingested
by larger predators such as the zooplankton (e.g., Schauer et al. 2006) with remark-
able effects on the carbon flux throughout the trophic food web. Despite these
favorable traits and the fact that filamentous bacteria can account for up to 50% of
the total bacterial biomass (Corno et al. 2008), filament-forming colonies do at times
represent a low percentage of the total FL abundance, and their direct ingestion by
the zooplankton represents an energetic shortcut toward the upper trophic levels
(since each trophic level involves a loss of energy in the C-/biomass transfer).
Another mechanism of defense against predation is the production of toxins, e.g.,
microcystins from Cyanobacteria, that are triggered by the presence of grazers (Jang
et al. 2003; Ger et al. 2016).
Genome streamlining is typical in FL bacteria (Ghylin et al. 2014) (Fig. 2.2) and
generally involves the reduction of regulatory genes (e.g., σ factor) and an increase
in relative abundance of core genes (Giovannoni et al. 2014). However, some
relevant exceptions have been reported, e.g., for the Prochlorococcus strains
MIT9303 and MIT9313 (Kettler et al. 2007). Living in a highly diluted environment,
FL bacteria encounter a reduced variety of exploitable compounds, and several
ancillary genes might be poorly expressed. When a gene became “useless,” because
of the lack or the ephemeral nature of the triggering stimuli, the occurrence of a
possible disruptive mutation will not cause any functional disadvantage to the
2 Relationship Between Lifestyle and Structure of Bacterial Communities. . . 23

Fig. 2.2 The figure reports the relative genome size among different categories of bacteria
(horizontal gray bars) and correlates their occurrence in the microhabitats (vertical black bars)

carrying organisms. The absence of a negative selection, therefore, will not decrease
the fitness of these bacteria, leading eventually to loss of functional genes (Ochman
and Davalos 2006 and references therein). Having a streamlined genome allows FL
bacteria to reduce their growth and replication costs (Ochman and Davalos 2006), a
feature that endorses for their dominance in such nutrient-depleted environments.
A remarkable example is the Alphaproteobacteria SAR11, which dominates the
bacterial communities in the oligotrophic ocean thanks to a streamlined genome, an
optimal surface-to-volume ratio, and the presence of several high-affinity membrane
transporters (Lauro et al. 2009). Sometimes genome streamlining is so pronounced
that even core genes can be lost in the presence of well-established mutualistic
interactions among different species. This process has been conceptualized as the
Black Queen Hypothesis that explains positive selection of bacteria with disruptive
mutations of core genes when these genes code for functions that are costly and
leaky in the community (Morris et al. 2012). Vitamins and amino acids are some of
these “public” goods whose production and availability become essential for several
streamlined bacteria leading them to the so-called auxotrophy for these compounds
(Garcia et al. 2015). Genome streamlining, however, is not a compulsory require-
ment for FL bacteria, and relevant differences in the ancillary-core gene ratio have
been reported for the same clade due to long-term adaptation processes. Lauro et al.
(2009) reported on how genomes of cyanobacteria dwelling in the open ocean have
more oligotrophic traits than do cyanobacteria from freshwater ecosystems. In
contrast, the copiotrophic gammaproteobacterium, Photobacterium angustum, has
a broad genetic repertoire of genes to sense and react to environmental cues such as
24 L. Zoccarato and H. P. Grossart

sudden nutrient influx or depletion; it is even able to switch its lifestyle and
metabolic pathways in response to depletion of nutrient patches, e.g., by changing
from a PA to a FL lifestyle (Lauro et al. 2009). Among FL bacterial communities,
there are several other examples of copiotrophic bacteria often reported as abundant
species in their ecosystem. The relative abundance of the Betaproteobacteria
Limnohabitans and Polynucleobacter may result from their ability to exploit patches
of low molecular weight compounds such as simple organic acids, monosaccharides,
and photooxidated humic compounds, and by virtue of these species having rela-
tively fast growth rates (see Salcher 2014). Under extremely oligotrophic conditions,
some bacteria have specifically adapted to share available but limiting resources
(Salcher et al. 2013; Garcia et al. 2015).
Bacteriochlorophyll and proteorhodopsin are common traits of FL bacteria as
these pigments are involved in processes which help FL bacteria cope with
extremely oligotrophic conditions. Bacteriochlorophyll A is characteristic for AAP
bacteria which allows them to create a transmembrane reductive potential harvesting
green light. However, these organisms do not contain Rubisco and consequently
fully rely on organic carbon for growth (Yurkov and Csotonyi 2009). It has been
estimated that the AAP bacteria represent about 24% of total bacteria in most
oligotrophic aquatic environments (Lami et al. 2007). The coding potential for the
proteorhodopsin, a transmembrane light-driven proton pump known to support some
of the most costly cellular energetic processes such as the uptake of vitamin B1
(Gómez-Consarnau et al. 2016), has been detected in up to 75% of marine bacteria
(especially, Gammaproteobacteria and Bacteroidetes).
Generally, FL bacteria present marked seasonal fluctuations in population size
since they are directly exposed to environmental fluctuations (while particles and
hosts represent relatively stable bacterial refuges). Whenever environmental condi-
tions result in changes of the present micro-niches, the existing FL bacteria popula-
tion experiences a drastic decrease in favor of those bacteria equipped with the
respective metabolic machinery to efficiently exploit the new conditions (e.g.,
Fuhrman et al. 2006).
Among free-living bacteria, there is increasing evidence for the presence of
organisms shifting from a passive to an active chemotactic lifestyle exploiting
patches of OM and nutrients, and some otherwise free-living bacteria might also
be involved in Par and Bio microhabitat colonization (Grossart 2010). It has to be
kept in mind that the conditions allowing for this “microhabitat jump” are expected
to be quite stringent since the metabolic potential of FL bacteria differs significantly
from that of typical PA bacteria due to the high degree of specialization and also the
genome streamlining of FL bacteria (Ghylin et al. 2014; Giovannoni et al. 2014).
Perhaps the “traditional” concept of FL bacteria, which would suggest them to be
mostly passive and waiting for their food particles to arrive, needs to be
reconsidered. Genomes of FL oligotrophic-adapted bacteria are still poorly explored
due to the historical cultivation limitations which recently have been overcome
thanks to SAG approaches. SAG analyses are unveiling the extreme adaptation of
these bacteria by revealing not only their substrate specialization (Rinke et al. 2013)
but also some “unexpected” versatility; hereinafter we briefly highlight two
2 Relationship Between Lifestyle and Structure of Bacterial Communities. . . 25

interesting examples of specialized bacteria and one example of generalist FL

bacteria. Genome analyses of the widespread FL Actinobacteria acI have described
its preference for carbohydrate and N-rich organic compounds highlighting a further
level of specialization for the clade acI-A and acI-B in substrate utilization (Ghylin
et al. 2014). The Alphaproteobacteria Caulobacter crescentus has a dimorphic life
cycle that separates a chemotactic behavior in the presence of nutrients and an
immotile but reproductive stage. This species is consequently equipped with genes
that mediate the environmental sensing of carbon and ammonium or amino acid
starvation and then favor chemotactic behavior and genes that mediate adhesion to
nutrient patches (Boutte and Crosson 2013). The proteomic insight of the
Alphaproteobacteria Ruegeria pomeroyi DSS-3 revealed that 30% of its genes
encode for basal living function (housekeeping genes), while 20% are responsible
for physiological responses to the induced environmental perturbations within the
“natural” ranges of variability for these factors (salinity, temperature, UV, aromatic
compounds). Interestingly, 50% of its proteome was never expressed during exper-
imental treatments suggesting that very specific physiological responses are required
to exploit different substrates and thus express a generalist lifestyle (Christie-Oleza
et al. 2012).

2.3.2 Marine and Freshwater Particles (Par)

2.3.2.1 Physiochemical Characterization and Major Dynamics

The Par microhabitats are extremely heterogeneous environments as particle com-

position can be significantly different depending on their origin, chemical composi-
tion, and age (Fig. 2.1b). Marine habitats are mainly characterized by the occurrence
of autochthonous POM (produced by organisms inhabiting that ecosystem), while
freshwater systems can experience a significant input of allochthonous terrestrial
POM depending on the degree of the aquatic-terrestrial coupling. Furthermore,
autochthonous OM in aquatic systems differs in bioavailability and turnover
depending on its mode of production: (1) direct (excretion and exudates) or (2) indi-
rect (body fragment, carcasses).
Importantly, Par microhabitats are quickly and intensively colonized by bacteria
whose fraction can represent 25% of the total bacterial abundance in the epipelagic
zone, showing a tight correlation with algal abundance and a fast decrease with depth
(PA bacteria have a relative abundance <4% in mesopelagic layers; Mével et al.
2008). Immediately after the attachment, bacteria are able to activate a transcrip-
tional response and increase their enzymatic activities (Grossart et al. 2007).
Although they do not represent the majority of the bacterial community in most
aquatic environments, PA bacteria usually account for >50% of the total bacterial
activity (Ghiglione et al. 2007; Grossart et al. 2007; Rösel and Grossart 2012;
Stocker 2012), and extremely high values were recorded in areas characterized by
elevated trophic status (e.g., up to 80% of the total bacterial activity in a mesotrophic
26 L. Zoccarato and H. P. Grossart

area of the NE Mediterranean Sea; Ghiglione et al. 2007). The reason for these high
fractions of PA bacteria lies in the tight coupling with the available substrate in the
Par microhabitats and the likely metabolic cooperation among PA bacteria (Wahl
et al. 2012).
Marine particles are mainly represented by autochthonous POM, and its compo-
sition is, therefore, strictly linked to the local organisms producing it. Three major
categories of autochthonous POM can be distinguished: (1) exudates, (2) excretions,
and (3) detritus and carcasses. Exudates (1) are mainly composed of polymers such
as carbohydrates, proteins, and glycoproteins which are continuously released by
algae (e.g., diatoms, cyanobacteria) and that can reach remarkable concentrations in
the surface waters during algal bloom events. The chemical composition of these
substrates is related to both algal physiology and metabolism, which can vary for the
same species living in different environments (Becker et al. 2014). The composition
of the exudates eventually shapes the bacterial community by favoring that guild of
bacteria which can most efficiently exploit and degrade the aggregates; a clear
example is the tight coupling between the algae and bacteria successions during
bloom events (Bunse et al. 2016; Needham and Fuhrman 2016). In turn, some
bacteria are capable of interacting with the algae and deeply influence the bloom
dynamics controlling both bloom development and aggregation processes (Grossart
et al. 2006; Amin et al. 2015). Organic matter excretions (2) represent almost one
third of the ingested food of marine organisms such as zooplankton (e.g., copepods
and salps) and fish. Particles such as fecal pellets are rapidly colonized by PA
bacteria but also present relevant abundances of enteric bacteria which were released
from the digestive tracts of the host (Bio microhabitats, see below). These Par
microhabitats are characterized by several factors such as a different physical
structure and origin (e.g., copepod excretions are enveloped by a peritrophic mem-
brane which delays their remineralization process) that affect their sinking rates (i.e.,
small-size excretions are mostly degraded by bacteria in the water column, while
larger and heavier excretions are involved in sedimentation processes). This largely
influences OM colonization and degradation (Turner 2002; Tang et al. 2010).
Detritus and carcasses (3), e.g., of phyto- and zooplanktonic organisms share the
same fate of the excretions (e.g., Tang et al. 2014) and due to their high density are
involved in the biological carbon pump (Jiao et al. 2010) as well as in the dispersion
process of attached bacteria. Particulate-associated bacteria can cover longer dis-
tances and spread across physical barriers otherwise impenetrable for FL bacteria
(e.g., thermocline and water masses fronts) (Grossart et al. 2010).
In addition to a pronounced autochthonous fraction in limnetic habitats, freshwa-
ter particles generally contain significant portions of terrestrial POM, which is often
recalcitrant, being hard to degrade due to the incorporation of minerals (e.g., Ortega-
Retuerta et al. 2013), lignocellulose compounds, and humic matter. Lignocellulose
degradation on land is carried out mostly by fungi; however, their function is not as
well known in the aquatic environments where they appear to be abundant but less
diverse and of similar importance for OM cycling as are heterotrophic bacteria
(Crowther and Grossart 2015; Grossart et al. 2019). Humic matter represents a
class of molecules with a high degree of chemical complexity; hence their
2 Relationship Between Lifestyle and Structure of Bacterial Communities. . . 27

degradation understandably might be slow, energetically demanding, requiring the

presence of specific metabolic pathways and likely favoring the co-occurrence of
specific bacterial species (Habe and Omori 2003; Grossart 2010). A remarkable
boost in the degradation of these refractory substrates in the presence of more labile
OM can be related to co-metabolism processes. This co-metabolism is known as a
“priming effect” (Horvath 1972) and is represented by mainly three hypotheses:
(1) extracellular enzymes produced to degrade the labile fraction can also cleave a
part of the refractory OM; (2) by-products formed by the degradation of labile OM
support the energetic costs of refractory OM degradation conducted by more specific
bacteria; and (3) bacteria degrading labile OM acquire enough energy to produce
specialized enzymes and thus are able to cleave the refractory OM (Guenet et al.
2010). Although the theory of priming is still largely debated, it can explain the
consumption of recalcitrant OM discharged by a river in the presence of exudates
produced by a phytoplankton bloom and possibly also the remineralization of
refractory OM originating from the deep sea in upwelling zones (Bianchi 2011).
Throughout time, all Par microhabitats evolve and change their tridimensional
structures, chemical composition, and internal gradients, along with the microbial
degradation processes that play a major role in particle turnover. The new architec-
ture in turn can trigger a succession of PA bacteria communities on particles with a
sequential selection for species with the most suitable metabolic capabilities that
further degrade and evolve in a continuous, mutualistic, and dynamic process.
Lecleir et al. (2014) found a rapid change—within 24 h from sample collection—
in the PA microbial community retrieved through sediment traps, and Fontanez et al.
(2015) with a similar approach report on a shift from DOM- to POM-associated
bacterial groups (Alteromonadales and Flavobacteriales) to pathogens and sapro-
phyte groups (Vibrionales and Campylobacterales). In addition, fungi may have a
relevant impact on OM turnover, in particular on larger particles (Grossart
et al. 2019).
Initial microhabitat colonization processes on particulates might be random and
dependent on variables that include seasonal and abiotic factors which control the
composition of the pool of potential PA bacteria waiting in DifP and ColP micro-
habitats and also be affected by the population inhabiting other Par microhabitats
(serving as seeding hotspots). Once settled onto a particulate, PA bacteria can have
energetic advantages that allow them to prevent the settlement of competitors
through activities such as antibiotic production (Long and Azam 2001; Wahl et al.
2012). These dynamics might explain why clear patterns in the composition of the
PA bacteria community are still missing, although we suggest that this variability
more likely can be due to the high heterogeneity of particles as highlighted by
Ortega-Retuerta et al. (2013) who demonstrated that the bacterial community com-
position is more correlated with the particle quality than quantity.
Their remarkable associated bacterial activity and the relatively high substrate
bioavailability make Par microhabitats pivotal hotspots for processes affecting
biogeochemical cycles, in particular OM export and remineralization of nutrients
(Simon et al. 2002). The need for a better and more comprehensive knowledge of
particle diversity and bacterial enzymatic capacity is evident, as emphasized by
28 L. Zoccarato and H. P. Grossart

demands for new, more focused investigations. The application of novel methodol-
ogies such as “omics”, NMR, and Fourier transform ion cyclotron resonance
(FTICR) mass spectrometry coupled with an improved sampling design which
takes into account the heterogeneity of Par microhabitats will refine the description
of global biogeochemical cycles.

2.3.2.2 Bacterial Adaptations

In the past years, the number of studies addressing differences between PA and FL
bacteria communities has enormously increased, and the high-throughput
metabarcoding approach is providing a more comprehensive and detailed descrip-
tion of their in-depth composition. Two important obstacles for generalizing the
obtained results are cases of organisms being able to efficiently commute between
FL and PA life strategies (Lauro et al. 2009) and the unrepresentative size fractioning
for FL and PA bacteria neglecting the true size structure (see Sect. 2.2). However,
based on the most recent literature, we suggest the existence of a large number of
guilds of copiotrophic organisms whose genomes have evolved and been selected
for a PA lifestyle. Even though these species are considered generalists, each guild
has developed specific adaptations providing a higher fitness which, therefore, leads
to their dominance on different kinds of Par microhabitats as has been described
above. In order to test for the existence of these specific bacterial guilds, we reviewed
the results of several studies evaluating the PA bacteria community composition
across different Par microhabitats, and here, we try to highlight the underlying
phylogenetic and functional patterns.
Rieck et al. (2015) analyzed the PA bacteria diversity in the Baltic Sea which
represents one of the largest brackish basins in the world due to a limited water
exchange with the open ocean and high freshwater loads from the Scandinavian rivers
entering the system in the north. The authors highlighted the dominance of
Planctomycetes and Betaproteobacteria on freshwater particles (oligohaline) and a
dominance of flavobacteria on marine particles. Among flavobacteria, Polaribacter,
Fluviicola, and Formosa genera are known to be associated with particles rich in OM
as they have several enzymes for polysaccharide cleavage (Walsh et al. 2013; Biži-
ć-Ionescu et al. 2014), and Formosa and Planctomycetes are specifically involved in
the remineralization and degradation processes of algal biomass (Pizzetti et al. 2011;
Teeling et al. 2012). These findings agree with the general seasonal patterns of Par
microhabitats. Whereas during summer autochthonous particles are produced by
phytoplanktonic blooms, allochthonous particles are introduced in the oligohaline
area by high riverine runoff during the rainy seasons in fall/winter. The dominance of
PA flavobacteria in marine areas is usually shared with other bacterial groups such as
Verrucomicrobia (Ortega-Retuerta et al. 2013) and especially Gammaproteobacteria
(Alteromonadales, Oceanospirillales, and Vibrionales) (Bižić-Ionescu et al. 2014;
Fontanez et al. 2015). Limnetic systems have a different PA community composition
revealing a dominance of Alphaproteobacteria and Betaproteobacteria over
Gammaproteobacteria, Flavobacterium, and Planctomycetes. Moreover, limnetic
2 Relationship Between Lifestyle and Structure of Bacterial Communities. . . 29

PA bacterial communities show a higher degree of specialization than do marine PA

communities (Bižić-Ionescu et al. 2014).
In the marine environment, an increasing water depth is usually associated with
strong gradients in nutrient availability, oxygen concentration, and temperature
greatly affecting bacterial abundance and community composition. However, the
diversity of PA bacteria seems to be highly correlated with particle composition
rather than with abiotic variables of the surrounding water. Studies on sinking
particles are still rare, but there is increasing evidence for their enrichment in
Epsilonproteobacteria carrying out sulfur oxidation (Sulfurimonas, Sulfurovum,
and Sulfuricurvum) as well as sulfur and nitrogen reduction processes
(Sulfurospirillum) in the absence of molecular oxygen (Fontanez et al. 2015).
Some Epsilonproteobacteria are associated with metazoan surfaces and their diges-
tive tracts (Arcobacter, Wolinella, and Campylobacter) (Gugliandolo et al. 2008)
explaining their presence with the death and sinking of the host. Of further signif-
icance is the fact that particles represent micro-niches with oxygen concentrations
that often are lower than in the surrounding water. Gradients of decreasing oxygen
concentrations were shown to be correlated with the high microbial activity taking
place on Par microhabitats that is even sufficient to maintain anoxic condition within
large particles (e.g., fecal pellets or copepod carcasses; Alldredge and Cohen 1987;
Ploug et al. 2008). The resulting micro-niches can thus host ephemeral loci for
anoxygenic processes such as nitrogen fixation, even in oxic zones of the water
column (Riemann et al. 2010; Glud et al. 2015). Indeed, Ganesh et al. (2014)
reported for Par microhabitats collected at the oxygen minimum zone an enrichment
in genes involved in the last steps of the denitrification process.
Based on these findings, we speculate on the existence of phylogenetic base
guilds of generalistic copiotrophic PA bacteria well-adapted to degrade and exploit
particles characterized by different chemical composition and architectures. The
taxonomic resolution provided for these guilds, however, is still low (phyla to
order); thus, it is difficult to describe specific functional and metabolic patterns
(some examples are given by Ganesh et al. 2014; Fontanez et al. 2015). Here, we
identified two major problematic issues: (1) methodological biases in the size
fractioning procedures which prevent a clear characterization of FL and PA bacterial
communities (particle disruption, wrong mesh size, etc.) and (2) high heterogeneity
of Par microhabitats which generate even greater differences between micro-niches
hosting a huge diversity of associated bacteria (e.g., Amin et al. 2012).
Despite these limitations, it is possible to identify some essential traits of PA
bacteria which indicate their capability to exploit the complex mixtures of polymers
forming the Par microhabitats. What is considered essential for PA bacteria are the
motility and chemotactic behaviors allowing the organisms not only to find the Par
microhabitat but also to dynamically attach onto and detach from surfaces in
accordance with their physiochemical properties (Grossart et al. 2001; Fenchel
2002; Raina et al. 2019). Once settled onto the particles, many bacteria show a
high production of antibiotic substances that act as deterrent against possible com-
petitors (Long and Azam 2001; Grossart et al. 2004) although they still experience
the top-down control by many specialized grazers (Kiørboe et al. 2004; Corno et al.
30 L. Zoccarato and H. P. Grossart

2013) and of viral infection (Suttle 2007). The genomes of PA-associated bacteria
are generally larger than the genomes of FL bacteria (Giovannoni et al. 2014),
presumably representing the fact that PA-associated bacteria often have a higher
number of genes related to their greater capability for polymeric substrate degrada-
tion. Furthermore, PA bacteria have a wide repertoire of genes coding for extracel-
lular and membrane-attached enzymes such as those for the digestion of
phytoplankton exopolysaccharides (Smith et al. 2013) and other carbohydrates
(α-mannosidase, α-L-fucosidase, and L-fucose permease; Teeling et al. 2012;
Kappelmann et al. 2019). The PA bacteria can also express a wide range of trans-
porters (e.g., TonB-dependent transporters) for the uptake of amino acids, nucleo-
tides, and coenzymes cleaved from the particles (Smith et al. 2013). A broad set of
PA bacteria-associated genes mediates bacterial anchoring to the substrate and
allows the microbes to colonize different types of particles (as well as organisms
and hosts) (Ganesh et al. 2014). Among the large fractions of unclassified genes
recognized for PA bacteria are several putative sequences for the mobilization of
DNA expression for signaling-related pathways (Allen et al. 2013). Although the
synthesis of proteorhodopsins and actinorhodopsins represents a typical trait of FL
bacteria, the AAP are also abundant among PA bacteria (e.g., Lami et al. 2009;
Simon et al. 2014). This trait has deep implications for the bacterial metabolism,
particle turnover ratio, and eventually carbon flux as the APP organisms are capable
of doubling their efficiency of organic carbon assimilation in comparison to strict
heterotrophic bacteria (Yurkov and Cssotonyi 2009). Finally, the high density of
bacterial cells which colonize Par microhabitats (as well as Bio microhabitats, see
below) allows bacteria to stay in close contact, a condition that is quite rare in DifP
and ColP microhabitats. The proximity of bacterial cells favors processes like
conjugation and lateral gene exchange (Paul 1999) rendering particles as possible
seeding spots for genetic exchange which further increases the variability and
functionality of PA bacteria (Ganesh et al. 2014).
Overall, Par microhabitats have been often overlooked in the past, and so far, the
effect of PA bacteria on microbial diversity and functionality in aquatic ecosystems
has been greatly neglected. Nowadays, the increasing utilization of flow cytometry
to estimate bacterial abundances might represent a possible new source of bias; while
this technique provides unquestionable advantages in terms of resolution and anal-
ysis time, it completely ignores PA bacteria that have to be counted “manually”
which many scientists avoid doing. Generally, the PA fraction of the microbial
community (including also PA fungi and protists) exhibits higher metabolic activ-
ities than do microbes inhabiting the DifP and ColP microhabitats. Therefore, PA
microbes have a significant impact on the balance of several biogeochemical cycles
(e.g., carbon and nitrogen). As briefly reported above, some studies have started to
shed light on the dynamics of biological processes associated with Par microhabitats
although it will be crucial to further focus our research efforts on the functioning and
efficiency of these processes in order to provide more accurate and reliable estima-
tions on the role of aquatic systems in global C, N, and P fluxes.
2 Relationship Between Lifestyle and Structure of Bacterial Communities. . . 31

2.3.3 Living Biosphere (Bio)

2.3.3.1 Physiochemical Characterization and Major Dynamics

Bio microhabitats represent the most diverse and dynamic range of micro-niches
(Fig. 2.1c). Their high complexity is determined by the variable nature of the
“substrate” exploited by the bacteria, i.e., the host organism. These consist mainly
of eukaryotes ranging from protists to whales (although relevant associations have
been reported among bacteria, cyanobacteria, and archaea, e.g., Huber et al. 2002;
Aharonovich and Sher 2016). The host’s size scales up the size and diversity of the
associated bacterial community and introduces a different time scale for changes in
the microhabitat conditions (especially for the bigger and long-lived eukaryotes).
Each host species has its own physiology, behavior, and life stage development that
eventually affect bacterial colonization via changes in surface properties (external or
internal) for such variables as nutrient concentration and chemical gradients (e.g.,
Grossart et al. 2010; Scavotto et al. 2015).
The associated bacteria can be found at the host’s surface (epibionts) and within
some of its internal organs (endobionts). The epibiont-endobiont distinction is
pivotal since it has severe implications in terms of bacterial traits and ecology and
allows us to compare bacterial characteristics associated with the microhabitats
described in this chapter. Epibionts are accounted as PA bacteria because they
mainly exploit OM released by the host, while endobionts share several character-
istics more typically associated with FL bacteria since they live in a more homeo-
static environment leading to relevant genome streamlining.
Bacteria inhabiting Bio microhabitats undergo the same dynamics of random
colonization reported for PA bacteria (see Sect. 2.3.2) although for some tight and
specific associations, a recurrent pattern of the associated bacterial community has
been shown (e.g., Sharp et al. 2007). Thereby, the recruitment process mainly
accounts for epibiotic communities which change and evolve in composition over
time with continuous addition (and attempts of addition) from the surrounding
environment. In contrast, endobiotic communities tend to be more stable (Wahl
et al. 2012). The distinction between epibiotic and endobiotic characteristics can be
related to the specific conditions of micro-niches that favor bacterial species which
have evolved to maximize their fitness at the given conditions.
Host and bacteria can directly interact with each other by releasing attracting
chemicals and inhibitory substances among the latter being antibiotics and toxic
compounds (e.g., Tang et al. 2010; Amin et al. 2012). Host-bacteria interactions can
span over a wide range of symbiosis types, from positive such as mutualism and
commensalism to negative such as parasitism and saprophytism. These types of
interactions differ in their specificity and evolutionary history (e.g., mutualism
presumably requires a long coevolution and is more species-specific as compared
to commensalism; Kirchman 2012).
In a highly diluted oligotrophic environment, the host represents an exceptionally
dense and diverse hotspot of OM and nutrients supporting increased bacterial growth
32 L. Zoccarato and H. P. Grossart

rates and specific microbial processes absent in the surrounding water. Conse-
quently, OM-associated bacterial communities can reach extremely high volume-
specific densities, especially on zooplankton (up to 1011 cells ml1; Tang et al.
2010). Such high bacterial densities often lead to intra- and interspecies competitions
with severe functional consequences. The associated effects of bacterial metabolism
may have important implications for OM nutrient cycling and hence influence
trophic transfer efficiency as well as the well-being of the host. Understanding the
capability of bacteria to sense their population density through so-called quorum
sensing mechanisms (QS) is crucial to understanding the dynamics of host-bacteria
interactions and their ecological consequences (Miller and Bassler 2001). This type
of cell-to-cell communication relies primarily on the detection of small molecules,
so-called autoinducers (AI) (Dobretsov et al. 2009), excreted into the environment
by each bacterial cell, reaching a certain threshold concentration when a critical
bacterial cell concentration has been reached. At this threshold of saturation, a
signaling cascade is promoted by the bacterial cell detector mechanisms that even-
tually evoke a genetically coded response (Case et al. 2008). Examples of bacterial
strategies adopted for competition with each other are (1) the responses to the
reduction of available anchoring surface (Reid et al. 2001), (2) the depletion of a
specific substrate or nutrient essential for the metabolism of a competitor, (3) the
alteration of the chemical environment (pH, oxygen), and (4) the production of
antagonistic substances (e.g., antibiotics; Wannamaker 1980). Bacterial antagonistic
compounds thereby produced can also affect the host (Steinberg et al. 2011).
Phytoplankton provide several different OM compounds via exudation, thus
supporting the energetic requirements of the associated bacteria, and this represents
one of the most important host contributions to the complex biotic interactions (Sey-
mour et al. 2017). One of the most abundant phytoplankton exudates consists of
transparent exopolymeric polysaccharides (TEP), which have been shown to be
rapidly colonized and degraded by bacteria (Grossart et al. 2006). This class of
OM compounds is released especially during exponential algal growth (e.g.,
blooms), and these compounds have been suggested as a way in which phytoplank-
ton attract and promote bacterial settlement (Amin et al. 2012); Thalassiosira
pseudonana, for example, releases a sulfonate compound whose catabolic genes
are poorly distributed in the ocean, but it can be utilized by a co-occurring
Roseobacter bacterium (Durham et al. 2015). Bacterial colonization of phytoplank-
ton grants permanent substrate supply in the phycosphere, the boundary layer
surrounding the phytoplankton cell. On the other hand, bacteria can also mediate
this process by releasing proteins or polysaccharides which create a tridimensional
anchoring structure (Rinta-Kanto et al. 2012). Recently, host-bacteria interactions
within the phycosphere have been shown to be finely tuned by complex mechanisms
of communication. Recognition from the host of the approaching bacteria and vice
versa is essential for these interactions (Teplitski et al. 2011). Such inter-kingdom
communication is made possible by the evolution of small signaling molecules (e.g.,
the AI) and of specific binding receptors on the association active surfaces. Each host
and bacteria have developed different signaling compounds during their evolution;
however, they all share some chemical features, and in order to limit the dispersion,
2 Relationship Between Lifestyle and Structure of Bacterial Communities. . . 33

these molecules are typically hydrophobic and lipid-based. Moreover, allochthonous

signaling receptors have been discovered on different organisms involved in inter-
kingdom interaction, supporting the idea of long coevolution processes between the
host and specific bacterial strains (see Amin et al. 2012).
Similar to phytoplankton, zooplankton hosts can provide nutrient-rich microhab-
itats to the bacteria but have greatly been neglected by aquatic microbial ecologists.
As predators, they can accumulate high OM concentrations which can be well
exploited by both endobiotic and epibiotic bacterial communities. The latter have
a convenient proximity to the source of nutrients originating from either sloppy
feeding or of excreted matter (Møller et al. 2003), and, for instance, nitrogen-rich
excretions have been shown to increase the growth of epibiotic bacteria (Carman and
Dobbs 1997). Moreover, the exoskeleton itself can be considered a favorable
substrate since the inability of many microbes to effectively store carbohydrates
leads to low C:N and C:P ratios (Beers 1966). A substantial amount of bacteria are
more or less passively ingested by zooplankton, and some of these bacteria can well
survive the ingestion-digestion process (see Tang et al. 2010) effectively rendering
those ingested microbes as endobiotic commensals. This specific microenvironment
is characterized by several remarkable constrains that determine the composition of
the endobiotic bacterial community such as rapid changes in pH, oxygen, and OM
concentrations, as well as a short gut passage time. These properties require essential
bacterial adaptations for successful bacterial settlement within the host enabling a
rapid effective response to external stress and require an efficient anchoring strategy.
Their particular lifestyle is reflected in their extraordinary resistance to standard
disinfection procedures which may even result in a serious threat for public health
(Bichai et al. 2008). Eventually, some of the endobiotic bacteria are excreted via
fecal pellets and consequently have to survive a shift from Bio to Par microhabitats.
This abrupt change of microhabitat conditions may negatively select for specialist
bacterial with streamlined genomes, which do not alter the fitness of transient PA
bacteria.
An extra degree of complexity of Bio microhabitats is represented by the phys-
iology of the organismic hosts, both phyto- and zooplankton. Responses to fluctu-
ations in environmental factors or other stressors (e.g., pollutants) as well as different
life stages and physiological conditions of the host can imply remarkable changes in
microhabitat conditions (see Wahl et al. 2012). These changes favor different
bacterial species and induce shifts in the composition of the associated bacterial
community; some species might also change their behavior and thus render different
kinds of trophic interactions (e.g., from commensalism to parasitism). As an exam-
ple, in response to warming stress (temperature and UV), the red macroalga Delisea
pulchra loses its capability to quench the QS mechanism of associated bacteria via
the production of furanones; therefore the bacteria evolve a pathogenic behavior
inducing tissue damage and bleaching (Steinberg et al. 2011). Dispersal and differ-
entiation of the associated bacteria can be induced by the presence of nitric oxide
(NO), a typical biologic signal released by diatoms (Steinberg et al. 2011). While the
presence of NO in the phycosphere can act as repellent for most bacteria, it will
create micro-niches for those bacteria which are capable of detoxifying it. During
34 L. Zoccarato and H. P. Grossart

stress conditions the release of NO increases induction of the detachment of some

associated bacteria while attracting scavenger bacteria capable of sensing NO
concentrations (DeGroote and Fang 1995).
In general, Bio microhabitats have been largely overlooked by microbial ecolo-
gists. “Environmental” properties of the micro-niches can be well defined for most of
the hosts based on information about physiology, life cycles, and effects of external
stressors, although we still have relatively little information for some host groups,
e.g., protists. Ecological effects induced by changes associated with these host
properties on bacterial community are still largely neglected, and thus future inves-
tigations should tackle possible host-mediated consequences for bacterial species
composition and functionality. We already know that the host can affect bacterial
dispersion by acting as a dispersal courier in a similar way as do the sinking particles
in the Par microhabitats. However, organismic hosts provide an increased dissem-
ination possibility for the associated bacteria by not only sedimenting downward but
also by active movement in the water column (e.g., Grossart et al. 2010).

2.3.3.2 Bacterial Adaptations

Similar to PA bacteria, bacteria associated to living organisms are usually sorted out
during sampling (by size fractionation) or are underrepresented in the sample (as the
volume of this specific micro-niche is usually only a few mL; Grossart 2010). Most
of the available data on the host-associated bacteria stems from investigations that
targeted sponges (e.g., Sharp et al. 2007) although some information is also available
for zooplankton and algal hosts (Møller et al. 2007; Tang et al. 2010; Egan et al.
2013; Behringer et al. 2018). Bacteria associated with these organisms are involved
in a long-lasting coevolution process with their host which can be still seen in the
host genomes (see Amin et al. 2012). Our main knowledge gap, however, is that we
still do not know or poorly understand the metabolic functioning of the host-
associated bacteria (Wahl et al. 2012; Durham et al. 2015; Amin et al. 2015).
Generally, epibiotic bacteria are characterized by mechanisms which are QS
dependent, and the most relevant trait is the production of antibiotic compounds.
Bacteria associated to the host surface represent a main producer of antibiotics in
seawater (Wahl et al. 2012), but they also produce secondary metabolites, e.g., acids
from sugar fermentation to stave off potential competing bacteria by inducing
negative chemotaxis (Madigan and Martinko 2006) or inducing permanent binding
of the competitor cells to the substrates (Boyd et al. 1999). The capability of
producing antibiotic compounds is not limited to specific phylotypes or bioregions
but rather represents a widely spread trait, and many bacteria can modulate the
production of various compounds when necessary (Bode et al. 2002). Generalist-
epibiotic bacteria such as Pseudoalteromonas and Streptomyces tend to produce
active molecules affecting a broad range of species as they are mostly associated
with crowded microhabitats, while specialized-endobiotic bacteria might produce
less antibiotics and differently target specific competitor species (Hibbing et al.
2010). An alternative hypothesis relates the production of antibiotics to their
2 Relationship Between Lifestyle and Structure of Bacterial Communities. . . 35

functioning as signaling molecules (Sengupta et al. 2013) rather than as being

harmful compounds since their production cost is usually elevated and the targeted
species can quickly evolve to overcome this inhibition (Hibbing et al. 2010).
Epibiotic bacteria can actively find and colonize their hosts thanks to chemotactic
behavior which allows them to follow chemical gradients and also possible hydro-
dynamic cues (Carman and Dobbs 1997; Raina et al. 2019). The major phyla
associated with phytoplanktonic hosts are Bacteroidetes and Proteobacteria with
some genera having been particularly found associated with diatoms such as
Sulfitobacter, Roseobacter, Alteromonas, and Flavobacterium (see Amin et al.
2012). Interestingly, while consistent patterns in microalgal-associated bacterial
communities are found at high taxonomic levels (e.g., phylum), no similarities are
observed at lower ranks (i.e., genera or species) (Egan et al. 2013); this can be related
to methodological limitation but might also be correlated with specific and selective
host-bacteria interactions. Some studies provide evidences that the degrees of
variability of bacterial communities associated with the same algal species are also
reflected in community functionality rather than composition (Burke et al. 2011).
Aharonovich and Sher (2016) have shown that associations of Alteromonas
macleodii with different strains of the cyanobacteria Prochlorococcus can lead to
completely different interactions. For example, the presence of the heterotrophic
bacteria reduces the cyanobacterial expression of several core genes in the strain
MED4, e.g., those involved in the biosynthesis of amino acids, purines, pyrimidines,
vitamin B12, fatty acids, and phospholipids as well as of genes responsible for DNA
replication and cell division. MED4, however, showed an increasing growth rate in
the presence of Alteromonas denoting a synergic relationship with the heterotrophic
bacterium providing available basic substrates to the associate cyanobacterium. On
the contrary, the Prochlorococcus strain MIT9313 showed a competitive relation-
ship with Alteromonas which included an increasing expression of the gene for
uptake of small compounds and also the production of antimicrobial or signaling
compounds by Prochlorococcus (Aharonovich and Sher 2016).
In some cases, FL bacteria also can be associated with dinoflagellate and diatom
blooms, e.g., Roseobacter (Alphaproteobacteria), which can take up and utilize
some of the algal osmolytes, especially dimethylsulfoniopropionate (DMSP) (see
Egan et al. 2013).
Several studies have tried to characterize the community composition of bacteria
associated with zooplankton although it is not a trivial task to separate epibionts and
endobionts. According to the theory proposed by Harris (1993), endobionts are more
affected by the feeding activity, and thus the resident bacteria can be distinguished
from transient ones by analyzing the stability of the community composition. This
can be obtained by feeding the zooplankton with an axenic culture and subsequently
tracking the decreasing richness of the associated bacterial community composition
until it is stable. However, feeding experiments with xenic prey cultures may indicate
the presence of several transient bacterial groups which are capable of anchoring
within the organism’s stomach exploiting the rich OM pool, and these bacteria mainly
belong to the groups of Pseudoalteromonas, Sulfitobacter, and Roseobacter (Tang
et al. 2009). The bacterial community composition of crustacean zooplankton in lakes
36 L. Zoccarato and H. P. Grossart

has been observed mainly to be composed of 36 different phylogenetic groups

belonging to 6 major bacterial taxa: Actinobacteria, Firmicutes, Bacteroidetes,
Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria (Grossart
et al. 2009). The community composition of the sole endobionts appears less rich
and is represented by Bacteroidetes, Alphaproteobacteria, Betaproteobacteria, and
Gammaproteobacteria (mainly Pseudoalteromonas) (Peter and Sommaruga 2008).
As for phytoplankton, a generalization of the bacterial community composition on
zooplankton is only possible at high taxonomic levels since (as pointed out above) the
environmental conditions of the colonizing surface can drastically change between
host species.
Zooplankton endobionts can rely on high numbers of nutrient particles which
allow them to thrive on OM substrates which are otherwise extremely diluted in
seawater, e.g., DMSP-consuming bacteria (Diaz et al. 1992). Environmental DMSP
concentrations can be particularly high during the senescence of phytoplanktonic
blooms, but release of DMSP by active phytoplankton is almost negligible. Com-
pounds such as DMSP can reach μM or mM concentration in the zooplankton guts,
being released from the digestion of the phytoplankton prey (see Tang et al. 2010).
The Betaproteobacteria Limnohabitans exploiting the nutrient-rich conditions
within the gut of Daphnia magna was found responsible for 8% of the leucine and
9% of the N-acetyl-D-glucosamine uptake of the total bacterioplankton community
demonstrating the high metabolic activity of endobiotic bacteria. Although these
rates might be not astonishing, they have severe implications for ecosystem dynam-
ics since by ingesting a Daphnia, a predator will transfer also the carbon fixed by
Limnohabitans (Eckert and Pernthaler 2014). This “shortcut” in the food web
increases the efficiency of the carbon flux and eventually the energy budget of the
ecosystem. Due to the particular microenvironmental conditions within the guts,
specific processes can take place, which otherwise cannot occur in the surrounding
seawater. For example, the anoxic environment of full copepod guts allows for
nitrogen fixation by the cyanobacteria UCYN-A, while gammaproteobacterial nifH
sequences (gene candidate to identify nitrogen fixation in environmental samples)
were highly represented in starved copepods. The Gammaproteobacteria might be
represented by Vibrio spp. which often is associated with the exoskeleton and gut
lining of living copepods (Rawlings et al. 2007) from where it can retrieve the energy
necessary to support the energetically demanding nitrogenase activity (Scavotto
et al. 2015). Another example is the occurrence of denitrification in zooplankton
carcasses in an otherwise fully oxic water column with profound implications for
aquatic nitrogen cycling (Glud et al. 2015).
In summary, bacterial endobiont organisms are capable of penetrating the host
tissue and may survive the host’s digestion tract thus often establishing a long-
lasting relationship with their host. This process has been described several times for
different hosts (Kikuchi 2009; Archibald 2015) demonstrating its apparent evolu-
tionary advantage (although there are similar cases of associations with pathogens
and saprophytes). A general characteristic shared by all endobionts (resident species)
is their extreme genome streamlining (Fig. 2.2); this process is even more empha-
sized than in FL bacteria (genome of 5–10 Mb) and leads to the typically small
2 Relationship Between Lifestyle and Structure of Bacterial Communities. . . 37

genomes of pathogenic species (2–5 Mb) and often even smaller genome sizes for
symbionts (0.5–1.5 Mb). Symbionts have a stable genome organization without
lateral gene transfer (Ochman and Davalos 2006), and their minimal genomic size
is a direct effect of the extreme stability of the environment where several core
functions and metabolites are provided by the host. In exchange, symbionts may
provide their hosts either the capability to thrive on formerly unusable energy
sources (diet shift) or to cope with otherwise nutritionally deficient diets. Terrestrial
bacteria-host associations have shown loss of essential genes (information
processing, energy metabolism, cell envelope synthesis) which possibly provides
evidence for phylogenetic specific coevolution processes (Bennett et al. 2014).
Driven by the technological improvements, there is an increasing awareness of the
critical importance of the microbiome and its implication for the host
metabolome (Fischer et al. 2017). The unexpected diversity of bacterial species
present in several Bio microhabitats such as the human body (Eloe-Fadrosh and
Rasko 2013) raises questions on their possible interaction with the host. These
studies reveal that bacteria can significantly influence host processes, even being
entirely responsible for some host processes, and can have deep implications on the
dynamics of interactions among environment, host, and bacteria (Leonard et al.
2015).
It is worth remembering that bacteria inhabiting Bio microhabitats can be also FL
bacteria since they need to maintain their population in the absence of a host.
Epibionts can be involved in the exploitation of carcasses or fecal pellets of their
host and thus extend the life-span of their microhabitat beyond the lifetime of the
host; these bacteria can also actively attach and detach from the host thus being able
to survive also as FL bacteria (Møller et al. 2007). The endobionts, however,
experience drastic changes in their micro-niches upon the host’s death, and some
hosts can also release the associated bacteria into the surrounding seawater, which is
the case of bacterial associations between the squid Euprymna scolopes and the
bacterium Vibrio fischeri (Nyholm 2016). Therefore, these bacteria need to adopt ad
hoc strategies in order to rapidly colonize another host. Genome streamlining of the
endobionts is a relevant obstacle to their survival outside of the host, and the vertical
transmission to the host’s progeny can be a valid strategy.

2.4 Human-Mediated Implications

Following a size-based gradient, we would like to highlight some major concerns

related to human impacts on the structure and complexity of aquatic microhabitats as
earlier described in this chapter. We tried to provide some clues for the present and
future transformation dictated from human activities onto marine microhabitats. The
DifP and ColP microhabitats are mostly affected by the continuous discharge of
DOM, heavy metals, and toxic compounds (antibiotics, drugs, new chemicals)
(Fig. 2.3a); these products enter aquatic ecosystems mainly through drainage into
rivers, lakes, and coastal areas which represent the most affected biomes. While
38 L. Zoccarato and H. P. Grossart

Fig. 2.3 Effects of the

major anthropogenic
stressors on the complexity
of the aquatic microhabitats:
the diffusion-controlled
water phase (a), colloidal
phase (b), freshwater and
marine particles (c), and
living biosphere (d).
Question marks are added to
size and time axes because
the future effects are still
unpredictable

these pollutants have often lethal or weakening effects on large organisms, they
might represent an exploitable resource for bacteria and archaea. In particular, DOM,
heavy metals, and chemical compounds can increase the chemical and physical
complexity of DifP and ColP microhabitats, thus affecting the spatial heterogeneity
and selecting for different bacterial species and guilds (e.g., increasing sulfur-
reducing Verrucomicrobia species in the presence of Hg and MeHg (methylmer-
cury); Vishnivetskaya et al. 2011). During the last decade, we also started to feed
these microhabitats with a novel category of compounds, the nanoparticles. One of
the most used nanomaterials consists of silver nanoparticles which have been shown
to release Ag ions as consequence of the interactions with humic acids. Silver
nanoparticles and dissolved Ag ions have inhibitory effects on bacterial growth
(a potential oxidative stressor) and represent a critical toxic risk when they are
2 Relationship Between Lifestyle and Structure of Bacterial Communities. . . 39

discharged into natural environments (Fabrega et al. 2009). Although the full
environmental consequences of several types of nanoparticles remain unclear,
these compounds are capable of affecting bacterial community functions especially
in DifP and ColP microhabitats where the bacteria can hardly escape from dissolved
toxic compounds.
The major anthropic impacts on Par microhabitats are represented by the tremen-
dous number of micro-plastic particles (Fig. 2.3b) which are released into the
environment. The accumulation of this marine litter in the ocean is dramatically
increasing in terms of diversity, and the abundances of these microhabitats provide a
significant quantity of new colonizable surfaces. The photodegradation process
converts plastic wastes trapped inside the principal oceanic gyres into micro- and
nanoscale pieces which might represent formidable surfaces for microbial biofilm
development. Microbial associations with micro-plastic particles are characterized
by extremely high cell densities and high rates of metabolic activities (see Sect.
2.3.2); therefore, these particles have the potential to boost microbially mediated
processes like OM remineralization. The micro-plastic itself can represent a source
of energy for some bacteria which are capable of degrading it (Skariyachan et al.
2016; Urbanek et al. 2018), and the elevated abundance of micro-plastic might
strongly shape the bacterial community composition selecting for specific guilds
(Zettler et al. 2013; Arias-Andres et al. 2018) which can eventually overwhelm and
potentially even replace the natural microbial community in some areas of the open
oceans. However, while for bacteria micro-plastic can represent favorable resources
and colonization sites, an increasing number of studies are suggesting negative
effects of these particles upon macrofauna. Plastic microparticles will negatively
affect many Bio microhabitats (Fig. 2.3c). Zooplankton organisms as well as fish can
ingest these compounds and consequently experience decreased growth rates and
fitness, energetic depletion over time, behavior alterations, and reduced survival
rates (Cole et al. 2015; Lönnstedt and Eklöv 2016). In addition, as a result of micro-
plastic ingestion, these particles end up in fecal pellets and can be rapidly exported to
deeper water layers (Cole et al. 2016). This process further increases the heteroge-
neity of Par microhabitats but might also significantly affect carbon fluxes and the
energetic budget of carbon-limited bacterial communities—typically for the deep
ocean. Furthermore, the microbial niches associated with Par microhabitats are likely
the most impacted by eutrophication and soil erosion which are mainly connected
with agricultural activities, deforestation, and changes in land use. While the
increase in nutrient load affects all microhabitats in coastal and freshwater environ-
ments (e.g., Smith 2003), the concomitant input of terrestrial particles abruptly shifts
quantity and quality of Par microhabitats. The allochthonous particles have been
shown to reshape bacterial communities by providing new exploitable surfaces and
substrates (Traving et al. 2017) but also potentially seeding the local communities
with allochthonous microorganisms. The problem of alien species is further
increased by anthropogenic water pollution, e.g., by ballast water input (Williams
et al. 1988), directly adding and selecting for species which in a pristine environment
are not common or even not present. Thereby, bacteria and other microorganisms
preferentially survive different ballast water treatments on particles (Tang et al.
40 L. Zoccarato and H. P. Grossart

2011), which can make them invasive and alien species within profound but so far
unstudied consequences for microbial diversity and functioning.
Bio microhabitat complexity is further threatened by several stressors. The
introduction of invasive species usually implies a reduction of the diversity of
local communities since the successful establishment of an alien tends to overrule
local species. As a result, a drastic reduction of Bio microhabitat diversity can be
observed with tremendous consequential effects on the endobiotic bacteria. The
generalist-epibiotic bacteria likely can colonize the new resident species, but most
of the specialized-endobiotic bacteria will perish or became ecologically inactive as
resisting stage. Shifts in functioning of the host-associated bacteria are largely
unpredictable since they are largely dependent on specific characteristics of the
new hosts. Moreover, additional large-scale and unpredictable effects that are
triggered by overfishing result in the removal of specific groups of organisms with
severe implications for food web dynamics, e.g., due to alterations of inter- and
intraspecies guild competition, top-down trophic cascades (Hessen and Kaartvedt
2014), and reduction in diversity and abundance of Bio microhabitats. The least but
not the last stressor is the human-mediated increase of pathogen dispersal such as
viruses, bacteria, fungi, and other protists. The impact of a new pathogen in the
environment can be predicted for its designated host; however, similar to what is
discussed above for alien species, the overall consequences at an ecosystem or
regional level can be hardly conceived (e.g., cascade effects, species-barrier cross-
ing, etc.) (García-Vásquez et al. 2017).

2.5 Scaling Up to the Biomes

At a first glance, if one looks into lake or seawater, one might think that these
systems represent exclusively homogeneous habitats in which everything is diluted
and no relevant differences exist between them. However, each single aquatic
environment is composed of specific microhabitats as described above, and some
major differences can already be ascribed by the comparison of freshwater and
marine areas. In this last section, we will try to highlight a gradient in microhabitat
composition among different aquatic systems and upscale the implications of these
microhabitats for ecosystem functioning as a whole.
Small lakes experience the strongest impacts from land inputs of OM due to their
high ratio between land/water interface area and the relatively smaller volume of the
basin. The trophic state of these biomes is subjected to abrupt changes in their
physical and chemical features as their relatively small volume grants only a low
buffering capacity against environmental fluctuations (some might be even too small
to undergo thermal stratification such as polymictic lakes). The terrestrial-particle
load exerts strong effects on the ecosystem by triggering shifts in both the bacterial
community composition and the community’s associated functionalities (e.g., Biži-
ć-Ionescu et al. 2014). Small lakes thus tend to be mostly composed by Par
microhabitats of terrestrial origin whose degradation often leads to anoxic events
2 Relationship Between Lifestyle and Structure of Bacterial Communities. . . 41

in the deeper layers due to oxygen consumption associated with OM settlement. Part
of the OM recycled at the surface also supports the growth of primary producers
increasing the Bio microhabitats, especially during periods of high rainfall (Tranvick
and Jansson 2002); a remarkable enrichment in inorganic nutrient concentrations can
lead to microbial bloom events that eventually sink and thus further increase the
oxygen consumption at the lake bottom.
The coastlines of large lakes are still highly influenced by terrestrial input;
however, due to the much higher volume of these basins, their terrestrially related
processes contribute less extensively toward the overall microhabitat composition of
the ecosystem. Likewise, marine coastal areas also benefit from allochthonous input
of Par microhabitats. In the sunlit layers of both biomes, POM remineralization,
coupled with nutrient input from the deep-water layers (mixing events in spring and
fall seasons, upwelling), supports the growth of phytoplankton populations which in
turn enrich the environment with new autochthonous Par microhabitats (exudates,
fecal pellets, and later on of carcasses) and Bio microhabitats (phytoplankton itself
and zooplankton grazers). These marine and freshwater Par microhabitats, however,
are colonized by different bacterial species since they differ in chemical composition
and physical properties; for instance, in lakes there exist specific bacterial guilds,
e.g., for the degradation of humic compounds, while in marine areas species adapted
to diatom and cyanobacteria, OM degradation are favored. The different lability of
bacterial substrates implies different microbial turnover rates and, therefore, differ-
ent OM fluxes. Moreover, marine and freshwater systems select for different phy-
toplankton and zooplankton communities increasing the dissimilarity of Bio
microhabitats between the two environments. Abnormal dynamics occur in special
water bodies such as estuaries and lagoons where direct contact between freshwater
and seawater takes place. Mixture of both water types leads to the formation of Par
microhabitats (“turbidity maximum”) with different origins that imply the presence
of bacterial communities with exceptionally different composition and functionali-
ties and thus might lead to peculiar dynamics such as the priming effect (Treignier
et al. 2006).
The inner part of big lakes, which in terms of volume is the dominant portion of
these habitats, generally shares the same dynamics as open sea environments. The
surface layers of lakes (epilimnion) and of the open sea (epipelagic) are dominated
by DifP and ColP microhabitats as the consequence of persistent oligotrophic
conditions. These microhabitats are pervaded by rare environmental fluctuations
and thus are more stable, characterized by long-term seasonal variations contrary to
Par and, especially, Bio microhabitats that can experience extremely high dynamics
(i.e., process rates measured in hours rather than days). The relatively small size and
low bacterial activity of the DifP and ColP microhabitats in big lakes and open sea,
however, can pair and even overcome the bio-process rates occurring in the larger
and more active Par and Bio microhabitats. The reason for this lies in the huge
extension of DifP-ColP-dominated lake and open ocean biomes which represent
more than 80% of all aquatic habitats accounting for ca. 40% of total primary
producer biomass and 70% of net primary production (Valiela 1995).
42 L. Zoccarato and H. P. Grossart

Similarly, the deep layer of large lakes (meta- and hypolimnion) and marine areas
(meso- and bathypelagic layers) is dominated by DifP and ColP microhabitats
although the ColP microhabitats typically represent mostly unproductive and recal-
citrant spots (Williams et al. 1969). Both Par and Bio microhabitats are mostly
absent or extremely rare (diluted) in these deep layers, with the occurrence of
microhabitats largely dependent upon distance from the coast, the above presence
of productive epipelagic areas, and the oceanographic dynamics related to circula-
tion patterns. Offshore deep regions are thus unlikely to experience the presence of
nutrient-rich microhabitats since the sunlit layers of oligotrophic open oceans are
populated by small-size primary producers (mainly Synechococcus and
Prochlorococcus) whose allochthonous exports are rapidly consumed in the upper
part of the water column. Constrained in allochthonous inputs and in autochthonous
OM production, the deep ocean represents the most oligotrophic biome. The bacte-
rial activity and biogeochemical processes in this biome are, therefore, limited
although relevant in a global perspective due to the tremendous volume of the
deep sea (Arístegui et al. 2009). Recent studies indicate that these microbial com-
munities are not inactive as previously thought and that the gap between energy
requirements and OM inputs in these biomes might be covered by autochthonous
production of OM by chemoautotrophic bacteria (Swan et al. 2011). Anyhow, these
environments are dominated by DifP and refractory ColP microhabitats harboring
little diverse and active microbial communities.

2.6 Future Perspectives

We believe that in the near future, several exciting advances will be achieved
regarding comprehension of the extreme and vibrant heterogeneity of bacterial
microhabitats. Technological improvements coupled with a more representative
experimental design likely will provide high-resolution data useful to upscale our
knowledge regarding ongoing biological dynamics from the nano- and microscales
to the macro- and global scales. Technologies such as SAG, MS-MS, and DESI-MS
are providing us the capability to unveil the genetic potential and metabolically
characterize even individual cells. Our core perspectives will be refined with the
development of future instrumental and methodological improvements. A special
effort should be invested to cover two major critical knowledge gaps: (1) the missing
coupling between biochemistry and molecular techniques for fine-scale analyses of
the environmental-biological diversity and (2) the development of biogeochemical
models which can take into account the bacterial heterogeneity of microhabitats and
scale up to ecosystem and global levels. In this way it will be possible to eliminate
some of our present uncertainty which nullifies the study of processes occurring at
extremely limited spatial and temporal scales; these methodological errors magnify
when being scaled up to broader ecosystem levels and thus still keep our estimation
of global biogeochemical fluxes far from realistic.
2 Relationship Between Lifestyle and Structure of Bacterial Communities. . . 43

Compliance with Ethical Standards

Funding This study was funded by HFSP (grant number RGB 0020/2016).
Conflict of Interest Luca Zoccarato declares that he has no conflict of interest. Peter Grossart
declares that he has no conflict of interest.
Ethical Approval This article does not contain any studies with human participants or animals
performed by any of the authors.

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Chapter 3
Biofilms: Besieged Cities or Thriving Ports?

Otini Kroukamp, Elanna Bester, and Gideon M. Wolfaardt

Abstract From a humble beginning, with less than 50 articles published per year with
the term “biofilm” in the title prior to 1990, research output on this topic has grown
dramatically with concurrent improved understanding of this form of microbial exis-
tence. While remarkable advances in molecular techniques perhaps enabled the major
share of this growing knowledge base, we argue in this chapter that due consideration
of the biofilms’ physical environment in our experimental design, measurements and
interpretation of results, is needed. For instance, the effect of flow, and its effect on
nutrient and metabolite flux, is markedly different for cells attached to the surface
(impacted by both the hydrodynamic effect and physicochemical properties of the
surface) compared to those in the quiescent zone of low flow close to the surface
(impacted by only the hydrodynamic effect of the surface) and the moving bulk fluid
further away (little or no effect of the surface). The quiescent zone bordering a biofilm
presents an area where planktonic cells can remain, roam around the attached biomass,
and increase in density due to reduced flow resulting in dilution rates that are lower
than cell growth rate, thus potentially playing an important role in both immigration
into and emigration from the biofilm. This may yield a notably different progression of
biofilm development and maintenance and thus a higher degree of fluidity than the
discrete stages as depicted by the classical view of biofilm development.

3.1 Introduction

The recognition that biofilms, defined as surface-associated communities of micro-

organisms, are the prevalent mode in which microbes exist has, in addition to the
myriad of areas where biofilms intersect with human interests, resulted in an

O. Kroukamp (*) · G. M. Wolfaardt

Department of Chemistry and Biology, Ryerson University, Toronto, ON, Canada
e-mail: mkroukam@ryerson.ca
E. Bester
Department of Microbiology, University of Stellenbosch, Matieland, Stellenbosch, South Africa

© Springer Nature Switzerland AG 2019 53

C. J. Hurst (ed.), The Structure and Function of Aquatic Microbial Communities,
Advances in Environmental Microbiology 7,
https://doi.org/10.1007/978-3-030-16775-2_3
54 O. Kroukamp et al.

exponential increase in research efforts. A recent search of Elsevier’s Scopus

database (www.scopus.com) for articles published between 1976 and 2017 with
the term “biofilm” in the title yielded 16,286 documents. Whereas 50 articles were
published in 1990, this number grew consistently in subsequent years to 182 (2000),
504 (2005), 974 (2010), and 1790 (2016). As could be expected, the main subject
areas represented by these publications were immunology and microbiology
(36.1%), medicine (32.7%), biochemistry, genetics, and biology (29.7%), followed
by environmental science (22.5%) and agricultural and biological sciences (17.9%).
The remainder consisted of publications in fields as diverse as dentistry, chemical
engineering, physics and astronomy, materials science, and mathematics, to name
but a few. While this illustrates the multidisciplinary nature of biofilm-related
research, it should also provide a sobering reality for newcomers to the field; the
incorporation of fundamental principles from multiple, unrelated disciplines typi-
cally is required to adequately investigate biofilms. This naturally implies that
investigators must not only be aware of but also become conversant in subjects
that may fall outside of their primary training. Furthermore, by design and necessity,
microbiological research remains firmly entrenched in homogenous, pure culture
studies involving planktonic suspensions of microbes, despite the fact that spatially
and temporally heterogeneous, surface-associated aggregates, consisting of multiple
microbial species are more representative of prokaryote existence outside of the
laboratory. In this chapter, we aim to illustrate how critical it is to foster an adequate
awareness and understanding of the impact of the environment on biofilms. To
achieve this, we focus on key physical aspects of aqueous environment and their
influence on both individual bacterial cells and biofilms. Judicious use of analogies
is advocated as tools to aid in the simplification of complex concepts to, firstly,
convey information, secondly, facilitate understanding, and, thirdly, allow selection
of the most crucial parameters to incorporate in experimental studies.

3.2 Rationale

3.2.1 Living Beings Interact with the Environment

Living beings are influenced by their environment and impact their environment in
return. These interactions and resultant reciprocal changes may be minuscule or
significant. In the field of biological sciences, we usually try to gain an increased
understanding of a focus area by taking measurements during a carefully designed
experiment. These collective measurements are then repackaged into descriptions,
models, theories, and representations to (often incrementally) answer the questions
of “what” and “how” that inspired our curiosity in the first place. For example, Quinn
and Keough (2002) show in their first chapter that the scientific method involves
stating a research hypothesis (as opposed to statistical hypothesis testing for infer-
ential statistics) and how, with proper experimental design involving the measure-
ment of copepod (predator) mortality, it is possible to postulate a model formulating
a probable reason why dinoflagellates (prey) luminesce when the water is stirred.
3 Biofilms: Besieged Cities or Thriving Ports? 55

The particular model mentioned by Quinn and Keough (2002) was referred to as a
“burglar alarm” where the experimenters predicted that dinoflagellate biolumines-
cence would attract fish (copepod predators) leading to increased copepod mortality
and indirectly to greater dinoflagellate survival. In this example, the experimenters
seemingly considered sufficient role players (fish, copepods, luminescent and
nonluminescent dinoflagellates) to address their research hypothesis after measuring
copepod mortality. What was inherent, but perhaps invisible and therefore not
considered in the experiment, was the particular environment where everything
took place.
David Foster Wallace (2008) opened his Kenyon College commencement
address of 2005 as follows:
There are these two young fish swimming along and they happen to meet an older fish
swimming the other way, who nods at them and says “Morning, boys. How’s the water?”
And the two young fish swim on for a bit, and then eventually one of them looks over at the
other and goes “What the hell is water?”

As experimenters we remain vulnerable to let the obvious go unnoticed. In

addition, we may also be lacking either in the ability to measure some (or all)
environmental parameters, or being unable to accurately simulate these in an exper-
iment. The latter could be because we do not fully understand which parameters, or
interactions between parameters, are most relevant or because it is either physically
impossible or too expensive to adequately re-create those relevant conditions within
the laboratory. Naturally, the particular environment under which we conducted the
experiment may not have influenced our design and eventual conclusion—but even
in such cases it is best practice to describe the environment as thoroughly as possible
rather than to appear ignorant about its influence. Since the influence from the
environment is a given, the question is therefore simply whether the magnitude of
influence is relevant or not. For small organisms such as microbes, a particular
environment will affect the metabolism, growth, motility, gene expression, and
behavior. This is true for single microbes as well as extracellular polymeric sub-
stance (EPS) matrix-encased biofilms which exist in close proximity as well as
attached to surfaces. In this chapter we argue why the physical environment of
aqueous biofilms should be considered in our experimental design, measurements,
and interpretation of results.

3.2.2 Points of Consideration for the Next Generation

of Biofilm Researchers

Biofilm research occurs and is perceived from many vantage points, including
engineering, physics, and biological sciences, with each field making a unique and
important contribution to the whole. Novice researchers entering the field of biofilm
research will bring along with them an understanding and vocabulary native to their
field. They will describe and interpret their findings through the lenses of their
experience. While engineers and physicists are trained with an acute awareness of
56 O. Kroukamp et al.

physical and chemical environmental contributors, biologists often have minimal

such awareness, and it is precisely this limitation that may hinder progress in the
field.
When encountering any new field of study, students often are encouraged to use
analogies as a method and tool for simplifying complex scenarios. This tool has
deservedly stood the test of time as both a communication aid and a simplified means
of fostering understanding. However, despite our best intentions, the potential exists
that the similarities and correspondence with the original may break down at some
point or that our intuition may fail us.
In this chapter we will indeed introduce analogies, while attempting to be mindful
of our assumptions, to gain insight into the physical environment encountered by
microbes in and around a biofilm. We hope to achieve this by first assembling a list
of key aspects which will be considered parameters relating to the aqueous biofilm
environment with special emphasis on those aspects most affecting of microbes. In
the final section, these parameters will be applied to an example where awareness of
the environment may aid in experimental design and interpretation of experimental
results, thus leading to an increased understanding of the world around us.

3.3 Background

3.3.1 Microbiology Legacy

Human beings are inextricably intertwined with microbes—perhaps in no way more

evidently than the fact that each living human body houses more microbial genes and
cells than it does those of the human host (Qin et al. 2010). On any given day, the
human path intersects with microbes or microbial functions that directly or indirectly
influence the quality of life in areas as diverse as human health, enjoyment (food),
and environmental sustainability (nutrient cycles). It is no wonder then that humans
are motivated to understand and control microbial growth, death, and function.
Although the collective effects of microbes have been observed for millennia, their
existence went largely undetected until the invention of the microscope. Since
microbes were originally interesting to humans by virtue of their effects (disease,
fermentation, etc.), and measurable (noticeable) effects usually require large num-
bers of microbes, these organisms were and still are grown to sufficient quantities to
satisfy the resolution of the experimental techniques of the day. For example, the
well-mixed, pure culture batch flask allowed early researchers to learn more about a
particular microbial species under specific growth conditions (e.g., at a specific time
point) by assuming that the measured parameter of a homogenous collective was a
representative average of each individual member. Even though the population’s
metabolism may change over time, for example, when switching from one carbon
source to another (diauxic shift) or from one electron acceptor to another, the
microbes will all display similar behavior on average. In contrast, systems that are
not well-mixed such as static batch cultures and biofilms (more about the latter in
3 Biofilms: Besieged Cities or Thriving Ports? 57

upcoming sections) and the responses of their constituent microbes to gradients of

various forms (e.g., nutrients, redox potential) are expected to develop over time
(Stewart 2003). It should be noted, however, that it may be hard to distinguish
whether gradients develop first, followed by changes in microbial physiology, or
whether microbial activity leads to gradients of environmental parameters (or a
combination of both processes occurring simultaneously). Since each microbe is
acutely affected by its immediate environment (e.g., temperature, solute concentra-
tion, viscosity, etc.), the assumption of spatial homogeneity is a very convenient one
from an experimental perspective, especially given that an empirical result can be
reproduced fairly predictably under similar conditions. It is therefore not surprising
that the legacy of studying microbiology from a spatially homogenous viewpoint
still remains in textbooks and undergraduate training. Outside the laboratory, how-
ever, microbiology occurs in spatially heterogeneous systems such as biofilms
(Geesey et al. 1978).

3.3.2 Spatial and Temporal Heterogeneity Associated

with Biofilms

Despite the observation that biofilms formed by various bacterial species proceed
through similar steps and exhibit comparable properties, researchers have been
unable to determine whether a biofilm-specific genetic program is in operation or
if the resultant properties represent the culmination of the response and adaptation of
individual cells to local environmental change (Kjelleberg and Givskov 2007). The
inability to identify a biofilm-specific program may in fact be due to the acknowl-
edged spatial heterogeneity of biofilms (Stewart and Franklin 2008). The physical,
chemical, and biological properties of microenvironments within biofilms fluctuate
continually due to a combination of various factors, which may include nutrient and
oxygen diffusion gradients, sloughing, predation, etc. Individual cells respond and
adapt to the prevailing conditions, leading to the establishment of physiologically
differentiated subpopulations within a biofilm.
Due to its nature, a biofilm (even a pure culture biofilm) will be spatially
heterogeneous in terms of:
• Chemical environment, e.g.:
– Nutrients (both electron acceptor and donor)
– Excreted products
– Hydrogen ion (pH) concentrations
• Physiological manifestation
– Gene expression
– Mode of metabolism
– Motility
58 O. Kroukamp et al.

The resulting spatial heterogeneity complicates a study since the emergent prop-
erties of the interactions encountered may be difficult or impossible to foresee or
delineate. While this spatial heterogeneity is evident even in pure culture biofilms, it
is not a big leap to conclude that the complexity may be much more pronounced in a
multispecies environment. As a result, the strategy to research biofilm cells and their
behavior has had to be adapted, when necessary, to address this lack of experimental
resolution. Depending on whether viewed from an engineering (applied) or funda-
mental perspective, one could focus on global effects (overall mass balances or
functional measurements) or conversely shift toward experimental techniques
adapted to microscale to study local subpopulations within the biofilm with regard
to their gene expression and immediate surroundings (De Beer et al. 1996; Rani et al.
2007). Fortunately, researchers are starting to recognize how heterogeneity stem-
ming from single cell behavior affects collective function (Martins and Locke 2015).
Even more so than is the case for planktonic operation, explicit experimental
conditions [e.g., experiments conducted under static flow conditions at solid-air
interface (i.e., colony biofilms) or solid-liquid interface (i.e., microtiter plate biofilm
assays), or flow conditions (i.e., flow cells or moving bed biofilm reactor)] should be
taken into account when interpreting any biofilm-related experimental results given
the added complexity resulting from granular spatial heterogeneity. Lewandowski
and Beyenal (2013) argue for the need to consider the different levels of focus,
whether it involves the very close scrutiny of microelectrodes or comparing the
efficiency of different biofilm carriers in wastewater treatment.
While all of this might seem daunting to new researchers in the field, we hope to
show that familiarization with a few key concepts will aid substantially in both
understanding and communicating results and interpretations. In the next section, we
will address certain areas related to how microbes respond to their physical envi-
ronment, with a focus on aqueous systems, and how mastery of these building blocks
can act as implements in a toolbox where the researcher can use some and neglect
others to make sense of real-life observations.

3.4 Appropriate Analogies and Rubrics

A preferred means to convey new and complex concepts is by way of analogies.

Analogies are based on prior knowledge, thereby making it easier for the reader
(or listener) to categorize, classify, and position the new knowledge. In addition to
facilitating understanding, analogies have the added advantage that complex con-
cepts can be communicated parsimoniously, e.g., a bean being described as kidney-
shaped and vice versa.
Once these analogies have been related, it can be assembled into rubrics or lenses
for the researcher (or reader) to communicate. Given that the environment affects
microbial behavior, it is important to avoid drawing conclusions from generalized
inductive reasoning without regard for the experimental conditions that contribute
toward the measured microbial behavior. Conversely, an undiscerning, exhaustive
3 Biofilms: Besieged Cities or Thriving Ports? 59

listing of experimental conditions may be counterproductive in that it may limit

understanding or be too cumbersome for the reader to wade through. The benefit of
these rubrics is that areas of observation (whether on the scale of a research study or a
measured parameter) can be evaluated rapidly and effectively allow for the capturing
of the relevant important contributing factors without having to be exhaustive. For
example, Flemming et al. (2007) describes the extracellular polymeric substances
(EPS) as “The house of biofilm cells.” With our understanding of a house, this
mental picture makes it easier to conceptualize and explain different biofilm behav-
iors resulting from the presence of EPS such as structural stability, exchange of
genetic material (communication and transfer of knowledge), and retention of
nutrients and crucial enzymes. Similarly, Watnick and Kolter (2000) use the analogy
of “biofilms being cities of microbes” to explain biofilm development and spatial
arrangement by correlating it to geographical settling patterns and zoning laws found
in a city.
Analogies can be of great benefit during the process of gaining understanding in
an unknown or related field. However, caution should always be exercised in that
one should remain aware of terminology use as well as the explicit and inherent
assumptions—especially those that are valid in the progenitor field or system—that
can perhaps indiscriminately be carried over to the new endeavor. For example,
when we attempt to explain microbial behavior by using anthropomorphic termi-
nologies such as altruism, it is often challenging to remain neutral about the human
components associated with this term such as choice, compassion, goodwill, willing
sacrifice, etc. Referring to microbial altruism and cooperation, West et al. (2007)
stated:
However, progress is often hindered by poor communication between scientists, with
different people using the same term to mean different things, or different terms to mean
the same thing. This can obscure what is biologically important, and what is not. The
potential for such semantic confusion is greatest with interdisciplinary research.

With this in mind, an analogy to describe biofilm development under aqueous

conditions is presented in the next section. Our aim is to provide a framework to
evaluate biofilm experimental results by focusing on the differences in environmen-
tal conditions that result in many of the discrepancies observed between different
biofilm studies conducted in aqueous environments. We hope to show that by simply
being aware of whether or not the biofilm under consideration developed under flow
versus stagnant conditions, the reader will become cognizant of how these differ-
ences influence viscosity, nutrient concentration, motility, and association with a
surface and a biofilm, to name but a few parameters.

3.4.1 Thriving Port City or Besieged City?

According to this analogy, an aqueous biofilm surrounded by flowing conditions can

be envisaged as a port city with its harbor. Pertinent to this analogy, one can consider
60 O. Kroukamp et al.

three distinct spatial zones related to the flow-surrounded biofilm and discuss how
these zones interact via space and time to influence characteristic biofilm behavior.
The three zones under consideration include the free-flowing bulk liquid, the fluid
adjacent to the surface or biofilm, and the gel-like biofilm itself which consists of
microbial cells and the EPS matrix. The characteristics of a port city naturally lends
itself as a focal point for the initiation of colonization, but even long after the
establishment of thriving communities, the same “gateway” character of these cities
maintains their strategic importance. For example, when considering an established
coastal country, we are usually aware of the importance of port cities especially as it
relates to world commerce (as well as immigration and emigration during the time
prior to air travel). But it is sometimes easy to forget about the strategic and crucial
early roles that these port cities played in the establishment of the particular country
or nation—e.g.:
• Regional trade between coast and hinterland
• Transit points for goods and people
• Possible entry points for invasions
• Immigration and emigration
• World commerce (Gilbert 2006)
During the days of naval exploration, natural harbors were prized as access
points connecting the motherland (via the ocean) to the new land to be explored. The
sheltered nature of the natural harbor provided safety and rest after being exposed
and vulnerable during the ocean voyage, as well as access to the potential renewal of
sustenance available on land. As previously mentioned, the comparison of a biofilm
to a port city and its associated harbor will involve the recognition of three spatially
differentiated zones. Each of these zones will be discussed in terms of physical
characteristics (e.g., viscosity, diffusion, and advection), the impact of these char-
acteristics on microbial motility, and how both the aspects of spatial zones and
physical characteristics relate to emigrating from and immigration to microbial life
on surfaces.
While port cities epitomize the concept of flow and facilitation of maintenance
and growth, both in terms of resources and residents, a besieged city demonstrates
the opposite. The absence of flow (or severely hindered movement) engenders
conditions of stress, starvation, stagnation, refuse buildup, dormancy, and death.
Compared to the flow system, its stagnant counterpart will conceptually be divided
into two zones: the biofilm attached to a solid surface and the overlaying stagnant
bulk fluid. Continuing with our analogy, the stagnant system could be considered a
city in a swamp where all inhabitants and food will enter or exit at a much slower rate
compared to a flow system, to the extent that stress and starvation conditions will be
the eventual outcome if population growth is not controlled.
It should be noted that the two analogies of port cities and besieged cities were
arbitrarily chosen and are by no means all-encompassing descriptions of biofilm
environments but rather very commonly occurring scenarios. Prior to continuing
with the above analogy and describing how the physical conditions in each of these
zones impact on various aspects of microbial life at or near surfaces, it is essential to
3 Biofilms: Besieged Cities or Thriving Ports? 61

first look at how a microbe experiences a watery environment. This may seem
unnecessary if we inadvertently assume that a microbe will behave in a manner
analogous to humans or another macroscopic organism’s swimming behavior.
However, in the course of the following sections, the relevance of doing so should
become evident.
The selection of microbial swimming as a focus area is intended to demonstrate
how this seemingly simple, often observed bacterial parameter can be influenced by
its environment.

3.5 Key Physical Parameters

3.5.1 Microbes in Aqueous Solutions

General biology training usually does not include a comprehensive study of fluids
either at rest or in motion (fluid mechanics), and even when included in the
curriculum, it must be recognized that applying fluid mechanics to biological
systems requires facing unique assumptions and challenges. For example, not all
“rule of thumb” numbers such as transition values for Reynolds numbers and
boundary layer thickness are applicable, as reviewed by Alexander (2016), and
therefore this subject matter might be better taught with specific applications and
constraints in mind (Loudon 1999) rather than as broad generalities only.
Here, we do not attempt to act as an authoritative text on fluid mechanics but will
rather point the reader to worthwhile resources while at the same time hoping to raise
an increased awareness of key concepts (Persat et al. 2015). As an example, due to
the microscopic size of a microorganism, the impact of a watery environment is quite
distinct from that experienced by a macroscopic organism (e.g., a fish or a human).
The ability of a bacterium to propel itself relative to its surroundings when sub-
mersed in an aqueous solution is equivalent to a human being’s ability to move
around in honey. This is a prime example of an instance where attempting to
interpret a scenario through the lens of a known experience, obtained either through
observation or personal experience (e.g., swimming), could result in erroneous
interpretation of experimental results and thus reaching an incorrect conclusion.
In the next few sections, the dimensionless Reynolds number will provide a lens
to understand the challenges encountered by microbial swimmers.

3.5.2 The Reynolds Number

Physicists, mathematicians, and engineers have long attempted to understand their

objects of study and translate this understanding into models, theories, or laws that
could be encoded into a mathematical formula or theorem. Ideally, it would be
possible to fully describe every object or process in the natural world with elegant
62 O. Kroukamp et al.

mathematical formulas for which all parameters and variables are known or could be
measured. In practice, however, in many instances scientific efforts lead to the
development of unwieldy equations with mathematical operators that require
advanced training to understand and solve. To overcome these limitations, assump-
tions are made that allow simplification of the formulas to address relevant questions
related to a particular aspect of the object of study. The choice of which parameters
to include or exclude in experimental observations and measurements is usually
dictated by some characteristic that will satisfy a practical need for the investigator,
for example, excluding parameters that are not deemed to contribute significantly.
In the case presented here, the object of observation is either a fluid moving
relative to a rigid object such as a fluid moving in a conduit such as a pipe or a
swimming body moving relative to its surrounding fluid. The question that we wish
to answer relates to whether the characteristics of the fluid and the characteristics of
the rigid object influence the movement of one another (if at all). The Reynolds
number, which describes the ratio of inertial to viscous forces of a fluid under defined
conditions, can be applied to answer this question.

3.5.2.1 Inertial Force of a Fluid

When a fluid element (i.e., a small volume or unit of water) moves through space, an
inertial force is ascribed to it which will resist a change in momentum; the latter
could be either a change in direction or velocity. This inertial force is directly
proportional to the density of the fluid and the square of the characteristic velocity
meaning that when the fluid element has higher mass (as represented by the density
term) or higher velocity, greater force will be required to stop it. The inertial force
(Finertia) is derived from Newton’s second law of motion:

F inertia ¼ ma ð3:1Þ
m ¼ ρV ¼ ρl3 ð3:2Þ
2
U Ul U
a¼ ¼ ¼ ð3:3Þ
t tl l
F inertia ¼ ρU 2 l2 ð3:4Þ

where m is mass, a is acceleration, ρ is the liquid density, V is the volume, l is the

characteristic length, U is the velocity of the fluid, and t is time.

3.5.2.2 Viscosity of a Fluid

Another force that may play a role in the movement of a fluid element is that of
viscosity (Fviscous). Fluid sticks to itself, so if one pictures adjacent water elements as
sheets of paper, the viscosity would be an indication of the stickiness between these
3 Biofilms: Besieged Cities or Thriving Ports? 63

sheets or the “glueyness” of the fluid (Vogel 1994). Viscosity can also be viewed as
fluid friction, with friction being an indication of the resistance generated upon the
relative movement of two solid objects that are in contact with each other. Key to
understanding viscosity as fluid friction is the realization that neighboring fluid
elements are moving relative to each other at different velocities. For example, a
sheet of water moving at a faster velocity will be slowed down by an adjacent, slower
moving sheet. Conversely, the faster moving sheet will tend to drag the slower
moving sheet at an increased velocity and if the adjacent sheets of water are moving
at the same velocity, the viscous force between these will disappear.
With regard to viscosity, we have thus far only considered fluid moving relative
to itself; however these observations can be extended to the case where a fluid moves
relative to a solid object. In addition to fluid elements sticking to one another, the
fluid will also stick to a solid object that it is in contact with. In fact, the extent of this
affinity is so great that this phenomenon is referred to as the “no-slip” condition. This
means that this layer of fluid is considered to have zero velocity relative to the
surface of the object (i.e., both are moving at the same velocity). By implication, we
can infer that a liquid velocity gradient will extend from the solid surface into the
bulk liquid phase. A simple example to demonstrate this is by observing debris
floating down a slow flowing river that travel slower when close to the bank
compared to those floating in the middle of the stream. From this we can conclude
that solid surfaces in contact with a fluid, whether a static pipe wall or a solid
swimming body immersed in a fluid, will always result in viscous forces being
present in the layers close to the surface where these velocity gradients exist.
The derivation for the equation for viscous force (Fviscous) can be found in most
fluid mechanic textbooks and is given by

μUl2
F viscous ¼ ð3:5Þ
l

where μ is the dynamic viscosity of the fluid.

Terminology can sometimes be confusing, especially when a word might have
one meaning in everyday speech but a different or nuanced meaning in a certain
research field or when particular background knowledge is required to avoid misin-
terpretation. An example of the latter is found in fluid mechanics literature, where the
fluid layer close to a surface is referred to as the viscous layer or the “region where
viscous flow occurs” as opposed to inviscid flow further away from the surface. A
person familiar with the field will realize that the dynamic viscosity for an incom-
pressible Newtonian fluid such as water is indeed a constant (as is evident from the
derivation of the shear stress equation—see any fluid mechanics textbook for an
example), but a non-expert may assume that the viscosity decreases with increasing
distance away from the surface.
When considering the movement of a fluid element, the analogies from Vogel
(1994) are very useful: he describes inertial forces as reflecting the “individuality” of
these fluid elements, while the viscous force reveals their “groupiness.” In practice,
64 O. Kroukamp et al.

this can be viewed as follows: when inertial forces dominate, fluid movement can be
described by the progress of a milling crowd, while the movement resembles that of
a disciplined march when viscous forces prevail (Vogel 1994).

3.5.2.3 Reynolds Number Equation

For specific flow conditions and geometries, it is therefore possible to comment on

whether the flow conditions are dominated by inertial or viscous forces by consid-
ering the ratio of these forces acting on a fluid element. This ratio, represented by the
dimensionless Reynolds number (Re), is indeed frequently used in the field of fluid
mechanics to make a distinction between inertia-dominated flow (turbulent flow) and
viscosity-dominated flow (laminar flow).

F inertia ρl2 U 2 ρlU

Re ¼ ¼ 2 ¼ ð3:6Þ
F viscocity μl U=l μ

with the characteristic length, l, and characteristic velocity, U, being specific to

different flow geometries and conditions.

3.5.2.4 Points for Consideration When Applying the Reynolds Number

In reference to the ideal scenario of having appropriate mathematical formulas to

assist our understanding of the physical environment, Vogel (1994) praises the
usefulness of the abovementioned ratio by calling the:
Peculiarly powerful Reynolds number the centrepiece of biological fluid mechanics.

And Vogel goes on to say that this “almost magical variable” is “the nearest thing
we have to a completely general guide to what is likely to happen when solid and
fluid move with respect to each other.”
After reading these commendations from an expert in the field of fluid mechanics,
a biologist might conclude that he or she has encountered some sort of equational
jackpot—which it indeed can be—but this is only true after careful consideration of
how the Reynolds number should be understood and used.
In our case, we described the Reynolds number as the ratio of forces acting on a
fluid element. However, it should be noted that there are various physical interpre-
tations of the Reynolds number, four of which are mentioned by Lauga and Powers
(2009). For the purpose described in this chapter, the main function of the Reynolds
number is to provide an indication under which flow conditions inertial (turbulent) or
viscous (laminar) forces will dominate, given the characteristics (e.g., density,
viscosity, velocity, etc.) of the fluid under consideration. Rather than attaching
importance to the specific numeric value of the Reynolds number, close attention
should be paid to the critical Reynolds number, which indicates when flow
3 Biofilms: Besieged Cities or Thriving Ports? 65

transitions from laminar to turbulent. For example, the transition Reynolds number
for flow in a circular pipe starts at approximately 2000, and turbulent flow can be
expected at a Reynolds number of 4000 (Brading et al. 1995). It is important to note
that the transition Reynolds number is dependent on the geometry and flow condi-
tions. For example, each geometry results in a different definition for the character-
istic length, l, and the characteristic velocity, U; e.g., for a circular pipe, the
characteristic length is equal to the inner diameter of the pipe, whereas the charac-
teristic velocity is the average fluid velocity. In essence, the Reynolds number
indicates the character of the flow regardless which of the contributing variables
cause the change. A tenfold reduction in characteristic length will increase relative
viscous effects by a factor of 10, similar to what would happen if the viscosity
increased tenfold (Vogel 1994).

3.5.2.5 Reynolds Number for a Swimming Body Such as a Bacterium

Osborne Reynolds (after which the Reynolds number was named) investigated the
transition from laminar to turbulent flow in pipes, which represent a fluid moving
past a stationary surface. However, the same reasoning regarding the ratio of inertial
forces to viscous forces can be applied to a rigid body moving through stationary
fluid, given that the appropriate characteristic length and characteristic viscosity are
defined. For a bacterial cell moving through a fluid, the characteristic length is taken
as the greatest length of the swimming body in the direction of flow, and the
characteristic velocity is the difference in velocity between the swimmer and the
velocity of the bulk fluid. In stationary fluid, the bulk fluid velocity will be reduced to
zero and the characteristic velocity used to calculate the Reynolds number will be
equal to the velocity of the swimmer.
The Reynolds number for a swimming microorganism can therefore be deter-
mined by using the following approximate values (Lauga and Powers 2009); the
density and dynamic viscosity of water can be given by ρ  103 kg m3 and
μ  103 Pa s, respectively, the characteristic length of microbes ranges between
1 and 10 μm, and the characteristic speed is 10–30 μm s1. These values will yield
a Reynolds number of approximately 105 to 104; these very low numbers indicate
that a microorganism swimming in water will experience strong viscous resistance.
In comparison, a 2-m-tall human, swimming at 1–2 m s1 in water, would be
equivalent to a Reynolds number in the order of 105 to 104, thus indicating a
dominance of inertial forces. Only if a human would attempt to swim in honey,
with a dynamic viscosity roughly 10,000 times that of water, would this person
experience viscosity-dominated laminar swimming conditions similar to what bac-
teria experience when moving in water.
Related to whether viscous forces will dominate or not, we can use the same
equations and reasoning to determine what distance a swimmer will coast (i.e., float)
after all activity has stopped. Lauga and Powers (2009) derived equations to show
that the coasting distance, d, under inertia-dominated flow conditions is given by
66 O. Kroukamp et al.

ρswimmer
dl ð3:7Þ
ρfluid

where l is the characteristic length of the swimmer and ρ the density of the swimmer
and fluid, respectively. This equation indicates that a human swimmer will coast for
a few meters in the water before coming to a standstill.
Under conditions where viscous forces dominate, the coasting distance is given
by

ρswimmer
d  lRe ð3:8Þ
ρfluid

where l is again the characteristic length and Re is the Reynolds number calculated
as before, indicating that a bacterium will slow down after about 0.1 nm, within a
time frame in the order of microseconds (Purcell 1977).

3.5.3 Reduced Flow Velocity Near a Surface

The liquid flowing near a wall, such as the inner surface of a pipe, will be dominated
by viscous forces under both laminar and turbulent conditions. This region is
associated with a gradient in flow velocity, with increasing fluid velocity observed
further away from a surface. While the velocity profile in this region is strictly
speaking parabolic, the assumption of linearity is acceptable if only a small distance
is considered (Rao 2010). Engineers refer to this region as the hydrodynamic
boundary layer, and conceptually it separates this fluid from the zones further
away from the surface where viscous forces are negligible compared to inertial
forces (Brading et al. 1995). Under turbulent flow conditions, the flow close to the
surface is referred to as the “laminar sublayer” or “viscous sublayer” (Brading et al.
1995). From a biological perspective (e.g., biofilms growing on a pipe surface), the
region close to the surface can be viewed as a “surface microenvironment” (Caldwell
and Lawrence 1988; Watnick and Kolter 2000) as opposed to the macro-
environment encountered in the bulk fluid flow further away from the surface.
For smooth surfaces a conservative thickness, δ, for this sublayer can experimen-
tally be determined as

5v
δ ¼ pffiffiffiffiffiffiffiffiffiffiffi ð3:9Þ
τW =ρ

where v(v ¼ μ/ρ) is the kinematic viscosity of the fluid, ρ is the density of the fluid,
and τw is the shear stress at the wall (Rao 2010; Agrawal 2012). The thickness of δ is
determined experimentally from velocity profile graphs (the regions where these
profiles dominate and where they intersect) portraying the different layers that occur
3 Biofilms: Besieged Cities or Thriving Ports? 67

under turbulent flow conditions. Depending on which portions of a particular

equation or specific intersection of two profiles are used, some textbooks will
provide a δ with a different constant (Allan 1995; Rubin and Atkinson 2001), e.g.:

11:6v
δ ¼ pffiffiffiffiffiffiffiffiffiffiffi ð3:10Þ
τW =ρ

More detailed explanations for this discrepancy can further be explored elsewhere
(White 2010). Despite the use of these different constants, an approximation of the
thickness of the viscous sublayer can be garnered from reported values, although
scarce in the current literature. Rough approximations for industrially (Rubin and
Atkinson 2001) and environmentally relevant conditions (Kumarasamy and Maharaj
2015) estimate this thickness to be in the order of 102 μm. This would suggest that
the viscous sublayer may provide sufficiently retarded flow close to the surface for
microbes to attach and form biofilms even under turbulent flow conditions. The latter
has been reported for Listeria innocua (Perni et al. 2006), mixed species biofilms
(Percival et al. 1999), and E. coli (Teodósio et al. 2011).

3.5.4 Flow in Non-Newtonian Fluids Such as a Biofilm

Up to now we have mostly considered Newtonian fluids (e.g., water) where the
viscosity does not change when the rate of shear strain (change of strain or defor-
mation with respect to time) is increased or decreased. However, the behavior of
Newtonian fluids can change drastically when small particles are suspended in the
liquid or when macromolecules are dissolved in the liquid (Brown and Jaeger 2011).
The focus now turns to two aspects related to this nonlinear fluid behavior as it
relates to biofilms, i.e., shear strain rate-induced nonlinearities (shear-thickening and
shear-thinning) and viscoelastic behavior.
Some suspensions will be liquid-like when slightly perturbed, but once exposed
to higher impact, an increase in viscosity will harden the liquid to such an extent that
it could support a person running across it without sinking into it (Waitukaitis and
Jaeger 2012). This phenomenon is termed shear-thickening, whereas a decrease in
viscosity upon an increased rate of shear strain is called shear-thinning. Since a
particular fluid may change its behavior depending on the shear rate applied, a fluid
cannot be unequivocally classified as either shear-thinning or shear-thickening.
These terms should therefore rather be considered as dependent on the flow condi-
tions, as opposed to a characteristic of the fluid itself (Mewis 2012). Furthermore, the
definitions of shear-thinning and shear-thickening (often portrayed as a graph of
shear stress vs strain rate) represent another example where the terminology may be
misleading since these terms refer to shear, while the definitions require strain rate to
be the controlling factor (Mewis 2012). Biofilms or EPS matrix encapsulated
microbial cells (Flemming and Wingender 2010) have been described as a complex
68 O. Kroukamp et al.

fluid (Wilking et al. 2011) and recorded to display shear-thinning behavior (Houari
et al. 2008; Billings et al. 2015; Patsios et al. 2015).
Some sources in the literature may also refer to “strain hardening” or “shear
stiffening” (functional definitions will be mentioned later in this paragraph) (Barai
et al. 2016), but these terms should not be confused with shear-thickening or shear-
thinning (Mewis 2012). Rather, the former terms are used to indicate the occurrence
of viscoelasticity, which is normally determined from a plot of shear stress vs strain
(Fabbri and Stoodley 2016), as opposed to using plots of shear stress vs strain rate to
visualize shear-thickening or shear-thinning behavior (Stoodley et al. 1999a, 2002a;
Klapper et al. 2002; Peterson et al. 2015). A viscoelastic material will display both
viscous and elastic properties, meaning that it deforms when placed under stress.
Once the stress is removed, the material returns to its previous state, which may
seem similar, but not necessarily identical, to the original state (Peterson et al. 2015;
Fabbri and Stoodley 2016).

3.6 Bacterial Locomotion

In the previous section, key interactions between the movement of fluids and solid
surfaces or bodies relative to one another were discussed. In the following section,
focus is shifted to bacterial motility with the aim of understanding how microbes
reach and interact with surfaces, since this is a crucial step in biofilm development. It
should be noted that while bacterial motility plays a role in surface association, not
all microbes are capable of locomotion and furthermore that other factors, such as
fluid hydrodynamics, do play a role in translocating microbes to surfaces.
Prokaryotes can propel themselves in different ways through liquids, including
swimming, swarming, gliding, twitching, or floating, among others as reviewed by
Jarrell and McBride (2008). Those authors detailed various mechanisms for move-
ment, including the use of surface appendages such as flagella and pili, or internal
structures such as the cytoskeleton and gas vesicles. The method of propulsion and
mechanism does not always correspond; for example, swimming with or without
flagella or gliding with or without pili is possible. Since the scope of this chapter is
limited to aqueous environments, the main focus of this section will be flagellar
swimming. Most bacterial flagella are thin (20–50 nm diameter) helical structures
(Berg 2003) that can extend from the cell body for several cell lengths. Information
regarding flagellar structure, assembly (Macnab 2003), and regulatory genes has
been reviewed by Jarrell and McBride (2008).

3.6.1 Single Cell Swimming

While flagellar-mediated swimming is a common means of microbial propulsion,

there are a few subclassifications of this mode of locomotion that become relevant to
3 Biofilms: Besieged Cities or Thriving Ports? 69

the discussion of near-surface swimming. In particular, we will consider differences

in flagellar number, location or arrangement on the cell body, and swimming
method.
The number and location of prokaryotic flagella vary among different species
(Merino et al. 2006). Examples include single (monotrichous) or multiple
(lophotrichous) polar flagella as found in Pseudomonas aeruginosa and Pseudomo-
nas putida, respectively (Theves et al. 2013), and multiple flagella distributed
uniformly over the cell body (peritrichous) such as seen for Escherichia coli and
Salmonella enterica (Berg 2003; Merino et al. 2006). The specific swimming
method strongly influences microbial behavior when an organism is interacting
with its environment, such as when it encounters surfaces or nutrient gradients.
Two important components of the method of swimming are speed and the ability to
change direction, with the mechanism of the latter varying substantially among
different flagellar arrangements.
Bacterial swimming speeds vary widely; e.g., Bdellovibrio bacteriovorus with a
single polar flagellum (Lambert et al. 2006) was observed to swim at 160 μm s1,
whereas P. putida with multiple polar flagella was able to swim at speed of
75 μm s1 (Harwood et al. 1989), and E. coli was capable of reaching speed of
35 μm s1 with peritrichous flagella (McCarter 2005).
Microbes with a single polar flagellum, such as P. aeruginosa, propel themselves
forward in a pusher motion by counterclockwise (CCW) rotation of their flagella or
reverse their direction in a straight trajectory by clockwise (CW) rotation (puller
motion; Cai et al. 2016). Peritrichous bacteria, such as E. coli, bundle their flagella,
and CCW rotation similarly drives the cells forward (called a “run”), while clock-
wise rotation disrupts the flagellar bundle and results in a “tumble” (Jarrell and
McBride 2008; Cai et al. 2016) or “twiddle” as the original authors pointed out (Berg
and Brown 1972). It should be noted that the direction of motion resulting from CW
or CCW rotation depends on the handedness of the flagella of a particular species.
CCW rotation of the left-handed filaments of E. coli drives the cell forward, whereas
the single, right-handed helical filament of the bacterium Caulobacter crescentus
move the cells forward by rotating CW and reverse the cell by CCW rotation (Lele
et al. 2015). Thus, a switch between CCW and CW flagellar rotation brings about
both propulsion and direction changes for peritrichous bacteria, while alternating
between CCW and CW rotation for bacteria with a single flagellum will merely
move the cells forward or backward in a straight trajectory (in the absence of a
nearby surface).
While not a focus area in this chapter, eukaryotic flagella are considered to be
distinct from prokaryotic flagella (Berg 2003) in terms of location, structure, func-
tion, and swimming patterns (Polin et al. 2009; Guasto et al. 2012; Moran et al.
2014). Among the eukaryotes, Chlamydomonas (algal unicellular flagellates) are
capable of beating their flagella in two different ways: a breaststroke and an
undulatory (waves travelling down the flagellum) stroke (Tam and Hosoi 2011).
70 O. Kroukamp et al.

3.6.2 Collective Swimming

While only single cell swimming has been considered thus far, collective swimming
by numerous cells results in interesting deviations from the norm and may influence
physical fluid parameters such as viscosity and diffusion. For example, Lushi et al.
(2014) investigated self-organizing patterns exhibited by dense suspensions of
confined bacteria. However, the interactions between microbes and fluid parameters
are of greater relevance to the present discussion and will remain the focus area.
Sokolov and Aranson (2009) measured the shear viscosity in thin films resulting
from swimming Bacillus subtilis using two complementary experimental approaches
[shear viscosity can be determined experimentally as the ratio of the tangential shear
stress and the shear rate (Sunthar 2010)]. The first approach involved measuring the
deterioration of a large vortex, whereas the second determined the viscous torque on
a rotating magnetic particle in the presence of various concentrations of B. subtilis
cells. The authors report a decrease in liquid viscosity of up to a factor of 7; this
decrease was relative to the viscosity of a fluid containing non-motile or no bacteria,
and it was dependent on the bacterial concentration and swimming speed (Sokolov
and Aranson 2009). It seems, however, that the above observation was particular to
pusher-type swimmers such as B. subtilis since Rafaï et al. (2010) showed experi-
mentally that puller-type swimming by the unicellular microalgae, Chlamydomonas
reinhardtii, resulted in an increase in fluid viscosity compared to the same concen-
tration of dead cells.
Ishikawa (2009) discussed how puller-type swimmers will repel each other when
swimming side by side, while pusher-type swimmers will attract each other. The
author goes on to describe how suspensions of swimming microbes can affect both
fluid rheology and diffusive properties (enhancing the diffusion of dissolved
chemicals in the suspension) by influencing these fluid properties on a mesoscale
(between microscopic and macroscopic; Ishikawa 2009). Underhill et al. (2008)
expand on the latter as it relates to the mode of swimming and showed that the pusher
motion enhanced the diffusion of tracer molecules more so than did puller-type
swimming.
Experimental simulations of a bath containing swimming E. coli indicated a
significantly larger effective diffusion coefficient for a tracer molecule suspended
in the fluid, compared to the thermal effective diffusion coefficient exhibited by a
fluid containing only passive particles (Morozov and Marenduzzo 2014). The
authors concluded that this increase in effective diffusion is dependent on the
concentration of bacteria, their swimming speed, as well as a characteristic velocity
field created by a single swimming bacterium (Morozov and Marenduzzo 2014).
Wolgemuth (2008) investigated the formation of transient jet and vortex patterns in
the presence of high densities of B. subtilis using a theoretic model. According to the
simulations performed, active swimming by the bacteria resulted in turbulent mixing
of the fluid, with fluid velocities exceeding the maximum swimming speed set for
individual bacteria.
3 Biofilms: Besieged Cities or Thriving Ports? 71

3.6.3 Surface Interaction

3.6.3.1 Swimming Close to a Surface

The tendency for swimming cells to remain in close proximity to surfaces in a time
frame ranging from seconds to minutes has been ascribed to hydrodynamic effects
(Vigeant et al. 2002; DiLuzio et al. 2005; Conrad 2012), as was shown for
non-tumbling E. coli (Berke et al. 2008).
Confirmation of the role of bacterial locomotion in surface association was
provided by Galajda et al. (2007). The authors established that swimming rather
than non-swimming microorganisms were trapped along a wall with micro-
fabricated funnel-shaped openings. Pusher cells strongly migrate toward nearby
boundaries or surfaces when few in number. However, at higher cell concentrations,
this coherence is disrupted by large-scale interaction between swimming cells
(Hernandez-Ortiz et al. 2005).
The accumulation of cells at a surface has also been demonstrated to depend on
swimming speed and cell length, with faster and longer cells accumulating to a
greater degree than slower and shorter ones (Li et al. 2011). In addition, the tumbling
frequency of peritrichous bacteria was reduced by 50% within 20 μm of a surface
and has been proposed to contribute to near-surface cell trapping (Molaei et al.
2014).
The proximity of a surface is known to influence microbial swimming behavior
such as a change in the microbe’s trajectory or a reorientation of a cell. For example,
E. coli swims in a CW, circular motion near a solid boundary; rotation of E. coli’s
left-handed helix will cause the cells to turn to the right (CW) (Lauga and Powers
2009). The circular motion observed when monotrichous bacteria swim in reverse
near a surface has been described as “run-and-arc” swimming (Karimi et al. 2015)
and has been reported for a number of monotrichous bacteria including Caulobacter
crescents and P. aeruginosa. Pusher cells have been found to reorientate themselves
parallel to a surface, while pullers will orientate themselves at a right angle with
respect to the wall, thereby appearing to swim into it (Lauga and Powers 2009).
While the singly flagellated Vibrio alginolyticus usually swims backward and
forward by alternating the rotation direction of its flagella, it behaves differently
near a surface. Its forward and backward swimming speeds have been observed to
differ significantly and the swimming motion to trace a more circular trajectory. The
latter effect was explained by fluid dynamic interactions between the cell and the
rigid boundary (Goto et al. 2005).

3.6.3.2 Surface Sensing

The question may arise as to how a bacterium senses that it is near a surface. Cells
are able to sense direct contact with a surface or experience surface-associated
changes in hydrodynamic conditions via different mechanisms, one of which
72 O. Kroukamp et al.

involves the restriction in flagellar rotation (Harapanahalli et al. 2015). Belas (2014)
investigated the role of rotating flagella in surface sensing and found that rotating
flagella are used as mechanosensors to detect subtle changes in the operation of their
motors when they near a surface (Belas 2014), specifically via the motor stators (Lele
et al. 2013). Obstruction of flagellar rotation may trigger adhesion and surface-
associated motion (Ellison and Brun 2015). Cairns et al. (2013) discussed how the
inhibition of flagellar rotation triggers a signal transduction cascade in B. subtilis. In
addition to the differential expression of genes linked to motion, the association with
surfaces and the related mechanical interactions may influence virulence factor-
related gene expression. The latter was shown by Siryaporn et al. (2014) for
P. aeruginosa after attachment to various, chemically distinct surfaces and is
worth considering in the study of biofilms’ role in infection.
However, it should be noted that not all surface-sensing pathways involve
flagella, such as the case of non-motile bacteria (McClaine and Ford 2002; Belas
2014). Some bacteria sense a surface when experiencing adhesion force-induced
deformation of the cell wall (Harapanahalli et al. 2015).

3.6.3.3 Shear Trapping

In the absence of flow, the density of a dilute suspension of non-tumbling, peritri-

chous B. subtilis was found to be uniform, whereas the introduction of flow led to the
accumulation of cells at surfaces (Rusconi et al. 2014). An increase in flow rate or
shear corresponded to an increase in the concentration of cells at the surface and was
termed “shear trapping” (Rusconi et al. 2014; Rusconi and Stocker 2015). This
phenomenon required motile cells, since dead cells did not show any variation in
spatial density (similar to the instance of no-flow) (Rusconi et al. 2014). Shear
trapping not only increases surface attachment but also suppresses chemotaxis
(Rusconi et al. 2014) and the ability of a cell to respond to external stimuli (Bearon
and Hazel 2015).
Similar results were observed for P. aeruginosa (Lecuyer et al. 2011) where the
time that the bacteria remained adhered to the surface increased nearly linearly as a
result of an increase in wall shear stress from zero to 3.5 Pa. The authors
furthermore observed that while the absence of type I pili, type IV pili, the flagellum,
or EPS building block production affected the frequency with which cells could
become either adhered or detached, none of these factors could account for the
average increase in adhesion time correspondent with shear (Lecuyer et al. 2011).
Since the same result was observed for tumbling B. subtilis and the monotrichous
P. aeruginosa, it appears plausible that this behavior occurs irrespective of the
swimming motion. Early reports by Molaei et al. (2014) indicated an increased
incidence of shear trapping under conditions without flow (Molaei et al. 2014). In a
subsequent study, the group (Molaei and Sheng 2016) used digital holographic
microscopy to track swimming E. coli near surfaces under shear. Under these
conditions, the authors found that shear mitigated the tumbling inhibition, which
in turn reduced cell trapping close to the surface.
3 Biofilms: Besieged Cities or Thriving Ports? 73

3.6.3.4 Swimming Upstream

Contrary to what may be assumed, microorganisms are able to swim and translocate
over surfaces against the prevailing direction of flow (Hill et al. 2007; Rusconi and
Stocker 2015). For example, P. aeruginosa cells are initially orientated along the
direction of flow, after which the retraction of the polar type IV pili allow upstream
twitching (Shen et al. 2012).
Kaya and Koser (2012) observed that E. coli can swim upstream under flow
conditions at speeds exceeding 20 μm s1. In a previous section, it was mentioned
that E. coli swims in circles when encountering a surface under quiescent conditions.
In this mode the cell experiences an increased hydrodynamic drag which rotates and
dips the front of the cell body, thereby keeping the bacterium pointed toward the
surface with the flagella oriented away from the surface (Kaya and Koser 2012).
Under flow conditions the drag on the flagella oriented further away from the surface
than the cell body will rotate the cell to face upstream. The authors concluded that
this upstream swimming behavior is not unique to the propulsion mechanisms of
E. coli but merely requires a swimming microorganism moving freely over a surface
under moderate flow conditions (Kaya and Koser 2012). Korber et al. (1989)
compared surface colonization of motile and non-motile P. fluorescens under two
flow velocities, 8 and 120 μm s1. They found that flagellated cells not only attached
more rapidly than did nonflagellated cells under both velocities but also migrated
upstream against the laminar flow.

3.7 Dissolved Nutrients

In this chapter the main focus is on how microbes interact with their physical
aqueous environment. As a result, the question of how microbes interact with their
chemical environment, especially as it pertains to breathables (electron acceptors)
and edibles (electron donors) (Nealson 2003), the gradients of these chemicals and
how it relates to chemotaxis will only be discussed briefly—its importance, however,
should always be considered when interpreting biofilm behavior.
For biofilms growing on surfaces, a mass transport or diffusion boundary layer
exists within the hydrodynamic boundary layer (Lewandowski and Beyenal 2013).
This mass transport boundary layer can be demonstrated by the nutrient concentra-
tion gradient increasing with increasing distance from the surface (assuming that
nutrients found in the bulk is being consumed as it approaches the biofilm on the
surface); at distances close to the surface where fluid velocity is low, mass transport
is diffusion dominated, whereas the higher flow velocities prevailing at greater
distances from the surface result in convective mass transport.
74 O. Kroukamp et al.

3.7.1 Diffusion and Advection

If one considers a stationary, nutrient-filled fluid surrounding a spherical stationary

microbial cell that assimilates all nutrients reaching its surface, a nutrient concen-
tration of zero will result at the cell’s surface (Dusenbery 2009). The difference in
nutrient concentration between the cell surface and the bulk fluid will generate a
concentration gradient which in turn will be the driving force for diffusion from the
bulk to the cell surface. Given that the concentration of the particular nutrient is zero
(at the cell surface) under these conditions, the question is whether the microbe
would be able to increase its rate of nutrient uptake by swimming? To attempt an
answer to the above question, some rudimentary calculations will provide insight
into the relative contributions of the two main mechanisms of solute transport
through a solution, namely, flow and diffusion. Under conditions of flow (advective
transport) at velocity U, the time, ta, required for a solute to move a distance l can be
approximated (Purcell 1977; Dusenbery 2009) by

l
ta  ð3:11Þ
U

For diffusion the time, td (Purcell 1977; Dusenbery 2009), for transport can be
approximated as

l2
td  ð3:12Þ
D

where D is the diffusion coefficient. One consequence of time being proportional to

the squared distance is that diffusion times are very short over small distances; a
small molecule will diffuse across a bacterial cell (1 μm) in approximately a
millisecond (approximating the diffusion coefficient in water as 109 m2 s1).
Both of the above approximations of transport time are derived from the one
dimensional advective diffusion equation for an incompressible fluid where variable
sizes have been estimated on the level of orders of magnitude rather than precise
values. If the ratio of diffusion transport time to that of flow (advection) transport
time is taken, we arrive at the dimensionless Péclet number (Vogel 2004; Dusenbery
2009):

t d Ul
Pe ¼ ¼ ð3:13Þ
ta D

Similar to the Reynolds number discussed earlier, the Péclet number is used to
comment on the occurrence of certain regimes of flow (transport dominated by
advection or diffusion) and exact values are of lesser importance, especially given
that the Péclet number is not taking any geometrical details into account and has
3 Biofilms: Besieged Cities or Thriving Ports? 75

been derived from order of magnitude assumptions rather than exact variables
(Vogel 2004).
From our earlier discussions into how an individual swimming microbe experi-
ences a watery environment, it is evident that “at low Reynolds numbers you cannot
shake off your environment” (Purcell 1977). Purcell (1977) and Vogel (2004)
approximated a Péclet number of 102 for a microbe swimming in water, thereby
indicating that swimming will not increase nutrient transport to the cell. On a
practical level this implies that a swimming microbe carries a layer of fluid around
with it, and the only means whereby nutrients can cross the fluid is via diffusion.
This phenomenon is succinctly summarized by Dusenbery (2009) as follows: “they
carry a halo of fluid depleted of nutrient around with them.”

3.7.2 Chemotaxis

Since tumbling by an E. coli cell will result in a random change of direction, the
question arises on how this microbe manages to navigate in a desired direction, e.g.,
toward a higher concentration of a food source or another chemoattractant. Chemo-
taxis is facilitated by regulating the run length, which in effect means that the runs in
between tumbling events will be longer when moving in the direction of a
chemoattractant (Cai et al. 2016). In contrast, the presence of a chemorepellent
will increase the tumbling frequency resulting in a net change in overall swimming
direction. The chemotactic ability of monotrichous bacteria such as P. aeruginosa,
especially as it relates to directional changes, has been ascribed to a number of
different strategies. Firstly, a change in direction has been attributed to random
derailing due to Brownian motion of the fluid (Li et al. 2008). Secondly,
P. aeruginosa can increase the likelihood of moving toward a chemoattractant by
prolonging movement in a particular direction (Cai et al. 2016). It was found that
P. aeruginosa can use either the forward or reverse motion to move up a
chemoattractant gradient (Cai et al. 2016). The switch between CCW and CW
rotation is separated by a pause, with the length of the latter positively correlated
with the size of the turning angle (Qian et al. 2013). Thirdly, some uniflagellate
bacteria such as Vibrio alginolyticus can turn or “flick” (Son et al. 2013) by utilizing
a strain-induced collapse in its flagellum, which the authors call “an example in
nature of a biological function stemming from a controlled mechanical failure.”
Taylor and Stocker (2012) argue that microbes should derive sufficient nutritional
benefit given the energy expenditure required for chemotactic swimming motility
(speed). Maintaining optimal swimming speeds to balance this trade-off will be
particularly important in nutrient-poor environments such as ocean water (Stocker
et al. 2008). The authors found that the monotrichous (Gauthier et al. 1995) marine
bacterium Pseudoalteromonas haloplanktis was able to engage in a chemotactic
response that was >10 times as rapid as E. coli. This swift response was mainly
attributed to the faster swimming speed of P. haloplanktis (68 μm s1). Stocker
(2011) mentions that the chemotactic motility pattern of monotrichous bacteria
76 O. Kroukamp et al.

(a hybrid of forward and reverse movement with flicking) outperforms the chemo-
tactic response of the run-and-tumble moving pattern exhibited by, for example,
E. coli. Interestingly, of 600 motile marine bacteria isolated, approximately 90%
had a single polar flagellum (Leifson et al. 1964). The ability to respond rapidly may
result in a competitive advantage given the transient microscale nutrient patches in
the ocean.

3.8 Evaluating the Port City Analogy

To integrate the preceding discussions of the interactions between microbes and

surfaces in a dynamic fluid environment, especially as it relates to bacterial motility,
we will restrict the evaluation of our port city analogy to initial adhesion to a surface
or biofilm (immigration), the association of motile cells with an existing biofilm, and
detachment or dispersion from a biofilm (emigration).
When considering a port city with its harbor, we could recognize three zones,
namely, the city, the harbor, and the ocean. These regions can in turn be related to the
three zones of a biofilm in flowing conditions: the surface-attached biofilm, the
quiescent zone of low flow close to the surface, and the moving bulk fluid further
away from the surface (Fig. 3.1). Vigeant et al. (2002) similarly defined three zones
but based these on the distance from the solid surface, namely, the near-surface
constrained region, the near-surface bulk region, and the bulk fluid. The near-surface
constrained zone is impacted by both the hydrodynamic effect and physicochemical

Fig. 3.1 A diagram of the proposed zones associated with a biofilm under flowing conditions
(images taken from Bester et al. (2013) and not drawn to scale). According to the port city analogy,
the biofilm corresponds to the city (purple cells and brown EPS matrix), whereas the region with
reduced flow rates equates to the harbor (white background). In this zone, the reduced flow rates
allow for freely motile cells to swim upstream and around the biomass. The zone furthest away from
the surface (blue background) represents the ocean or bulk liquid flow where surface-associated
hydrodynamics have no influence on the characteristics of the flow
3 Biofilms: Besieged Cities or Thriving Ports? 77

properties of the surface, the near-surface bulk phase experiences only the hydrody-
namic effect of the surface, while in the bulk fluid, no effect from the surface is
evident.
According to the classical dogma, the formation of a biofilm is considered to be a
developmental process which proceeds through four or five stages from attachment
to maturation and detachment or dispersion (Sauer et al. 2002; Stoodley et al.
2002b). While this model provides a useful framework, it does not capture the
complexity of surface-associated microbial growth. The particular focus of this
model on the attached, sessile cells has the potential to restrict our perspective and
understanding of surface-associated microbial communities.
For example, this model does not take into account the “harbor” region of the port
city analogy where planktonic or freely motile cells can remain, meander around
biofilm biomass, and multiply indefinitely due to reduced flow rates. This “low-
flow” zone may be critically important in both immigration into and emigration from
the biofilm. By extension, this may yield a notably different progression of biofilm
development with a higher degree of fluidity than the discrete stages as depicted by
the classical biofilm model.
An additional example of the inherent, but perhaps unrecognized bias of the
biofilm developmental model is evident from an investigation into Legionella
pneumophila biofilm formation. Mampel et al. (2006) found that continued plank-
tonic cell replication (rather than sessile cell division) was necessary for biofilm
formation under static conditions. L. pneumophila was furthermore unable to form
robust biofilms under conditions of flow, but effectively associated with existing
biofilms comprised of certain environmental bacterial species (Microbacteria and
Acinetobacter baumannii), while other biofilms consisting of Klebsiella
pneumoniae, Pseudomonas spp., or Corynebacterium glutamicum resisted
L. pneumophila colonization (Mampel et al. 2006).

3.8.1 Biofilm Formation or Association: Immigration

Flow conditions can impact nutrient uptake and the rate of cell-to-cell encounters,
while shear in particular can influence the attachment of bacteria to the surface as a
first step in the process of microcolony and later biofilm formation (Brumley et al.
2015). Lemon et al. (2007) compared wild-type, flagellum-minus, and paralyzed-
flagellum mutants of Listeria monocytogenes and found that flagellar motility was
essential for initial surface attachment and biofilm formation in this species.
Vigeant et al. (2002) comments on the different forces at play when considering
the classic biofilm development dogma. According to this theory, reversibly adher-
ing cells originate from planktonic cells trapped by hydrodynamic forces after
swimming close to the surface for extended periods. The subsequent, irreversible
attachment of these cells can be explained by DLVO (Derjaguin, Landau, Verwey,
and Overbeek) electrostatic interactions.
78 O. Kroukamp et al.

Much of the boundary effect theory is based on the no-slip wall condition which
considers that at a solid boundary the fluid velocity relative to the boundary will be
zero. Hu et al. (2015) predicted that E. coli can sense nanoscale surface slip as
opposed to the usual no-slip assumption occurring at boundaries. Surface slip occurs
at surfaces modified by, e.g., adsorbents and hydrophobic compounds (Lauga et al.
2007; Rothstein 2010). An increasing slip length, as could be achieved experimen-
tally by coating a surface with the EPS polymer alginate, implies CCW circles of
decreasing radius which may lead to increased surface adsorption due to a prolonged
local residence time (Hu et al. 2015).
Reports such as the one by Barken et al. (2008) investigated whether the mere
presence of certain organelles versus the presence of functional organelles
influenced the outcome of a study deserve further consideration. The authors showed
that both type IV pili and flagella are required for the development of mature
multicellular structures in P. aeruginosa biofilms. Whereas motile, and thus func-
tional flagella were necessary for this process, type IV pili were only required to be
present but not necessarily functional, perhaps indicating a structural role in adhe-
sion (Barken et al. 2008). Wood et al. (2006) reported similar findings in E. coli
where the poorest biofilm formation, with respect to both architecture and thickness,
was observed for strains with impaired motility.
Once a swimming cell adheres to a surface and biofilm formation is initiated, one
could ask what happens to the appendages involved in motility. While a comparison
of global gene expression between biofilm and planktonic P. aeruginosa only
revealed a 1% difference, the expression of genes involved in pili and flagella
synthesis were repressed in biofilms (Whiteley et al. 2001). In contrast Domka
et al. (2007) showed that 20 motility-related genes are induced throughout all stages
of E. coli biofilm development, ranging from early attachment stages to fully mature
biofilms. Kalmokoff et al. (2006) demonstrated via proteomic analysis that the
flagella motility complex plays an important role in initial attachment of Campylo-
bacter jejuni. In addition, the continued expression of the flagella motility complex
even into the stages of a mature biofilm was noted. While motility is important for
the initial attachment of Vibrio cholerae, the expression of the flagella filament, flaA
decreases in mature biofilms, as reviewed by Guttenplan and Kearns (2013). It is
however unknown whether, after surface attachment, the flagellum remains func-
tional or if it is lost and degraded or if it acts as a structural component of the biofilm
(Teschler et al. 2015). Guttenplan and Kearns (2013) investigated the four model
systems of Bacillus, Vibrio, Pseudomonas, and Escherichia and proposed that
flagellar motility in the biofilm is regulated in two stages: firstly a fast inhibition
stage at the level of flagellar function and, afterward, a slow inhibition at the level of
flagellar gene transcription.
Nadell et al. (2015) investigated V. cholerae attached to surfaces and showed that
the extracellular matrix prevented biofilm invasion, where non-motile cells were
incapable of entering the existing biofilm and motile cells were only capable of
colonizing and growing on the biofilm exterior.
3 Biofilms: Besieged Cities or Thriving Ports? 79

3.8.2 Motility in or Around the Biofilm

Li et al. (2014) demonstrated that near-surface microbial swimming behavior differs

in viscoelastic fluids, such as biofilms, compared to Newtonian fluids. Proteins and
polysaccharides associated with the biofilm EPS matrix disperse into the surround-
ings, thereby imparting viscoelastic properties to the ambient fluid (Li et al. 2014).
The viscoelastic environment allows puller swimmers to escape more readily from
the biofilm environment, while the pusher swimmers appear to be perpetually
trapped (Li et al. 2014). Disparate swimming strategies will interact differently
with the viscoelastic environment, leading to increases or decreases in the swimming
speed of both single and collective swimming bacteria. For example, helical bacteria
demonstrate faster swimming speeds in a viscoelastic fluid compared to a Newtonian
fluid with the same viscosity (Li et al. 2014).
Martinez et al. (2014) measured E. coli motility in polymer solutions and
concluded that the flagella experienced a lower local viscosity than the cell itself.
In this sense the flagella not only act as small rheometers able to detect local
non-Newtonian behavior but also provide corridors of lower viscosity that will
make it easier for another bacterium to follow in its wake (Martinez et al. 2014).
Planktonic bacteria in close proximity to a biofilm can tunnel deep into the
biofilm by creating transient pores (Houry et al. 2012). These pores could benefit
the biofilm by enhancing nutrient transport or harm it by allowing the penetration of
antimicrobials (sometimes produced by the tunnelling bacteria themselves) leading
to supplanting of the original biofilm occupants (Houry et al. 2012).

3.8.3 Biofilm Detachment: Emigration

The proposed advantages of detachment from biofilms are numerous and include
(1) the ability to escape deteriorating environmental conditions once nutrients are
depleted due to increased competition, or the presence of an antimicrobial agent,
(2) the potential to reach and make use of new habitats, and (3) the opportunity to
generate genetic variation (McDougald et al. 2012). To these advantages should be
added the fact that detachment provides a mechanism to maintain metabolically
active biofilms that are in equilibrium with the carrying capacity of the local
environment, by balancing cell formation with accumulation. The continuous pro-
duction and release of cells, both during biofilm development and after steady state is
attained, has been proposed to be a mechanism for prokaryote proliferation and
transmission (Bester et al. 2009). A prerequisite for the dissemination of microbes
from biofilm-colonized habitats requires the capacity to detach and be actively
propelled or passively transported to a different environment, whereafter
reassociation with an existing biofilm or primary surface colonization can take
place. Several biofilm detachment modes have been identified and described
80 O. Kroukamp et al.

empirically, but a clear distinction between the different mechanisms and consensus
regarding the underlying mechanisms and ecological purpose remains to be reached.

3.8.3.1 Modes of Biofilm Detachment

The mechanisms of biofilm detachment have been defined both in terms of the
particle size of the detached biomass and the frequency of detachment (Bryers 1988).
Grazing by higher organisms such as protozoa or nematodes may result in the
physical disruption of the biofilm, and additional loss of attached biomass can be
expected to occur as a result of abrasion by solid particulates. Both of these
mechanisms may remove biomass ranging in size from small clumps to large
aggregates containing numerous microbial cells and EPS. Substantial reductions in
the amount of biomass may also occur at random intervals, due to the sloughing off
of large aggregates from the biofilm. While the underlying mechanisms resulting in
sloughing have not been identified, it is thought to occur when shear forces exerted
by flowing liquid exceeds the cohesive strength of the biofilm matrix.
Biofilms can move over surfaces (Stoodley et al. 1999b) and through porous
media under both laminar and turbulent flow conditions (Stoodley et al. 2005).
Under turbulent flow, ripple-like structures form and move downstream (Stoodley
et al. 1999b). Furthermore, the formation of filamentous biofilm streamers has been
reported under turbulent as well as laminar flow conditions (Stoodley et al. 1998;
Rusconi et al. 2010, 2011), with the latter being dependent on complex flow patterns
created by varied channel geometries. An increase in turbulent flow velocity led to
the breakage or detachment of some filaments (Stoodley et al. 1998), whereas
spontaneous sloughing off of filaments occurred at unpredictable intervals and
completely constricted laminar flow (Drescher et al. 2013). While it was thought
that the development of filamentous streamers under laminar flow required the
presence of unusual channel geometries such as corners, Parvinzadeh Gashti et al.
(2015) observed the formation of streamers in straight microchannels. The accumu-
lation of dense ridges of biofilm biomass was correlated to the abrupt partial
detachment of one end of a ridge, leading to the formation of streamers. The authors
determine a corresponding detachment rate of 0.15 events mm2 h1 for a single
species Pseudomonas biofilm.

3.8.3.2 Erosion, Planktonic Cell Yield, and Seeding Dispersion

The detachment of single cells from the biofilm has similarly been attributed to
various processes, including liquid-mediated erosion of cells from the biofilm
surface, active growth, cell division and release of progeny cells by the biofilm,
and most recently seeding dispersion. Despite evidence to the contrary, the detach-
ment of single cells from biofilms continues to be ascribed primarily to liquid-
mediated erosive action. Under conditions where substrate availability is governed
by flow velocity, the observed detachment rates are often better correlated to
3 Biofilms: Besieged Cities or Thriving Ports? 81

substrate availability or biofilm growth rates, rather than shear removal forces
(Peyton and Characklis 1992; Stewart 1993; Bester et al. 2009, 2013).
The rate at which single cells detached from various 120-h-old Pseudomonad
biofilms, exposed to bulk liquid flow, was quantified at approximately
107 cells cm2 h1, with an increase in the detachment rate evident as early as 6 h
after the introduction of the cells into a sterile environment (Bester et al. 2009). The
number of detached cells increased alongside biofilm development until both
parameters reached a maximum after 96–120 h. The removal of the carbon source
from 5-day-old Pseudomonad biofilms subjected to continuous flow resulted in a
40%, 94%, and 99% decrease in detachment rate after 1, 5, and 24 h, respectively.
During the ensuing 8-day period of carbon starvation, the rate of cell detachment
cells decreased by 1–2 orders of magnitude, compared to pre-starvation. A rapid
increase in the number of detached cells was observed upon a reintroduction of
carbon into the system, with a complete recovery of pre-starvation detachment rates
within 24 h (Bester et al. 2011). The observed response in cell detachment was
mirrored by the metabolic activity of the biofilm and clearly demonstrated the
contribution of active biofilm carbon consumption and growth to the number of
detached planktonic cells. To differentiate between shear-related erosion of attached
biomass and active attached cell growth and release of progeny into the environment,
the term “biofilm-derived planktonic cell yield” has been proposed (Bester et al.
2009).
Observation of the active dispersal of planktonic cells from within P. aeruginosa
biofilm microcolonies (Sauer et al. 2002; Purevdorj-Gage et al. 2005) has been
designated as the final stage of the biofilm development life-cycle model (Stoodley
et al. 2002b). During dispersion, actively swimming planktonic cells are observed to
exit from the interior of biofilm microcolonies, while a sessile, hollow structure
remains behind (Purevdorj-Gage et al. 2005). Prior to cell dispersal taking place, an
increase in cell lysis and death within the colonies have been reported. The latter is
likely due to an accumulation of reactive oxygen and nitrogen species as well as
bacteriophage-mediated cell lysis (Webb et al. 2003; Barraud et al. 2006). Interest-
ingly, the dispersed population for both P. aeruginosa (Kirov et al. 2007) and
Pseudoalteromonas tunicata (Mai-Prochnow et al. 2006) displayed significant var-
iation in a number of phenotypic traits, such as colony morphology, motility, biofilm
formation, metabolic activity, production of quorum sensing molecules, and viru-
lence factors. The authors drew an analogy between this observed increase in
phenotypic differentiation within a prokaryote and known eukaryotic dispersal
strategies, which generate variation to enhance the probability of successful surface
colonization under a variety of environmental conditions (Mai-Prochnow et al.
2006). While seeding dispersal has been documented for a number of bacterial
species, not all strains within a species behave in this manner as evidenced by
conflicting reports on the ability (or inability) of mucoid clinical isolates of
P. aeruginosa to disperse (Purevdorj-Gage et al. 2005; Kirov et al. 2007). The
relative contribution of each of these mechanisms to the extent of biofilm detachment
will likely vary in space and time and depend on environmental conditions as well as
biofilm-specific factors.
82 O. Kroukamp et al.

3.8.3.3 Rates and Extent of Biofilm Detachment

In addition to the abovementioned, others have attempted to quantify detachment

rates from single species, defined consortia, and multispecies biofilms. In an elegant
study by Stoodley et al. (2001), the rates at which aggregates detached from a
defined four-species consortium biofilm and an undefined tap water biofilm, both
cultivated under turbulent flow, were quantified. Detachment rates from the tap
water biofilm were estimated at 3.7 detaching clusters mm2 h1 with an analysis
interval of 1 h, up to 80.2 detaching clusters mm2 h1 when data was acquired at
20-min intervals. The majority of detached particles were single cells or small cell
aggregates, whereas large clusters consisting of multiple cells detached less fre-
quently but accounted for a significant fraction of the overall amount of biomass lost.
The detachment rate from P. aeruginosa PAO1 biofilms has been estimated at
5.14  104 events min1 mm2. Single cells accounted for a minimum of 70% of
these events, whereas the remaining 30% corresponded to events consisting of 2 to
>1000 cells, with aggregates consisting of >100 cells accounting for less than
0.28% of the total detachment events but containing up to 37% of the total number
of detached cells for PAO1 (Wilson et al. 2004). A detachment rate of 1.8  107
CFU cm2 h1 quantified for 7-day-old Staphylococcus aureus biofilms. In contrast
to P. aeruginosa biofilms, it was found that aggregates containing 11–100 cells
detached at the greatest frequency (20% of all events) and contained the largest
fraction of all detached biomass (54%) (Fux et al. 2004).

3.9 Conclusion

The recognition that microbes are predominantly associated with aggregates or

biofilms in natural environments has naturally led to a shift in the perception of
microbial processes, along with a concomitant increase in research efforts to address
our lack of understanding. Extensive investigation into biofilm development by a
selected number of “model” microorganisms has indeed greatly enhanced this
understanding, and a notable outcome from these efforts was the establishment of
the classical four- or five-stage model of biofilm development. This model, under-
standably, focuses on the attached, matrix-encased sessile cells and associated
microenvironments, whereas planktonic cells are only seen to contribute to biofilm
processes during initial attachment to an uncolonized surface and eventual detach-
ment from a mature biofilm. This limitation has the potential to restrict our perspec-
tives and understanding of surface-associated microbial communities.
Sessile cells acquire genetic and phenotypic characteristics during biofilm for-
mation that distinguish them from planktonically derived cells. However, a number
of authors have commented on the existence of a third phenotype (Bester et al.
2005). This phenotype is displayed by cells that detach from the biofilm and have
been characterized with respect to differences in adhesion (Rollet et al. 2009),
3 Biofilms: Besieged Cities or Thriving Ports? 83

virulence (Uppuluri and Lopez-Ribot 2016), and antimicrobial susceptibility (Boles

and Horswill 2008).
In addition, the biofilm or surface-associated region with greatly reduced flow
rates may facilitate the persistence of an independently-replicating planktonic pop-
ulation alongside the attached biomass. Delineating the interactions between these
attached and motile subpopulations, along with determining their relative contribu-
tions to global processes, would require concerted future research efforts.
It can be expected that the particular environment experienced by bacterial
populations, whether conditions of plenty or scarcity, will drastically influence
their behavior and productivity. In order to learn more about an individual cell in
its habitat, it is critical to be aware of the characteristics of the environment in which
it is studied, a good example being consideration that different conditions can turn
cooperator into cannibal (González-Pastor et al. 2003). It can be argued that learning
about an individual or species in its environment is only of interest to fundamental
researchers. However, in the case where humans are dependent on the well-being
versus demise of a microbial community, the importance of understanding the
relationship that the community has with its environment becomes much more
than fundamental curiosity, especially if it must be manipulated to achieve a desired
outcome.

List of symbols with definitions and units

Mathematical
symbol Definition Units
a Acceleration m s2
l Characteristic length m
ρ Density kg m3
d Coasting distance m
δ Thickness of viscous sublayer m
D Diffusion coefficient m2 s1
F Force N or kg m s2
Finertia Force inertial N or kg m s2
Fviscous Force viscous N or kg m s2
m Mass kg
ν Kinematic viscosity m2 s1
Pe Péclet number Dimensionless
Re Reynolds number Dimensionless
t Time s
ta Time required for a solute to move a distance l under advective s
transport
td Time required for a solute to move a distance l under diffusive s
transport
τ Shear stress Pa
τw Shear stress at the wall Pa
μ Dynamic viscosity Pa s
U Velocity m s1
V Volume m3
(continued)
84 O. Kroukamp et al.

Mathematical
symbol Definition Units
Shear stress, τ The parallel component of the applied force, Fk, divided by the F
τ ¼ Aok
initial area of the sample, A0
Shear rate, γ_ Various definitions dependent on physical configuration but γ_ ¼ Ul
for a parallel plate system defined as the velocity of the top
plate relative to the stationary lower plate divided by the
distance between the plates
Newtonian For a Newtonian fluid the shear stress, τ, is directly propor- τ ¼ μγ_
fluid tional to the shear rate, γ_ , with the dynamic viscosity, μ, as the
slope of the straight line

Compliance with Ethical Standards

Conflict of Interest Otini Kroukamp declares that he has no conflict of interest. Elanna Bester
declares that she has no conflict of interest. Gideon M. Wolfaardt declares that he has no conflict of
interest.

Ethical Approval This article does not contain any studies with human participants or animals
performed by any of the authors.

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Chapter 4
Complex Structure but Simple Function
in Microbial Mats from Antarctic Lakes

Ian Hawes, Dawn Sumner, and Anne D. Jungblut

Abstract Microbial mats growing under the permanent ice cover of Antarctic lakes
occupy an exceptionally low-disturbance regime. Constant temperature, the absence
of bioturbation or physical disturbance from wind action or ice formation allow mats
to accumulate, as annual growth layers, over many decades or even centuries. In so
doing they often assume decimetre scale, three-dimensional morphologies such as
elaborate pinnacle structures and conical mounds. Here we combine existing and
new information to describe microbial structures in three Antarctic lakes—simple
prostrate mats in Lake Hoare, emergent cones in Lake Untersee and elaborate
pinnacles in Lake Vanda. We attempt to determine whether structures emerge simply
from uncoordinated organism-environment interactions or whether they represent an
example of “emergent complexity”, within which some degree of self-organisation
occurs to confer a holistic functional advantage to component organisms. While
some holistic advantages were evident from the structures—the increase in surface
area allows greater biomass and overall productivity and nutrient exchange with
overlying water—the structures could also be understood in terms of potential
interactions between individuals, their orientation and their environment. The data
lack strong evidence of coordinated behaviour directed towards holistic advantages
to the structure, though hints of coordinated behaviour are present as non-random
distributions of structural elements. The great size of microbial structures in Antarc-
tic lakes, and their relatively simple community composition, makes them excellent
models for more focused research on microbial cooperation.

I. Hawes (*)
University of Waikato, Tauranga, New Zealand
e-mail: ian.hawes@waikato.ac.nz
D. Sumner
Department of Earth and Planetary Sciences, University of California, Davis, CA, USA
A. D. Jungblut
Life Sciences Department, Natural History Museum, London, UK

© Springer Nature Switzerland AG 2019 91

C. J. Hurst (ed.), The Structure and Function of Aquatic Microbial Communities,
Advances in Environmental Microbiology 7,
https://doi.org/10.1007/978-3-030-16775-2_4
92 I. Hawes et al.

4.1 Introduction

Photosynthetic microbial mats (biofilms) frequently show consistent physical pat-

terns that hint at organisation. At one level, organisation is exemplified by metabolic
zonation, which typically occurs on mm length scales (Franks and Stolz 2009). Such
zonation results in organisms capable of oxygenic photosynthesis concentrated at the
top of a mat, where light is most abundant, while deeper in the mats, lower irradiance
and depleted oxygen enable the growth of organisms capable of a cascade of
anaerobic metabolisms (Kühl and Fenchel 2000). Layering is often visible as colour
changes, with an orange surface from abundant light-protective carotenoid pigments,
transitioning to green light-harvesting phycobilin and chlorophyll pigments and pink
layers corresponding to photosynthetic sulphur bacteria in anoxic zones. There are
many examples of this metabolic “organisation” in the literature, and the close
proximity of interacting metabolisms allows diffusion to be an effective coupling
mechanism that provides key advantages to this mat structure (Seckbach and Oren
2010; Nadell et al. 2016). Viewed as an integrated unit, the microbial mat shows
elegant metabolic coupling, facilitated by a one-dimensional, organised structure
that allows a more efficient utilisation of resources than any one organism could do
alone (Paerl and Pinckney 1996; Stahl 1995; Franks and Stolz 2009). Indeed, some
argue that the organised complexity and physiological cooperativity of a microbial
mat is analogous to that of eukaryotic tissues (Webb et al. 2003; Guerrero and
Berlanga 2007; Stahl et al. 2018).
When microbial mat communities grow in low-disturbance environments, a
higher level of organisation can occur, whereby elaborate three-dimensional struc-
tures emerge on scales much larger than those of either component organisms or
metabolic zonation (Bosak et al. 2013). Such emergent structures are most fre-
quently described from environments where conditions preclude metazoa large
enough to physically disrupt developing microbial communities (Fenchel 1998).
The most frequently described emergent microbial structures are from quiescent
geothermal or hypersaline waters, where physical disruption is minimal and insects
and other invertebrates are temperature- or salinity-excluded (Des Marais 1995;
Oren 2010). There, a variety of macroscopic mat forms occur, overlain on metabolic
zonation, which in quiescent locations often take the “ridge-pinnacle” form of
regular-spaced cones of cm-scale size and spacing, connected by narrow ridges
(Walter et al. 1976).
Microbial mats thus show emergent organisation on at least two levels: a three-
dimensional arrangement of organisms within the mat that appears to produce an
efficient metabolic unit and a three-dimensional organisation of the mat matrix to
form spatially complex structures. In modern concepts of emergent organisation
(Halley and Winkler 2008), the former would be termed a simple emergence,
computable through a reductionist understanding of organism-environment interac-
tions and close to an (dynamic) equilibrium (Corning 2002; de Wolf and Holvoet
2005). Whether the three-dimensional organisation is an example of complex emer-
gence, within which some degree of self-organisation occurs to convey a holistic
4 Complex Structure but Simple Function in Microbial Mats from Antarctic Lakes 93

functional advantage, or whether it too is driven by simple organism-environment

responses is not clear. At small scales, there is accumulating evidence for holistic,
coordinated behaviour in biofilms (Stahl et al. 2018), perhaps controlled by cell to
cell signalling and “programmed death” that would allow a mechanism for complex
emergence of structure, though to date we are aware of no definitive examples from
nature where coordinated behaviour for holistic benefit is shown (Battin et al. 2007).
One of the few recent studies to suggest a holistic function for macroscale ridge-
pinnacle structure suggests that a mechanism exists to space pinnacles to optimise
overall access to water column resources by minimising inter-pinnacle competition
(Petroff et al. 2010). This degree of self-organisation requires information transfer
amongst pinnacles to achieve the overall optimum condition, which the authors
propose via resource depletion “shells” around structures. Other authors argue
for mechanisms of complex structure emergence that require much less self-
organisation, suggesting that cones initiate as surface irregularities that are conse-
quences of un-oriented movement (Shepard and Sumner 2010) that then propagate
vertically either through phototaxis (Stahl 1995) or through differential growth of
tips due to enhanced access to limiting resources (Tice et al. 2011). The question of
whether 3-D microbial mat structures derive from internal or external drivers, or a
mix of the two, extends beyond semantics; it relates to whether ridge-pinnacle
structure is the inevitable outcome of individual behaviour in response to environ-
mental drivers and has no holistic function or whether individual behaviour within
microbial mats has, over time, been structured to facilitate enhanced overall function
through a self-organised structure.
In this contribution, we examine the extent to which microbial mat structures
are organised and consider whether this organisation is essentially mechanistic, with
organisms responding to environmental stimuli or whether there is evidence that
organised structures contribute to holistic functions that enhance overall perfor-
mance. As case studies, we focus on similarities and differences between mats in
three perennially ice-covered Antarctic lakes (Fig. 4.1). Antarctic lakes are particu-
larly favourable locations for the development of complex microbial structures
and contain some of the best developed modern examples (Hawes et al. 2011,
2013; Mackey et al. 2015; Sumner et al. 2016). They share with geothermal and
hypersaline waters the lack of bioturbation from macro-invertebrates, due in this
case to the combination of extreme environmental conditions and geographic isola-
tion (see discussion in Karanovic et al. 2014), but furthermore, the ice cover ensures
that lake water is exceptionally still, over long time periods, with no wind-induced
turbulence (Wharton et al. 1993; Andersen et al. 2011; Hawes et al. 2013) that could
generate sufficient shear stress to disturb mat communities (Tice et al. 2011).
Consequently, large-scale structures, overlain on an internal mat zonation, reach
decimetre proportions in these lakes (Fig. 4.2; Andersen et al. 2011; Hawes et al.
2011, 2013). Microbial mats with obvious internal one-dimensional structure are
also common in shallow Antarctic habitats (Howard-Williams et al. 1989; Vincent
2000; Jungblut et al. 2012), but their prolonged winter freezing and summer water
movement appear to prevent the development of large-scale three-dimensional
organisation, and thus our focus remains on lake mats developing in perennially
liquid, under-ice habitats.
94 I. Hawes et al.

Lake Untersee

Lakes Hoare
and Vanda

Fig. 4.1 The location in Antarctica of the three lakes primarily referred to in this chapter. Public
domain image produced by the US National Aeronautics and Space Administration

Fig. 4.2 Examples of contrasting microbial mats in perennially ice-covered Antarctic lakes. (a)
Complex emergent structures at 22 m depth in Lake Vanda, Antarctica. (b) Microbial mat with little
emergent complexity at 11 m depth in Lake Hoare. The checkers are 1 cm in both images, which are
by courtesy of Tyler Mackey

We focus on three lakes that allow comparison of three distinctly different

macroscale mat topographies: Lake Hoare where prostrate forms prevail (Hawes
et al. 2014, 2016); Lake Untersee where a unique conical growth form is widespread
(Andersen et al. 2011); and Lake Vanda, where dense fields of tall cuspate pinnacles
4 Complex Structure but Simple Function in Microbial Mats from Antarctic Lakes 95

dominate (Fig. 4.2; Hawes et al. 2013; Sumner et al. 2016). Although early literature
refers to Antarctic lake mats rather generically as “modern stromatolites” (e.g. Parker
et al. 1981), for the most part, this is misleading. Though laminated, they are mostly
unlithified (Mackey et al. 2015), being entirely built from alternating annual lami-
nations of sediment and microbially derived organic material (Hawes et al. 2001;
Sutherland and Hawes 2009). Thus, the potential structuring effects of spatially
constrained mineral precipitation (Petroff et al. 2013) are not in play, and Antarctic
lakes instead present some of the best developed microbe-soft sediment accretion
structures on modern Earth. Lake Vanda has the added advantage that it has
undergone recent increases in lake level, with newly inundated areas of lake bed
providing dated underwater landscapes allowing an examination of the evolution of
structural complexity over time (Hawes et al. 2013; Sumner et al. 2016).

4.2 Lake Environments

4.2.1 Lake Hoare

Lake Hoare (77.63
S, 162.88
E) is a closed-basin lake near the eastern end of Taylor
Valley in southern Victoria Land, Antarctica. The lake is 4.2 km long and 1.0 km
wide and has maximum and mean depths of 34 and 14 m (Spigel and Priscu 1998). It
is dammed to the north-east by the Canada Glacier, which provides an inflow of
glacial meltwater (Wharton et al. 1992). Other sources of inflow come from Ander-
sen Creek entering the north-east corner of Lake Hoare and drainage from Lake
Chad in the south-east. No outflows from Lake Hoare exist, and water loss is
restricted to sublimation of ice and evaporation of meltwater during summer
(Doran et al. 1994). The ice cover of Lake Hoare is perennial, except for small
areas at the lake margins, which melt during summer. The ice cover was 3.5 m thick
in 1983 (Wharton et al. 1992), but the thickness has increased to ~5 m since that time
(Doran et al. 2002). There are 3 months of complete darkness during winter and
3 months of continuous light during summer (Dana et al. 1998). Howard-Williams
et al. (1998) summarised the optical properties of the ice cover and the water column.
Net transmission of photosynthetically active radiation (PAR) in the lake centre
ranged from <1% to 3%, with a spectral transmission peak at wavelengths between
450 and 550 nm. Vertical extinction coefficients for downwelling PAR within the
water column from beneath the ice to a depth of 33 m were typically 0.12–0.22 m1
(Howard-Williams et al. 1998), resulting in a 1% irradiance depth of between just
below the ice cover and 9 m.
Lake Hoare shows weak density stratification in the upper waters, and although
there is controversy over the extent of mixing of surface water (Spigel and Priscu
1998; Tyler et al. 1998), the density gradient is continuous to at least 13 m depth.
There is a pronounced inflexion in the density-depth profile at 13–15 m from the
surface (the depth of the lake varies temporally), which divides the lake into upper
and lower compartments. The upper compartment is characterised by low
96 I. Hawes et al.

concentrations of dissolved nutrients, particularly nitrate, which is probably limiting

planktonic production (Lizotte and Priscu 1992). Upper waters also contain lower
bicarbonate, with a higher pH (up to 8.6, Cathey et al. 1981) than the lower
compartment (pH 7.9). The lake is anoxic below 25–26 m.
Benthic microbial mats, comprised primarily of cyanobacteria, diatoms and other
bacteria, with various microbial eukaryotes at low abundance, line the lake from the
edge into the anoxic zone at 25 m (Wharton et al. 1983; Hawes and Schwarz 1999).
Mats in Lake Hoare are often prostrate, with little surface elaboration, though in
some shallow areas rounded pinnacles and lift-off structures can form (Wharton et al.
1983). The matrices of the mats are dominated by narrow, filamentous
cyanobacteria, attributable on morphological and molecular bases to the genera
Leptolyngbya and Pseudanabaena with occasional Phormidium (Sutherland and
Hawes 2009; Zhang et al. 2015). Diatoms comprise 10–15% of the mat community,
though this increases at depth. Sixteen taxa of diatoms have been recognised, with
Psammothidium chlidanos, Diadesmis contenta var. parallela and Navicula
gregaria the most frequent (Sutherland and Hawes 2009). These mats are adapted
to low irradiance through efficient light harvesting and utilisation, and compensation
points of <1 μmol photons m2 s1 allow net production to occur to >20 m depth
(Hawes and Schwarz 1999, 2001; Vopel and Hawes 2006).

4.2.2 Lake Untersee

Lake Untersee (Fig. 4.1) is located at 71.33
S 13.75
E in the Otto-von-Gruber-

Gebirge (Gruber Mountains) of central Dronning Maud Land. The lake is 563 m
above sea level, with an area of 11.4 km2, and is the largest surface lake in East
Antarctica. Lake Untersee has two subbasins; the largest, 160 m deep, lies adjacent
to the Anuchin Glacier and is separated by a sill at 50 m depth from a smaller,
100-m-deep basin in the southwest corner (Wand et al. 1997). The deep basin is
close to homothermal and well oxygenated to the bottom. In contrast the shallow
basin is density stratified below the sill depth (c. 50 m) and anoxic at its base. Profiles
of conductivity, temperature and dissolved oxygen indicate that the deep basin and
upper part of the anoxic basin are part of the same body of well-mixed water (Kaup
et al. 1988; Haendel et al. 1995; Wand et al. 1997, 2006; Andersen et al. 2011). The
unusually high pH of this mixed water mass (pH 10.4) has been attributed to
weathering of the predominant anorthosite rock in the lake’s catchment in the
absence of an effective connection to the atmosphere (Kaup et al. 1988; Andersen
et al. 2011). The lake ice transmits approximately 5% of incident irradiance and
vertical extinction coefficient for scalar PAR of 0.033 m1, (Andersen et al. 2011)
resulting in a 1% surface irradiance depth of ~50 m.
Benthic microbial mats occur to at least 100 m in Lake Untersee and comprise
mostly oscillatorian cyanobacteria primarily as Phormidium autumnale and
Leptolyngbya spp., with some unicellular Chamaesiphon and nitrogen-fixing Nos-
toc, with few chlorophytes and almost no diatoms (Andersen et al. 2011; authors’
4 Complex Structure but Simple Function in Microbial Mats from Antarctic Lakes 97

unpublished molecular data). These microbial mats form two distinct macroscale
morphologies, pinnacles and large cones, with the latter, which can be up to 50 cm
tall, being most widespread. Cones are built from sub-mm scale laminations of fine
glacially derived sediments and organic material, and radiocarbon dating suggests
that they accumulate over centuries (Andersen et al. 2011). As in lakes Vanda and
Hoare, these cyanobacteria are rich in phycoerythrin, and mats have an overall
purple-pink appearance.

4.2.3 Lake Vanda

Lake Vanda (Fig. 4.1) lies in the Wright Valley, one of the McMurdo Dry Valleys of
southern Victoria Land (77.50
S, 161.67
E). It occupies a closed basin with a
perennial ice cover 3.5–4.0 m thick; though for several weeks each summer, the
ice around parts of the lake shore melts to produce a discontinuous, open-water
moat. In recent years inflow has exceeded outflow, and the lake level has risen by
12 m between 1960 and 2012 (Hawes et al. 2013). The endorheic nature of the lake
and historical changes in water balance have left Lake Vanda with an unusual
physical structure, with an inverse temperature gradient stabilised by increasing
solute content with depth. In December 2013, temperature and conductivity were
constant from 4 m to 23 m depth at ~4
C and ~560 μS cm1, increasing abruptly as
a pycnocline from 23 to 28 m depth, then stabilising at 6
C and 1020 μS cm1
between 28 and 45 m. These two upper cells are individually mixed by thermohaline
convection (Spigel and Priscu 1998), while below 45 m, there is a continuous
gradual increase in temperature and conductivity. The ice cover transmits 15–20%
of incident photosynthetically active radiation (PAR), biased to wavelengths below
550 nm (Hawes and Schwarz 2001). The lake water is extraordinarily clear, with a
vertical extinction coefficient for downwelling scalar PAR of 0.06 m1 (Howard-
Williams et al. 1998), resulting in a 1% surface irradiance depth of 48–63 m. Low
concentrations of dissolved reactive phosphorus (DRP) appear to limit phytoplank-
ton growth (Vincent and Vincent 1982).
In Lake Vanda, benthic microbial mats are widespread, attain high biomass, form
elaborate pinnacles and extend to at least 50 m depth (Figs. 4.2 and 4.3; Wharton
1994; Hawes and Schwarz 2001; Hawes et al. 2013). These mats comprise mostly
cyanobacteria, particularly species of Phormidium and Leptolyngbya (Sumner et al.
2016), with pennate diatoms (species of Navicula, Nitzschia, Caloneis and
Stauroneis) throughout and occasional strands of moss below the 25 m pycnocline
(Love et al. 1983). Lake Vanda mats appear to have a higher cyanobacterial diversity
than Lake Hoare, with up to 12 compared to 8 operational taxonomic units (OTUs)
based on clone library analysis (Zhang et al. 2015). Vanda mats are annually
laminated, with the upper 2–4 laminae constituted an orange-brown zone, rich in
myxoxanthophyll and dominated by intertwined Leptolyngbya trichomes. Below the
upper zone, green-/pink-pigmented subsurface zones are present, containing up to
six phycobilin-rich laminae, also colonised by Leptolyngbya, Oscillatoria and
98 I. Hawes et al.

A B C Oxygen Concentration (mol/L)

1200 1400 1600 1800

-3

Depth into mat (mm)

3

12

15

Fig. 4.3 A vertical section of microbial mat from 10 m depth in Lake Hoare, 2010. (a) A
photograph showing mm-scale, annual laminations and the zonation from pink-brown at the
surface, though pink, to colourless. (b) A false-colour composite fluorescence image (blue excita-
tion, red emission) of a similar section. False colour scale is in arbitrary units of chlorophyll
fluorescence. The scale bars in (a) and (b) represent 10 mm. (c) An in situ dissolved oxygen profile
through a mat at the same depth from which the sections were obtained, shown on a similar vertical
scale

Phormidium morphotypes (Hawes et al. 2013, 2016; Sumner et al. 2016). Recent,
well-constrained, increases in lake level have allowed mats from different depths to
be examined as a proxy for rate of development (Hawes et al. 2013). At depths
inundated for <30 years, mats were accreting, adding one lamina (0.3 mm) and
~0.2 μg cm2 of chlorophyll-a per year. At depths that have been submerged for
>40 years, mat chlorophyll-a appeared to have reached an equilibrium and did not
increase over time. The older mats formed tall pinnacles that were only beginning to
emerge in shallower mats.

4.3 One-Dimensional Structures

Lake Hoare microbial mats frequently lack well-developed, complex three-

dimensional structures (Fig. 4.2), though one-dimensional organisation is well
developed. The properties of 1-D structure in these mats take two forms; lamination
and zonation (Hawes et al. 2014). Lamination, composed of alternating sediment-
rich and sediment-poor bands, is a product of annual accumulation (Hawes et al.
2001). Sediments enter the lake in summer via streams that typically flow for
1–2 months, and settle as a distinct optically dense band over autumn-winter when
darkness prevents photosynthetic growth. The resumption of growth the following
spring, when light returns but inflows are not running, forms a hyaline band (organic
4 Complex Structure but Simple Function in Microbial Mats from Antarctic Lakes 99

accumulation without sediment) overlying the relatively opaque (sediment without

growth) band formed in winter (Fig. 4.3a). Hawes et al. (2001) used confocal laser
scanning microscopy to show that trichomes are relatively sparse and oriented
mostly vertically in the hyaline bands, including at the surface in summer, but tend
to be more abundant, intertwined and oriented parallel to the mat surface in the
opaque bands. Such laminations through trapping and binding of episodic deposi-
tions of sediment are reported from microbial mat communities in other sedimentary
environments such as marine stromatolites (e.g. Reid et al. 2000).
Zonation is overlain on this annual banding pattern and in Antarctic lakes is
recognisable by pigmentation bands that comprise multiple laminae. The orange-
brown surface zone contains a higher proportion of carotenoids and live diatoms
than the lower zone, where carotenoids are rare and cyanobacterial phycobilin
pigments are responsible for pink coloration (Hawes et al. 2016). However,
cyanobacteria dominate biovolume throughout the pigmented zone, with a similar
morphotypic composition, dominated by Leptolyngbya in both zones, with
Pseudanabaena present in both but most abundant in the upper pink-brown zone
(Sutherland and Hawes 2009; Hawes et al. 2016).
In addition, Lake Hoare mats have two unusual features that are not common in
temperate microbial mats: (1) the >18 mm thickness of the pigmented zone, which
occupies 9 years of vertical accumulation (Fig. 4.3a, b) and (2) the persistence of
dissolved oxygen concentrations exceeding that of the ambient water column to
more than 15 mm into the mat (Fig. 4.3c). These are despite the mat receiving <1%
of lake-incident irradiance (Vopel and Hawes 2006; Hawes et al. 2014). Fluores-
cence imaging (Fig. 4.3b, see Vopel and Hawes 2006 for methods) shows that
chlorophyll-a is present throughout the pigmented zone of the mat, only reaching
the limits of detection at 15 mm or more below the mat surface. The bulge in oxygen
concentration associated with the pigmented zones is indicative of a broad photo-
synthetic zone, but also that oxic conditions extend well below the region containing
pigments associated with oxygenic photosynthesis. This is different to what is
normally seen in temperate mats, and even in shallow water (seasonally frozen)
mats in Antarctica (Hawes et al. 1999), where oxygenic photosynthesising organ-
isms are usually concentrated in the upper 1–2 mm, and a sharp oxycline is present at
the base of the oxygenic, pigment-containing zone. In temperate system, the
oxycline within the mat can be associated with an increase in sulphide and a shift
in dominant metabolism from oxygen- to sulphur-based and moves vertically in
response to diel changes in irradiance (e.g. Denis et al. 2012). Indeed, in temperate
mats, this redox change is the basis of the 1-D metabolic structure that is often
interpreted as having the functional role of facilitating nutrient cycling by position-
ing all components of nutrient cycling pathways within diffusion distance of each
other (e.g. Paerl and Pinckney 1996).
The unusual oxygenation features of Lake Hoare mats may in part reflect the 24-h
daily irradiance that they receive, albeit at low photon fluxes. Estimates of photo-
synthesis in Lake Hoare show that photic zones of microbial mats export oxygen to
the water column and to underlying mats during summer and that oxygen consump-
tion in mats below the photic zone quickly falls to close to zero despite an abundance
100 I. Hawes et al.

of organic carbon (Hawes et al. 2014). Vopel and Hawes (2006) demonstrated that
the rate of photosynthesis is linearly related to irradiance and argued for a compen-
sation photon flux as low as 0.1 μmol m2 s1. Hawes et al. (2014) went on to
confirm persistent light-limitation of photosynthesis but also showed that net flux of
oxygen to the water column continued through the 24-h light cycle. However, the
export of dissolved oxygen at low irradiances around midnight was due to ongoing
effusion of dissolved oxygen produced during brighter parts of the day: under the
night-time photon flux of ~1 μmol photon m2 s1. Gross photosynthesis was close
to zero, and a net consumption of oxygen was actually occurring when flux was
integrated through the mat, implying that ~1 μmol photon m2 s1 may be closer to
the effective community compensation point than 0.1 μmol photon m2 s1.
The ability of the Lake Hoare microbial mat to be photosynthetic at low irradi-
ance is enhanced by efficient absorption and utilisation of light. Hawes and Schwarz
(2001) showed how the spectral absorption characteristics of ice (which absorbs red
light strongly) results in light that penetrates to 10 m depth in Lake Hoare having a
dominant wavelength of close to 500 nm and an overall blue-green hue. They
showed how phycoerythrin allowed benthic mats to capture effectively these green
wavebands and absorb up to 50% of light incident to the mat using these photosyn-
thetic pigments. A variety of methodologies have shown how the cyanobacteria
function at close to the maximum quantum yield of oxygenic photosynthesis, fixing
the equivalent of 1 mol carbon per 12 mol photons absorbed (Hawes and Schwarz
1999, 2001; Vopel and Hawes 2006; Hawes et al. 2014). Low respiration rate is,
however, also essential to both low-compensation irradiance and deep-penetrating
oxygen (Hawes and Schwarz 1999; Hawes et al. 2014). The low respiration rate,
despite an apparent abundance of oxygen and organic carbon in the mat matrix
(Sutherland and Hawes 2009), is unexpected. It has been suggested that low
respiration rate is due to the recalcitrant nature of the cyanobacterial sheaths and
extracellular polymeric material that is suspected to form much of the organic
laminae (Sutherland and Hawes 2009). What happens to dissolved oxygen concen-
tration in mat communities over the 4 months of winter darkness is not yet known.
While we can hypothesise that the unusual oxygen profiles in Lake Hoare mats
reflect the absence of release of labile organics from the photosynthetic mat com-
ponents to the underlying mat, it is clear that the 1-D structure of these Antarctic lake
mats is not based around redox gradients that facilitate coupling of microbial
processes. The absence of an oxycline within the mats implies the absence of
processes that link aerobic and anaerobic metabolisms to the carbon acquisition
processes at diffusion-efficient distance scales—at least during the summer months.
Nutrient accumulation does occur in these mats, with concentrations of all inorganic-
and organic-dissolved forms of N and P increasing severalfold from the water
column to the mat (Quesada et al. 2008), but the absence of abrupt redox gradients
means that benefits from closely linked, diverse metabolisms are unlikely, and the
enigma of apparently abundant carbon resources in oxygen-rich, aphotic lower zones
of microbial mats remains incompletely resolved.
So does the one-dimensional arrangement of cyanobacteria in the mat have
another function? It seems unlikely that the structure of the mat, with pigments
4 Complex Structure but Simple Function in Microbial Mats from Antarctic Lakes 101

arranged within a sequence of laminae representing up to 10 years of growth

(Fig. 4.3), has the function of enhancing the ability to harvesting light. The most
effective arrangement of pigments to intercept light would be to concentrate them in
a narrow layer at the mat surface, as this would minimise attenuation due to
non-photosynthetic mat components (Kühl and Fenchel 2000). Thus, one might
expect these potentially motile organisms to migrate vertically to the upper, better
illuminated part of the mat. At present the reason for the absence of migration is not
clear, but we have previously argued (Hawes et al. 2014) that the only available cue
for migration is light and that the rapid scattering of light within mats (Kühl and
Fenchel 2000) and the low component of the red wavelengths that appear to be
important in phototaxis (Ng et al. 2003) prevent coordinated vertical migration from
occurring. The alternate hypothesis then is that pigments in deeper, older laminae are
persistent legacies of previous growth and that the 1-D structure seen in these mats—
annual lamination and some zonation of pigments and dominant morphotypes—has
no holistic function; rather it is simply a consequence of how these mats grow
and age.

4.4 Three-Dimensional Structure

4.4.1 Lake Untersee

Above we argue that the 1-D structure that is evident in vertical sections of microbial
mat from Lake Hoare, at 10 m depth, is largely a consequence of the way that the mat
grows and confers no advantage in the acquisition of the key-limiting resource at that
location. In the second example considered here, Lake Untersee, Dronning Maud
Land, the mats have a 1-D laminated structure that superficially resembles that of
Lake Hoare, but this 1-D structure is folded into the emergence of conical macro-
scale structures (Andersen et al. 2011). Here we will consider how these structures
may form and how this formation may create a holistic advantage for the capture of
otherwise limiting resources.
Lake Untersee actually contains two types of emergent structure, pinnacles and
cones (Andersen et al. 2011; Fig. 4.4). Surveys using drop cameras and divers have
shown that the cones are the most frequent structures seen in the lake and that these
are present from less than 15 m depth (the minimum depth surveyed) to in excess of
100 m, equivalent to optical depths of ~3–0.25% of surface incident irradiance
(author’s unpublished data). This encompasses the optical depth of the mats
discussed above from Lake Hoare (~1% surface light). Cones themselves are all
sizes, but often large, up to 50 cm tall, and rise steeply (average slope 60
) from the
surrounding sediment with a spacing length of metres (Andersen et al. 2011). A
characteristic feature is a fibrous cluster of a broad-trichome cyanobacterium (iden-
tified by sequence analysis of 16S rRNA gene as Phormidium autumnale; author’s
unpublished data) on the apex of the cones, particularly evident when viewed from
above (Fig. 4.4b). In fact, 16S high-throughput sequencing showed that Phormidium
102 I. Hawes et al.

A C

5 mm

Fig. 4.4 (a) A field of conical mounds at 20 m depth in Lake Untersee. For comparison with other
lakes, 20 m physical depth in Lake Untersee is the depth to which 2–3% of incident irradiance
penetrates. (b) A close-up of a small conical mound, with a nascent structure in the upper right. The
scale bar shows cm. (c) Cross section of the cone imaged in (b) after return to the surface, section
close to “shoulder” of the cone. All images are taken with no artificial lighting

dominates the cyanobacterial assemblages on the sides and top of the cone from
where the fibrous cluster of a broad-trichome cyanobacterium protrudes (authors’
unpublished data). This is quantifiable as an elevated concentration of photosyn-
thetic pigments at the cone apex (Table 4.1).
Vertical sections of cones reveal that the cone structure is built as a modification
of the simple laminar accrual process described in Lake Hoare, but the internal
structure is quantitatively different to that seen in Lake Hoare (Fig. 4.4c). In
particular, zonation is restricted to a single pink-pigmented, upper zone less than
1 mm thick, overlying an unpigmented organosedimentary zone, and the scale of
lamination in Lake Untersee is much finer, sub-mm scale laminated clay-organic
sediment. Although arranged differently, the absolute abundance of pigments per
unit area was similar to lakes Vanda and Hoare (Table 4.1). Potential benefits of the
conical geometry include an increase in mat surface area for a given area of lake bed.
Given that the concentration of pigments on the cone walls are similar to those on the
adjacent flat areas (Table 4.1), the increase in surface area of cones relative to flats
4 Complex Structure but Simple Function in Microbial Mats from Antarctic Lakes 103

Table 4.1 Comparison of pigment concentrations in Lake Untersee at 25 m depth, Lake Hoare at
10 m and Lake Vanda at 20 and 30 m
Lake Location Chlorophyll (μg cm2) Phycoerythrin (μg cm2)
Untersee Top of cone 24.2  12.1 38.0  14.6
Side of cone 11.9  2.3 8.1  4.4
Flat mat 9.5  3.2 5.6  3.5
Hoare Flat mat 14.5  0.4 36.0  5.3
Vanda Flat mat (20 m) 11.9  1.2 24.7  1.5
Flat mat (30 m) 15.1  0.5 46.6  0.5
Pinnacle (20 m) 17.8  2.8 30.5  3.4
Pinnacle (30 m) 33.9  9.1 103.0  32.1
N ¼ 3 (Untersee—from Andersen et al. 2011) or N ¼ 5 (Hoare and Vanda—from Hawes and
Schwarz 2001, and unpublished)

will inevitably increase the overall biomass per unit lake bed. A regular-truncated
cone that is 20 cm tall, with a 60
wall slope and a flat apex 6 cm across would have
more than twice the surface area of an equivalent flat disc (996 vs. 380 cm2).
Allowing for enhanced pigment concentration at the apex, the overall average
chlorophyll-a concentration per unit bed area of a cone would be approximately
35.5 μg cm2 compared to 9.5 μg cm2 for flat mat.
The very thin laminations in Lake Untersee compared to Lake Hoare suggest a
lower rate of sediment and organic carbon accrual, even though the irradiance in
Lake Untersee is higher than in Lake Hoare. New estimates (author’s unpublished
data) of sedimentation in Lake Untersee, based on four replicate passive traps at
20 m depth, deployed for 2 years, yield an accumulation rate of 6 g m2, which
compares to 41 g m2 for Lake Hoare (Wharton et al. 1989). New in situ measure-
ments of oxygen concentration profiles at 20 m depth and a photon flux of
30 μmol m2 s1 in Lake Untersee (Fig. 4.5), obtained as described in Vopel and
Hawes (2006), show that the zone of net oxygenic photosynthesis is restricted in
Lake Untersee to ~1 mm, much narrower than Lake Hoare at 10 m depth (Fig. 4.5).
However, the flux of oxygen to the overlying water calculated from this profile was
190 μmol m2 h1 or approximately half of that in Lake Hoare at 10 m depth with an
irradiance of 3 μmol photons m2 s1 (Vopel and Hawes 2006). Thus Lake Untersee
mats show lower gross photosynthesis and photosynthetic efficiency than Lake
Hoare mats. As in Lake Hoare, there is no evidence for a sharp oxycline below the
zone of oxygenic photosynthesis; instead there is a steady decline deep into the mat,
repeating the apparent paradox of coincident oxygen and organic carbon in the
aphotic mat seen in Lake Hoare.
Two likely reasons for the lower accumulation of extracellular organic carbon in
Lake Untersee compared to Lake Hoare are geochemical and taxonomic differences.
Lake Untersee has an unusual water chemistry, with a pH of >10.4 generated by
weathering of anorthosite rock floor under an ice cover that effectively eliminates
atmospheric contact (Kaup et al. 1988; Andersen et al. 2011). High pH persists some
distance into the sediment, with a maximum coincident with the photosynthesis-
induced dissolved oxygen maximum at the mat surface (Fig. 4.5). Recently, using
104 I. Hawes et al.

Fig. 4.5 Profiles of pH

dissolved oxygen and pH
10.30 10.35 10.40 10.45 10.50 10.55 10.60
through the surface layer of 4
a cone from Lake Untersee.
Profile obtained using a

Depth into sediment (mm)

manual micromanipulator, 2
and freshly calibrated
Unisense Ltd. (http://www.
unisense.com/) 0
microelectrodes connected
to a Unisense UW-M Oxygen
–2
underwater picoammeter pH

–4

–6
200 300 400 500 600 700

Dissolved oxygen (µmol L–1)

techniques described by Hawes et al. (2011), we measured concentrations of

dissolved inorganic carbon (DIC) in water from Lake Untersee at 12 μmol L1,
amongst the lower values recorded in natural waters. At pH 10.4, less than 50% of
DIC would be in a photosynthetically available form, e.g. as bicarbonate. Thus the
difference between the rate of accumulation of lamina thickness between Lake
Untersee and Lake Hoare may be due to limitation by both sediment deposition
and carbon accrual rates.
A second substantial difference between the lakes is the dominance of
Phormidium taxa in the phototrophic community in Lake Untersee, whereas in
Lake Hoare mats are dominated by a mix of Leptolyngbya and diatoms. Phormidium
tends to be a highly motile group, occurs at high pH in Antarctic lakes (Mackey et al.
2015) indicating an affinity for bicarbonate and is characterised as a “surface-
smoothing” taxon (Mackey et al. 2015). In contrast Leptolyngbya is less motile,
but is associated with topographical enhancement through vertically oriented growth
(Reyes et al. 2013; Mackey et al. 2015). The surface topography, with filaments of
Phormidium autumnale projecting from the top of each mound in a loose, woolly
surficial layer, may represent a behavioural response to optimise carbon accrual at
low DIC availability; the ability of cyanobacteria to move along light and DIC
gradients is well known (see review by Stahl 1995). At low irradiance a tendency for
trichomes to accumulate at the surface of topographic highs, through orientation
towards the light, and then to extend vertically through a pH and DIC gradient, is
consistent with known behaviours. Enhanced accumulation of sediment, on the
flattened apices of cones is also likely due to the enhanced trapping ability of the
“woolly cap” of P. autumnale trichomes and may also contribute to the differential
accumulation of mass on the apex of the cone compare to the cone sides and
surrounding lake floor.
4 Complex Structure but Simple Function in Microbial Mats from Antarctic Lakes 105

Fig. 4.6 Vertical image of

the floor of Lake Untersee at
45 m depth obtained with a
low-resolution SeaView
(http://www.seaview.com/)
drop camera lowered
through a hole in the ice.
The dark spots are the apices
of mounds, and the streaks
trailing from the apices are
trails of cyanobacteria. The
bar represents ~2 m

Once a biomass maximum is established on a cone apex, the morphology is likely

to become a self-reinforcing feature. Bosak et al. (2012) showed that the tips of
pinnacles grow faster than the surrounding bases because of higher density of active
cells and, like many other authors, suggested that this reflects enhanced access to
water column nutrients for vertical elements projecting though the hydraulic laminar
sub-layer. This advantage is most effective in situations where near-laminar flow
occurs at the lake bed. Unlike many ice-covered lakes, most of the water column of
Lake Untersee is mixed at least some of the time (Wand et al. 1997). Mixing in the
lake is indicated by homogenous conductivity profiles with depth through the
>140 m water column and is thought to be driven by a mix of solar heating and
the generation of cold, dilute and buoyant water through subsurface melting of the
Anuchin Glacier that forms the northern boundary of the lake (Wand et al. 1997;
Andersen et al. 2011). Thus water movement, albeit slight, occurs in the lake, and
sufficient water movement may occur to result in significant near-bed velocity
gradients. Evidence in favour of slow flow at the lake bed that interacts with cone
apices is provided by the apparent unidirectional drift of trichomes from apical
concentrations of P. autumnale evident in vertical images of the lake floor taken
with a drop camera (Fig. 4.6). Divers operating in the lake have never detected any
evidence of water movement, even when disturbing bottom sediments during sample
collection, and we suspect that at most flows are slow and laminar rather than
turbulent. However, it is possible that the presence of tall, smooth cones within
moving water may enhance nutrient supply to taller “canopy” elements of benthic
communities over what would accrue on flats as they would be placed in higher
velocity parts of the laminar flow gradient (Ghisalberti et al. 2014).
Nepf (2012) reviewed the hydrodynamics of vegetated channels, providing an
understanding of the role of canopy density on near-bed turbulence regime. She
noted that sparse canopies, as in Lake Untersee, resemble a simple boundary layer
regime with a velocity gradient predicted by distance from the channel bed, enhanc-
ing the probability that the cone apices would experience higher velocity
106 I. Hawes et al.

environments than cone bases. While the conical structures allow an increase in
surface area for uptake of nutrients, a possible specific functional advantage of the
conical structures in Lake Untersee may thus emerge as placing the most actively
growing part of the microbial population in the location where they are most
hydrodynamically favoured to exploit otherwise limiting nutrients, in this case
perhaps DIC.
The growth of large conical structures in Lake Untersee may be the consequence
of the responses of the constituent organisms growing under unusual field condi-
tions. Cones may naturally develop on local topographic irregularities, which prop-
agate through enhanced growth and sediment accumulation on the apex. This model
implies no complex, holistic organisation of structure. However, one observation
challenges this simple interpretation. The spacing of cones shows marginally sig-
nificant regularity, based on analysis of six down-looking images of cones, using
nearest neighbour analysis (Clarke and Evans 1954). Cone spacing was on scales of
decimetres or more, yet the Rn statistic (n-sample nearest neighbour risk) of the
nearest neighbour analysis for the image in Fig. 4.6 was 1.29 (where 1 is random
and >1.2 indicates a significant element of regularity—analysis was at N > 200 and
p > 0.05).
Overall the Rn for the six analysed images was 1.36  0.06 (average  s.d.).
While this statistic suggests a low level of ordering, any emergence of regular
spacing may imply information transfer between cones, and how this may occur is
not clear. The potential for competitive interaction for water column resources to
result in regular spacing in pinnacle mats has been argued for on the basis of elegant
experimentation and modelling by Petroff et al. (2010). Those arguments suggest
that under still conditions, spacing on cm distance scales will result from pulsed
activity on a 24 periodicity (i.e. photosynthesis). Even in Antarctic lakes, with 24 h
of daylight, sufficient day-night variation in irradiance exists to drive a diel photo-
synthetic pattern (Hawes et al. 2014). Thus in still water, this mechanism might
explain a cm scale of spacing of pinnacles (Andersen et al. 2011) but not a greater
than dm-scale spacing of large cones, a point noted by Petroff et al. (2010).
However, as discussed above, laminar flow of water is suspected in Lake Untersee,
and Petroff et al. (2010) went on to argue that larger-scale spacing patterns may be
propagated by the competitive interaction mechanism in directional flowing water.
They suggest that laminar flow would result in longitudinal regularity, or ridges,
rather than the distribution of cones seen in Lake Untersee.
As with Lake Hoare, the emergent structures in Lake Untersee, here a mixture of
one-dimensional laminations elaborated to large conical forms, appear to largely
result from uncoordinated responses of organisms to growth conditions. However,
the enigmatic tendency for cones to be spaced in a non-random pattern over dm
distances is unexplained. A mechanism whereby communication between cones
could occur over such distances is not clear, and given the expectation of minimal
competitive interaction at such scales, neither is any holistic advantage that such
communication may confer.
4 Complex Structure but Simple Function in Microbial Mats from Antarctic Lakes 107

30.0 60 35
A B C
25.0 50 30
Spacing (mm)

Height (mm)

Height (mm)
20.0 25
40
20
15.0 30
15
10.0 20
10 R² = 0.9299
5.0 10 5
0.0 0 0
6 8 10 14 18 22 26 6 8 10 14 18 22 26 0 5 10 15 20 25
Depth (m) Depth (m) Spacing (mm)

Fig. 4.7 (a) Spacing and (b) height of pinnacles along a depth gradient in Lake Vanda. (c) The
relationship between spacing and height at each sample depth. Spacing and height are not normally
distributed, and the median and quartiles are shown. Modified after Hawes et al. (2013)

4.4.2 Lake Vanda

If there is a “model” emergent structure for microbial mats that has dominated recent
literature, it is that of cm-scale spaced, cuspate pinnacles, associated with narrow-
trichome cyanobacterial taxa (Petroff et al. 2010; Tice et al. 2011; Bosak et al. 2012).
This morphology and taxonomic dominance is common in Lake Vanda (Hawes et al.
2013; Zhang et al. 2015; Sumner et al. 2016). For logistic reasons, published
information is largely limited to the upper part of the lake, from 6 to 26 m depth
where a time series of mat age that has resulted from a gradual increase in lake level
allows insights into the development of these microbial pinnacles (Hawes et al.
2013; Sumner et al. 2016). The number of annual growth laminations and accumu-
lation of biomass along the depth gradient in the upper water column of Lake Vanda
was consistent with an accumulation of mat material over the duration of inundation
calculated from rising water level (Hawes et al. 2013). The gradual increase in height
and spacing of pinnacles with depth (Fig. 4.7) was therefore consistent with temporal
development. Recently, we deployed drop cameras across Lake Vanda and found
that pinnacles extend to depths of at least 55 m (Fig. 4.8), though most pinnacles at
this depth appear to have collapsed, perhaps a result of declining irradiance over time
as the lake level has risen. The potential for insights into the mechanisms of pinnacle
growth from Lake Vanda are high because pinnacles can be ordered by age, are
particularly large and are readily measured. Thus, we focus on both the organisation
of fields of pinnacles and possible functions of the pinnacle structure itself.

4.4.2.1 Size and Spacing

Distributions of pinnacle height at any given depth are skewed by the presence of
exceptionally large pinnacles at all depths (Fig. 4.9). There was a close, positive
correlation between median size at each depth and median spacing (Fig. 4.7c, n ¼ 7,
r2 ¼ 0.96, p < 0.0005). Nearest neighbour analysis of the Vanda pinnacles showed,
as in Lake Untersee, a slight tendency away from random, towards even spacing. For
108 I. Hawes et al.

17 m 17 m
(7.8%) (7.8%)

41 m 55 m
(1.5%) (0.7%)

Fig. 4.8 The appearance of pinnacle mats from a range of depths in Lake Vanda. The physical
depth and optical depth, as % incident irradiance at noon, are indicated. Where visible, the red laser
dots are 3 cm apart

14 m 22 m
30
45 n= 259
25 n= 165 40
Frequency (%)

Frequency (%)

35
20 30
15 25
20
10 15
5 10
5
0 0
0–4
4–8
8–12
12–16
16–20
20–24
24–28
28–32
32–40
>40

40
10 0

80 0
0
0

12 20
10 00

0
20 0
0
1

–8
–5
–4

–6

14
–2

–3
0–

–1

>1
0–
30

0–
50

60
40

Size range (mm) Size range (mm)

26 m 25
25
n= 110
Frequency (%)

20
Frequency (%)

20
15
15
10 10

5 5
0 0
10

0
0
0

90 0

11 10
0
10 00
0

20
0
40
0

80 0
10 0

0
0

0
10 00

12 20
0
20 0

–4
–5
–2

–9

12
–3

–7
–6
–5

–8
1

–4

–6

14
–3
–2

–1

1
0–

>1

0–

1
–1

>1
0–

0–
30

50
40

60

50
20

60
10

80

0–
0–
30
40

Size range (mm) Spacing(mm)

Fig. 4.9 Log-normal distributions of pinnacle height at three depths in Lake Vanda and of spacing
distance at 21 m depth
4 Complex Structure but Simple Function in Microbial Mats from Antarctic Lakes 109

Fig. 4.10 Pinnacle mats at 22 m water depth in Lake Vanda. At the centre, a cluster of large
pinnacles shows a tendency to merge at the base but for the pinnacles themselves to diverge

three quadrats taken at 21 m depth, Rn was calculated from 80 measures of nearest

neighbour difference as 1.25  0.01, while for three quadrats at 8 m depth Rn was
1.25  0.02 (N ¼ 3, mean  s.d.), suggesting a weakly significant effect ( p > 0.05).
When pinnacle spacing was examined more closely, it was apparent that deviation
from randomness was largely driven by truncation of the distribution at the closely
spaced end. This was evident for both the larger, well-spaced pinnacles at 22 m depth
(Fig. 4.9) and also the small, closely packed pinnacles at 8 m (8 m data not shown).
Two simple ways to explain this observation are that (1) at short separation, it
is not possible to detect accurately small spacing between pinnacles, due to their
finite basal diameter, or (2) interactions lead either to divergence or merging of
pinnacles at short range. Figure 4.10 shows how closely associated tall pinnacles
can tend to both merge at the base and lean away from each other, suggesting that
both mechanisms may be in play at least some of the time. Divergence of closely
associated pinnacles is consistent with the concept of competition for nutrients from
the water column, or perhaps to minimise mutual shading. In contrast, the correlation
between spacing and size is consistent with the basal merging of pinnacles to
produce fewer, larger individuals as the microbial mat ages. The distribution of
pinnacles in Lake Vanda provides only partial support for the view of emergence of
structural complexity driven by inter-pinnacle competition for water-column
resources. Rather the simplest explanation is of random initiation (see Sumner
et al. 2016), merging as basal area increases, with direct inter-pinnacle interactions
only being evident at close range. This type of distribution pattern can be fully
110 I. Hawes et al.

accommodated by simple interactions between organisms and environment rather

than needing to invoke a more holistic mechanism for pinnacle growth.

4.4.2.2 Structure

While data from Lake Vanda suggest that pinnacle fields tend to be structured by
simple mechanisms that occur at the individual pinnacle level, the prevalence of the
pinnacle structure across a range of optical depths spanning at least 1–15% incident
irradiance implies that it has some functional advantage. It is noteworthy to compare
the pinnacles of microbial mats in Lake Vanda at 1.5% surface irradiance (45 m
depth, Fig. 4.8), with the much flatter structure of Lake Hoare at similar optical depth
(Fig. 4.2a). It appears that, based on molecular taxonomy, the two lakes share
dominant cyanobacteria (Zhang et al. 2015), and thus behavioural differences, as
proposed for Lake Untersee, are less likely. What are the functional advantages that
the formation of pinnacles confer on the microbial communities in Lake Vanda, and
why is this advantage enhanced for a small subset of the pinnacle population
resulting in the frequent occurrence of a few “outsize” pinnacles?
The evolution of pinnacle morphology and zonation in Lake Vanda is described
by Sumner et al. (2016). In brief, all pinnacles and flat mats share a laminated and
zoned structure; the outer 3–4 laminae are orange-brown in colour, with green and
purple subsurface laminae. The transition from the surface orange-brown zone to the
green and purple zones is abrupt and only occasionally crosses lamina boundaries
(Fig. 4.11). The outer laminae were typically less than 0.5 mm thick each (except at
pinnacle tips), whereas the thicknesses of green/purple subsurface laminae ranged
from less than 1 to greater than 3 mm thick. The shapes of the green/purple
subsurface laminae towards the tips of pinnacles differed from those of the orange-
brown laminae; the tops of these laminae were conical to geniculate (Fig. 4.11),
rather than cuspate. Thus, the overall thickness of a single green/purple lamina was
more uniform across the pinnacle than any orange-brown laminae were, and no
green/purple laminae mimicked the cuspate morphology of pinnacle tips. Sumner
et al. (2016) noted that at young stages, pinnacles only comprised the outer orange-
brown zone, with the green and purple zones only extending into the pinnacles at
later stages of development. However, once the pinnacles contain green and purple
zones, the interior laminae grow and thicken, which causes the lateral expansion of
pinnacles and results in a columnar pinnacle morphology (Fig. 4.12; Sumner et al.
2016). 16S rDNA clone library techniques show the orange-brown laminae in the
pinnacles contain almost exclusively Leptolyngbya ribotypes (80% L. antarctica),
while the green and pink zones contain 40–50% L. antarctica and the remainder
larger ribotypes, primarily Phormidium and Tychonema (Sumner et al. 2016).
As indicated in Fig. 4.12, tall, columnar pinnacles take considerable time to
emerge, and it is important to recall that pinnacle growth is a function of the
differential between growth of the apex and the surrounding base. Hawes et al.
(2013) calculated that during the first decades of growth, prostrate mat accumulated
at approximately 0.33 mm year1, while pinnacle apices extended at 0.25 mm
4 Complex Structure but Simple Function in Microbial Mats from Antarctic Lakes 111

Fig. 4.11 Pinnacles from 19 m depth in Lake Vanda. (a) A pinnacle developing “columnar”
attributes with the exterior dominated by orange-brown laminae. The vertical section on the right
shows the thick purple and green subsurface zones that cause the columnar shape. (b) A smaller,
cuspate pinnacle that has similar exterior orange-brown laminae. The vertical section on the right
illustrates the near absence of purple and green subsurface zones except near the base and near
sediment at the crest

year1, a net growth rate of 0.48 mm year1 for pinnacles. Thus the differential
between the rate of growth of pinnacles and the base mat is slight—on average.
However, some pinnacles increased in height by more than 2 mm year1 over a
decade (Sumner et al. 2016). Thus, the large size of pinnacles in Lake Vanda attests
to both their age and variations in their growth rate. Hawes et al. (2013) calculated
that the pinnacles at 20 m depth may be the result of 60–80 years of undisturbed
growth.
Positive differential growth relative to background mat requires that the pinnacle
morphology must provide a growth advantage to the apex. This may relate to
interactions between flow of water around the pinnacle fields and nutrient supply,
as discussed above for Lake Untersee. As in Untersee, parts of Lake Vanda are
convectively mixed, and currents of up to 1 cm s1 have been reported in the lake
(Ragotzkie and Likens 1964). While DIC is not depleted in Lake Vanda as in Lake
Untersee, dissolved reactive phosphorus is thought to be limiting (Vincent and
Vincent 1982). Ghisalberti et al. (2014), like others, argue that tall organisms—or
in this case pinnacles—benefit by extracting more nutrients from water columns due
to the effect of the near-bed velocity gradient.
 112

Fig. 4.12 Sketch of pinnacle geometry and growth history. After several years of prostrate growth, microbial tufts nucleate in orange-brown laminated prostrate
mats and grow into small cuspate pinnacles. Once thick enough, a green/pink cyanobacterial community colonises the interior of the mat, and over time more
deeply buried layers begin to degrade. Apical growth exceeds the flat surrounding mat, and as pinnacles continue to grow, the green/pink community colonises
the interiors of pinnacles and begins to swell. Formerly cuspate laminae become geniculate as biomass increases within the laminae. A shoulder develops on the
I. Hawes et al.

pinnacle and propagates upward through time. Eventually, the cores of pinnacles begin to degrade. Modified after Sumner et al. (2016)
4 Complex Structure but Simple Function in Microbial Mats from Antarctic Lakes 113

Table 4.2 Organic N:P molar ratios for pigmented layers of microbial mat taken from pinnacles
and flat mats at two depths in Lake Vanda
Depth (m) Flat Pinnacle
16 38.4  7.0 34.3  5.1
18 44.4  10.5 42.0  8.4
For flat mats N ¼ 8, for pinnacles N ¼ 16; mean  s.d.

Nepf (2012) points out that the movement of water within a canopy decreases
proportionally to the density of the canopy elements, enhancing this effect. Canopies
significantly affect turbulence, and when the product of volume-specific frontal area
of canopy elements and their height exceeds 0.23, the canopy slows water sufficient
to generate a shear layer at the top of the canopy (Nepf 2012). The product of
volume-specific frontal area and height in Lake Vanda pinnacle fields at 20 m is
0.3—exceeding the critical value of Nepf (2012). Thus, a shear layer is likely near
the tops of pinnacles. If limiting nutrients are being derived from the water column,
the density of the pinnacle field in Lake Vanda is sufficient to affect water turbu-
lence, leading to enhanced nutrient supply and thus growth to pinnacle apices.
Indeed, the presence of a velocity gradient displaced from the lake bed to the level
of the median pinnacle height may also provide a growth advantage to isolated extra-
tall pinnacles and thus reinforce the differential between median and quartile sizes.
While this model is attractive, there is little evidence to suggest that enhanced
nutrient availability is effective. The N:P ratios of pigmented outer layers taken from
pinnacles and from adjacent flat mats are both high, consistent with P limitation, but
there are no significant differences that would suggest an enhanced nutrient supply,
relative to growth, for pinnacles over flat mats (Table 4.2). Conditions in Lake
Vanda, where currents, though measured, are slight (no visible movement of dis-
turbed sediment has ever been seen by divers operating in Lake Vanda), may not be
well suited to hydraulic enhancement of growth of pinnacles. Indeed, it has been
shown that nutrient concentrations in microbial mat interstitial waters can exceed
that in overlying water, suggesting that internal recycling is a significant potential
source of nutrients (Quesada et al. 2008) and this may be expected to be most
effective over short-length scales rather than in elongated pinnacles.
An alternative growth model is differential photosynthesis rates as observed by
Bosak et al. (2012). To determine whether net photosynthetic capacity per unit area
increased towards the apices of the pinnacles, we collected three pinnacles and
returned them to a lakeside laboratory and maintained them at ambient photon flux
(40 μmol m2) and temperature (4
C) in static lake water for 3 h to reach
equilibrium with respect to oxygen concentration profile. Oxygen profiles were
measured with a Unisense microelectrode system and the gradient of dissolved
oxygen at equilibrium was measured at approximately 1 cm intervals along the
length of each pinnacle, and this used to calculate the diffusive flux of oxygen out of
the microbial mat using Fick’s law (methods fully described in Vopel and Hawes
2006). No consistent evidence emerged for any enhanced net rate of photosynthesis
along the length of the pinnacles (Fig. 4.13). Similarly, Sumner et al. (2016) did not
114 I. Hawes et al.

Fig. 4.13 Oxygen efflux rates at simulated ambient light and temperature conditions at approxi-
mately 1 cm intervals along three representative pinnacles from Lake Vanda

observe increased photosynthetic potential near pinnacle tops using PAM fluores-
cence techniques.
While photosynthesis potential per unit area is not enhanced towards pinnacle
apices, there is no doubt that surface area is. As calculated for Lake Untersee, the
increase in surface area conveyed by the pinnacle morphology results in an increase
in pigment biomass per unit of lake bottom (Table 4.1) and, if sufficient irradiance is
received, will result in a greater overall accumulation of carbon per unit lake bottom
area than for flat mats. The simplest interpretation of cumulative data on pinnacles in
Lake Vanda is that the functional benefit of forming elaborate pinnacle structures is
to enhance surface area. The pinnacles are physiologically and compositionally
simply flat mats that have been stretched and with a more vertical orientation.
Photosynthetic tissue is thus spread over a larger area than in flat oriented mats,
resulting in the same number of thinner laminae each accumulating less falling
sediment per unit area, due to steeper slope. Reduced sediment load minimises
competition for light absorption between sediment and photosynthetic pigments,
high surface area allows ready access to water column nutrients, and thin laminae
allow ready access to nutrients regenerated in decomposing material below. Within
these stretched outer pinnacle laminae, trichomes of Leptolyngbya are oriented
parallel to the axis of the pinnacle (authors unpublished data and Sumner et al.
2016), often intertwined to form the network of meshlike skeletons that if growing
by extension will naturally result in enhanced apical growth. We argue that the small
growth advantage that pinnacles have over flat mats relates to this process of
increased surface area and oriented extension.
The surface area mechanisms will only be effective if the steep sides of pinnacles
receive adequate illumination. The angular distribution of light in Lake Vanda at
10 m depth is moderately diffuse due to the scattering nature of the lake ice cover
(Sumner et al. 2016). Radiance is maximal at across a 60
arc about the vertical, and
substantial light is still present at angles close to 60
from zenith. The light reaching
4 Complex Structure but Simple Function in Microbial Mats from Antarctic Lakes 115

the steeply sloping faces of pinnacles is only slightly less than that reaching
horizontal surfaces, particularly as the thinning of pigmented laminae will increase
the penetration of light through the pinnacle walls (c.f. Hawes and Schwarz 2001).
As in Lake Hoare and Lake Untersee, the pinnacles in Lake Vanda appear to be
predictable and to emerge on the basis of simple organism response to environmental
variables, over long periods of stable conditions.

4.5 Conclusions

In this brief review of the function of structure in microbial mats in Antarctic lakes,
we have focused on features of these unusual systems that may provide insights into
general features of microbial mats. Structures formed in Antarctic lakes can be
particularly well developed, due to the extreme lack of disturbance that allows
them to accumulate over very long time periods (decades perhaps to centuries).
Structure is evident on both 1-D and 3-D bases. 1-D structure comprises two
elements, an annual banding due to temporal separation of sedimentation and carbon
accrual and a pigment zonation superficially similar to the metabolic zonation seen in
some more typical mats, though lacking any linkage to oxygen and sulphide
gradients. Instead the pigment zonation can be reconciled to the persistence of
organisms, particularly cyanobacteria, in buried laminae for many years, and pene-
tration of just enough light to allow these to survive and even grow in some
instances. There is no clear indication in data available to date that this zonation
provides any holistic advantage to the mat components through, for example,
cascades of nutrient cycling elements. Further investigations are needed to better
understand the bacterial and microbial eukaryote assemblages as well as functional
potential of the communities, using in-depth environmental genomics analyses.
Antarctic lakes provide excellent opportunities to increase understanding of the
emergence of complex 3-D structures in microbial mats, because they display a wide
range of such structure most of which are non-lithifying and which attain substantial
sizes across environmental gradients. Limited data available to date confirm findings
from elsewhere that the interaction of dominant organisms present and growth
conditions are important in determining the outcome of 3-D emergence. We note
that elaboration occurs slowly, resulting from small differences in growth rate in
various parts of a microbial mat community. For example the spectacular pinnacles
in Lake Vanda, which reach over 10 cm high, accrue at annual rates on the order of
mm or less, but the rate of emergence of complex features in other less stressful
locations may scale with growth rate. Thus rather small differentials in growth may
be responsible for emergence of structures. Functional benefits of these structures
may relate to enhanced resource availability through increased surface area, and
possibly interactions of structures with water movement to enhance delivery of
resources.
While we were able to show how the increase in surface area of elaborated
structures over flat ones allows greater biomass and overall productivity on a lake
116 I. Hawes et al.

bed area basis, the structures that formed could be easily understood in terms of
potential interactions between individuals, their orientation and their environment.
The data lack strong evidence of coordinated behaviour, directed towards holistic
advantages to the structure. The possibility of some level of coordinated behaviour
is, however, suggested by the spacing of the structural elements. Initiation of
structures in the lakes considered here was close to random, and the growth of
pinnacles in Lake Vanda appears to result in a gradual increase in spacing, following
a log-normal distribution, due to merging of near neighbours. However, though a
hint of regular spacing in both Lake Vanda and Lake Untersee does argue for some
inter-communication between pinnacles, more focused research will be required to
investigate this intriguing possibility. The large size of structures in Antarctic lakes
make them particularly suitable to investigation of any role of cell to cell signalling
over short or long spatial scales in structural emergence that occur on small spatial
scales in biofilms (Battin et al. 2007; Decho et al. 2010).

Acknowledgments The information presented in this contribution is derived from field and
laboratory work that would have been impossible without the support of many people and
organisations. Logistic support was provided by Antarctica New Zealand, the US Antarctic
Program and Antarctic Logistics Centre International. We thank all of our Antarctic field col-
leagues, in particular Drs Dale Andersen and Tyler Mackey, without whom the underwater research
would not have been possible. We also thank the reviewers and editors for helpful comments and
hard work to see the volume to production.

Compliance with Ethical Standards

Funding This study was funded by NASA Astrobiology: Exobiology and Evolutionary Biology
(NNX08AO19G and NN13AE77A), the New Zealand Ministry of Business Innovation and
Employment (CO1605 and UOWX1401), the National Science Foundation (MCM-LTER grant
number 1115245) and the Tawani Foundation.
Conflict of Interest Ian Hawes declares that he has no conflict of interest. Dawn Sumner declares
that she has no conflict of interest. Anne D. Jungblut declares that she has no conflict of interest.
Ethical Approval This article does not contain any studies with human participants or animals
performed by any of the authors.

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Chapter 5
Fungal Decomposers in Freshwater
Environments

Vladislav Gulis, Rong Su, and Kevin A. Kuehn

Abstract Streams, rivers, and freshwater marshes often depend on plant litter as a
source of carbon, nutrients, and energy that drive ecosystem processes. Decompo-
sition of this organic matter, such as leaves, wood, or emergent macrophytes, is
mediated mostly by fungi, whereas the role of bacteria is minor. Fungal colonization
leads to enzymatic breakdown of major plant polymers and fungal biomass accrual
(often around 10% of total detrital dry mass), which makes decaying plant material
more palatable to detritivorous invertebrates. Representatives of almost all major
groups of fungi can be isolated from decaying plant litter collected in freshwater
ecosystems or detected using molecular techniques; however, ascomycetes, includ-
ing their asexual stages (e.g., aquatic hyphomycetes in streams), predominate. In
recent years, utilization of radioisotopic approaches (e.g., acetate incorporation into
ergosterol) to estimate fungal growth rates and production has facilitated the con-
struction of partial carbon budgets for decaying plant litter that illustrate the impor-
tance of fungal decomposers in both lotic and lentic systems. For example, some
estimates suggest that 23–60% of leaf litter carbon loss in streams can be explained
by fungal assimilation (production plus respiration), which does not include fungal-
mediated losses as fine particulate or dissolved organic carbon. Estimates of fungal
contribution to plant carbon loss can be even higher (47–65%) in standing-dead
emergent macrophyte systems in wetlands. The effects of environmental variables
on fungal activity and plant litter decomposition in freshwaters, including inorganic
nutrient availability and eutrophication, have also received considerable attention in
the recent years. Molecular approaches are now becoming increasingly important in
both streams and wetlands to assess the effects of environmental variables on litter-
associated fungal assemblages. However, there are considerable differences in

V. Gulis (*)
Department of Biology, Coastal Carolina University, Conway, SC, USA
e-mail: vgulis@coastal.edu
R. Su · K. A. Kuehn
Department of Biological Sciences, The University of Southern Mississippi,
Hattiesburg, MS, USA

© Springer Nature Switzerland AG 2019 121

C. J. Hurst (ed.), The Structure and Function of Aquatic Microbial Communities,
Advances in Environmental Microbiology 7,
https://doi.org/10.1007/978-3-030-16775-2_5
122 V. Gulis et al.

fungal dynamics and assemblages between streams and freshwater wetlands, which
are discussed here in detail.

5.1 Introduction

Streams and rivers as well as inland ecosystems with standing waters (wetlands,
ponds, littoral zones of lakes) receive considerable input of allochthonous terrestri-
ally derived organic carbon (C) in the form of leaf litter and wood from riparian trees
(Webster and Meyer 1997; Benstead et al. 2009) as well as dead autochthonous
organic matter originating from submerged and emergent macrophytes (Mitsch and
Gosselink 2007). Increasing lines of evidence indicate that fungi are key players in
the decomposition of this plant litter under aerobic conditions, as indicated by the
high rates of fungal secondary production observed in plant litter and the concom-
itant accrual of fungal biomass (Gulis and Suberkropp 2003c; Suberkropp et al.
2010; Kuehn et al. 2011; Kuehn 2016).
However, historically, comprehensive assessments of fungal-mediated decay
processes have been hindered by the inability to quantify fungal biomass and rates
of fungal production in field-collected plant material. The major impediment to such
data has been the pervasive, intimate association of fungi with plant litter they
colonize, which conceals their filamentous somatic body (hyphae) and makes
estimation of their biomass and growth rates extremely difficult. However, compel-
ling evidence has accumulated on the usefulness of ergosterol, a membrane lipid
found in higher fungi (Weete et al. 2010), as a biomarker for quantifying fungal
biomass in decaying plant matter and soils (Newell et al. 1988; Gessner and Chauvet
1993; Gessner 2005; Gulis and Bärlocher 2017). Additional techniques have also
been developed for measuring instantaneous growth rates of fungi via incorporation
of [1-14C]-acetate into ergosterol, which allows for the estimation of fungal growth
and production rates (Newell and Fallon 1991; Suberkropp and Gessner 2005; Gulis
and Bärlocher 2017). Both of these methods have been increasingly used within a
variety of aquatic and terrestrial ecosystems, where they have yielded important
information concerning the role and contribution of fungi to ecosystem carbon and
nutrient cycling pathways (Gessner et al. 2007; Rousk and Baath 2007; Gulis et al.
2008; Suberkropp et al. 2010; Kuehn et al. 2011; Clemmensen et al. 2013;
Wallander et al. 2013; Kuehn 2016).
Despite the critical roles that fungi play in carbon, nutrient, and energy flow in
freshwater ecosystems, there are considerable differences in fungal communities,
biomass and production dynamics, and litter processing between lotic (i.e., flowing)
and lentic (i.e., standing water) ecosystems. This can be explained by differences in
environmental conditions and microhabitats where fungi-driven decomposition of
plant litter typically takes place. In streams, aquatic fungi are mostly associated with
benthic coarse particulate organic matter. In wetlands dominated by emergent
macrophytes, fungi start decomposing senescent plant litter in the standing-dead
position before it ultimately collapses to the benthic sediments where decomposition
5 Fungal Decomposers in Freshwater Environments 123

continues. Thus, the dynamics of fungal decomposers in streams and wetlands are
fundamentally different and need to be discussed separately. Fungi are also common
within the pelagic zone of lake ecosystems (Wurzbacher et al. 2010, 2014); however,
data concerning their functional role in ecosystem processes is limited in comparison
to streams and wetlands. As a consequence, they are not discussed here.

5.2 Fungi in Running Waters

5.2.1 Aquatic Hyphomycetes

Representative of almost all major groups of fungi (Chytridiomycota, Zygomycota,

Ascomycota, and Basidiomycota) can be isolated from submerged plant litter col-
lected in streams and rivers or detected using molecular techniques (Tsui and Hyde
2003; Nikolcheva and Bärlocher 2004; Shearer et al. 2007). However, some of the
species reported from running waters are transient organisms, occurring as dormant
propagules or displaying little metabolic activity and, hence, contributing little to
ecosystem processes. Aquatic hyphomycetes are arguably the best-known group of
fungi associated with decaying leaf litter in streams where they complete their life
cycle under submerged or amphibious conditions. They are an ecological group of
~300–320 species, with phylogenetic affinities to ascomycetes or basidiomycetes
(Webster 1992; Descals 2005; Shearer et al. 2007; Duarte et al. 2013). Molecular
analyses of environmental samples indicate that ascomycetes dominate plant litter-
associated fungal communities in streams in terms of both the number of operational
taxonomic units (OTUs) and relative abundance (Nikolcheva and Bärlocher 2004;
Seena et al. 2008). The majority of aquatic hyphomycetes that were cultured and
subjected to phylogenetic analyses (based mostly on 28S and ITS rDNA sequences)
have been placed in the class Leotiomycetes, with fewer species associated with
Dothideomycetes (Baschien et al. 2013; Duarte et al. 2013). However, sexual stages
are known for less than 10% of the species (Shearer et al. 2007); instead, aquatic
hyphomycetes reproduce mainly via asexual spores (i.e., conidia) which often have
unique morphologies (Fig. 5.1). Due to the characteristic morphology of conidia in
many species, it is often possible to identify taxa by their individual spores within
environmental samples (Gulis et al. 2005). The shape of the spores is an adaptation
to lotic environment, allowing conidia to attach firmly to their substrate as well as
facilitating dispersal and concentration of conidia in the foam at the water surface
(Webster and Descals 1981; Descals 2005). As autumn-shed leaves or twigs enter
the water, they are quickly colonized by conidia from the neuston or by conidia
drifting in the water column.
124 V. Gulis et al.

Fig. 5.1 Conidia of aquatic hyphomycetes demonstrating the variety of shapes and sizes.
(1) Varicosporium elodeae. (2) Tetracladium marchalianum. (3) Tetrachaetum elegans.
(4) Triscelophorus sp. (5) Goniopila monticola (or Margaritispora aquatica). (6) Tricladium
kelleri. (7) Heliscella stellata. (8) Heliscus lugdunensis. (9) Heliscina antennata. (10) Culicidospora
gravida. (11) Anguillospora longissima

5.2.2 Litter-Associated Fungal Biomass, Production,

and Sporulation

Allochthonous organic matter in the form of leaves, twigs, branches, dissolved

organic matter (DOM), etc. fuels forested stream ecosystems, with some estimates
suggesting up to 99% contribution to stream carbon and energy budgets (Fisher and
Likens 1973; Webster and Meyer 1997). Annual streambed litter inputs are typically
on the order of 500 g dry mass m
2 year
1, but may exceed 1000 g m
2 year
1 in
small forested headwater streams (Webster and Meyer 1997; Benstead et al. 2009).
Decomposition of this plant litter is a key ecosystem process, which is driven mostly
by fungi. Multiple concurrent estimates of fungal and bacterial biomass associated
with decomposing leaf litter in streams suggest that fungi contribute 88–99.9% of
total microbial (fungi plus bacteria) biomass (Baldy et al. 1995, 2007; Baldy and
Gessner 1997; Hieber and Gessner 2002; Gulis and Suberkropp 2003c; Duarte et al.
2010; Suberkropp et al. 2010; Tant et al. 2013). A similar pattern of fungal
dominance has also been reported for submerged wood across streams in the USA,
while bacterial participation is more important on fine particulate organic matter
(FPOM) (Findlay et al. 2002a; Tant et al. 2013).
Leaf litter entering streams typically has low levels of fungal colonization by
epiphytic fungi and early terrestrial saprotrophs. Shortly after submergence, spores
of aquatic fungi attach and germinate usually within several hours. Molecular
5 Fungal Decomposers in Freshwater Environments 125

evidence suggests that fungal diversity may be somewhat higher during early stages
of colonization than at later decomposition stages due to the presence of terrestrial
fungi and spores of some aquatic fungi that may be unable to establish on a particular
type of substrate (Nikolcheva et al. 2003). Once established, hyphae of aquatic fungi
pervasively extend within the leaf matrix, and the increase in fungal biomass
associated with plant litter and their rates of growth and production can be detected
using ergosterol-based methods (see above). In general, the dynamics of fungal
biomass and parameters of fungal activity, such as sporulation, growth rate, and
production, depend on the type of plant litter (e.g., leaves vs. wood), the intrinsic
litter quality (e.g., nutrient and lignin content) (Gessner and Chauvet 1994; Ferreira
et al. 2006b; Gulis et al. 2008), and environmental variables including water
chemistry (see Sect. 5.2.4). Fungal biomass often increases sharply at early stages
of decomposition, reaches a maximum, and then either levels off or decreases
slightly during later stages of leaf decay (Fig. 5.2, see also Ferreira et al. 2006a;
Gulis et al. 2006). Peak litter-associated fungal biomass is typically attained within
2–10 weeks after litter submergence depending on litter type and environmental
conditions and can reach as high as 23–25% of total detrital dry mass (Table 5.1).
However, typical peak values for decomposing leaves of deciduous trees are lower,
on the order of 8–12%. Fungal biomass associated with randomly collected leaf litter
from streams during various seasons (i.e., a composite of litter of different species at
different stages of decomposition) averages around 4–6% of litter dry mass (Methvin
and Suberkropp 2003; Carter and Suberkropp 2004; Suberkropp et al. 2010).
Estimating fungal growth rates and production may give a better understanding of
the importance of fungal decomposers in facilitating carbon losses from decaying
plant litter, since estimates of fungal biomass alone do not provide any insights into
carbon losses as conidia, mycelial fragments, through respiration or due to inverte-
brate feeding and hyphal death. Fungal growth and production rates, as determined
from 14C-acetate incorporation into ergosterol, usually peak early after litter sub-
mergence when fungal biomass is still relatively low (Suberkropp and Weyers 1996;
Gessner and Chauvet 1997). Fungal growth rate estimates from decaying leaf litter
range from <0.01 to 0.64 day
1 (Methvin and Suberkropp 2003; Carter and
Suberkropp 2004; Suberkropp et al. 2010; Gulis et al. unpublished), which are
generally lower than growth rates of bacteria. However, due to much higher litter-
associated fungal biomass (typically >90% of microbial biomass), fungal produc-
tion is often much higher (1–627) than bacterial production in all studies where
both groups were followed simultaneously (Weyers and Suberkropp 1996; Baldy
et al. 2002; Pascoal and Cassio 2004; Pascoal et al. 2005; Suberkropp et al. 2010).
After litter submergence, sporulation rates of aquatic hyphomycetes typically
peak earlier than fungal biomass (Fig. 5.2), often within 1–2 weeks, suggesting
that aquatic hyphomycetes tend to invest considerable resources into reproduction
early. Some estimates show that these fungi can invest up to 46–80% of their
production into conidia (Suberkropp 1991) and convert up to 7% of initial plant
litter carbon into spores (Suberkropp 1991; Hieber and Gessner 2002; Ferreira et al.
2006b). Since large amounts of spores are released from decomposing litter, the
concentration of conidia in stream water can reach up to 25,000 spores L
1 in some
126 V. Gulis et al.

Fig. 5.2 Dynamics of 120

Fungal biomass (mg g-1 AFDM)

fungal biomass (top panel),
sporulation rates of aquatic
100
hyphomycetes, and litter
mass loss (bottom panel)
associated with two types of 80
decomposing leaves in a
Portuguese stream. All data 60
are on the basis of litter
ash-free dry mass (AFDM).
Symbols indicate means 1 40
Alder
SE (n ¼ 4–8). Note the
Oak
relatively fast litter 20
breakdown and fungal
dynamics due to high water 0
temperature and relatively 0 10 20 30 40 50 60
high dissolved nutrient
concentrations. Data from 8000
Gulis et al. (2006) and
unpublished data from the
(conidia mg-1 AFDM d-1)

same authors
6000
Sporulation rate

4000

2000

0
0 10 20 30 40 50 60
100 Time (days)
Litter AFDM remaining (%)

80

60

40

20

0
0 10 20 30 40 50 60
Time (days)
5 Fungal Decomposers in Freshwater Environments 127

Table 5.1 Estimates of fungal biomass associated with decomposing plant litter in streams
Fungal biomass No. of Litter
(mg g
1 detrital mass)a streams typeb References
61–158 2 LB (7)Suberkropp et al. (1993), Gessner and Chauvet
(1994)
78–226 4 LB (1) Methvin and Suberkropp (2003), Carter and
Suberkropp (2004)
34–73 6 RCL-A Methvin and Suberkropp (2003), Carter and
Suberkropp (2004), Suberkropp et al. (2010)c
17–104 2 RCL-M Suberkropp et al. (2010)c
1–249 10 WV Simon and Benfield (2001), Stelzer et al. (2003),
Gulis et al. (2004, unpublished), Ferreira et al.
(2006b)
2–25 4 WS Diez et al. (2002), Spanhoff and Gessner (2004)
5–56 9 RCWS Gulis et al. (2008, unpublished)
a
Ergosterol concentrations were converted to fungal biomass assuming an average concentration of
5.5 mg g
1 dry mass (Gessner and Chauvet 1993) unless more specific data was available
b
LB ¼ leaves in litter bags with the number of leaf types in parentheses (maximum fungal biomass
from decomposition experiments is given); RCL ¼ randomly collected naturally occurring leaf litter
(RCL-A ¼ annual means, RCL-M ¼ monthly means); WV ¼ wood veneers and WS ¼ wood sticks
(range of fungal biomass from decomposition experiments); RCWS ¼ randomly collected naturally
occurring wood sticks 7–40 mm diam
c
Data from 6 years of monthly sampling in two streams

streams during the autumn-winter season when in-stream leaf litter standing stocks
are at their highest. These spores can be easily captured on membrane filters,
identified, and counted, an approach often employed in ecological studies of aquatic
hyphomycetes (e.g., Suberkropp 1991; Gulis and Suberkropp 2004; Bärlocher
2005).

5.2.3 Contribution to Plant Litter Decomposition

Fungal decomposer communities in streams are well equipped to break down all
major plant polymers. Enzymatic capabilities of many aquatic hyphomycetes have
been assessed in pure cultures (see review by Shearer 1992). Enzymes that hydrolyze
cellulose and hemicelluloses are common, while ligninolytic capabilities of many
aquatic hyphomycetes may be limited. Nevertheless, some aquatic hyphomycetes
have been reported to degrade lignin-like substrates (Zare-Maivan and Shearer 1988;
Abdullah and Taj-Aldeen 1989), while other freshwater fungi are known to solubi-
lize lignin from wood (Bucher et al. 2004). Overall, there is little specialization
among species indicating that aquatic hyphomycetes are a generalist group; how-
ever, some substrate preferences and distinct communities associated with leaves
vs. wood have been reported (Gulis 2001; Ferreira et al. 2006b). In field studies, the
128 V. Gulis et al.

activity of several lignocellulolytic enzymes was positively related with litter-

associated fungal biomass (Tank et al. 1998; Gulis et al. unpublished), suggesting
that the enzymes were likely derived from fungi rather than other microorganisms.
The activity of pectin-degrading enzymes that correlated with higher fungal activity
has been shown to be crucial for litter mass loss, since it facilitates the maceration of
leaf litter by releasing whole plant cells (Jenkins and Suberkropp 1995).
The different fates of plant litter C and the proportion of C channeled through
fungi as litter decomposes can be estimated by constructing partial litter decompo-
sition budgets. In general, a fraction of initial litter C is converted into microbial
(including fungal) production, while some C is lost as CO2 due to microbial
respiration. Unfortunately, litter-associated respiration measured in the field cannot
be easily partitioned among microbial groups colonizing plant litter (i.e., fungal
vs. bacterial). However, experiments in laboratory microcosms simulating stream
conditions (Suberkropp 1991) with plant litter colonized by pure cultures of aquatic
hyphomycetes show remarkably similar decomposition patterns to those found in
streams (Gulis and Suberkropp 2003a, b; Ferreira and Chauvet 2011). In these
studies, fungal assimilation (i.e., production + respiration) accounted for 23–56%
of plant carbon loss depending on temperature and dissolved nutrient availability.
However, these values are likely underestimates of fungal contribution, since cumu-
lative fungal production was estimated from accumulated biomass and C losses as
conidia (i.e., not via [14C]-acetate-to-ergosterol approach), which would not include
hyphal losses with FPOM. In addition, fungal-generated FPOM and DOM losses
were also not taken into account. In streams, when litter-associated fungal produc-
tion is accurately measured, it is possible to calculate fungal respiration and assim-
ilation by using estimates of fungal growth efficiency obtained in laboratory
microcosms (24–60%). Such estimates resulted in fungal assimilation accounting
for 29–39% of leaf litter C losses, while bacteria contributed only 4–13% (Pascoal
and Cassio 2004). The low contribution of bacteria is especially surprising since the
experiment was conducted in a polluted river where bacteria may also metabolize
DOM from the water column. The values for fungal contribution to litter decompo-
sition given above are clearly underestimates since they also did not account for
fungi-mediated FPOM and DOM losses from leaf litter, which may be comparable to
respiratory losses (Gessner et al. 2007). When these losses were estimated, fungi
accounted for up to 98% of the microbial contribution to leaf mass loss (Baldy et al.
2007).
Several studies have noted strong significant relationships between litter-
associated fungal biomass, sporulation rate of aquatic hyphomycetes, percentage
of initial litter C converted into conidia, and rates of litter decomposition (Gessner
and Chauvet 1994; Gessner et al. 2007; Gulis et al. 2009). Recently, we found that
fungal parameters (biomass or production) measured at early stages of decomposi-
tion (day 28) can be used to accurately predict final decomposition rates of leaf litter
and wood after 4–5 months (Fig. 5.3; Gulis et al. unpublished). Cumulative spore
production by the end of the experiment also showed a strong relationship with litter
decomposition rates (Fig. 5.3), suggesting that fungi are major decomposers of plant
litter in streams.
5 Fungal Decomposers in Freshwater Environments 129

Maple leaves
R² = 0.72, p=0.016 R² = 0.75, p=0.012

-1
on rate (d-1)

Rhododendron leaves

0.01 Wood veneers 0.01

n
R² = 0.92, p<0.001
R² = 0.86, p<0.001

R² = 0.97, p<0.001
0.001 0.001 R² = 0.84, p<0.001
dec

0.0001 0.0001
0.01 0.1 1 10 100 0.01 0.1 1 10 100
Fungal production (mg g-1 litter AFDM d-1)
(conidia µg-1 initial litter AFDM)

Fig. 5.3 Relationships between final plant litter decomposition rates and fungal production at early
stages of breakdown (day 28) (left panel) and cumulative spore production of aquatic hyphomycetes
from plant litter by the last day of decomposition experiment (day 111 for maple leaf litter, day
144 for rhododendron leaves and wood veneers) (right panel). Data points are means from seven
dissolved inorganic nutrient treatments set up in replicated streamside channels. R2 and p values of
linear regressions based on log-log transformed data are shown. Gulis et al. unpublished

5.2.4 Effects of Environmental Factors, Including Dissolved

Nutrients, on Fungal Activity and Organic Carbon
Processing

A variety of parameters can potentially affect litter-associated microbial activity and

decomposition rates. Intrinsic parameters of the organic substrate include the type of
plant litter (e.g., leaves of deciduous trees vs. grasses vs. wood) and litter quality,
such as lignin content (or carbon quality) and the concentrations of nitrogen (N) and
phosphorus (P), i.e., litter C:N and C:P ratios. In general, fungal activity and litter
decomposition are negatively affected by high lignin content and high initial C:
nutrient stoichiometric ratios of the substrate as was summarized earlier (e.g., Stelzer
et al. 2003; Ferreira et al. 2006b; Gessner et al. 2007). The major external factors
exerting control over fungal activity and breakdown rates are water temperature
and water chemistry. The effects of stream pollution on aquatic fungi including
nutrient enrichment, acidification, effects of heavy metals, organic xenobiotics,
nanoparticles, and thermal pollution have been recently summarized by Ferreira
et al. (2014, 2015). Here we address the effects of dissolved nutrients as this has
received considerable attention in the recent years, since in most streams affected by
human activities, the magnitude of their effects on fungal processes and litter
decomposition is greater than that of other environmental parameters.
Pollution of streams and rivers often leads to elevated concentrations of dissolved
inorganic nutrients. Fungi colonizing plant litter can obtain N and P from both the
substrate they grow on and the water column (Suberkropp 1995; Cheever et al. 2013).
130 V. Gulis et al.

Mining N and P from organic substrates requires considerable energy and resource
expenditures to produce extracellular enzymes to attack recalcitrant organic mole-
cules in order to cleave amino or phosphate groups (Sinsabaugh et al. 2014). Thus,
from an energetic perspective, fungi should preferentially use dissolved inorganic
nutrients. In addition, plant litter C:N and C:P ratios are considerably higher than
C:nutrient ratios of microbial, including fungal, biomass (Danger and Chauvet 2013;
Grimmett et al. 2013; Gulis et al. 2017). The mismatch between the resource
stoichiometric ratios and microbial biomass elemental ratios or nutrient limitation
of fungal activity can be alleviated by external nutrient supply. Indeed, experiments
in laboratory microcosms simulating stream conditions and whole-stream nutrient
addition experiments have shown that elevated dissolved nutrient concentrations
stimulate fungal activity (growth and sporulation rates, maximum fungal biomass
attained, cumulative production, respiration) and plant litter decomposition
(Suberkropp 1998; Gulis and Suberkropp 2003a, b, c; Suberkropp et al. 2010;
Ferreira and Chauvet 2011). In some studies, the relationships between nutrient
concentrations and fungal parameters or decomposition rates followed asymptotic
saturation-type models (e.g., Michaelis-Menten) where large responses were trig-
gered by relatively small increases in nutrients (Rosemond et al. 2002; Ferreira
et al. 2006b). Stimulation of fungal activity by inorganic nutrients is generally more
pronounced for low-quality (low N and P, high lignin) substrates, such as wood
(Stelzer et al. 2003; Gulis et al. 2004, 2008; Ferreira et al. 2006b), pointing to more
severe nutrient limitation of fungi on these substrates. Correlative studies based on
natural or anthropogenic gradients of dissolved nutrients show mixed results, since
in many cases nutrient enrichment was accompanied by the presence of other
pollutants (pesticides, heavy metals, etc.) with corresponding negative effects on
fungal activity. However, a recent pan-European study of 100 streams (Woodward
et al. 2012) and a meta-analysis by Ferreira et al. (2015) suggest that stimulation of
microbially driven decomposition by dissolved nutrients is widespread.
Most studies that dealt with the effects of nutrients on aquatic fungi have involved
enrichments by both N and P; however, very little is known about how N and P
concentrations and varying N:P ratios affect fungal activity. The situation is com-
plicated by the ability of fungi to obtain these nutrients from both the substrate and
the water. Recent studies suggest that fungal activity and litter decomposition may
be stimulated to a greater extent by dissolved N rather than P availability (Fig. 5.4;
see also Cheever et al. 2012, 2013). This phenomenon may be related to fungal
biomass stoichiometry and elemental homeostasis. Until recently, little was known
about C:N and C:P ratios of mycelium in aquatic fungi, since it is impossible to
separate it from the substrate for direct measurements. Indirect estimates of fungal
elemental stoichiometry can be obtained from field studies by plotting increases in
litter-associated fungal carbon vs. increases in detrital N or P (Fig. 5.5), assuming
that changes in nutrient content of detritus are due to fungal immobilization of
inorganic N and P. Such estimates are generally in agreement with data from
laboratory experiments in liquid cultures (Danger and Chauvet 2013; Grimmett
et al. 2013). Our recent experiments performed in defined media and, more
5 Fungal Decomposers in Freshwater Environments 131

0.7
T. chaetocladium
H. lugdunensis
0.6
6-species assemblage
Fungal growth rate (d-1)

0.5

0.4

0.3

0.2

0.1

0.0
10 100 1000 1 10 100
Dissolved NO3-N (μg L-1) SRP (μg L-1)

Fig. 5.4 Effects of nitrate-N and soluble reactive phosphorus (SRP) concentrations on growth rates
of two aquatic hyphomycetes and six-species aquatic fungal assemblage associated with submerged
decomposing maple leaf litter in experimental microcosms simulating stream conditions. Data
points are means for 15-day-old samples from nine dissolved inorganic nutrient treatments. Fungal
growth rates were determined based on 14C-acetate-to-ergosterol approach (Gulis and Bärlocher
2017). For the left panel, the effects of NO3–N on fungal growth rate were significant (TC R2 ¼ 0.88,
p < 0.001; HL R2 ¼ 0.88, p < 0.001; assemblage R2 ¼ 0.76, p ¼ 0.002). Data are from Gulis et al.
(2017)

importantly, with fungi growing on plant litter in laboratory microcosms (Gulis et al.
2017) suggest that aquatic hyphomycetes are homeostatic with respect to their C:N
ratio (i.e., fungal C:N does not change with varying dissolved N availability), but are
not homeostatic with respect to their C:P ratio (Fig. 5.6). This indicates that fungi
may be able to take up and store P in excess when it is available. Overall, fungal
biomass accrual and concomitant immobilization of dissolved nutrients during litter
decomposition have important implications for higher trophic levels in stream
ecosystems (see below).

5.2.5 Fungal Importance at the Ecosystem Scale

The majority of studies that followed the dynamics of fungal biomass, production,
and sporulation have dealt with decomposition of a single type and cohort of plant
litter, typically enclosed in litter bags and placed in a stream. However, streams
naturally contain a high diversity of litter types at different stages of decay due to the
lateral litter input (“blow-in”) occurring throughout the year. Thus, periodic mea-
surements of standing stock of plant litter on areal basis (per m2 of stream bottom)
combined with periodic estimates of fungal parameters per g dry mass of naturally
occurring plant litter would provide insights into fungal importance at the ecosystem
scale. Estimates of leaf litter-associated fungal biomass on areal basis range from <1
132 V. Gulis et al.

Fig. 5.5 Relationships

between increases in fungal

Increase in detrital N (mg g-1 leaf AFDM)

C and litter N content
(top panel) and fungal C and
litter P content
(bottom panel) of
rhododendron leaf litter at
Coweeta Long Term
Ecological Research site in
North Carolina and alder
leaf litter in a Portuguese
stream. Slopes of
regressions were used to
calculate fungal elemental
ratios (molar), C:N ¼ 4.3, C:
P ¼ 91, and N:P ¼ 21
for rhododendron, and
6, 278 and 50 for alder litter,
respectively. Data are from
Gulis et al. (2006), Ferreira
et al. (2006b) and Tant et al.
(2013)
Increase in detrital P (mg g-1 leaf AFDM)

Incerease in fungal carbon

(mg g-1 leaf AFDM)

to 23 g C m
2 and are highly seasonal in temperate streams due to seasonal patterns
of deciduous leaf input and, hence, in-stream litter standing stocks that peak in
autumn (Suberkropp 1997; Methvin and Suberkropp 2003; Carter and Suberkropp
2004; Suberkropp et al. 2010). Mean annual fungal biomass estimates in these
studies ranged from 1 to 9 g C m
2. Fungal biomass associated with small wood
(7–40 mm diam) was estimated to average 4–7 g C m
2 in two streams with little
seasonal variation (Gulis et al. 2008).
5 Fungal Decomposers in Freshwater Environments 133

Fig. 5.6 Relationships 100

between dissolved inorganic T. chaetocladium
N:P ratios (molar) and C:N H. lugdunensis
or C:P ratios of fungal 6-species assemblage

Fungal biomass C:N ratio

biomass. Data are from
experiments in laboratory
microcosms simulating
stream conditions with fungi
(two monocultures and a
six-species fungal 10
assemblage) grown on
maple leaf litter (Gulis et al.
2017). NaNO3 and KH2PO4
were used as dissolved N
and P sources in microcosm
solutions. Data points are
means of three replicates.
For the bottom panel, the 1
slopes of regression of 1 10 100
log-log transformed data are
different from zero 1000 Dissolved N:P ratio
suggesting that fungi are not
homeostatic with respect to
Fungal biomass C:P ratio

their C:P elemental ratio

(TC R2 ¼ 0.89, p < 0.001;
HL R2 ¼ 0.76, p ¼ 0.011;
assemblage R2 ¼ 0.72,
p ¼ 0.004)
100

10
1 10 100
Dissolved N:P ratio

Areal estimates of annual fungal production associated with leaf litter in five
streams ranged from 8 to 23 g C m
2 year
1 (Suberkropp 1997; Methvin and
Suberkropp 2003; Carter and Suberkropp 2004). Taking into account annual leaf
litter input into these streams (~190–246 g C m
2 year
1) and fungal growth
efficiency (33%, Suberkropp 1991; Gulis and Suberkropp 2003b), annual fungal
assimilation (production + respiration) accounted for 10–29% of annual litter input.
However, these streams exhibited relatively low litter retention, i.e., considerable
amount of leaf litter tends to be flushed from these streams during winter storm
events. In contrast, in two small highly retentive streams with high litter inputs that
maintained mean annual litter standing stocks up to 450 g C m
2, annual fungal
134 V. Gulis et al.

Fig. 5.7 Relationship 350

between litter-associated
fungal biomass and detrital Wood veneers
C:N ratios (molar) at later 300 y = 770.4x-0.58, R² = 0.82
stages of decomposition
(day 77) for three substrate Rhododendron leaves
250

Detrital C:N ratio

types. Data (Gulis et al. y = 964.2x-0.70, R² = 0.97
unpublished) are fitted
200 Maple leaves
separately using a power
function; R2 are shown for y = 1859.6x-0.86, R² = 0.58
each substrate. When data 150
for all substrates are
combined, linear regression
100
based on log-log
transformed data gives
R2 ¼ 0.91, p ¼ 9.0  10
12, 50
and n ¼ 21
0
0 20 40 60 80 100 120
Fungal biomass (mg g-1 litter AFDM)

production reached 49–290 g C m
2 year
1 with estimated fungal assimilation of

35% to >100% of the annual leaf litter input (Suberkropp et al. 2010).
Corresponding annual bacterial production from leaf litter in these streams ranged
from 7 to 13 g C m
2 year
1, which is more than an order of magnitude lower than
annual fungal production. Estimates of annual fungal production associated with
small wood in these same two highly retentive streams were 13–17 g C m
2 year
1,
which translated into a fungal assimilation of 45–57% of annual wood inputs to these
streams (Gulis et al. 2008, unpublished). These findings further emphasize the key
role that fungi play in carbon and energy flow from submerged plant litter in stream
ecosystems.
Increases in fungal biomass as litter decomposes (see above) and concomitant
fungal uptake and immobilization of dissolved N and P lead to fungi exerting strong
control over the C:N and C:P stoichiometry of decomposing plant litter (Fig. 5.7; see
also Tant et al. 2013; Manning et al. 2015, 2016). These increases in N and P content
of plant litter together with the digestion of recalcitrant plant polymers by extracel-
lular fungal enzymes improve the resource quality and palatability of plant litter to
stream invertebrates (Suberkropp 1992), which lead to increased secondary produc-
tion of some metazoan groups (Cross et al. 2006). N- and P-rich fungal biomass have
been shown to directly contribute to diets of detritivorous invertebrates (Cross et al.
2007), thus facilitating the transfer of C and nutrients to higher trophic levels in
aquatic ecosystems. In some cases, incorporation rates of fungal C accounted for up
to 100% of daily growth rates of aquatic invertebrate larvae (Chung and Suberkropp
2009).
5 Fungal Decomposers in Freshwater Environments 135

5.3 Fungi Associated with Emergent Macrophytes

in Freshwater Wetlands

5.3.1 Characteristics of Freshwater Marshes and Emergent

Plant Decay

Freshwater marshes are widely regarded as important transition zones that occur at
the interface of terrestrial and aquatic habitats. A common feature of these freshwater
ecotones is the presence of emergent macrophytes, such as Phragmites, Typha, and
Juncus. These wetland-adapted plants exhibit high aboveground biomass production
frequently exceeding 1 kg C m
2 year
1. As a result, emergent marsh plants
constitute an important reservoir of carbon and nutrients and are often the principal
carbon and nutrient pool depicted in most wetland elemental budgets (Mitsch and
Gosselink 2007; Reddy and Delaune 2008).
Most of the plant biomass produced in marshes is not consumed by herbivores but
instead enters the detrital pool where microbial decomposers (bacteria and fungi) and
detritus-feeding consumers (macroinvertebrates) participate in its breakdown and
mineralization. Despite the well-known importance of plant detritus in these eco-
systems (Moore et al. 2004; Hagen et al. 2012), natural plant decomposition in
freshwater marshes and the role of litter-associated fungal decomposers have rarely
been investigated. Most studies examining emergent plant decomposition have
focused on microbial decay processes occurring either at or within the wetland
surface sediments (e.g., Polunin 1984; Webster and Benfield 1986). This approach
continues today (e.g., Rothman and Bouchard 2007; Fennessy et al. 2008; Gingerich
and Anderson 2011) and is likely grounded in the false perception among wetland
researchers that emergent plant decomposition occurs solely at or within the marsh
sediments, where it is driven by aerobic and anaerobic bacterial communities
(Gutknecht et al. 2006; Kayranli et al. 2010; Morrissey et al. 2014). As a result,
our understanding of fungal importance in wetland biogeochemical cycles has not
been fully realized and today remains notably absent in conceptual or quantitative
models describing wetland carbon and nutrient cycles (Batzer and Sharitz 2006;
Mitsch and Gosselink 2007; Reddy and Delaune 2008). Such a paradigm contrasts
sharply with our knowledge of litter decomposition in streams (see above), where
fungi are widely recognized as a key microbial group that drives litter decomposition
and facilitates carbon and nutrient transfer to invertebrate consumers (Gessner et al.
2007; Gulis et al. 2009; Findlay 2010).
Fundamental details to recognize when examining emergent plant decomposition
are both the spatial and temporal conditions under which plant material naturally
decomposes. In most emergent macrophytes, abscission and collapse of plant matter
to the sediments or overlying surface waters do not occur immediately following
plant senescence and death. A significant portion of the plant detrital mass remains in
a standing-dead position (Asaeda et al. 2002; Christensen et al. 2009) where it can
undergo considerable initial microbial transformation and decay before it collapses
to the water or sediments (Gessner 2001; Kuehn et al. 2011; Su et al. 2015). As a
136 V. Gulis et al.

result, emergent plant decay is a complex sequential process that involves two
distinct spatial phases separated in time: an initial decay phase under aerial
standing-dead conditions followed by a second decay phase under submerged or
surface sediment conditions after the eventual collapse of standing litter. When plant
litter decomposition studies have closely simulated these natural decay conditions,
then fungi have been found to be important drivers of emergent macrophyte decom-
position (Gessner et al. 2007; Kuehn 2008, 2016).

5.3.2 Fungal Diversity in Freshwater Marshes

Mycologists have known for over a century (Saccardo 1898) that filamentous fungi
pervasively colonize and reproduce on and within both standing and collapsed litter
of emergent macrophytes (Kuehn 2016). To date, much of our knowledge of fungal
diversity associated with plant litter comes from traditional studies where fungal
reproductive structures (e.g., ascomata) were microscopically observed and identi-
fied either directly from field-collected material or after culturing fungi in the
laboratory. To date, very few published studies have employed molecular-based
techniques to characterize litter-associated fungal communities in freshwater
marshes (e.g., Buesing et al. 2009).
Unlike stream systems where aquatic hyphomycetes typically dominate on sub-
merged leaf litter (see above), fungal communities inhabiting emergent plant litter
are often quite diverse. For example, an earlier compilation of literature by Gessner
and Van Ryckegem (2003) found that over 600 species of fungi had been recorded
from the common reed (Phragmites australis) alone, with members of the
Ascomycota being the most common. Many studies have documented distinct
temporal changes in fungal taxa during litter decomposition (Pugh and Mulder
1971; Apinis and Taligoola 1974; Van Ryckegem and Verbeken 2005b, c; Van
Ryckegem et al. 2007). For example, Pugh and Mulder (1971) observed that
prevalent fungi during the standing decay phase of Typha latifolia consisted of
common phylloplane taxa, such as Aureobasidium, Cladosporium, and Epicoccum.
These initial fungal colonizers were followed by an increased occurrence of asco-
mycetes (e.g., Leptosphaeria spp.). Predatory nematode-trapping fungi,
Arthrobotrys and Dactylaria, were the most common taxa observed following the
collapse of standing litter to the marsh benthic sediments.
In addition to temporal changes during emergent litter decay, fungal taxa may
also vary in their spatial colonization of plant litter. Several researchers have
reported that certain fungal taxa may preferentially occupy specific plant tissues,
such as leaves, leaf sheaths, or the nodes and internodes of culms (Pugh and Mulder
1971; Apinis et al. 1975; Poon and Hyde 1998; Van Ryckegem and Verbeken
2005a; Van Ryckegem et al. 2007), which is likely a result of changes in decay
conditions (aerial vs. submerged) and differences in the intrinsic quality of plant
litter, such as concentrations of recalcitrant compounds (lignocellulose) or nutrients
(N and P). For example, Van Ryckegem and Verbeken (2005a) examined the fungal
5 Fungal Decomposers in Freshwater Environments 137

communities associated with standing-dead culms of P. australis in four tidal

marshes that varied in salinity (freshwater to mesohaline). A distinct vertical distri-
bution of fungal taxa was observed along the P. australis shoot axis, which was
influenced by flooding height, frequency of inundation, and salinity.

5.3.3 Fungal Contribution to Litter Decomposition: Biomass

and Rates of Production

Despite the abundant evidence indicating fungal colonization of decaying plant

litter, our quantitative understanding of fungal decomposers in wetlands has lagged
behind the body of data on other microbial groups, such as bacteria, due primarily to
methodological limitations (see Sect. 5.1 above). However, application of
ergosterol-based methods in freshwater marshes has produced compelling evidence
that fungi are important participants in plant litter decomposition within these
systems, particularly during the initial standing decay phase. Significant accumula-
tion of fungal biomass has been reported in standing emergent plant litter, with peak
values often accounting for as much as 5–10% of the total detrital dry mass (Newell
et al. 1995; Bärlocher and Biddiscombe 1996; Kuehn and Suberkropp 1998b; Kuehn
et al. 1999, 2011; Gessner 2001; Findlay et al. 2002b; Newell 2003; Welsch and
Yavitt 2003; Ohsowski 2008; Su 2014; Su et al. 2015; Kuehn 2016). Recently,
Kuehn et al. (2011) and Su et al. (2015) estimated the contribution of fungal
decomposers during standing litter decay of Typha angustifolia and Typha
domingensis leaves in a temperate (Michigan) and subtropical (Alabama) freshwater
marsh, respectively. During senescence and early standing litter decay, significant
increases in both ergosterol and chitin (glucosamine) were observed in Typha leaves
(Fig. 5.8), which revealed the rapid colonization of Typha litter by fungal decom-
posers. Increases in both ergosterol and glucosamine concentrations correlated with
losses in leaf carbon, providing strong evidence that increased growth of fungal
decomposers coincides with accelerated losses in Typha detrital mass (see also
Gessner 2001). Analogous to spatial patterns in fungal diversity (above), differences
in fungal biomass have also been noted among specific plant tissues. For example,
Kuehn et al. (1999) quantified fungal biomass concentrations in Erianthus giganteus
during shoot senescence and early standing decomposition. Significantly higher
fungal biomass was observed in leaf versus culm litter, which was consistent with
the more recalcitrant nature and lower N and P concentrations of culm litter.
In addition to accumulating large quantities of biomass, fungal communities
inhabiting standing litter also exhibit high rates of secondary production. Using
the 14C-acetate-to-ergosterol incorporation method, Kuehn et al. (2011) reported
fungal biomass production rates ranging between 0.2 and 3.3 mg fungal C g
1
detrital C day
1 in standing T. angustifolia litter in a north temperate freshwater
marsh (Fig. 5.9). Significant differences in fungal production rates were observed
during the study period, with peak daily production occurring just after leaf
138 V. Gulis et al.

Fig. 5.8 Dynamics of 1.5

T. angustifolia - Michigan 120
fungal biomass (a) and A
glucosamine (b) T. domingensis - Alabama

(mg C / g detrital C)
(µg / g detrital C)

Fungal Biomass
concentrations in Typha 1.0
80

Ergosterol
angustifolia (Michigan) and
Typha domingensis
(Alabama) leaves during 0.5 40
standing litter
decomposition. Symbols
indicate means 1 SE
0.0 0
(n ¼ 6). Data from Kuehn
8
et al. (2011) and Su et al.
B
(2015)
(mg / g detrital C)

6
Glucosamine

0
0 100 200 300 400
Days

Fig. 5.9 Rates of fungal 6.0 0.6

production associated with

T. domingensis - Alabama
T. angustifolia - Michigan

(mg C / g detrital C/ d)
(mg C / g detrital C/ d)

T. angustifolia (Michigan)

Fungal Production
Fungal Production

and T. domingensis 4.0 0.4

(Alabama) leaves during
standing litter
decomposition. Symbols
2.0 0.2
indicate means 1 SE
(n ¼ 6). Data from Kuehn
et al. (2011) and Su et al.
(2015) 0.0 0.0
0 100 200 300 400
Days

senescence in late fall (74 days, November) and again during the summer season
(300 days, July). Similar production patterns were also reported by Su et al. (2015)
for fungal communities inhabiting T. domingensis in a subtropical freshwater marsh,
with peak production rates being observed in early summer (~300 days, June).
However, in contrast to Kuehn et al. (2011), fungal production associated with
standing T. domingensis leaves was roughly an order of magnitude lower than
production rates observed in T. angustifolia (Fig. 5.9), which was consistent with
the lower accumulation of fungal biomass in standing T. domingensis leaf litter
(Fig. 5.8).
When integrated over the entire study period, estimated cumulative production of
fungi associated with standing T. domingensis and T. angustifolia leaf litter totaled
39 and 123 mg fungal C per g initial detrital C, respectively, indicating that a large
5 Fungal Decomposers in Freshwater Environments 139

Table 5.2 Partial litter decay budgets, associated microbial parameters, and estimated contribution
of fungal decomposers during standing-dead leaf litter decay of T. angustifolia and T. domingensis
Michigan Alabama
Parameter T. angustifolia T. domingensis
Total leaf mass loss (mg C g
1 initial leaf C)a 556  60 371  39
Cumulative fungal production (mg C g
1 initial leaf C)b 123  10 39  4
Cumulative respiration (mg C g
1 initial leaf C)b – 135  23
Mean fungal biomass (mg C g
1 initial leaf C)a 33  11 17  1
Fungal production to biomass (P:B) ratio 3.5  1.3 2.3  0.1
Turnover time of fungal biomass (day) 99  34 145  7
Fungal yield coefficient (%)c 22  3 11  1
Fungal contribution to overall leaf C loss (%)d 69  5e 47  5
Data from Kuehn et al. (2011) and Su (2014)
a
Values for total leaf mass loss and fungal biomass are means 1 SD
b
Values for cumulative fungal production and respiration are means 1 SD, as estimated using
Monte Carlo simulation analysis
c
Fungal yield coefficient (%) ¼ total net fungal production/total leaf mass loss 100
d
The contribution of fungi to overall carbon loss from standing-dead leaf litter was determined by
dividing total fungal assimilation (cumulative production + respiration) by the total leaf C mass loss.
Assumes that respiratory activity is entirely due to fungal organisms
e
Assumes fungal growth efficiency of 32% (Gulis and Suberkropp 2003b)

portion of the observed leaf carbon lost was converted into fungal biomass (see
fungal yield, Table 5.2). Furthermore, estimates of cumulative fungal production
associated with Typha litter were significantly related with cumulative losses in leaf
litter carbon (Fig. 5.10), providing additional evidence that fungi are likely the
primary drivers of emergent plant decomposition during the standing decay phase.
If respiratory activities in standing litter are taken into account, which are likely from
fungi (see below), then total fungal assimilation (production + respiration) could
account for even a greater proportion of leaf carbon losses (Table 5.2).
After standing litter collapses, commencement of the benthic decomposition
phase is often accompanied by a notable shift in the environmental conditions
(e.g., increased water availability, exogenous nutrients, decreased temperature fluc-
tuations), which may lead to shifts in litter-associated microbial communities and
concomitant changes in the biomass and production of both fungi and bacteria.
Earlier, Kuehn et al. (2000) observed a rapid decrease in litter-associated ATP
concentrations, fungal biomass (ergosterol), and production rates of fungi following
the transition of standing Juncus effusus litter to a submerged environment. This
initial decrease was followed by an increase in fungal biomass and production rates
during later stages of submerged litter decomposition, reflecting a possible shift in
fungal community to taxa better adapted to an aquatic or semiaquatic existence (see
fungal diversity above). Similar initial decreases in fungal biomass and activity have
also been reported for P. australis litter following its transition from an aerial to a
submerged environment (Tanaka 1991; Kominkova et al. 2000; Van Ryckegem
et al. 2007).
140 V. Gulis et al.

Fig. 5.10 Relationship 600

between cumulative fungal A T. angustifolia - Michigan
production and cumulative
litter carbon loss of (a)

Cumulative Leaf Litter Mass loss (mg C / g initial detrital C)

T. angustifolia (Michigan) 400
and (b) T. domingensis
(Alabama) leaves during
standing litter
decomposition phase.
200
Symbols indicate means 1
SE (n ¼ 6). Data from r 2 = 0.91, p<0.001
Kuehn et al. (2011) and Su
et al. (2015)
0
0 50 100 150
600
B T. domingensis - Alabama

400

200

r 2 = 0.92, p<0.001

0
0 10 20 30 40
Cumulative Fungal Production
(mg C / g detrital C)

Despite litter submergence and initial declines in fungal biomass, fungi continue
to be a quantitatively important microbial group on and within decaying plant litter.
Simultaneous estimates of fungal and bacterial biomass associated with submerged
plant litter reveal that fungal decomposers often account for >90% of the total
microbial biomass (Findlay et al. 1990, 2002b; Newell et al. 1995; Sinsabaugh
and Findlay 1995; Kominkova et al. 2000; Kuehn et al. 2000, 2014; Su et al.
2007). Furthermore, several studies have reported that rates of fungal production
are often comparable to or greatly exceed corresponding rates of bacterial production
(Newell et al. 1995; Kuehn et al. 2000, 2014; Findlay et al. 2002a, b; Su et al. 2007,
but see Buesing and Gessner 2006). For example, Su et al. (2007) simultaneously
quantified bacterial and fungal biomass and production and extracellular degradative
enzyme activities during the decomposition of collapsed benthic leaf litter of
T. angustifolia in two hydrologically different (i.e., permanently inundated
vs. exposed) Lake Erie coastal marshes. Microbial biomass and production associ-
ated with decaying Typha litter were dominated by fungi at both sites, accounting for
greater than 94% and 99% of the total microbial biomass and production,
5 Fungal Decomposers in Freshwater Environments 141

respectively. Dynamics of extracellular microbial enzymes involved in the acquisi-

tion of detrital C, N, and P were similar to observed patterns in fungal biomass and
production, suggesting that litter-inhabiting fungal decomposers are responsible for
a large portion of extracellular enzymes produced (see also Romani et al. 2006).
Kuehn et al. (2000) also found that rates of fungal production associated with
J. effusus litter greatly exceeded corresponding rates of bacterial production through-
out early stages of submerged litter decomposition. Estimates of fungal and bacterial
contributions indicated that fungal assimilation could explain a substantial portion
(68%) of the observed J. effusus litter mass loss, providing evidence that fungi are
also important players in the transformation and decay of plant litter under sub-
merged or surface sediment conditions (Kuehn et al. 2000).

5.3.4 Factors Affecting Fungal Activities on Emergent Plant

Litter

Plant litter decomposition in freshwater marshes involves a complex array of biotic

and abiotic processes that result in the production of decomposer biomass (microbial
and invertebrate consumers), release of CO2 and nutrients (N and P) through
mineralization, and also release of DOM and FPOM (Gessner et al. 1999; Kuehn
2008). From a microbial perspective, the rates of these processes are strongly
influenced by the response of microbial communities to the environmental condi-
tions and the intrinsic quality of the detrital resources they metabolize (Gessner et al.
2007). Being osmotrophic, both bacteria and fungi acquire detrital resources via the
extracellular digestion of complex organic matter by degradative enzymes. The
production and release of extracellular enzymes represent a substantial energy cost
for microorganisms. As a result, microbes will regulate the production and release of
extracellular enzymes in accordance with detrital resource availabilities (C, N, P),
which serves to optimize their assimilatory return on investment (Sinsabaugh and
Follstad Shah 2012; Sinsabaugh et al. 2014). Collectively, this regulation can affect
outcomes related to fungal community performance (biomass, growth, reproduction)
and hence the decomposition and mineralization of plant detritus.
Similar to deciduous leaf litter in streams, emergent plant litter in freshwater
marshes typically has low nutrient concentrations and, hence, high C:nutrient ratios
(e.g., C:N > 80, C:P > 4000). The nutrient stoichiometry (C:N:P) of fungal biomass
is typically lower than that of the plant litter they colonize. As a consequence, the
biomass and activities of fungi are often limited by N and P availability. These
nutrients can be obtained either from the substrate they inhabit or from an exogenous
source, such as dissolved nutrients in the water after the collapse of standing litter.
For example, Kuehn et al. (2011) and Su et al. (2015) observed rapid increases in
fungal biomass (ergosterol) during senescence and early standing leaf litter decay in
T. angustifolia and T. domingensis. Despite similar patterns of increase, total fungal
biomass accumulation in standing T. angustifolia leaf litter was notably higher than
142 V. Gulis et al.

Fig. 5.11 Relationships 140

between litter-associated A T. angustifolia - Michigan
120 T. domingensis - Alabama

Carbon : Nitrogen Ratio

fungal biomass and (a)
detrital C:N ratios and (b)
detrital C:P ratios of 100
y = 345x-0.412 , r2 = 0.61
T. angustifolia (Michigan)
and T. domingensis 80
(Alabama) leaves
60
throughout standing litter
decomposition phase. Data 40
were fitted using a power
function 20
7000
B
Carbon : Phosphorus Ratio

6000

5000
y = 18813x-0.469 , r2 = 0.60
4000

3000

2000

1000
0 20 40 60 80 100 120
Fungal Biomass (mg C / g detrital C)

in T. domingensis (Fig. 5.8). Furthermore, daily and cumulative production of fungi

in T. angustifolia were also markedly higher than in T. domingensis, which was
consistent with the higher rates of mass loss observed in T. angustifolia (55%) versus
T. domingensis (37%) leaf litter (Table 5.2). The contrasting performance and
overall contribution of fungal decomposers to leaf decay in these studies may be
explained by differences in litter nutrient content between these Typha species. Su
et al. (2015) found that C:N and C:P ratios of T. domingensis leaf litter averaged
88 and 4352 throughout the post-senescent stages of standing litter decomposition,
respectively. In contrast, Kuehn et al. (2011) observed much lower C:N and C:P
ratios in standing T. angustifolia leaves, which averaged 67 and 2583, respectively.
Fungal biomass in both T. angustifolia and T. domingensis leaf litter were negatively
related to litter C:N and C:P ratios (Fig. 5.11), implying that fungal communities
may have experienced considerable stoichiometric constraints on growth
(Sinsabaugh et al. 2015) and, hence, litter decomposition rates.
A number of studies conducted in both subtropical and temperate freshwater
marshes have also established that water availability is a key factor controlling the
activity of microbial decomposers in the standing litter decay phase (Kuehn et al.
1998, 1999, 2004; Kuehn and Suberkropp 1998a; Welsch and Yavitt 2003). Kuehn
et al. (2004) examined the effects of environmental conditions on rates of microbial
respiration (CO2 flux) from standing P. australis litter in two temperate freshwater
marshes in Switzerland. Under field conditions, rates of microbial respiration from
5 Fungal Decomposers in Freshwater Environments 143

(µ g C / g detrital AFDM / h)
Fig. 5.12 Diel changes in 250
Leaf-blades

Microbial Respiration
rates of microbial respiration Leaf-sheaths
200
from standing-dead leaves
and leaf sheaths of 150
Phragmites australis within
a temperate freshwater lake 100
littoral marsh (top panel). 50
Corresponding diel changes
in temperature and relative 0
humidity patterns and plant 25

Relative Humidity (%)

litter water potentials and 100

Temperature (˚C)
20 Temperature
precipitation are also Relative Humidity 90
illustrated (middle and 15
bottom panels). Symbols 80
10
indicate means 1 SE
(n ¼ 3). Data from Kuehn 5 70
et al. (2004)
0 60
0.25
0.0
Water Potential MPa

Precipipation (mm)
Leaf-blades 0.20
-2.0
Leaf-sheaths
-4.0 0.15
Precipitation

-6.0 0.10

-8.0 0.05

-10.0 0.00
8 11 14 17 20 23 2 5 8 11
Time (h)

standing litter exhibited a diel periodicity, with the highest rates occurring at night
when water becomes available to litter-inhabiting microbial assemblages via dew
formation (Fig. 5.12). In contrast, microbial respiration virtually ceased during the
day as a result of increased desiccation stress. These results were remarkably similar
to the diel microbial respiratory patterns reported earlier from standing litter in a
subtropical freshwater marsh (Kuehn and Suberkropp 1998a), where temperature-
driven increases in relative humidity and dew formation were identified as important
mechanisms controlling nighttime increases in litter water potential (water availabil-
ity) and associated microbial respiration. Additional research found that fungi
inhabiting standing litter likely survive these diel periods of desiccation by accumu-
lating intracellular compatible solutes (Kuehn et al. 1998).
Similar to observed patterns in fungal diversity, biomass, and production, distinct
differences in rates of microbial respiration have also been observed among different
plant litter substrates. Kuehn et al. (2004) reported significant differences in micro-
bial respiration rates among standing litter fractions of P. australis (leaf blades
vs. leaf sheaths vs. culms), which were consistent with differences in water absorp-
tion patterns, structural characteristics (lignocellulose), and the degree of fungal
colonization. Maximum rates of respiration among litter fractions were positively
correlated with litter-associated fungal biomass (ergosterol, r ¼ 0.72). Similar
findings by other researchers (Kuehn and Suberkropp 1998a; Su 2014; Su et al.
144 V. Gulis et al.

2015) lend support to the idea that respiratory activity in standing litter can be mostly
attributed to fungal decomposers.
Under submerged or surface sediment conditions, rates of microbial respiration
associated with emergent macrophyte plant litter are comparable to the maximum rates
reported for standing-dead litter (Kominkova et al. 2000; Buesing and Gessner 2006;
Su et al. 2007). Buesing and Gessner (2006) examined microbial respiration rates
associated with submerged P. australis litter at the same littoral marsh site used by
Kuehn et al. (2004). Respiration rates ranged between 31 and 319 μg CO2–C g
1 litter
organic mass h
1 throughout the year, with fluctuations closely following changes in
lake water temperature. Since litter-associated microbial biomass is predominantly
fungal (see above), it is plausible that a large portion of the respiratory C losses from
collapsed plant litter may also be due to the metabolic activities of fungal decomposers.
In addition to chemical and physical factors described above, microbial perfor-
mance and litter decomposition in freshwater marshes (and streams) may also be
influenced by the myriad of biotic interactions that occur within the detrital land-
scape (Gessner et al. 1999, 2007). For example, under submerged conditions
decaying plant litter often develops complex microbial biofilms (Battin et al.
2007), which can contain a diverse community of both autotrophic (algae) and
heterotrophic (bacteria, fungi, protists) microorganisms. The close spatial proximity
of these microbial groups on and within submerged plant litter suggests the potential
for biotic interactions (e.g., Mille-Lindblom and Tranvik 2003; Francoeur et al.
2006; Mille-Lindblom et al. 2006; Danger et al. 2013; Kuehn et al. 2014). For
example, Kuehn et al. (2014) provided intriguing evidence that photosynthetically
active algae may elicit a “priming effect” on heterotrophic bacterial and fungal
decomposers colonizing submerged emergent plant detritus (see also Danger et al.
2013). Well-established in terrestrial ecosystems, the priming effect describes a
natural phenomenon whereby the microbial decomposition and mineralization of
recalcitrant soil organic matter are enhanced by pulsed or continuous inputs of labile
carbon, such as plant root exudates (Blagodatsky et al. 2010; Kuzyakov 2010).
These carbon inputs produce hotspots and hot moments of soil-rhizosphere micro-
bial activity, where microbial heterotrophs are provided energy-rich compounds
that increase their metabolic capabilities (e.g., enzyme production) to degrade and
mineralize refractory soil organic matter. In the presence of periphytic algae, Kuehn
et al. (2014) and Francoeur et al. (2006) documented that short-term light exposure
(400 μmol m
2 s
1 photosynthetically active radiation, PAR) rapidly stimulated
rates of bacterial and fungal production (Fig. 5.13) and extracellular hydrolytic and
oxidative enzyme activities in decaying plant litter, respectively. Furthermore,
experimental incubations of decaying litter with 14C- and 13C-bicarbonate
established that inorganic C fixed by algal photosynthesis was rapidly transferred
to and assimilated by heterotrophic microbial decomposers (Kuehn et al. 2014),
which was likely due to labile DOC exudation from co-occurring periphytic algae.
These findings underscore that microbial interactions are important for our under-
standing of key ecological processes, such as plant litter decomposition. Further-
more, these findings also strengthen the contention that the priming effect
phenomenon may be relevant within aquatic ecosystems (Guenet et al. 2010),
5 Fungal Decomposers in Freshwater Environments 145

Fig. 5.13 Production rates 180

of (a) bacteria (3H-leucine A Dark Light

(µ g C/g detrital C/h)

Bacterial Production
incorporation into protein)
and (b) fungi (14C-acetate
incorporation into 120
ergosterol) in natural
T. angustifolia detrital-
periphyton communities
when exposed to short-term 60
light (400 μmol m
2 s
1
PAR, UV-free) or dark
conditions in the laboratory. 0
T. angustifolia detrital-
periphyton was collected 180
10 and 29 days after litter B
(µ g C/g detrital C/h)
Fungal Production

submergence. Bars indicate

means 1 SE (n ¼ 5). Data 120
from Kuehn et al. (2014)

60

0
10 29
Day

where algal presence and activity within the detrital microbial landscape may
facilitate microbial decomposition and mineralization of detrital organic matter
(see also Rier et al. 2007, 2014; Danger et al. 2013; Hotchkiss et al. 2014).

5.3.5 Fungal Importance at the Ecosystem Scale

To date, most studies examining fungal activities in freshwater marshes have

focused on quantifying fungal biomass and production during specific litter decom-
position periods. In this regard, plant litter of known age and type (e.g., leaves) was
enclosed in litter bags or tagged in a standing position and sampled through time
(Newell et al. 1995; Bärlocher and Biddiscombe 1996; Kuehn and Suberkropp
1998a, b; Kuehn et al. 1999, 2000, 2011; Komínková et al. 2000; Gessner 2001;
Su et al. 2015). This approach has been useful in examining fungal dynamics during
the litter decomposition process (e.g., Fig. 5.8) and for constructing partial litter
decay budgets (e.g., Table 5.2) that focused on quantifying the contribution of fungal
decomposers to litter mass loss. However, to assess the impact of fungi or other
microbial assemblages on ecosystem-scale carbon and nutrient cycling, it is neces-
sary to have areal (m
2) estimates of microbial biomass and production associated
146 V. Gulis et al.

Fig. 5.14 Fungal biomass 40

Fungal Biomass (g C / m 2)
associated with standing-
dead plant litter per m2 of
marsh surface area in a 30
mixed C. jamaicense and
J. roemarianus marsh stand
(Alabama). Bars indicate 20
means 1 SE (n ¼ 6). Data
from Su (2014)
10

0
M J J A S O N D J F M A
Month

with naturally occurring plant litter, which would include plant detritus in various
stages of decay. Currently, very few studies have estimated the ecosystem-scale
contribution of fungi to carbon and nutrient cycling in freshwater marshes (Buesing
and Gessner 2006; Ohsowski 2008; Su 2014; Kuehn and Gessner unpublished).
Recently, Su (2014) estimated annual fungal biomass and production associated
with naturally occurring standing-dead litter in a subtropical marsh dominated by
Cladium jamaicense. Living fungal biomass per gram of plant litter carbon remained
fairly constant over the study period, averaging 30 mg C g
1 detrital C. Because of
appreciable standing litter accumulation, corresponding standing stock estimates of
fungal biomass per square meter of marsh were considerable, averaging 18 g C m
2
over the annual study period (Fig. 5.14). Substantial rates of fungal production on
areal basis were also observed. When integrated over the study period, estimated
annual fungal production in standing litter totaled 90 g C m
2 year
1 (Table 5.3),
which was equivalent to ~14% of mean annual standing litter mass (643 g C m
2). If
annual estimates of microbial respiration are taken into account (CO2 flux, 124 g C
m
2 year
1), which is most likely fungal, then total fungal assimilation could
account for ~33% of mean annual standing litter mass. Comparable estimates of
fungal production in standing-dead litter in other freshwater marsh systems
(Table 5.3) suggest that fungal-mediated processes may be a significant pathway
of carbon flow at the ecosystem scale.
High areal fungal production has also been reported for benthic plant detritus
(Table 5.3; Buesing and Gessner 2006; Ohsowski 2008). Buesing and Gessner
(2006) estimated annual production of both bacterial and fungal communities
associated with naturally occurring benthic litter of P. australis in temperate lake’s
littoral marsh in Switzerland. Annual fungal production on submerged P. australis
detritus totaled 93 g C m
2 year
1, indicating that ~15% of the annual aboveground
P. australis production (603 g C m
2 year
1) was transformed into fungal biomass.
However, in this study, despite certain reliance on dissolved organic carbon, annual
bacterial production was considerably higher (661 g C m
2 year
1), suggesting that
litter-associated bacteria may play an important role in carbon transformation once
standing litter collapses to the overlying marsh surface sediments.
5 Fungal Decomposers in Freshwater Environments 147

Table 5.3 Estimates of annual production, production to biomass (P:B) ratios, and turnover times
of fungi associated with standing-dead and benthic plant litter in freshwater marshes
Annual fungal Annual Turnover
Marsh production (g C m
2) P:B time (day) Reference
Weeks Bay (Alabama)
Cladium jamaicense/Juncus roemarianus marsh
Standing-dead leaf 90.3  4.4 4.9 74 Su (2014)
and stem litter
Independence Lake (Michigan)
Typha angustifolia marsh
Standing-dead leaves 78.0  5.5 4.0 90 Kuehn (unpublished)
Standing-dead stems 56.1  3.8 8.2 44
Paint Creek (Michigan)
Typha angustifolia marsh
Standing-dead leaves 7.9  0.6 1.0 342 Ohsowski (2008)
Standing-dead stems 34.5  2.2 2.7 137
Benthic litter 112.3  1.1 7.3 50
Lake Hallwil (Switzerland)
Phragmites australis marsh
Standing-dead leaves 7.5  4.3 3.8 103 Kuehn (unpublished)
Standing-dead sheaths 11.9  6.2 7.2 53
Standing-dead culms 2.5  4 6.2 62
Benthic litter 93.2  36.0 10.0 37 Buesing and Gessner
(2006)
Values for annual fungal production are means 1 SD

Observations of considerable fungal production and biomass in emergent plant

litter imply that fungi are also efficient in acquiring and immobilizing detrital N and
P to meet their stoichiometric demands for growth and reproduction. This may be
particularly true in standing litter decay systems where access to additional nutrients
from exogenous sources is likely to be limited. Using a fungal stoichiometric ratio of
C100N9P1 (Gulis et al. 2017) and data from Su (2014), we estimated that fungal
decomposers could immobilize ~25 and up to 100% of the detrital N and P,
respectively, in naturally occurring standing litter in a C. jamaicense marsh. Similar
observations in other freshwater marsh systems (Findlay et al. 2002b; Van
Ryckegem et al. 2006; Kuehn et al. 2011; Su et al. 2015) suggest that fungal
decomposers play an important role in the cycling of N and P in these systems.

5.4 Conclusions

Many freshwater environments, such as streams, rivers, and wetlands, depend on

inputs of dead plant material as a source of carbon and other nutrients to fuel
ecosystem processes. Fungi in these environments are key players in decomposition
148 V. Gulis et al.

of plant litter, where they facilitate the transfer of energy and nutrients to higher
trophic levels, such as aquatic invertebrates and ultimately vertebrate animals. It is
not uncommon for fungal biomass to contribute around 10% of detrital mass, annual
fungal production to reach tens to hundreds of g m
2, and direct fungal assimilation
of plant carbon to account for about one third of annual litter input. However, only
relatively recent advances in methodology have allowed us to uncover the critical
importance of fungi in decomposition of plant litter in these systems. Nevertheless,
new approaches are needed to quantify the effects of global change (temperature,
pollution, precipitation) on fungi and microbially driven processes in freshwaters.
Since changes in fungal activity and community structure may affect ecosystem
functioning, new molecular approaches, including the next-generation sequencing,
hold promise to further our understanding of fungi in freshwaters.

Compliance with Ethical Standards

Funding Financial support from the National Science Foundation (DEB 0919054, DEB 1655797
to VG and DBI 0420965, DBI 0923063, DEB 0315686, DEB 1457217 to KAK) is gratefully
acknowledged.
Conflict of Interest Vladislav Gulis declares that he/she has no conflict of interest. Rong Su
declares that he/she has no conflict of interest. Kevin A. Kuehn declares that he/she has no conflict
of interest.
Ethical Approval This article does not contain any studies with human participants or animals
performed by any of the authors.

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Chapter 6
The Ecology of Methanogenic Archaea
in a Nutrient-Impacted Wetland

Andrew Ogram, Hee-Sung Bae, and Ashvini Chauhan

Abstract Wetlands are the largest natural sources of methane, and many wetlands
are subject to nutrient enrichment due to runoff from adjacent agricultural and urban
lands. Methanogenic archaea are responsible for much of the methane produced in
terrestrial wetlands and participate in a range of additional activities including
nitrogen fixation and mercury methylation. Nutrient enrichment may impact the
dominant metabolic groups of methanogens, such that the fundamental activities of
methanogens may be associated with the nutrient status of the wetland. Regions of
the Everglades, a large marsh in the southern part of Florida, in the USA, are subject
to nutrient enrichment and are characterized by a gradient in available phosphorus,
organic carbon, and sulfate concentrations. This marsh provides an outstanding
system in which to study the impacts of nutrient enrichment on the distribution
and activities of methanogens. Competition for acetate with sulfate-reducing pro-
karyotes plays an important role in structuring methanogenic consortia in nutrient-
impacted regions, and the potentials for methanogenic nitrogen fixation and mercury
methylation differ along the nutrient gradient.

6.1 Introduction
6.1.1 Background

Terrestrial wetlands are the largest natural source of the greenhouse gas methane and
are responsible for the production of over 150 Tg of methane per year or approxi-
mately 20% of the total global methane budget (van Amstel 2012). Many of these
wetlands are subject to nutrient enrichment resulting from runoff from agricultural or

A. Ogram (*) · H.-S. Bae

Soil and Water Sciences Department, University of Florida, Gainesville, FL, USA
e-mail: aogram@ufl.edu; hsbae@ufl.edu
A. Chauhan
School of the Environment, Florida A&M University, Tallahassee, FL, USA
e-mail: ashivini.chauhan@famu.edu

© Springer Nature Switzerland AG 2019 157

C. J. Hurst (ed.), The Structure and Function of Aquatic Microbial Communities,
Advances in Environmental Microbiology 7,
https://doi.org/10.1007/978-3-030-16775-2_6
158 A. Ogram et al.

urban systems. This nutrient enrichment may impact a range of ecosystem processes,
including methane production. This review will use regions of the Florida Ever-
glades, a large freshwater wetland in the Southeastern USA, as a model system to
explore the impacts of nutrient enrichment on the microbial ecology of methane
production and processes related to methane production, such as fermentation,
sulfate reduction, and mercury cycling. Only the microbial ecology of methano-
genesis, and not methane consumption or total methane flux, will be considered in
this review. We also focus on the ecology of methanogens in peat within the
Everglades, and not in other environmental compartments where methanogens
may be active, such as the flocculent material and floating periphyton.
Most biological methane is the product of a strictly anaerobic metabolism
conducted exclusively by archaea that are only capable of metabolizing relatively
simple electron donors. However, the relationships that methanogenic archaea have
with their environment and with other microorganisms can be quite complex.
Methanogenic archaea respond to a variety of environmental conditions, including
nutrient and electron donor availabilities and redox potentials. They are largely
dependent on other microorganisms to provide electron donors and are typically
outcompeted for those electron donors in many other freshwater systems by more
efficient metabolic processes such as nitrate, iron, and sulfate reduction. Because the
archaea are dependent on other microbial groups for electron donors, the funda-
mental microbial ecology of methane production is not only dependent on numerous
environmental factors that affect the rates of methane production but is also depen-
dent on a variety of fundamental interactions, including those between different
functional groups at the cellular level, the nutrient limitations that control those
interactions, and the pathways through which methane is produced. The Everglades
provides a particularly good system for studying the relationships between nutrient
availability, alternative terminal electron acceptors such as sulfate, and the ecology
of methanogenic archaea.

6.1.2 The Everglades

The modern Everglades is comprised of 8000 km2 (not including the upper Ever-
glades that includes the Kissimmee River) of marshes and swamps that cover much
of the southern tip of the state of Florida. The Everglades provides an array of
ecological services, including habitat for numerous endangered species and migra-
tory birds, and recreation and water for the growing human populations of South
Florida. Approximately 3000 km2 south of Lake Okeechobee were drained in 1948
to form the muck farms of the Everglades Agricultural Area, which are responsible
for much of the domestic sugarcane crop. The Everglades was historically very low
in available phosphorus due to the limestone substrata, which also maintained a
relatively high pH (between 7 and 8). Fertilizers containing phosphate were applied
to the land, as was elemental sulfur (S) to modulate the pH. Elemental S is converted
to sulfate through the actions of sulfur oxidizing bacteria, which lowers the
6 The Ecology of Methanogenic Archaea in a Nutrient-Impacted Wetland 159

Fig. 6.1 Map showing location of the Everglades in the state of Florida, with Water Conservation
Area 2-A identified and total soil P concentrations

pH. Canals drain the land to maintain crop production, which in turn carried P and
sulfate from runoff into the Everglades through the Water Conservation Areas
(WCAs, see Fig. 6.1). Gradients in the concentrations of P in water and soil were
thereby established, with soil P concentrations ranging from 1.28 g per kg in the
northern part of Water Conservation Area 2A (WCA-2A) to approximately 0.25 g
per kg further south in WCA-2A (Bae et al. 2015).
The release of elevated concentrations of P into the northern Everglades effec-
tively fertilized the originally very low P soils, leading to a shift in the dominant
vegetation from sparse stands of sawgrass (Cladium sp.) to dense cattails (Typha
sp.). Greater primary productivity accelerated peat development adjacent to the
canals. Microbial activities are elevated in the P-impacted soils and include signif-
icantly higher production of methane and CO2 (Bae et al. 2015), higher rates of
nitrogen fixation (Inglett et al. 2011; Bae et al. 2018), and increased activities of
many extracellular enzymes that are associated with microbial carbon and nitrogen
acquisition than typically would be observed in soils that are not impacted by P
(Penton and Newman 2007). The microbial communities of the P-impacted peat
predictably exhibit characteristics of P-excess and mildly N-limited systems,
whereas microbial communities in soils further south in the less P-impacted exhibit
characteristics of highly P-limited systems (Morrison et al. 2016). A shallow gradi-
ent in surface water sulfate concentrations is also present in the WCAs, with
concentrations ranging from 56 mM observed in the P-impacted regions of
WCA-2A, down to less than 4 mM in regions of WCA-3A (Bae et al. 2015).
The amounts of P and sulfate entering the Everglades have been significantly
reduced in recent years; however, there is sufficient P and sulfate stored in the peat to
maintain significant internal cycling of these nutrients, such that these regions
remain impacted by the elevated nutrients. Both P and sulfate are being gradually
160 A. Ogram et al.

redistributed, however, with P and sulfate moving down stream toward the south of
the system.

6.1.3 Decomposition in Methanogenic Peatlands

There are notable aspects of the Florida Everglades that impact methane production
and the ecology of methanogens; however, decomposition of plant material in
methane-producing peatlands generally follows a common trajectory. The energy
that is typically available to anaerobic metabolism is significantly less than the
energy available to aerobic metabolism processes, such that more complex organic
molecules are metabolized via a series of coupled metabolisms. Decomposition of
organic carbon is slow in anoxic peat systems due to a number of factors, including
lack of terminal electron acceptors and the “peatland enzymic latch” (Freeman et al.
2001). The enzymic latch refers to the apparent importance of phenol oxidase, an
enzyme that requires O2, to decompose phenolics that would be inhibitory to
extracellular hydrolases required for increasing the availability of N and P, which
are necessary for organic matter decomposition. Peat therefore accumulates, with
most of the available carbon coming from decomposition products from the aerobic
zone above and the slow decomposition of polymers at greater depths. The general
flow of carbon and electrons in a methanogenic peat accumulating wetland such as
the Everglades is presented in Fig. 6.2, in which moribund and dead plant material as
standing biomass is colonized by saprophytic fungi. Decomposition then proceeds
through utilization of easily metabolized compounds and depolymerization of larger
molecules such as starches, cellulose, and lignin. The primary genus responsible for
depolymerization of cellulose to glucose and fermentation of those products in
anoxic regions of the Everglades appear to belong to the Firmicutes and are
represented by diverse members of the genus Clostridium (Uz et al. 2006). The
dominant clades within Clostridium shift along the gradient, likely due to changes in
carbon quality (Uz and Ogram 2006) as the dominant emergent vegetation shifts
from cattail in the nutrient-impacted area to sawgrass in the nonimpacted area.
Fermentation of carbohydrates typically results in production of a range of
products, including H2 and small organic acids such as propionate, butyrate, acetate,
and lactate. In the Everglades, as in many other wetlands, propionate is a dominant
fermentation product (Bae et al. 2015). Propionate and butyrate contain significant
energy, such that their fermentation is sufficient to provide energy for another group
of microorganisms that function as secondary fermenters. These secondary fer-
menters are frequently referred to as syntrophs due to the requirement that their meta-
bolisms be closely coupled with another group, such as methanogens, to successfully
scavenge either H2 or formate via interspecies electron transfer. The energy required to
regenerate oxidized dinucleotides such as FADH requires that H2 be maintained in very
low concentrations (Schink 1997), such that H2 must be removed quickly from the
immediate vicinity of the syntroph. The great majority of syntrophs described to date
are also capable of gaining energy via sulfate reduction, such that metabolism of
6 The Ecology of Methanogenic Archaea in a Nutrient-Impacted Wetland 161

WATER (AEROBIC)
Plant Detritus Monomers and Oligomers
Methanotrophs
ANAEROBIC Cellulolytic & Fermentative Bacteria

H2 and CO2 Short chain fatty acids

(propionate, butyrate, etc);
alcohols.
Homoacetogens
Syntrophic Bacteria

Acetate Acetate H2 and CO2 Acetate

Sulfate Reducing Bacteria Methanogens

S- and CO2 CH4

Fig. 6.2 The general pathway for decomposition of organic matter to methane in terrestrial
wetlands

fermentation products by syntrophic interactions may be bypassed when sufficient

sulfate is available. In addition to H2, acetate and formate are common secondary
fermentation products. In a typical methanogenic system, methanogenic archaea use H2
either to reduce available CO2 to methane or ferment acetate to methane in order to gain
energy (Conrad 1999). While it is not known at this time how much of the methane
produced is dependent on syntrophy, it is clear that both the physiologies of syntrophs
and their methanogenic partners are intimately linked and that the relationships may be
complicated by the presence of sulfate.
The physiologies and ecologies of methanogenic archaea in the context of the
Everglades will be discussed in the sections below, with potential implications of the
interactions between syntrophs and methanogens with respect to mercury cycling
included.
162 A. Ogram et al.

6.2 Methanogenic Archaea, Sulfate-Reducing Prokaryotes,

and Syntrophy

6.2.1 The Methanogens in the Everglades

Along with CO2, methane is the terminal product of carbon mineralization in many
anoxic systems. Methane is exclusively produced by members of the Euryarchaeota,
phylum of the Archaea. Methanogenic archaea are characterized by a strict anaerobic
metabolism and of being limited to a narrow range of potential electron donors. At
the time of this writing, methanogenic archaea are divided among seven orders, each
of which are phylogenetically distinct and most of which can be defined by their
range of electron donors and optimum temperatures (Borrel et al. 2013).
In general, the major physiologies of methanogens fall into two broad groups
based on their electron donors: the hydrogenotrophs, or those that can use H2 as an
electron donor to reduce CO2 with the production of methane, and the acetotrophs,
which use an acetoclastic pathway to ferment acetate to CH4. The energy available
per mole of methane produced by hydrogenotrophs is typically greater than that
available to acetotrophs (Nüsslein et al. 2001). The dominant methanogenic taxa in
the Everglades vary with the nutrient status (Castro et al. 2004, 2005; Bae et al.
2015). In the nutrient-impacted soils, the hydrogenotrophic Methanomicrobiales
were consistently more numerous than the acetotrophic family Methanosaetaceae
during 3 years of monitoring (Bae et al. 2015). Conversely, members of the
Methanosaetaceae outnumbered the Methanomicrobiales during the same period.
The redundancy analysis (RDA) presented in Fig. 6.2 indicates that numbers of
Methanomicrobiales are inversely correlated with Methanosaetaceae and less
strongly correlated with sulfate and total phosphorus concentrations. The differences
in dominant methanogenic order and metabolism (hydrogenotrophic vs acetoclastic)
suggest that the dominant pathway for methane production may also shift along
the nutrient gradient, with a greater proportion of methane produced through the
hydrogenotrophic pathway in the nutrient-impacted soils and less in the
nonimpacted soils (Bae et al. 2015). This is supported by δ13CH4 analysis of
methane emitted from soils along the gradient, which indicated a shift from approx-
imately 50% hydrogenotrophic in the nutrient-impacted soils to approximately 23%
hydrogenotrophic in the nonimpacted soils (Holmes et al. 2014).
On a global basis, most terrestrial methane is thought to arise from acetate via
acetoclastic methanogenesis. Stoichiometric conversion of carbohydrates to acetate
and H2 via primary fermentation (Fig. 6.2; fermentation of sugars to fermentation of
fatty acids and alcohols) and secondary fermentation (a fermentation of primary
fermentation products, such as fatty acids and alcohols) can only supply sufficient H2
for reducing CO2 to CH4 to account for 1/3 of the total methane produced (Conrad
1999). Thus, it has generally been observed that acetate fermentation accounts for
approximately 70% of the methane produced in terrestrial environments (Conrad
1999). A few notable exceptions to this paradigm have been described (Conrad
1999; Nüsslein et al. 2003), many of which favor the hydrogenotrophic pathway. As
6 The Ecology of Methanogenic Archaea in a Nutrient-Impacted Wetland 163

discussed above, the dominant pathway in the Everglades differs between the
nutrient-impacted and nutrient-nonimpacted soils. As will be discussed below, the
observed shift in methanogenic pathway is due to competition between methanogens
and sulfate-reducing prokaryotes (SRPs) in the nutrient-impacted area.

6.2.2 Interactions Between Methanogens and SRP Along

the Nutrient Gradient

The SRPs are distributed across a broad phylogenetic range of prokaryotes that spans
domains and they exhibit both diverse metabolisms and diverse ecological roles
(Castro et al. 2000). Sulfate concentrations at our study sites vary along the nutrient
gradient, with sulfate concentrations consistently greater than 39 μM being observed
in the nutrient-impacted soils and less than 4 μM in the nonimpacted soils (Bae et al.
2015). In a preliminary study utilizing terminal restriction fragment length poly-
morphism (TRFLP) analysis of the gene encoding dissimilatory bisulfite reductase
(dsrB), a gene that is frequently used a phylogenetic marker of SRP, Castro et al.
(2005) estimated that the dominant SRPs in WCA-2A soils are associated with the
genus Desulfotomaculum. Members of this genus are capable of forming endo-
spores, a trait that would be beneficial for strict anaerobes inhabiting soils with
fluctuating redox conditions (Castro et al. 2000, 2005). The water levels in WCA-2A
drop below soil surface each spring, and the surface soils shift from anoxic to oxic.
Usage of TRFLP analysis has methodological limitations (Prakash et al. 2014) in
its ability to resolve closely related phylogenetic groups. However, Castro et al.
(2005) noted that dominant dsrB TRFs consistently differed between the nutrient-
impacted and nutrient-nonimpacted soils in monthly samplings conducted over the
course of 1 year. The nature of SRP metabolism can be roughly divided into two
types based on the ability to metabolize acetate as an electron donor and source of
carbon, complete versus incomplete lactate oxidizers (Castro et al. 2000). Complete
oxidizers are capable of metabolizing lactate to CO2, while incomplete oxidizers
metabolize lactate to acetate. Incomplete oxidizers are not capable of utilizing
acetate as an electron donor. The genus Desulfotomaculum includes representatives
of both complete and incomplete oxidizers. Significantly, Castro et al. (2005)
reported that the majority TRFs associated with Desulfotomaculum sp. in nutrient-
impacted soils are characterized by a predicted complete lactate oxidation meta-
bolism, while predicted incomplete lactate oxidation dominated the Desulfoto-
maculum TRFs in the lower sulfate, nonimpacted soils. This suggested that SRP
may compete with methanogens for acetate in the impacted soils and to a lesser
degree in the nonimpacted soils. Potential competition between methanogens and
SRP for acetate and the implications of this competition are inferred from the
redundancy analysis (RDA) plot presented in Fig. 6.3, where the proportions of
hydrogenotrophic Methanomicrobiales (MM) in WCA-2A soils are inversely
164 A. Ogram et al.

Fig. 6.3 Redundancy analysis (RDA) representing the correlation among mcrA and dsrB copies
with geochemical parameters obtained from along the nutrient gradient. Arrows pointing in the
same direction indicate positive correlations and arrows pointing in opposite directions indicate
negative correlations. Arrow length corresponds to variance explained by the environmental
variable. The first two axes explain 88.3% of the total canonical eigenvalues, with a significant
Monte-Carlo test value (P < 0.05). From Bae et al. (2015)

correlated with the acetotrophic Methanosaetacae (MST) and weakly correlated with
the proportions of SRP (represented by dsrB) and sulfate concentrations.
To assess whether competition for acetate between SRP and methanogens may
control the dominant methanogenic pathways in the nutrient-impacted and nutrient-
nonimpacted soils, laboratory incubations of soil from the impacted area with the
sulfate reduction inhibitor MoO42 indicated a shift in the δ13CH4 toward the
acetoclastic pathway and an increase in the mRNA transcribed from the acetotrophic
family Methanosaetaceae (Bae et al. 2015). These results indicate that competition
with SRP for acetate shifted the major pathway away from the acetoclastic pathway
and toward the hydrogenotrophic pathway in the higher sulfate, nutrient-impacted
soils.
In addition to competition, many SRP may cooperate with methanogens as
syntrophs. Some SRP may shift from anaerobic respiration using sulfate as a
terminal electron acceptor to a fermentative metabolism. Bae et al. (2014) found
that the majority of dsrB transcripts clustered within the Deltaproteobacteria and
were associated with SRP capable of fermentative metabolism and of syntrophy with
methanogens. Depending on environmental factors such as carbon to sulfate ratios,
SRP may either compete with or cooperate with methanogens.
6 The Ecology of Methanogenic Archaea in a Nutrient-Impacted Wetland 165

6.2.3 Syntrophs in the Everglades

The term “syntrophy” has been used since the mid-twentieth century to describe a
mutually beneficial relationship in anoxic systems, frequently (although not exclu-
sively) between secondary fermentative bacteria (syntrophs) and their methanogenic
archaeal partners (methanogens). The syntrophy allows the metabolic conversion of
volatile fatty acids (VFAs) such as butyrate, propionate, and acetate into methane
(Schink 1997). Thermodynamically, such conversions are not feasible because ΔG
for the reaction is endergonic, but microorganisms facilitate such endergonic
reactions by engaging in interspecies electron transfer (IET) (Schink 1997) to
regenerate oxidized dinucleotides. The syntrophic members typically cluster with
Deltaproteobacteria (e.g., Syntrophus, Desulfovibrio) or Firmicutes (e.g.,
Syntrophomonas, Desulfotomaculum) (McInerney et al. 2008). Notably, the
deltaproteobacterial syntrophs are typically obligate anaerobes that ferment VFAs
when grown in co-culture with a partner; however, if sulfate is bioavailable, many
deltaproteobacterial syntrophs (e.g., Desulfovibrionales and Desulfuromonadales)
may utilize VFAs. Representatives of syntrophic Clostridia and Bacilli may be
either strict or facultative anaerobes; many are spore-formers and include typical
syntrophic genera such as Syntrophomonas and Pelotomaculum (Morris et al. 2013).
The prokaryotic groups that act as sinks to consume the by-products of syntrophic
metabolism include the hydrogenotrophic methanogenic Euryarchaeota.
The compositions and architectures of the syntroph-methanogen partnerships are
characteristic of the nutrient status of the site. Significant differences (P-
values < 0.001) were observed by our research group in the phylogenies of
syntrophs and archaea between microcosms with soil from nutrient-impacted, tran-
sitional, and nutrient-nonimpacted sites. Microcosms with soils from the different
respective nutrient regions were spiked with different VFAs and then observed with
regard to methane production, microscopic visualizations of the resultant syntrophic-
methanogenic consortia, most probable number estimations of microbial population
density, and 13C-DNA-based stable isotope probing (SIP) to investigate the path-
ways of VFA metabolism and the dominant syntrophs. Most propionate-utilizing
syntrophs in microcosms with soil from the nutrient-impacted and transition sites
clustered with Pelotomaculum spp. and Syntrophobacter spp. and for butyrate, either
Deltaproteobacteria or Firmicutes dominated, with Syntrophospora spp. and
Syntrophomonas spp. (Chauhan and Ogram 2006). In the nonimpacted soils,
Pelospora spp. and SRP dominated. Also noted in the nutrient-impacted and tran-
sition soils were 16S rRNA gene sequences clustering with the genus Smithella,
members of which may convert propionate to butyrate. The mechanism used by
Smithella propionica is to dismutate propionate to acetate and butyrate, followed by
β-oxidation (de Bok et al. 2001; Liu et al. 1999). In addition to the typical syntrophic
guilds, SRPs also constitute a significant proportion of the total relative phylotype
abundance within the impacted soils. This situation was quite different in the
nonimpacted microcosms, where SIP did not identify many known syntrophic
166 A. Ogram et al.

Syntrophs Methanospirillum

Methanosaeta

Fig. 6.4 Photomicrograph of syntroph-methanogen consortium isolated from enrichments of soil

taken from the nutrient-impacted regions of WCA-2A. From Chauhan et al. (2004)

bacteria; however, 16S rRNA gene sequences clustered with various SRP and
Pelobacter spp.
In lab-controlled microcosms spiked with VFAs, we observed an interesting
feature: the presence of “tripartite” rather than a “bipartite” microbial syntrophic
association which included a syntroph, hydrogenotrophic methanogen, and an
acetotrophic methanogen (Fig. 6.4). The nest-like structures characteristic of these
tripartite associations were only observed in enrichments of soil from the nutrient-
impacted and transition soils and not in the nonimpacted soils (Chauhan et al. 2004).
Similar associations have been reported in the literature and were primarily associ-
ated with enrichments in which VFAs were more rapidly consumed by tricultures of
Methanospirillum hungatei (hydrogenotroph), Methanothrix soehngenii (acetate
utilizer), and MPOB (mesophilic propionate-oxidizing bacteria) relative to
bicultures consisting of Methanospirillum with MPOB (Voolapalli and Stuckey
1999), suggesting that the low acetate and H2 concentrations were favorable for
syntrophy in many systems. It is very likely that the dramatic increase in the methane
production pathways and rates observed in the impacted Florida Everglades soils
(Castro et al. 2004, 2005; Chauhan et al. 2004; Ogram et al., unpublished) occur
because the microbiota in these systems have adapted and evolved into ecologically
beneficial “tripartite” associations in nutrient-impacted soils. As noted above, esti-
mations for methanogen numbers via quantitative PCR (qPCR) enumerations
revealed that hydrogenotrophic methanogen populations numbers were significantly
greater than those of acetotrophs, especially in the P-impacted soils (Chauhan et al.
6 The Ecology of Methanogenic Archaea in a Nutrient-Impacted Wetland 167

2004; Castro et al. 2004, 2005; Bae et al. 2015), adding support to the conclusion
that syntrophy may be a major route for methane production in the Everglades.
Sulfate concentrations fluctuate significantly in the Everglades (Bae et al. 2015),
with porewater concentrations ranging from 39 to 56 μM determined in three
samplings taken over 3 years. It is well-documented that SRP may outcompete
methanogenic archaea for H2 and acetate (Lovley et al. 1982; Schönheit et al.
1982; Robinson and Tiedje 1984), provided that sufficient sulfate is available. Little
is currently known of the potential impact of fluctuating sulfate concentrations on
syntrophy. As the level of bioavailable sulfate increases within a given habitat, we
must ask “Does the syntrophic-methanogenic relationship breaks apart such that the
syntrophs switch to sulfate reduction?” Most known syntrophs are capable of
gaining energy via sulfate reduction metabolism, and our study has found that
Deltaproteobacteria associated with the genus Syntrophobacter dominated in
microcosms from the nutrient-impacted and transition soils and Deltaproteobacteria
have been shown to metabolically perform dissimilatory sulfate reduction to produce
energy. Evidence that members of this genus prefer to release the resultant reducing
equivalents toward their syntrophic methanogenic partners or other SRPs has been
reported (Wallrabenstein et al. 1995; Harmsen et al. 1998). Currently, it is unknown
in what way fluctuating sulfate concentrations may impact the structures and func-
tions of syntrophic consortia in the Everglades.

6.3 Associated Activities

6.3.1 Methanogenic Nitrogen Fixation

Elemental cycles are typically coupled, and the intersection points of cycles of
environmental interest may interrelate within the metabolisms of individual
populations. In addition to their importance in the production of methane,
methanogenic archaea are also important contributors to the cycling of nitrogen in
Everglades peat (Bae et al. 2018). The ability of some methanogens to fix N2 has
been recognized for over 30 years (Murray and Zinder 1984; Belay et al. 1984);
however, the importance and potential for methanogenic nitrogen fixation to occur in
wetlands has been understudied.
During peatland development, relatively young systems are typically limited
in N, while N is put into the system by N-fixation, atmospheric deposition, and
other sources (Anderson 1964). With time, a shift in nutrient limitation is frequently
observed from N to P, such that older peatlands are typically limited by available
P. Nitrogen limitation can also be imposed on peatlands through anthropogenic input
of P, as has been observed in WCA-2A. In WCA-2A, nutrient runoff shifted the
primary nutrient limiting primary productivity away from P and back to N (Corstanje
et al. 2007). As expected, N-fixation rates are highest in the high P regions of
WCA-2A peat and lower down the N-P limitation gradient (Inglett et al. 2011).
168 A. Ogram et al.

The highest rates of N-fixation in the Everglades are likely due to the activities of
aerobic bacteria such as cyanobacteria in the water column and periphyton. The
nitrogen fixed by these organisms is probably cycled within the periphyton and
flocculent layer above the soil and within the top few centimeters of the peat, with
little available nitrogen moving deeper into the peat where light is low, redox
potentials drop dramatically, and anaerobic metabolisms such as methanogenesis
dominate (White and Reddy 2003). Much of the available nitrogen at these depths is
provided from the slow decomposition of organic matter and N-fixation by anaer-
obes (Inglett et al. 2011), with methanogens being among the dominant N-fixers at
the Everglades peat (Bae et al. 2018). Sequence analysis of nifH, the gene encoding
dinitrogenase reductase (critical to the nitrogen fixation process), showed that
between 6 and 44% of the nifH copies at the 0–4 cm depth belonged to methanogens.
It should be noted that these samples included surficial peat, such that many of the
nifH copies originated from cyanobacteria or other aerobes that would not be present
below the area where light could penetrate. Estimates of total copy numbers of nifH
per gram of peat ranged from 5.5  108 to 1.9  1010 copies per gram of dry soil.
The relative activities of methanogenic N-fixers in WCA-2A are inferred by the
proportion of mRNA transcribed from methanogen nifH; up to 49% of the total nifH
mRNA from these samples originated from methanogens, indicating that a signifi-
cant amount of the N fixed in WCA-2A peat is due to the action of methanogenic
Archaea. It is not known at this time how the nutrients that limit primary production
in WCA-2A influence the rates of methanogenic nitrogen fixation in peat, and more
work is required to fully understand the impacts of methanogenic N-fixation on
methane production and peat decomposition along the nutrient gradient.

6.3.2 Mercury Methylation by Methanogens

Many wetlands, including the Everglades, are subject to atmospheric deposition of

inorganic mercury, primarily in the form of Hg2+. Once in anoxic environments, Hg2+
may be methylated to more toxic CH3Hg+, or methylmercury, by diverse anaerobic
prokaryotes. Methylmercury is of particular concern because of its tendency to
bioaccumulate through the food chain, potentially resulting in neurological damage
and developmental problems. Fish in some areas of the Everglades are banned from
human consumption due to methylmercury concentrations, and methylmercury has
also been implicated in changes in the mating habits of wading birds that have led to
declines in their numbers (Frederick and Jayasena 2011).
The SRPs were initially identified as being responsible for the majority of
mercury methylation in the Everglades, with significant differences in methylation
rates observed along the sulfate and P gradients in the WCAs (Gilmour et al. 1998).
The known diversity of prokaryotic groups capable of mercury methylation was
greatly expanded with the identification of the genes responsible for methylation,
hgcAB (Parks et al. 2013; Gilmour et al. 2013). These genes encode a corrinoid
protein that is involved in transferring methyl carbanions from one substrate to
6 The Ecology of Methanogenic Archaea in a Nutrient-Impacted Wetland 169

another. It is likely that mercury methylation is a gratuitous reaction that occurs

during cellular metabolism and is not specifically directed toward mercury. All
organisms known to carry hgcAB have been shown to methylate mercury (Gilmour
et al. 2013), such that hgcAB may be used as a genetic marker for screening samples
for the presence and phylogeny of mercury methylators. This work has led to
identification of a great diversity of microbial groups representing different meta-
bolic strategies as mercury methylators and include fermenters, syntrophs, and
methanogens, as well as SRB.
Bae et al. (2014) reported that the dominant groups harboring hgcAB in the soils
of the WCAs were affiliated with the deltaproteobacterial syntrophs (which may also
function as sulfate reducers when in the presence of sufficient sulfate), Firmicutes,
and methanogens. The relative proportions of methanogens ranged from 8% in the
nutrient and sulfate-enriched soils of the northern WCA-2A to 27% in the
low-sulfate soils of WCA-3A and include both acetotrophs (Methanosarcinaceae)
and hydrogenotrophs (Methanomicrobiales). Subsequent investigations have found
that the relative proportions of methanogenic hgcAB to other groups increase down
the nutrient and sulfate gradients and may form a very significant proportion of
mercury methylators in periphyton and floc in low-sulfate environments (Bae et al.,
manuscript in preparation). The rates of methanogenic methylation compared to
methylation by sulfate reduction and other processes in the Everglades are not
known at this time. However, methanogens were shown to be the dominant meth-
ylators in periphyton at a site in Canada (Hamelin et al. 2011), and mercury
methylation by hgcAB-carrying syntrophic-methanogenic consortia may be signifi-
cant. It is likely that the quality and availability of carbon, in addition to the range of
available electron acceptors available to competitors of methanogens, will control
the dominant processes involved in mercury methylation in this complex Everglades
ecosystem.

6.4 Summary

Much of the microbial ecology, and hence biogeochemical cycling, in the Water
Conservation Areas (WCAs) of the Everglades is impacted by the input of nutrients
from the adjacent Everglades Agricultural Area (EAA). The Everglades was histor-
ically a very low-nutrient ecosystem, and input of nutrients (notably P) resulting
from runoff from the adjacent EAA for many years significantly impacted a range of
ecosystem processes in the Everglades. Notably, primary productivity increased
dramatically, resulting in higher amounts of organic carbon put into the soils. In
addition, sulfate runoff from the EAA resulted in a shallow gradient in sulfate
concentrations and stimulated carbon mineralization through sulfate reduction in
those areas. This in turn impacted the fundamental ecology of methanogens, which
compete with sulfate-reducing prokaryotes (SRPs) for electron donors. Notably,
SRP and methanogens compete for acetate in the nutrient and sulfate impacted
soils of the northern WCA-2A, which increases the proportion of hydrotrophic
170 A. Ogram et al.

methanogenesis relative to the typically more dominant acetoclastic pathway (Bae

et al. 2015; Holmes et al. 2014).
Much of the carbon in the Everglades, as in most peatlands, is likely to be
processed through methanogenic consortia involving secondary fermenters, or
syntrophs, and hydrogenotrophic methanogens. The architecture of the syntroph-
methanogen consortia involves three members: the syntroph, a hydrogenotrophic
methanogen, and an acetotrophic methanogen. These consortia differ from those
found in the nonimpacted soils, which include only the syntroph and a hydro-
genotrophic methanogen.
In addition to methane production, methanogens are responsible for other signifi-
cant processes, including nitrogen fixation and mercury methylation. The phylogeny
of the dominant nitrogen-fixing methanogens in the WCAs falls primarily within the
hydrogenotrophs, suggesting that the distribution of hydrogenotrophs and compe-
tition for electron donors along the nutrient gradient may impact methanogenic
nitrogen fixation rates. The potential relationship between N-limitation of general
microbial activities (Corstanje et al. 2007) and the rates at which methanogens fix N
is unknown at this time, as is the potential importance of methanogenic N-fixation to
the general microbial community. Methanogens may also play an important role in
the methylation of mercury in the Everglades, and the relative proportions of
methanogenic mercury methylators increases with decreasing sulfate inputs, which
would promote mercury methylation by SRP.
In summary, the distributions and activities of methanogens are impacted by
nutrient additions in the Everglades, much of which can be explained by the impacts
that the availabilities of electron donors have on dominant metabolic groups of
methanogens.

Compliance with Ethical Standards

Conflict of Interest Andrew Ogram declares that he has no conflicts of interest. Hee-Sung Bae
declares that he/she has no conflicts of interest. Ashvini Chauhan declares that he/she has no
conflicts of interest.
Ethical Approval This article does not contain any studies with human participants or animals
performed by any of the authors.

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Chapter 7
Briefly Summarizing Our Understanding
of Vibrio cholerae and the Disease Cholera

Christon J. Hurst

Abstract Vibrio cholerae is a naturally existing aquatic bacteria that lives in

association with the chitinous exoskeletons of crustaceans including copepods.
Cholera is an infectious disease of humans which is caused by ingesting those strains
of the bacteria Vibrio cholerae that carry both of two disease related factors, a toxin
gene coded by the bacteriophage CTXΦ which produces the cholera toxin, and the
toxin-coregulated pilus which both facilitates attachment of the bacteria to host cells
and also serves as the CTXΦ receptor. Cholera is considered a waterborne infection,
with the primary route of infection being ingestion of fecally contaminated water and
secondary transmission being caused by ingesting fecally contaminated food. Devel-
opment of mathematical modeling frameworks may help to provide an essential lead
time for strengthening intervention efforts to either prevent or ameliorate outbreaks
of cholera in regions where the disease is endemic.

7.1 Introduction

The Vibrionaceae are a family of heterotrophic bacteria found in oceanic environ-

ments. A few members of the species Vibrio have extended their range to occur in
brackish and freshwater environments (Takemura et al. 2014). The growth and
concentrations of Vibrio naturally found in coastal waters increases with warmer
water temperatures, often leading to a seasonal distribution of Vibrio infections in
temperate regions with most of those infections occurring from summer through
early autumn (Sinatra and Colby 2018).
Vibrio cholerae naturally exists in association with the chitinous exoskeletons of
crustaceans including copepods, and not surprisingly Vibrio cholerae produces
chitinases (Nalin 1976). Indeed, it is presumed that all Vibrio species produce

C. J. Hurst (*)
1814 Woodpine Lane, Cincinnati, OH 45255, USA
Universidad del Valle, Cali, Colombia

© Springer Nature Switzerland AG 2019 173

C. J. Hurst (ed.), The Structure and Function of Aquatic Microbial Communities,
Advances in Environmental Microbiology 7,
https://doi.org/10.1007/978-3-030-16775-2_7
174 C. J. Hurst

chitinases (de Magny et al. 2011) which degrade the crustaceans insoluble exoskel-
eton chitin polymer into soluble chitin oligosaccharides (Hayes et al. 2017). The
chitinases of Vibrio cholerae allow that bacterial species to use chitin as a sole
carbon and nitrogen source (Mondal et al. 2014) and, correspondingly, Vibrio
cholerae bacteria that are colonized onto the chitinous surface of copepods are
able to utilize the copepods chitin as a sole carbon and nitrogen source (Mondal
et al. 2014). One of the two known Vibrio cholerae chitinase enzymes, ChiA2, also
is secreted by Vibrio cholerae within the mammalian intestine and that enzyme
hydrolyzes intestinal mucin to release N-Acetylglucosamine. The released sugar
then is utilized by Vibrio cholerae for growth within the host intestine (Mondal et al.
2014).
Cholera is an infectious disease of humans which is caused by ingesting only
those strains of the bacteria Vibrio cholerae that carry both of two disease related
factors. The pathogenicity factors for Vibrio cholerae are the cholera toxin which is
encoded by a lysogenic bacteriophage CTXΦ, and a toxin coregulated pilus which is
encoded by a pathogenicity island (Faruque and Mekalanos 2012) and also serves as
the CTXΦ receptor (Krebs and Taylor 2011).
Cholera primarily is considered to be a waterborne disease with secondary
transmission routes that include fecally contaminated food. Vaccination is one of
our newest tools in fighting cholera. However, preventing deaths due to cholera will
require that we both reduce the risk of exposure to pathogenic strains of the bacteria
and that we successfully treat those individuals who contract the illness. Community
understanding, along with awareness and intervention, will allow us to resolve this
health problem. The goal is that John Snows wish (Snow 1849, 1854) of eliminating
the health risk asssociated with cholera finally will be realized.

7.2 Ecology of the Vibrios that Cause Infections in Humans

Understanding a pathogenic microbes ecology is a key factor in comprehending its

associated disease.

7.2.1 The Four Horsemen of Vibrio Disease

Human health concerns regarding the genus Vibrio encompass four environmental
species and those are Vibrio anguillarum, Vibrio cholerae, Vibrio parahaemolyticus,
and Vibrio vulnificus.
Vibrio anguillarum usually is considered to be a pathogen of fish, but it has a
wide host range that also naturally encompasses bivalve molluscs plus crustaceans
and extends to include larvae of the wax moth Galleria mellonella (McMillan et al.
2015). Vibrio anguillarum causes economic losses in the fishing and aquaculture
industries, and has produced bacteremia in humans as a consequence of people
consuming either fish or crustacean seafood which contained the bacteria (Sinatra
and Colby 2018).
7 Briefly Summarizing Our Understanding of Vibrio cholerae and the Disease Cholera 175

Both Vibrio parahaemolyticus (Letchumanan et al. 2014) and Vibrio vulnificus

are found in estuaries and coastal waters, where these bacterial species naturally are
concentrated into the tissues of filter feeding bivalve molluscs and cause an initially
gastrointestinal disease when the contaminated shellfish are consumed either raw or
undercooked (Raszl et al. 2016). The resulting human gastrointestinal infections
caused by Vibrio parahaemolyticus and Vibrio vulnificus (Park and Lee 2018) can
spread to the bloodstream producing bacteremia. Vibrio vulnificus additionally
causes disease via wound infections (Raszl et al. 2016).
An examination of archived formalin-preserved plankton samples that included
noting the presence of Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio
vulnificus, supplemented by using generalized additive models, revealed that long-
term increase in Vibrio abundance from 1958 to 2011 in the North Atlantic was
promoted by increasing sea surface temperatures and positively correlated with the
Northern Hemisphere Temperature (NHT) and Atlantic Multidecadal Oscillation
(AMO) climatic indices (Vezzulli et al. 2016).

7.2.2 Focusing on the Horseman Named “Pestilence” by

Understanding the Ecology of Vibrio cholerae

The natural habitat for Vibrio cholerae is the chitinous shells of crustaceans (Grimes
1991) including copepods. Attachment of Vibrio cholerae to the surface of live
copepods has been suggested as being important for ecological persistence of Vibrio
cholerae in natural water (Huq et al. 1983) and that attachment beneficially affects
multiplication of Vibrio cholerae (Huq et al. 1984). The beneficial effect of Vibrio
cholerae’s association with copepods has been determined to be greatest at an
alkaline pH of 8.5 with lesser pH levels of 6.5 and 7.5 also having been examined,
at 30  C with lower water temperatures of 5 through 25  C having been examined,
and with maximum attachment to copepods noted at 15 g/kg salinity as compared
against salinities of 5 and 10 g/kg (Huq et al. 1984). It should be remembered that
full salinity sea water is considered to be around 35 g/kg. Vibrio cholerae is present
throughout the year both in the gut and on the surface of its zooplankton copepod
hosts and in addition this bacteria has an association with some species of phyto-
plankton at least in Bangladeshi waters (de Magny et al. 2011). There additionally
have been findings of Vibrio cholerae associated with Acanthamoeba castellanii,
and of Vibrio cholerae attached to the mucilaginous sheath of the cyanobacteria
Anabaena (Almagro-Moreno and Taylor 2013). Vibrio cholerae, along with cope-
pods which are its presumed primary hosts, has been shown autochthonous to
riverine, estuarine, and coastal waters. Copepods have been found as part of the
microflora in drinking water distribution systems of even economically well devel-
oped countries (van Lieverloo et al. 2012) and this knowledge suggests that
microcrustaceans may represent a potentially supportive environment for Vibrio
contaminants that gain entrance to water distribution systems. Association of Vibrio
176 C. J. Hurst

organisms with the chitinous shells of micro crustaceans such as copepods, and
macrocrustaceans such as crabs, may facilitate survival of the microbes during
passage through the stomach, and that potential increase in gastric survival could
be an important factor in waterborne disease if the Vibrio cholerae in source waters
were attached to microcrustacean copepods when these microcrustaceans inadver-
tently get ingested along with the water (Nalin 1976; Nalin et al. 1978).

7.3 Characteristics of the Disease Cholera

Cholera is an acute diarrheal disease caused by toxin-producing strains of the

bacterial species Vibrio cholerae (Lippi and Gotuzzo 2014). More than 200 serotypes
of Vibrio cholerae have been identified, with several of those serotypes known to be
capable of causing mild to serious gastroenteritis including local outbreak situations
of diarrheal illnesses with “cholera-like” symptoms. However, it is the toxigenic
strains of Vibrio cholerae serogroups O1 and O139 that have been identified with
cholera epidemics (de Magny et al. 2011).
Cholera disease is noted for its severe watery diarrhea that can lead to dehydration
and, if untreated, the disease can result in death. Azman et al. (2013) have provided a
good general summary of the incubation period and symptomatology for cholera. It
takes between 12 h and 5 days for a person to show symptoms after ingesting either
contaminated water or food, and the disease then can kill within hours if left
untreated. Susceptibility to infection by Vibrio cholerae seems to be determined
by a combination of immunologic, nutritional, and genetic characteristics (Harris
et al. 2008). Presumably most of those people who are infected with Vibrio cholerae
will display no symptoms and yet those people still may have the bacteria present in
their feces and thus potentially represent a source of infection for other people. Most
of the people who do demonstrate symptoms have either mild or moderate illness
that can be treated with oral rehydration solution (World Health Organization 2006).
A minority of cholera cases will develop acute watery diarrhoea with severe dehy-
dration that can lead to death if left untreated.
Cholera is considered to be a waterborne disease, principally acquired by
ingesting fecally contaminated water, with there also being fecally associated sec-
ondary transmission (Snow 1849, 1854). It is fortunate that improved personal
hygiene standards and improved drinking water quality largely have resulted in the
disappearance of cholera from developed countries. Unfortunately, the menace of
this disease does continue in many regions of the world due to poor sanitation
accompanied by lack of important infrastructure and the complicating factor that
flooding results in fecal contamination of water supples (Almagro-Moreno and
Taylor 2013). The remaining endemnicity of cholera is in regions of the world
where inadequate sanitary practices commonly are associated with consumption of
contaminated water and food. It has been estimated that approximately 1.3 billion
people are at risk for cholera in endemic countries, with perhaps 2.86 million cholera
7 Briefly Summarizing Our Understanding of Vibrio cholerae and the Disease Cholera 177

cases (uncertainty range: 1.3 m–4.0 m) and a possible 95,000 deaths occurring
annually (Ali et al. 2015).

7.4 The Association of Cholera Incidence and Climatic

Conditions

The incidence rate of cholera disease has been found to correlate both negatively and
positively with rainfall patterns. In some studies is has seemed that dry weather
periods may result in people using riskier sources of drinking water. Oppositely,
surface runoff might overwhelm drinking water treatment systems and flood wells
during wet weather seasons. In the study by Camacho et al. (2018) there was a
positive correlation between the spring rainy season in Yemen and cholera
incidence.
Rainfall, in addition to temperature and salinity, has proven to be an important
factor in the ecology of Vibrio cholerae (de Magnya et al. 2008). Rainfall and daily
hours of sunlight, along with water temperature, water depth, and conductivity, are
characteristics that generally affect plankton populations. Phytoplankton blooms
naturally are followed by blooms of zooplankton, including copepods, and that has
accounted for the observation of an approximate 8 week lag time between the point
when a phytoplankton bloom peaks and when cholera cases subsequently appear
(Huq et al. 2005).
The results of a study by Fu et al. (2012) have suggested that a predictive model
based upon growth curve functions which combined environmental temperature and
organic nutrient levels might help to reliably predict Vibrio cholerae in the aquatic
environment. Ecological analysis research by Jutla et al. (2013) has shown that the
use of satellite based radiance observations of the difference between blue (412 nm)
and green (555 nm) wavelengths helps to assess the timing and presence of organic
matter in coastal waters which in turn can be related to likely incidence of seasonal
cholera. Development of such modeling frameworks may help to provide an essen-
tial lead time for strengthening intervention efforts to either prevent or ameliorate
outbreaks of cholera in regions where the disease is endemic.

7.5 The Role of Crustaceans in Some Other Diseases

Crustaceans are not associated with the transmission of only Vibrio cholerae.
Several species of crustaceans including freshwater copepods have long been rec-
ognized as intermediate hosts of helminths (Leiper 1936). Particularly notable
among those is the guinea worm Dracunculus medinensis whose larvae develop
within a copepod’s digestive tract before being transmitted to humans, and that
transmission occurs when humans ingest freshwater copepods infested with
178 C. J. Hurst

Dracunculus medinensis resulting in the disease dracunculiasis also called Guinea

worm disease (Centers for Disease Control and Prevention 2012). Marine copepods
and other aquatic crustaceans similarly also serve as primary hosts for many hel-
minth species, and the consumption of those infested crustaceans by fish then
produces a resultant infestation of the fish (Zander et al. 1994). Fish lice seem
paticularly notable as intermediate crustacean hosts for nematodes (Moravec et al.
1999). Additionally, parasitic crustaceans including copepods serve as both hosts as
well as vectors for viruses, and those vector relationships may play a role in
transmitting the pathogenic agents to other economically valuable crustaceans
among which are the penaeid shrimp (Overstreet et al. 2009).

7.6 Additional Transmission Routes for Cholera

In addition to Vibrio cholerae causing massive outbreaks of disease associated with

the consumption of contaminated water, food also has served as a vehicle for
transmission of cholera (Rabbani and Greenough 1999). Vibrio cholerae is partic-
ularly known to cause illness associated with the consumption of contaminated
macrocrustaceans such as crabs (Finelli et al. 1992), and indeed that could be
expected as a possible natural contamination of food given that the native habitat
of this bacterial organism is the chitinous shells of crustaceans (Grimes 1991). The
possibility that association of Vibrio cholerae with chitin may provide the bacteria
with a natural means of protection from stomach acids (Nalin et al. 1978) indirectly
may facilitate the spread of this diease. The natural contamination that occurs with
crustaceans is not avoidable but can be ameliorated by adequately cooking food to
kill the bacteria before consuming the food.
As with the disease typhoid (Hurst 2018) which is caused by Salmonella enterica
subsp. enterica serovar Typhi, previously named Salmonella typhi, it should be
presumed that for cholera ingestion of water contaminated with feces and sewage
serves as the primary, or initial, route of transmission (Snow 1849). Careless fecal
contamination of food likely is a secondary transmission route (Hurst 2018; Snow
1849). The transmission of cholera by food, aside from the instances of natural
contamination as mentioned above for macrocrustaceans such as crab, is an outcome
that easily could be avoided by adequate sanitation. Unfortunately, people often just
simply do not wash their hands even when adequate means are available for doing
that washing. The fact that cholera transmission can be associated with a failure to
wash hands and the consequent contamination of food, has been understood at last
since the time when it was explained by John Snow nearly two centuries ago (Snow
1849). Social behaviours relating to the sharing of food clearly may increase the risk
of gastrointestinal disease transmission. As an example of this, Camacho et al.
(2018) found that an increased risk of cholera transmission in Yemen seemingly
had been associated with events related to celebration of Ramadan, a month of the
Islamic calendar when there are large gatherings for meals in which people share
food. That connection of social activity with cholera transmission occured following
7 Briefly Summarizing Our Understanding of Vibrio cholerae and the Disease Cholera 179

spring rains which naturally would have increased the chance for Vibrio cholerae
contamination of water as a primary transmission route and associatively enhanced
the risks of food associated cholera as a secondary transmission route.
There also has been a suggestion that synanthropic flies could serve as mechan-
ical transmission vectors for cholera equally as they likey also transmit all other
infections that are spread by the fecal-oral route. Vectoring of disease by flies was
suspected as contributing to the transmission of the viral disease polio (Cirillo 2016)
and has been hypothesized for both bacteria (Graczyk et al. 2001; Junqueira et al.
2017) as well as protozoa (Graczyk et al. 2001).
Once an outbreak of cholera has occurred, Vibrio cholerae strains can be traced
geographically to understand the development of cholera disease outbreaks (Kiiru
et al. 2013). Efforts also have been made by Chao et al. (2014) and Nishiura et al.
(2017) to utilize disease transmission models for understanding the dynamics of
cholera outbreaks. A basic explanation of compartment modeling and risk estimation
for primary waterborne disease transmission accompanied by secondary routes of
disease transmission can be found with examples in Hurst (2018).

7.7 Concurrent Infections Can Worsen Gastrointestinal

Bacterial Disease

There always is the concern that malnutrition and also concurrent infections caused
by other pathogenic organisms, such as those which produce malaria and measles
and are known to increase the risk of severe outcome from other gastrointestinal
infections, similarly could worsen the severity of cholera. My suggestion of concern
relating to malaria is due to the fact that while non-typhoidal Salmonella serotypes
(NTS) often are associated with gastroenteritis in immunocompetent individuals,
individuals with severe pediatric malaria can develop bacteremic infections with
NTS during which symptoms of gastroenteritis are commonly absent (Mooney et al.
2014). My concerns about malnutrition and measles were summarized in 2018
(Hurst 2018). The probability of human death from gastrointestinal llness caused
by Cryptosporidium parvum in the general population is 0.0002 (100 deaths per
403,000 cases of illness) (Hurst 2018). Numbers from a publication by Crawford and
Vermund (1988) indicate that the probability of cryptosporidial illness leading to
death can be 0.14 (14%, or 1 in 7, or 2/14) if there is underlying malnutrition. The
risk of death from Cryptosporidium can be 0.20 in the case of underlying measles
(20%, or 1 in 5) (Crawford and Vermund 1988) which represents a 2000-fold
increase over the rate of death from Cryptosporidium infections in the general
population. By itself, illness caused by the measles virus Morbillivirus measles
morbillivirus is almost never fatal but it may be one of the most immunosuppressive
viruses that infect humans.
180 C. J. Hurst

7.8 Prevention of Cholera Including Vaccination

and “WASH”

Vaccination and community awareness ultimately will help us to defeat the pesti-
lence known as cholera.

7.8.1 Immunity to Cholera and Vaccination Against

the Disease

Vibriocidal antibody is an immunologic marker associated with protection from

Vibrio cholerae. It has been found that knowledge of the levels of serum IgA to three
specific antigens: the B subunit of cholera toxin, lipopolysaccharide, and the major
subunit of the toxin-coregulated pilus TcpA that induces mucosal and systemic
immunoglobulin A immune responses in patients with cholera caused by Vibrio
cholerae types O1 and O139, can be used to predict protection in household contacts
of patients infected with Vibrio cholerae O1 (Harris et al. 2008). Two types of killed-
cell vaccines currently are available for helping to prevent cholera (World Health
Organization 2017).

7.8.2 International Efforts to Control Cholera

The Global Task Force on Cholera Control (2017) has a goal of reducing cholera
deaths by 90% and expresses the hope that as many as 20 of the 47 countries
currently affected by cholera could completely eliminate cholera disease transmis-
sion by 2030. The task force strategy consists of interventions that include oral
cholera vaccines plus a group of water related activities to which they have assigned
the acronym “WASH”. That acronym is said to represent several points, although the
exact number and wording of those points seems to vary.
Firstly, “Basic water supply” is a concept that means having access to safe
drinking water, and could be represented by a community supplied potable water
distribution network which pipes safe drinking water to each household. Among the
alternative possibilities that are considered reasonable for providing an adequate
water supply are having access within a 30-minute round-trip to either a public
standpipe, borehole, protected dug well, protected spring, or rainwater collection
system. If the available water is not potable, meaning safely drinkable, then there
should be a provision for either household or community disinfection of the water.
That concept of basic water supply helps with preventing both primary transmission
and also secondary transmission of waterborne infections. I have addressed else-
where in this book the subject of providing microbiologically safe drinking water for
7 Briefly Summarizing Our Understanding of Vibrio cholerae and the Disease Cholera 181

populations ranging from municipalities to individual households (Chap. 9,

“Microbiome of Drinking Water Distribution Systems” pp. 261–311).
Secondly, “Basic sanitation”, meaning access to improved sanitation facilities as
represented by either having households connected to a public sewer, or a septic
system, or a pour-flush latrine, or a pit latrine, will help to prevent secondary
transmission.
Thirdly, “Basic hygiene”, meaning that every household has access to a hand-
washing station with soap and water. Appropriate care when preparing food, includ-
ing safely either peeling or washing ingredients, cooking things thoroughly, and then
protecting the prepared food against inadvertent fecal contamination, will help to
avoid secondary transmission. It also is helpful to establish effective community
engagement programs to promote safe hygiene.
One of the important aspects of public health programs is to carefully manage the
necessary monetary and physical resources that are required for achieving success in
disease elimination. There also is a necessity for creating adequate governmental
health agencies that will be assigned and comply with the task of monitoring the
quality of community water supplies including piped water distribution networks.
Disease surveillance and reporting, including the important step of confirming
Vibrio cholerae infections at the peripheral level for suspected cholera cases, and
conducting a monitoring program for identifying outbreaks, represent expensive but
helpful efforts that require access to laboratory culture capacity and rapid diagnostic
tests. Having an ability to test for antibiotic susceptibility of the causative bacteria
during oubreaks, and the ability to geographically track the bacterial strains associ-
ated with outbreaks, also are helpful (Camacho et al. 2018; Kiiru et al. 2013) because
accurate surveillance data can advance efforts towards achieving the goal of prior-
itizing preparedness.
The types of advance preparations which can be made for confronting cholera
outbreaks include pre-positioning supplies of oral rehydration salt (ORS) solution
for performing oral rehydration therapy, which is the administration of fluid by
mouth to prevent or correct the dehydration caused by the diarrhoea (World Health
Organization 2006), plus having available intravenous fluids for rehydration when
instances of diarrhea are so severe that oral rehydration would not be sufficiently
effective for saving life. Hypochlorite disinfectant solution should be available for
sanitation to prevent iatrogenic infections inadvertently associated with medical
examinations during the treatment of cholera patients. The health care system also
can be benefited by advance training of health workers to improve patient care and
benefited by education about reducing iatrogenic infections. Having dedicated health
care facilities which might be either Cholera Treatment Centers or Cholera Treat-
ment Units could further help to reduce the likelihood of nosocomial cholera
infections (Global Task Force on Cholera Control 2017).

Compliance with Ethical Standards

Conflict of Interest Christon J. Hurst declares that he has no conflict of interest.

Ethical Approval This article does not contain any studies with human participants or animals.
182 C. J. Hurst

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BF02366204
Chapter 8
Options for Providing Microbiologically
Safe Drinking Water

Christon J. Hurst

Abstract This chapter describes how to provide microbiologically safe drinking

water for different population groups, ranging from large municipalities to small
communities and households. Information is presented about potential sources of
treatable water including surface water, springs and seeps, groundwater, rainwater
and fog. Storage of rainwater harvested by ground catchments and roof catchments
also is explained. The appropriate processes for treating water are sedimentation,
coagulation, flocculation, filtration and disinfection. Those processes often are
achieved differently depending upon the population size that is being served. For
example, disinfecting water by boiling works well to centrally treat water at the
household level but trying to centrally boil enough water to supply a population of
one million people would be unmanageable. I also have included information about
safely distributing drinking water including the use of municipal plumbing networks,
tanker trucks, prepackaged bottled water, water vending machines, and water dis-
pensers. As a final topic, I explain how water is collected and recycled aboard the
International Space Station along with images which show the developmental testing
of that technology.

8.1 Introduction

Having safe drinking water available in our homes, accessible by simply opening a
faucet, is something which many of us have the good fortune of being able to take for
granted. Sadly, there are a great many people in the world who can only dream of
having such luxury. This chapter examines the approaches which may allow us to
reach the goal of successfully providing microbiologically safe drinking water for all
households and communities.

C. J. Hurst (*)
1814 Woodpine Lane, Cincinnati, OH 45255, USA
Universidad del Valle, Cali, Colombia

© Springer Nature Switzerland AG 2019 185

C. J. Hurst (ed.), The Structure and Function of Aquatic Microbial Communities,
Advances in Environmental Microbiology 7,
https://doi.org/10.1007/978-3-030-16775-2_8
186 C. J. Hurst

Perhaps the best starting point for discussing any aspect of aquatic microbiology
is a reminder of two basic truths about water and microbes. First of those would be
acknowledging that water and microorganisms naturally co-occur. Second of those
would be recognition that by its nature aquatic microbial life is persistent even when
we might wish it not to be. When we speak of either finding or creating microbio-
logically safe drinking water, we almost cannot intend that no microbes would be
present. Sometimes sustaining health does indeed require being able to medically
administer microbe free solutions, and we are able to do that for relatively small
quantities of liquid. Our practical goal with drinking water is to remove those
microorganisms that are pathogenic. Unfortunately even that goal becomes difficult
when we think about not only creating microbially safe drinking water but then also
needing to distribute the water without degradation of its quality.
Sometimes, what we consider to be microbial contaminants in water are environ-
mental microorganisms that will naturally be present in even the most pristine of
water sources, and many of those microorganisms are of health concern for us. More
often, however, the aquatic microbial burden with which we need to be concerned
originates from humans and animals that naturally reside either in or near to the
water. We acquire some waterborne infections by ingestion of contaminated water
and most often those infections are gastrointestinal. Many other water associated
infections are acquired from body surface contact with contaminated water, and that
category includes partially as well as fully immersive activities such as bathing.
Bathing activities typically result in infections of the skin, eyes, ears, and nose.
Bathing activities can result in gastrointestinal infections because water often gets
swallowed either accidently or intentionally when we bathe. Inhalation of aerosol-
ized water tends to cause respiratory illnesses, with those being a potential result of
shower bathing and also by encountering infectious aerosols arising from waterfalls,
vaporizers, cooling towers, and air conditioning units.
The infections which humans acquire from water recently have been summarized
elsewhere in this series (Hurst 2018). Table 8.1 lists a few of those diseases along
with their respective causal microorganisms. Many of the waterborne pathogens
found in source water, such as Legionella, Naegleria, and Vibrio vulnificus, pre-
sumably are naturally present environmental organisms and the diseases which they
cause in humans are unnecessary to the ecology which sustains the natural existence
of those microbes. Some of the other microbes on that list, such as Cryptosporidium
parvum and Giardia intestinalis, seem to exist naturally as pathogens of other
animals and while infection of humans by those microbes may be a part of the
microbes natural existence those infections of humans would be coincidental and not
a core part of the microbes existence. A few among the viruses, such as the human
enteroviruses, have an ecology that centers upon their infectiousness for humans. All
of these microbes successfully must be either removed from the source water or
rendered harmless in order for consumption of the water to become free from risk.
The most recently available estimates from the World Health Organization
(2018b) about availability of usable drinking water for human populations are that:
In 2015, 71% of the global population (5.2 billion people) used a safely managed drinking-
water service—that is, one located on premises, available when needed, and free from
contamination.
8 Options for Providing Microbiologically Safe Drinking Water 187

Table 8.1 Examples of infectious diseases associated with waterborne pathogens

Pathogen Type of
Exposure route group disease Causative microorganism(s)a
Ingestion (includes contami-
nated drinking water and other
beverages plus ice and water-
associated contamination of
foods)
Bacterial
Enteric fever Salmonella (especially Salmo-
nella enterica subsp. enterica
serovar Typhi, which causes
typhoid fever)
Enteritis Campylobacter, Shigella
(causes bacterial dysentery),
Vibrio (especially V. cholerae,
which causes cholera)
Febrile Francisella tularensis
syndrome
Septicemia Vibrio vulnificus
Protozoan
Enteritis Cryptosporidium parvum, Ent-
amoeba histolytica (causes
amebic dysentery), Giardia
intestinalis (includes the for-
merly named Giardia lamblia)
Viral
Encephalitits Enterovirus
Gastroenteritis Alphacoronavirus,
Mamastrovirus, Norovirus,
Rotavirus, Vesivirus
Hepatitis Vesivirus, Hepatovirus
Meningitis Enterovirus
Body Surface Contact (usually
associated with either personal
bathing, recreational, aquatic
activities or occupational
activities)
Bacterial
Enteritis Vibrio cholerae
Nephritis Leptospira interrogans
Wound Vibrio parahemolyticus, Vibrio
infections vulnificus
Metazoan
Worm Schistosoma
infestation
Protozoan
Encephalitis Naegleria
(continued)
188 C. J. Hurst

Table 8.1 (continued)

Pathogen Type of
Exposure route group disease Causative microorganism(s)a
Enteritis Entamoeba histolytica
Viral
Encephalitis Enterovirus
Gastroenteritis Alphacoronavirus,
Mamastrovirus, Norovirus,
Rotavirus, Vesivirus
Meningitis Enterovirus
Pharyngocon- Mastadenovirus
junctival fever
Inhalation
Bacterial
Pneumonic Legionella pneumophila
fever
a
If an organism is indicated by both its genus and species names, then the disease association is with
that particular species. If only a genus name is given, then the disease association is with more than
one species belonging to that genus

89% of the global population (6.5 billion people) used at least a basic service. A basic
service is an improved drinking-water source within a round trip of 30 min to collect water.
844 million people lack even a basic drinking-water service, including 159 million
people who are dependent on surface water.
By 2025, half of the world’s population will be living in water-stressed areas.
In low- and middle-income countries, 38% of health care facilities lack an improved
water source, 19% do not have improved sanitation, and 35% lack water and soap for
handwashing.

Those estimates about safely managed water services could allow us to feel
overly optimistic about how we are progressing towards the goal of reducing
waterborne human health risks, when in fact the product water which those services
provide very often contains microorganisms that would cause disease if the water
were consumed (Daud et al. 2017; Ghaderpoori et al. 2009; Hashmi et al. 2012;
Hurst 2018; Payment et al. 1991, 1997).
Community distributed water usually comes from sources of surface freshwater
where that is available. Otherwise, potentially drinkable water may be obtained from
either groundwater sources via wells, collection of rainwater where it is available in
sufficient quantity, or by desalinating brackish and marine water. Sometimes com-
munities are able to supply drinking water but not deliver it to individual households.
If it is not possible to install a pressurized water piping network for distributing the
water to individual households, then a good choice for managing the situation can be
a pressurized distribution network that makes the water available to community
standpipes. If a piping network is entirely not available then developing community
cisterns with multiple taps may be the answer for safely achieving storage and
community distribution of the treated water. Either way, if the community is served
by community standpipes or cisterns, then people in the community will need to
8 Options for Providing Microbiologically Safe Drinking Water 189

bring their own water carrying containers to those standpipes and cisterns, and the
people then must transport their drinking water home with them.
There are times and places where water successfully is delivered to households
through community plumbing systems but the quality of that distributed water is
inadequate, such that further household treatment of the delivered water is necessary
for microbial safety. All too frequently, the task of finding potentially drinkable
source water and then treating that water to make it safe for human consumption is
left entirely to the individual consumers, an aspect that is covered below in Sect. 8.5
of this chapter. Both of those scenarios result in our needing to rely upon drinking
water treatment techniques that have been developed for household usage. There
also are times when the only available source for generating potable water is directly
recycled wastewater, and that need will be particularly obvious in the section of this
chapter which addresses water management on the International Space Station.
One of the best documents that I have found on the subject of developing
available water sources is a publication on rural water supply which seems to have
been designed as the text for an engineering course (United States Agency for
International Development et al. 1982). It gives guidance for developing safe
water supplies, along with notes for providing drinking water treatment to small
communities and households. It also presents a very thorough compilation of
technical notes helpfully containing basic planning, design and construction infor-
mation. The surface water sources considered for utilization are ponds, lakes,
reservoirs, streams, and rivers. It also mentions using spring boxes for collecting
water from springs, and using seepage collection systems to obtain water from seeps,
both of which represent groundwater sources that either occur very near to the
surface or emerge onto the land surface. Information additionally is provided for
constructing different types of vertical and horizontal wells to obtain ground water
that is not emerging onto the nearby land surface. Rainfall harvesting by either roof
catchment or ground catchment also is included. That publication then explains how
to treat the obtained water for household and small community usage. Means for
accomplishing that water treatment include small community sedimentation basins,
slow sand filters, small community disinfection units represented by chlorinators,
boilers, household sand filters and other household treatment techniques, in addition
to household water storage. That document now is a rather old publication, but much
of the newer information available on the internet draws from that 1982 publication
and reproduces its illustrations.
The need for using treatment technology for producing microbiologically safe
drinking water is generally understood and accepted by the people who live in
communities that range in population size from large to moderately small. Part of
the challenge in trying to provide microbiologically safe drinking water to very small
communities and individual households in poorer and rural areas relates to such
factors as the people sometimes having received limited previous education regard-
ing the risks associated with unsafe water, overall community economic factors
including their possibly limited ability to pay for drinking water treatment and
perhaps a question of their willingness to pay, their level of water demand and
usages, the availability of chemicals and spare parts for sustaining treatment
190 C. J. Hurst

technology, plus raw water characteristics such as availability, source quantity and
source quality. All of those factors will together determine what level of treatment
technology can be accepted and used (Parr and Shaw 2019).

8.2 The Three Technical Tasks Set Before Us

There are three tasks to be accomplished. The first is to find usable water. The second
is treating the water as necessary to make certain that it will be safe to drink. The
third is, if possible, to help by safely delivering the treated water to those are in need
of it.

8.2.1 Finding a Suitable Water Source

Collection of source water often is done as a municipal function and delivering that
water to its users may also be a municipal function. Private companies both large and
small, often limited to being one-person businesses, do also play a part in obtaining
and delivering water. There are several options for finding suitable source water,
whether that be surface water, ground water, rain or fog. The most technologically
challenging choice for a water source would be either brackish or saline water that
needs to be desalinated prior to its consumption. The information that I am
presenting in this chapter presumes desalination is not necessary, although that
technology is indeed used to create safe drinking at the both the level of communities
and households. My suggestions for reference information to understand desalina-
tion and the microbial hazards associated with inadequately maintained desalination
process equipment are the publications by Nagaraj et al. (2017), World Health
Organization (2011b), and Yari et al. (2018).

8.2.1.1 Surface Water

The first of our tasks is to find a source which can provide a sufficient quantity of
water. Surface supplies of freshwater containing minimal sewage pollution are the
best options but can be hard to find in areas of high population and dry climates.
Figure 8.1 shows three of my students holding sampling bottles by the shoreline of
the Ohio River, at the Public Landing for Cincinnati, Ohio. The Ohio provides plenty
of raw, albeit turbid and sewage polluted, source water both for Cincinnati and for
numerous other towns and cities between that rivers official beginning in Pittsburgh,
Pennsylvania, and the point in Cairo, Illinois, where the Ohio River joins with the
Mississippi River.
8 Options for Providing Microbiologically Safe Drinking Water 191

Fig. 8.1 This image shows the Ohio River near the public landing in Cincinnati, Ohio. Slightly east
of that location is the water supply inlet for Cincinnati, Ohio. Use of this image is courtesy of the
author

8.2.1.2 Groundwater

The second best option would be a hopefully uncontaminated and non-saline

groundwater. Figure 8.2 shows two places near Cincinnati where groundwater
emerges and flows onto the surface. The top of that figure shows icicles where
water emerges between limestone layers at a roadcut. The bottom of that figure
shows a creek where the water is clear but always served as our positive control for
viruses, because during periods of low flow most of the water carried by that creek
comes from household septic tanks. Figure 8.3 shows an artesian well in Colombia,
South America, and also two bucket wells in southwestern Ohio. Hand operated
pumps serve for providing well water to very small communities and individual
households when the wells used are shallow and only low flow rates are required.
Deeper wells and wells intended to provide high rates of water flow will need
reliable power sources for motorized pumping. Figure 8.3 additionally shows three
pump wells in Southwest Ohio that provide different capacities. All of the wells in
Fig. 8.3 have their sides protected against contamination from surface flow and five
of the wells also have their top openings protectively covered The well head shown
in the bottom right image of Fig. 8.3 serves the city of Hamilton, Ohio and that well
head further is enclosed by a steel shed with a locking door. Figure 8.4 shows a
protected pump well which is used to fill an elevated storage tank, providing flowing
water for a nursing home facility near Cali, Colombia, that pump is enclosed and
protected by a locked gate. Figure 8.5 shows an unprotected shallow well in the
192 C. J. Hurst

Fig. 8.2 This image shows groundwater returning to the land surface. The upper image is of icicles
that resemble a frozen waterfall showing where groundwater has emerged between layers of
limestone at a roadcut in Highland Heights, Northern Kentucky. The lower image shows Clough
Creek in Hamilton County, Ohio slightly east of Cincinnati, Ohio. The water in the creek is
aesthetically pleasing due to its very low turbidity, but the water has a high virus level because it
receives leachate from buried septic tanks. Use of these images is courtesy of the author
8 Options for Providing Microbiologically Safe Drinking Water 193

Fig. 8.3 This figure shows wells that provide water for groups with different levels of need and the
wells show different levels of sanitary protection. Upper left is an artesian flow well in the Cauca
River valley region of Colombia. Center left is a covered bucket well under a roof shelter near
Oxford, Ohio, USA. Lower left is a capped bucket well near Cincinnati, Ohio, USA. Upper right is a
sealed well with a hand powered pump near Cincinnati, Ohio, USA. Center right is a sealed well
operated by the drive shaft of a farming tractor in Hamilton county, Ohio, USA. Lower right is a
sealed well within an enclosure in Butler County, Ohio, USA, and this view shows that water in the
well head was being sampled for possible viral contaminants. Use of these images is courtesy of the
author

seasonally dry bed of an African river. Figure 8.6 shows a spring box which collects
groundwater that emerges on a hillside near Cincinnati and, as with the creek in
Fig. 8.2, water being collected into and distributed from that spring box largely
194 C. J. Hurst

Fig. 8.4 This image shows

the water supply system for
a nursing home near Cali,
Colombia. The water source
is a protected well. The well
casement extends above the
ground surface and the
opening to that casing is
covered with a concrete lid.
A large stone provides
additional weight on top of
the concrete lid. A pipe
leads horizontally above
ground from the well casing
to a pump that is located in
the bottom of the tower.
That pump is protectively
housed behind a locked
gate. The water is stored in a
covered reservoir tank on
top of the tower. Use of this
image is courtesy of the
author

originates from septic tanks. Figure 8.7 shows a spring that has been intercepted by
means of a horizontal well in Ukraine. Figure 8.8 shows the collection of a water
from a seep in the United States. The publication by Meuli and Wehrle (2001)
discusses different types of springs, legal rights, water quality, water quantity, design
and construction of gravity fed catchments, plus the design and construction of
artesian catchments. The publication by United States Agency for International
Development et al. (1982) describes different types of wells plus catchment systems
for springs and seeps.

8.2.1.3 Rainwater

We often can capture rainwater when it is available and then store that water for use.
Figures 8.9 and 8.10 show ground collection systems. Water collected by the
drainage system of ITESM Campus, Ciudad de México, shown in Fig. 8.9 does
8 Options for Providing Microbiologically Safe Drinking Water 195

Fig. 8.5 This figure shows collection of turbid seepage which is being used as the source of
drinking water for Mwamanongu Village in Tanzania. This water has seeped into an open pit that is
called a seep well. It this instance, the well was dug in the bed of a dry river. The image is titled
“Mwamongu water source” by Bob Metcalf and is in the public domain. Seep wells also can be dug
a slight distance away from bodies of surface water so that water subsequently collected from the
seep well will have been filtered to some extent by passage through the ground, and that process is
similar to the engineered collection and processing of water by riverbank filtration

not seem to be purposefully used, and descriptive text published with that photo-
graph suggests the circular structure into which the water drains may be the modified
entryway of a sinkhole. Figure 8.10 shows a cistern located in Rajasthan, India,
which stores rainwater collected by ground catchment. The ground water collection
system for the cistern in Fig. 8.10 seems to show no protection against entrance of
contaminants except for a concrete labyrinthine entry weir which allows some
provision for settling. The water in that cistern serves as a source of drinking
water and is accessed by using a bucket that would be lowered through the cisterns
top opening. You will notice that the top opening is covered and has of been elevated
with protective sides to help preclude contamination of the cistern opening by any
water that may collect on the cisterns lid.
Figure 8.11 shows two rooftop rainwater collection systems and their storage
containers. Environmental contaminants including animal feces can get washed into
the rainwater storage containers and cisterns. The water storage tanks that are used
with rooftop collection systems can be protected against entry of contaminants by
any of several methods. One option is to wait until perhaps 5–10 min after a rainfall
event has begun before manually diverting flow of rooftop collected water into the
storage container. There is an option of using automatic ‘first flush’ diverters, which
196 C. J. Hurst

Fig. 8.6 These images show a spring water cistern, sometimes called a “Spring box”, which
intercepts and stores for distribution the water that emerges from a hillside spring near Cincinnati,
Ohio. Use of these images is courtesy of the author
8 Options for Providing Microbiologically Safe Drinking Water 197

Fig. 8.7 This image shows water from a spring which is being conducted by means of a horizontal
well pipe into a collection pool. This spring is the water source for the village Dzyhivka in Ukraine.
The image is titled “Siphot” by USchick and being used under a Creative Commons Attribution-
share alike unported license. 3.0 Generic license

attach to the collection downspouts and assure that the initial water collected, which
hopefully carries the greatest amount of contaminants such as animal feces, will not
enter the storage tank. There also are filtration units that can be mounted at the top of
the storage tanks and those filters will remove much of the contaminants that are
washed down from the roof. The publication by United States Agency for Interna-
tional Development et al. (1982) describes catchment systems for rainwater. Some
additional suggested references for rainwater catchment would be: Despins et al.
(2009), de Kwaadsteniet et al. (2013), Karim et al. (2005), Manitoba Conservation
and Water Stewardship (2014), PennState Extension (2016) which is particularly
well detailed, Rahman et al. (2014), Skinner and Shaw (2019a).

8.2.1.4 Fog

There even are some places where we similarly can capture the water contained in
dense fog (Dodson and Bargach 2015) as shown in Fig. 8.12.
198 C. J. Hurst

Fig. 8.8 This image shows collection of water from a seep. The water was collected using a
perforated polyvinyl chloride drainage pipe. Usage of the image is courtesy of Shawn and Beth
Dougherty https://onecowrevolution.wordpress.com/2018/05/13/spring-water-catching-a-seep/.
Accessed 10 Jan 2019

8.2.2 Understanding the Basic Processing Steps for Treating

Water

The second task is to assure that the water we do find and will then provide is safe for
human use, including its being drinkable. Very often the found water needs to be
treated before it safely can be consumed. Our concerns about the safety of drinking
water include not only the microbes which cause disease, but also turbidity, toxic
metals, and toxic chemicals (World Health Organization 2017). This chapter deals
with the first two of those, microbes and turbidity. The recent lead crisis in Flint,
Michigan, of the United States has made it very obvious that hazardous contami-
nants other than microbes can even originate from the materials with which we
construct water distribution systems (Ruckart et al. 2019; Zahran et al. 2017). Hurst
(2001) offers a good general summary of turbidity removal and water disinfection.
The publication by Parr and Shaw (2019) presents some discussion regarding the
process used for plain sedimentation which is simple gravitational sedimentation
done without adding either coagulants or flocculants, and also mentions roughing
filters, slow sand filters, rapid sand filters, aeration, coagulation, disinfection.
8 Options for Providing Microbiologically Safe Drinking Water 199

Fig. 8.9 This figure shows the receiving basin for a rainwater ground catchment system. The lower
image is a magnification of the upper image, and better shows the water inlet pipe. This image is
titled “03242012Taller sostenibilidad lore037” by Talento Tec and used under the Creative
Commons Attribution 3.0 Generic license. The text described this as being a cenote, which suggests
that it may be an adapted sink hole
200 C. J. Hurst

Fig. 8.10 This cistern is located in Rajasthan, India, and it is a traditional tank used for storing
rainwater collected by ground catchment. The image is titled “Rainwater harvesting tank, India” by
Spiritualfade and is in the public domain

8.2.2.1 The Processes of Sedimentation, Coagulation and Flocculation

It often is necessary to treat surface water, and sometimes to treat groundwater, in

ways that will aesthetically improve the waters acceptability. We can improve both
the visual appearance and taste of the water by reducing its turbidity. Some microbes
will be associated with the particles that contribute to turbidity of the water, and thus
removing those particles can by itself help to reduce the waters microbial burden.
Many of the things that cause turbidity can complicate the task of disinfection, and
so lowering turbidity for the sake of facilitating disinfection provides yet another
reason for turbidity reduction.
The simplest approach for turbidity reduction is plain sedimentation and an easy
procedure for performing that on a household basis is called the three pot technique.
The three pot technique consists of allowing water to rest stationary in containers so
that the suspended solids can settle to the bottom by gravity. Clearer water subse-
quently can be collected from the containers either by decanting, which is pouring
off liquid in a way that will not carry over the sedimented solids, or by collecting the
settled water using a siphon. This settling technique is performing by placing source
water into a storage container and allowing the water to sit there for one day. The
water then is carefully transferred from that first container into a second storage
8 Options for Providing Microbiologically Safe Drinking Water 201

Fig. 8.11 These images

show receiving containers
for rainwater roof catchment
systems. The upper image is
a rain barrel of which the lid
contains a hole for the
downspout pipe, courtesy of
the author. The lower image
titled “Rainwater harvesting
tank (5981896147)” is by
SuSanA Secretariat (The
Sustainable Sanitation
Alliance) and used under the
Creative Commons
Attribution 2.0 Generic
license

container where the water will be allowed to settle for a second day. On the third day,
the settled water is carefully collected from the second container into a third
container and then can be disinfected for use.
We can speed up the process of sedimentation by assisted it with coagulation and
flocculation. It also is important to note that while some natural turbidity components
202 C. J. Hurst

Fig. 8.12 This image shows a fog harvesting net above a water collection trough. The water which
condenses on fog nets drains into the collection troughs which are mounted beneath those nets. That
collected water then drains into either a storage tank or a piping system by which the water is
distributed to a community. The image is titled “Atrapanieblas en Alto Patache” by Pontificia
Universidad Católica de Chile and used under the Creative Commons Attribution 2.0 Generic
license

will sediment easily by gravity, other components of turbidity must be coagulated

and even flocculated to facilitate their removal. Although these two terms, coagula-
tion and flocculation, often are used synonymously they technically are distinct. A
coagulating agent promotes particle collision by neutralizing the electrostatic charge
of suspended particles. Flocculation is considered to be a physical process by which
fine particulates are caused to clump together into a floc without involving the
neutralization of charge. Typically, for the purpose of drinking water treatment, a
polycationic compound is used as a coagulant to help manage the removal of
negatively charged particles such as silt, clay, and bacteria. Flocculation often can
be achieved by adding polymers that cause small destabilized particles to aggregate,
in turn facilitating separation of those particles from the water. If the floc floats to the
top of the liquid (creaming) then it can be collected by skimming. If the floc settles to
the bottom of the liquid (sedimentation) then it can be collected by decanting the
overlying liquid. The floc also can be filtered from the liquid.
Salts of aluminium and iron are the most commonly used coagulants for treatment
of drinking water. Aluminium sulfate, which is a chemical compound with the
formula Al2(SO4)3, often is used for that purpose in the form of potassium aluminum
sulfate dodecahydrate and is called alum. Ferric sulfate is a form of iron commonly
and similarly used for drinking water treatment. Plant based coagulants used in
8 Options for Providing Microbiologically Safe Drinking Water 203

drinking water treatment include that made from Moringa oleifera. Moringa works
as a coagulant due to its positively charged, water-soluble proteins.
Powdered activated carbon is added to the water being treated for two purposes,
to bind organic contaminants and because it also may help with the removal of fine
particulate material during subsequent sedimentation and filtration. This type of
activated carbon can be added directly in its powdered form, although often the
powder is added in the form of a slurry. Powdered activated carbon is used at the
municipal level and also has been tried at the household level. Drinking water
treatment plants need to add powdered activated carbon to the water prior to adding
coagulants because coagulants can coat the powdered activated carbon and that
coating will reduce the effectiveness with which the carbon removes organic
compounds.

8.2.2.2 The Process of Filtration

Traditionally, filtration was used to remove suspended particulates. We still do use it

for that purpose, but we also apply the term filtration in reference to many other
selective removal processes.
Paper filtration is based upon particle size retention and uses the same technique
that is presented as part of the school experiments done by chemistry students. In
chemistry classes, a flat round sheet of filter paper is folded into a pleated paper cone
and that paper cone placed into a funnel shaped holder. Paper filtration, when used as
a water treatment technique, relies upon a rectangular flat sheet of filter paper which
has been pleated and packaged as an inline cartridge. Figure 8.13 shows a paper filter
cartridge in its protective housing that has designed for home use. These filters must
be discarded once they have clogged, as the filter paper cannot successfully be
washed and reused.
Cloth filtration, which consists of pouring water through cloth, is based upon
particle size retention and works for removing copepods and correspondingly
reduces health risks associated with both the bacterial species Vibrio cholera that
causes cholera (Chap. 7: “Briefly Summarizing Our Understanding of Vibrio
cholerae and the Disease Cholera” by Christon J. Hurst, pp. 173–184), and the
nematode species Dracunculus medinensis that causes dracunculiasis. Cloth filtra-
tion usually is done to produce small volumes of treated water for satisfying limited
needs. Sari cloth has been recommended with success for reducing the risk of
cholera (Colwell et al. 2003; Huq et al. 2010). Polyester 100 μm mesh has been
recommended for reducing the risk of dracunculiasis. Figure 8.14 presents the idea
of cloth filtration. Cloth filters can be washed and reused. Cloth filtration also can be
used to remove precipitated floc and natural suspended solids as a part of other
treatment processes noted below.
Sand filtration is based upon particle size retention called mechanical straining,
plus physical adsorption, and it is a drinking water treatment option that we can scale
in size to accommodate the needs of any human community. Figure 8.15 shows the
basic concept of rapid sand filtration using a filter created from a plastic bucket, some
204 C. J. Hurst

Fig. 8.13 This image shows a water filtration unit designed for whole house installation, meaning
that it would be installed as part of the household water piping just inside of the point where the
water line enters the house. This version utilizes a pleated paper filter for removing particulates.
Other water filtration units can be more complex, incorporating an activated charcoal cartridge,
perhaps a reverse osmosis cartridge, and additionally use ultraviolet light. Some under the counter
water treatment units also incorporate water storage tanks. Use of this image is courtesy of the
author

pipe fittings, plus fine gravel and fine sand. The filter is created as a layered filtration
bed. Gravel is used as a bottom layer to facilitate draining water from the bottom of
the filter, the sand is on top of the gravel. Either a diffuser plate or another layer of
fine gravel needs to be on top of the sand so that addition of water onto the sand will
not result in the upper layer of sand being agitated, because agitation would cause
fine particulates trapped in the upper sand to be resuspended and the outflow water
consequently become turbid until the filtration stabilizes to again produce clear
water. Sand filters can be washed by agitation which may be achieved either from
the top or by rapid upflow of filtered water through the filtration bed. Additional
materials such as crushed garnet, anthracite, and zeolites may be added to the
filtration medium either to supplement the sand or as partial substitutes for the
sand. When performed as rapid sand filtration at a municipal level, the goal usually
is for the filter to remove fine particles that did not settle during a preliminary
coagulation and flocculation treatment of the water. Slow sand filtration treatment
plants typically do not use preliminary coagulation and flocculation treatment, but
may use gravel filtration of the water to achieve large particle removal prior to
actually sand filtering the water. Those preliminary gravel filters are known as
roughing filters. Slow sand filtration relies upon a hypogeal biological layer called
a schmutzdecke which forms in the upper layer of the sand for achieving the removal
of fine particulates including some capability for removing bacteria. Rapid sand
8 Options for Providing Microbiologically Safe Drinking Water 205

Fig. 8.14 This image helps

present the concept of
filtering water through clean
cloth to remove large natural
particulate material and
coagulated fine particulate
material. The cloth would be
folded several times and
then placed over the top of
the bottle. Safely filling the
bottle would be done by
pouring water into it through
the cloth. Use of this image
is courtesy of the author

filtration usually is performed for larger communities, and slow sand filtration
usually is performed for smaller communities.
The use of sand filtration is very effectively for treating drinking water at the
municipal level. Table 8.2 uses data from a publication by Johnson (1916) to show
how effectively the implementation of municipal sand filtration for treating drinking
water reduced the typhoid fever death rate early in the twentieth century. The
publication of Yang et al. (2018) describes limitations faced when trying to complete
the task by developing effective vaccination against typhoid.
Riverbank filtration was perhaps the precursor of municipal sand filtration plants
and will again be mentioned later in this chapter. Riverbank filtration still is being
used municipally and consists of establishing wells with pumps to collect ground-
water from a nearby surface water source. The basic idea is similar to what is shown
in Fig. 8.5.
Ceramic filters and porous pot filters are a concept that goes back to at least
colonial times in the Americas. The earliest filters seem to have been made in the
206 C. J. Hurst

Fig. 8.15 This image shows a sand filter constructed from a 5 US gallon plastic bucket using some
brass plumbing fittings, fine gravel and fine sand. The image on top shows the components used for
creating the filter. The lower left image shows the filter with naturally turbid water on top of the
sand, the glass jar on the left holds a sample of that naturally turbid water before filtration, and the
glass jar on the right shows a sample of that same water after having passed through the sand filter.
The lower right image shows a frontal view of the filter, plus the samples of naturally turbid water
and filtered water. Use of these images is courtesy of the author

form of porous volcanic stone bowls and also in the form of ceramic bowls. This
process is based upon particle size retention. Water is placed into the bowl or pot and
slowly permeates through the porous material. The filtered water is collected in a
bowl which has less permeability. Modern porous ceramic filtration pots are made
using clay that has been augmented with fine organic material such as sawdust. The
organic matter burns away and leaves voids or pores when the clay pot is fired to
create ceramic. The upstream surface of the filter, which for a filter pot is the inside
surface, will become coated with a layer of solids which slows the filtration process.
That layer can be scrubbed away. Eventually, however, the internal pores of the filter
material will clog and the ceramic filter or stone pot filter will need to be replaced.
8 Options for Providing Microbiologically Safe Drinking Water 207

Table 8.2 Relation between increase in percentage of United States city population supplied with
filtered drinking water and decrease in overall city population typhoid death rates
Percentage of city population served by water Typhoid fevera death rates for
Year filtration utilities same cities
1900 8.7 36
1901 10.8 34
1902 11.9 37
1903 13.3 38
1904 16.0 35
1905 17.4 30
1906 20.5 33
1907 23.2 32
1908 23.3 25
1909 30.1 21
1910 34.6 24
1911 37.2 20
1912 42.4 16
1913 48.0 16
a
Death rates given per 100,000 population per year
Data is presented for those United States cities having accurate registration as to cause of human
death
Source: Johnson (1916)

Ceramic filters also are made as hollow tubes called ceramic candles that are closed
at one end with the other end serving as a drain. Filtration of water using ceramic
candles is based upon water flowing from outside of the filter to the inside, and the
outside of the filter is scrubbed to remove excluded particulates.
Reverse osmosis filters use thin membranes that often are manufactured as sealed
cartridges. Reverse osmosis filters act by passing water through the filter membrane
using hydrostatic pressure while the membrane excludes passage of charged ions.
The filters are used both commercially and in homes for water desalination. It is
necessary to remove turbidity from the water by some other type of preliminary
filtration before processing the water with reverse osmosis filters. Otherwise, without
preliminary turbidity removal, the reverse osmosis membranes quickly will clog.
After having once being wetted, the reverse osmosis membrane filters are not
allowed to dry because they may develop micro cracks which will allow contami-
nants to pass through the filter membrane. There also is a hazard of biofilm growth
on the downstream side of the reverse osmosis membranes and microbes contained
in that biofilm can contaminate the product water.
Activated carbon filters use either granular or powdered carbon which has been
made by charring cellulosic materials. The carbon has been treated in a way called
activation that produces micropores which increase its surface area. Activated
carbon filtration is used for retaining organic material by adsorption. There is a
problem in that microorganisms can create a biofilm growing on the activated
carbon, and that biofilm may contaminate the filtered water. Granular activated
208 C. J. Hurst

carbon can periodically be reactivated, and large water treatment facilities success-
fully do use reactivation as a means of saving cost. Powdered activated carbon is
discarded instead of being recovered and reactivated.

8.2.2.3 The Process of Disinfection

Chlorination is the most commonly used method for disinfecting large quantities of
water. The options most relied upon include: chlorine gas, sodium hypochlorite
liquid, calcium hypochlorite powder, and sodium dichloroisocyanurate powder.
Community drinking water treatment facilities sometimes substitute chloramination
in place of chlorination to reduce the formation of carcinogenic byproducts. Chlorine
and chloramines temporarily provide at least some residual disinfectant capacity
which reduces regrowth of those microbes that survived the physical water treatment
processes, and that residual disinfectant capacity also protects the treated water
against recontamination by microbes that enter the water through plumbing mishaps.
Electrolytic generation of mixed oxidants from sodium chloride brine has been used
for disinfecting water (Bajszár and Dekonenko 2010) with the disinfecting products
created by that process largely consisting of chlorine, hypochlorite, and
hypochlorous acid.
Ozone is popular in some regions as a drinking water disinfectant and the ozone is
generated on-site. Ozonation can improve both the taste and odor of the water. Using
ozonation in place of chlorination also avoids the problem of creating hazardous
chlorinated organic byproducts.
Ultraviolet light disinfection by germicidal lamps is used at some large scale
community water treatment facilities and also serves in many home treatment units.
Successfully achieving ultraviolet light disinfection requires that the water not
contain suspended particulates which could shield the microorganisms against
germicidal exposure. A disadvantage is that water treated by ultraviolet light can
be easily recontaminated because that water retains no residual disinfection capacity.
Thermal treatment is effective and commonly used at the household level but it is
not practical for use at large scale drinking water treatment facilities. Boiling water is
the safest thermal treatment from a disease prevention perspective and it is a very
commonly used technique for home treatment of drinking water even in some areas
of the world where community treated drinking water is delivered to individual
households by a distribution network. Pasteurization is achieved by heating water to
temperatures that are below the boiling point. Distillation also is effective as a means
of disinfecting drinking water, although distillation has limited practicality even at
small population scales. The design of a solar distillation unit will be described later
in this chapter. The fact that thermal treatments provide no disinfection residual is
critically important.
Skinner and Shaw (2019b) presents a summary of information about straining
water with cloth, the three pot system, boiling, chlorine disinfection, and solar
disinfection which is pasteurization that uses sunlight as a source of heat.
8 Options for Providing Microbiologically Safe Drinking Water 209

8.2.3 Generating and Distributing Drinkable Water at

the Municipal Level

The methodology used for preparing safe drinking water at the municipal level will
vary depending upon the source and quality of the raw water, and must be appro-
priately designed to meet the needs of the community’s population size.
Water from surface sources may have high turbidity, and for that reason surface
water often is sand filtering prior to distribution. Filtration can make the water
visually more acceptable, make successful disinfection easier, and avoids sedimen-
tation in the water distribution system. The options for sand filtration are either rapid
sand filtration, which mostly removes turbidity, or slow sand filtration which has the
additional benefit of offering better bacterial removal capability. Aeration and
coagulation may be incorporated into the treatment process, and I generally think
of those two processes as being used in conjunction with rapid filtration. Slow sand
filtration may include the use of prefiltration through roughing filters which are
composed of gravel. After either rapid sand filtration or slow sand filtration, the
water should be disinfected prior to its distribution. Parr and Shaw (2019) have
presented a discussion on plain sedimentation, roughing filters, rapid sand filters,
aeration, coagulation, and disinfection. A third option is riverbank filtration which
uses pumps located some distance away from a surface water source to pull water
from that surface source through an aquifer and the pumps then discharge that water
either into a public distribution system or into a water treatment facility. Riverbank
filtration is used as an alternative to constructing a sand filtration drinking water
treatment plant.
Groundwater sources and springs produce water that may have a very low
turbidity and therefore water from those sources might not be sand filtered prior to
distribution. Low turbidity surface water similarly may not be sand filtered prior to
distribution. Hopefully, some disinfectant such as either chlorine or ozone always
will have been added to the water before that water enters a distribution system.
Community distribution networks for drinking water may include pipelines that
extend to connections for individual households, or more simply deliver water only
to communal standpipes. The treated water in municipal drinking water distribution
systems is not always microbially safe (Hashmi et al. 2012). Using Iran as an
example, Ghaderpoori et al. (2009) reported on community distribution systems in
rural areas that often provide water from springs and from deep and semi-deep wells.
It was estimated that perhaps eighty eight percent of the water was safe. However,
much of the rural population drank water that was not disinfected for reasons that
included lack of chlorine availability. In one instance, the level of fecal coliforms
was indicated to have been 1100 MPN/100 ml (Ghaderpoori et al. 2009) when there
should be no fecal coliforms in the water.
210 C. J. Hurst

Fig. 8.16 This image shows the entrance to the Barranquilla, Colombia rapid sand filtration
drinking water treatment plant. Use of this image is courtesy of the author

8.2.3.1 Municipal Conventional Treatment

Conventional treatment of water means many different things. For purification of

surface waters, conventional treatment usually consists of treatment with alum to
achieve coagulation and flocculation, followed by sedimentation, rapid sand filtra-
tion, and disinfection. Conventional treatment including rapid sand filtration can be
adapted to serve populations numbering in the hundreds of thousands to millions of
people.
Figure 8.16 shows the entrance to the drinking water treatment plant “Estación de
Tratamiento de Agua Potable” in Barranquilla, Colombia which uses Rio Magdalena
as its source. It is a conventional treatment plant. Figure 8.17 shows the coagulation,
flocculation and sedimentation section of the treatment plant, where the alum
treatment causes coagulation of the suspended solids and the floc then is allow to
settle prior to filtration. Powdered activated carbon sometimes is added as a slurry to
aid with sedimentation of fine particulates and the powdered activated carbon also
will help with removal of organic compounds. Figure 8.18 shows the rapid sand
filters for that treatment plant. The normal flow of water through the filters is
vertically downward during which the water on top of the filters has a very calm
surface as shown in upper image of Fig. 8.18. When the rate of water flow through a
filter has decreased too greatly due to clogging of the filtration media, the filter is
cleaned by backwashing which means having filtered water flow rapidly upward
through the filter material which fluidizes the filter material and washes out
suspended particulates that were trapped in the filter material. During backwashing,
8 Options for Providing Microbiologically Safe Drinking Water 211

Fig. 8.17 This image

shows the basins for
flocculation and
sedimentation, also termed
clarification, at the
Barranquilla rapid sand
filtration drinking water
treatment plant. Use of this
image is courtesy of the
author

the clean filtered water flowing vertically up through the filter causes the top of the
filter to appear as if it were raining. The backwash water and suspended particulates
either may be discharged or returned to the beginning of the plants water treatment
process. The bottom image in Fig. 8.18 shows the appearance of a filter that is being
backwashed. The sand in the rapid sand filters periodically is removed and replaced.
Figure 8.19 shows a rapid sand filter tank at the Rio Cauca treatment plant near Cali,
Colombia, which draws its source water from the Rio Cauca. Most of the filtration
medium, plus the gravel support which underlays the filter medium, had been
removed from that filter tank for maintenance which allows us to see the water
collection system installed at the bottom of the filter. Chlorine gas is used to disinfect
the product water of the Barranquilla treatment plant, just prior to that water entering
the municipal distribution network. The Rio Cauca plant also uses chlorination of
their product water. The distribution networks of both cities provide water directly to
individual residences. A good brief explanation of rapid sand filtration accompanied
by illustrations which demonstrate its engineering has been prepared by Bruni and
Spuhler (2018a).
212 C. J. Hurst

Fig. 8.18 These images

show rapid sand filters at the
Barranquilla drinking water
treatment plant. The upper
image shows filters in which
the surface of the water is
calm, and that indicates the
water in those filters is
flowing downwards as
normal. The lower image of
a single filter shows a
rippling appearance on the
surface of the water as if it
either were raining or the
water were boiling. That
appearance of rippling
shows water is flowing
upwards through the filter
during its backwashing
cycle. Use of these images is
courtesy of the author

8.2.3.2 Municipal Slow Sand Filtration

Slow sand filtration typically is used for small to medium size communities. This
technology can serve large communities, although doing so may involve prohibitive
land costs because the filter surface area required to process the same volume of
water by slow sand filtration is greater than the surface area needed for rapid sand
filtration.
The images that I am providing are for the “El Retiro” slow sand filtration plant
which serves a community of that same name located near Cali, Colombia. That
treatment plant uses the Rio Pance as its source. Prefiltration at the El Retiro plant is
by means of roughing filters which utilize a combination of downward and horizon-
tal flow of the water through filter beds that are composed of gravel. Figure 8.20
shows the first course of prefiltration at top, and the second course at prefiltration at
bottom. After passing through the roughing filters, the water at this treatment plant is
slow sand filtered. Figure 8.21 shows the slow sand filters and you can see a
reflection of the sky in the bottom image of that figure. These slow sand filters are
8 Options for Providing Microbiologically Safe Drinking Water 213

Fig. 8.19 This image

shows maintenance being
performed on a rapid sand
filter at the Rio Cauca water
treatment plant in Cali,
Colombia. Most of the
filtration media has been
removed from this filter tank
which allows viewing of the
system of perforated pipes
through which water would
flow when it collects and
drains beneath the layer of
sand, gravel and other
filtration material that
otherwise would be in this
tank. Seeing this empty tank
allows us to understand the
relative surface area and
depth of a rapid sand filter.
Use of this image is courtesy
of the author

covered with metal roofing to reduce potential growth of algae and cyanobacteria on
top of the filter material.
Cleaning of slow sand filters when they clog is done by removing the uppermost
centimeters of sand in which the biological layer has developed. Most of the
filtration effectiveness occurs in that biological layer, but eventually the top layer
clogs and must be replaced with fresh sand. It can become necessary to entirely
replace the filter material. Figure 8.22 shows a closer view of a slow sand filter in the
top image, and the bottom image in that figure shows replacement of the sand in one
of the filters. In the bottom image of Fig. 8.22 the workman is standing on the floor of
the filtration tank, and by comparing that with Fig. 8.19 you can notice the difference
in relative depth of municipal rapid sand filters versus municipal slow sand filters.
The filtered water at this treatment plant is disinfection with sodium hypochlorite.
Figure 8.23 shows the clearwell at the El Retiro sand filtration drinking water treatment
plant. The treated water from the El Retiro plant enters a municipal distribution network
which delivers water directly to individual residences. Figures 8.24 and 8.25 show
214 C. J. Hurst

Fig. 8.20 This image shows roughing filters at the El Retiro slow sand filtration drinking water
treatment plant near Cali, Colombia. Use of these images is courtesy of the author

examples of municipal distribution networks in other places that deliver water to

community standpipes rather than extending the networks to individual homes.
The design and operation characteristics of the El Retiro treatment plant and also
of two additional slow sand filtration drinking water treatment plants are described in
8 Options for Providing Microbiologically Safe Drinking Water 215

Fig. 8.21 This image shows sand filters at the El Retiro slow sand filtration drinking water
treatment plant near Cali, Colombia. Use of these images is courtesy of the author

the reference authored by Federación Nacional de Cafeteros de Colombia et al.

(1988) along with some history of using slow sand filtration for water treatment. The
multi-stage slow sand filtration process used at the El Retiro treatment plant has been
described by Galvis Castaño et al. (1999). Chan et al. (2018) has studied the
microbial community in full scale slow sand filters. A good brief explanation of
216 C. J. Hurst

Fig. 8.22 These images show sand filters at the El Retiro slow sand filtration drinking water
treatment plant near Cali, Colombia. The lower image shows replacement of the sand from one of
the filters. The workman seen in this image is standing on the floor of the filter tank. An important
detail to notice is the difference in depth of this slow sand filter as compared to the depth of the rapid
sand filter shown in Fig. 8.19. Use of these images is courtesy of the author
8 Options for Providing Microbiologically Safe Drinking Water 217

Fig. 8.23 This image

shows the clear well at the
El Retiro slow sand filtration
drinking water treatment
plant near Cali, Colombia.
Use of this image is courtesy
of the author

slow sand filtration including illustrations which demonstrate its engineering has
been prepared by Bruni and Spuhler (2018b).

8.2.3.3 Municipal Riverbank Filtration

Riverbank filtration uses the suction provided by a pumping station to collect surface
water by pulling that water through a natural aquifer. The objective of riverbank
filtration is to use the subsurface matrix as a filter and this avoids collecting directly
from a potentially risky body of surface water. Jeyakumar et al. (2017) reviewed
studies of river bank filtration in India and discussed how this process of filtration is
affected by geological conditions and well types, plus the effect of bank filtration on
physical, chemical and biological qualities of the water. Gutiérrez et al. (2017)
addresses the engineering issues associated with river bank filtration and also
218 C. J. Hurst

Fig. 8.24 This image

shows a public standpost
that provides safe drinking
water for a community. The
image is titled “A drinking
fountain in Saint-Paul-de-
Vence” by Claritas, and
used under the Creative
Commons Attribution 3.0
Generic license

mentions its presumed historical role as the precursor technology for other types of
drinking water sand filtration methods.

8.2.3.4 Health Risk Associated with Those Microbial Contaminants that

Are Not Successfully Removed Before Drinking Water Is
Municipally Distributed

This section refers to the study of conventionally treated drinking water. Conven-
tional treatment is good at protecting human health although it is not perfect in terms
of either removing or destroying all of the microorganisms that are present in source
water. The residual level of microorganisms in treated water represents a quantifiable
risk of infectious disease for people who consume that water. This section is not
meant to imply that the other treatment options described in this chapter are more
effective than is conventional treatment for reducing the risk associated with residual
levels of microorganisms in the treated water.
One part of the health risk from water is due to microorganisms that are neither
removed nor successfully rendered harmless during municipal treatment processes
8 Options for Providing Microbiologically Safe Drinking Water 219

Fig. 8.25 This image

shows a public standpost
that is used for providing
drinking water to a
community. The image is
titled “Communal tap
(standpost) for drinking
water in Soweto,
Johannesburg, South Africa
(2941729790)” by author
SuSanA Secretariat and is
being used under the
Creative Commons
Attribution 2.0 Generic
license

(Hurst 2018) as mentioned above and in Table 8.1. Two epidemiological studies
have been performed in Montreal, Canada to assess that risk. Results of a 1991
epidemiological study (Payment et al. 1991) indicated that the annual risk to
consumers of acquiring gastroenteritis from ingesting microorganisms contained in
the community-distributed conventionally treated tap water of Montreal, Quebec,
Canada, was 0.26, equivalent to 26%, or roughly one illness per person every
4 years. The results of that study have been evaluated by Hurst (2018) using a risk
estimation technique with knowledge of the estimated level in the treated water of
virus potentially pathogenic for humans, plus the levels of Cryptosporidium and
Giardia. The corresponding bacteriological data available for the 1991 study by
Payment et al. were not for pathogenic bacteria, and thus the risk of illness due to
bacteria in the community distributed water was presumed to be all residual risk not
accounted for by virus and protozoa. The summary of findings for that validation of
epidemiologically determined annual risk of illness (designated validation exercise
1 in Hurst 2018) per individual consumer were: together all microbial contaminants
in the municipally supplied tap water had been responsible for an annual risk of 0.26
(100.0%), the estimated amount of that risk caused by virus in the municipally
supplied tap water was 0.20861 (80.2%), the estimated amount of that risk attribut-
able to Cryptosporidium in the municipally supplied tap water 0.00267 (1.0%), and
the estimated amount of that risk attributable to Giardia in the municipally supplied
tap water 0.00674 (2.6)%. The difference, or residual level of risk, between the
epidemiologically determined annual risk of illness and the level of risk predicted by
220 C. J. Hurst

this estimation technique using data for levels of virus and protozoa in the water was
0.04198 (16.1%) and presumed to have been the bacterial risk.
Several years later, after efforts to improve the efficiency of that community’s
drinking water treatment facilities, Payment and coworkers performed a second
epidemiological study (Payment et al. 1997) during which they found that individ-
uals ingesting the conventionally treated, community-distributed tap water had an
overall 0.66 annual risk for incidence of gastrointestinal illness. That contrasted with
an annual illness risk of 0.58 among a control group of consumers who either drank
bottled water or else drank tap water that originated from the same community
distribution system but which had gone through an in-home treatment system
including filtration prior to the water being ingested. The difference in annual risk
thereby attributed to microorganisms in the tap water during the second epidemio-
logical study was 0.08. The validation exercise presented by Hurst (designated
validation exercise 2, in Hurst 2018) for the second epidemiological study of
Payment et al. (1997) suggested that of the 0.08 annual risk of illness per individual
consumer, the amount which could have been due to virus in the drinking water was
0.068749 (85.9%), with protozoa of the genera Cryptosporidium and Giardia
respectively accounting for approximately 0.00118 (1.5%) and 0.00337 (4.2%) of
the observed illnesses. The data which were published by Payment et al. (1997)
concerning bacterial concentrations in the tap water for that study represented
indicator bacterial groups rather than known waterborne bacterial pathogens. As
such, the only way to estimate the proportion of observed infectious disease which
would have been attributable to bacterial pathogens contained in the water during the
time of the second epidemiological study again was by subtraction. Thus, pathogenic
bacteria presumably accounted for an annual risk of 0.00670 representing 8.4% of
the observed cases of illness for the second study by Payment et al. (1997).

8.2.3.5 Health Risk Associated with Microbial Contaminants that Are

Acquired by Municipally Treated Water During Distribution
of the Water

Having made water safe for human consumption we then need to protect its quality.
Unfortunately, the need for moving water from where it was treated and made safe to
where it will be consumed does involve a possibility that quality of the water will
degrade.

8.2.3.5.1 Contamination Occurring in the Water Distribution Network

Part of the risk associated with community distributed drinking water is caused by
microbial contaminants that are introduced after the water begins its path through the
water pipeline distribution network. Within that distribution network, microorgan-
isms originating from the source water will be joined by biological contaminants that
accidentally enter the distribution system in association with infiltrations related to
8 Options for Providing Microbiologically Safe Drinking Water 221

piping engineering failures. Accidental cross contamination of drinking water by

septage collection systems represents yet another source of microorganisms that will
be entering the drinking water distribution system. The total accumulation of
biomass and its ecosystem structure that exists inside of a drinking water distribution
system must be understood and controlled, because the biomass in drinking water
interferes with those chemical disinfectant processes upon which we rely for deliv-
ering safe product water to consumers. The complex microbiology of drinking water
distribution networks is discussed in Chap. 9 of this volume “Microbiome of
Drinking Water Distribution Systems” by authors “Laurence Mathieu, Tony Paris
and Jean-Claude Block, pp. 261–311”.

8.2.3.5.2 Microbial Safety Issues Associated with Delivery of Drinking

Water by Tanker Truck

If safe water is available but must be delivered in containers, then options for that
delivery include the use of either tanker trucks or bottling the water. The microbi-
ology of bottled water will be discussed below in Sect. 8.3.1.
Tanker trucks do offer an economical means of delivering treated drinking water
to small communities and groups of people. Water deliveries by tanker may be used
to fill community cisterns that have multiple taps, from which people subsequently
will be expected to fill their own water containers and then carry that water away for
household usage. Tanker trucks also can deliver drinking water for community
systems that include distribution pipelines. Such distribution systems may include
water transmission lines that feed only community standposts with multiple taps or
the distribution systems may have household connections that deliver water to
individual dwellings.
Sule et al. (2014) found that the water coming out of delivery tanks in Nigeria
generally had higher levels of bacterial contamination, including total and fecal
coliforms, than did the water going into the tanks, and the authors suggested that
this contamination may have represented the tanker trucks having being used for
other hauling instead of being reserved for transporting only treated drinking water.
Mendonça et al. (2017) performed bacteriological analysis of drinking water
marketed by pipe trucks in Brazil and determined that the water delivered by this
means did not meet portability requirements as assessed by total coliforms,
thermotolerant coliforms, Pseudomonas aeruginosa, and heterotrophic bacteria.
Mendonça et al. (2017) found that the microbial quality of the water decreased
during the time that it was in the truck tank, and that plastic containers were the worst
for water storage perhaps because they are more favorable to microbial attachment
which will start the development of a biofilm. The State of Queensland (2015) has
offered typical guidelines with their requirement that tanker trucks which are used
for transport of potable water cannot be used for hauling other materials, and stating
that the trucks as well as their pipes, hoses and fittings, must not only be cleaned but
also disinfected with chlorine. The Texas Commission on Environmental Quality
(2018) has classified water haulers as transient noncommunity public water systems
222 C. J. Hurst

because they have the potential to provide drinking water to individuals in various
locations at a variety of times. Texas considers that such a transient noncommunity
public water system may be treated as a consecutive system for purposes of
compliance under Texas Commission on Environmental Quality rules. Their regu-
lations require that the water must be from an approved source, the tank can only be
used for transporting drinking water and can never have been used for transporting
anything other than potable liquids. They also have regulations requiring the water
carrying tank to have a manhole, vent and drain. They additionally require that the
hoses and pump, as well as the tank itself, must be disinfected at least monthly,
microbiological sampling of water from the truck must be done at least monthly, and
the hauler must maintain a specified level of disinfectant in the water that is being
transported.

8.2.3.5.3 Microbiology of Water Dispensers

We often reply upon public water fountains as well as beverage dispensers in

restaurants and other institutions to provide individuals with drinking water. How-
ever, even in places that are considered quite modern, the quality of water from those
dispensers may fail to meet microbiological standards and the dispensed water can
containing opportunistic pathogens (Al Moosa et al. 2015).

8.3 Bottled Water and Water Vending Machines

The use of bottled water and water vending machines are common options but a
sense of care must accompany our reliance upon them.

8.3.1 Bottled Water

There are two main source categories of commercially distributed bottled water. The
first of those would be water collected from springs. Spring water may be captured
where the spring naturally emerges at the land surface. At other times, a spring’s
water is obtained by using a well to intercept the flow of that water underground at a
point before the water can reach the surface. The other source for bottled water is
conventional tapwater that has been further purified.
The quality of bottled water varies widely. Fewtrell et al. (1996) sampled natural
mineral water and other bottled water, finding that some of the water contained total
coliforms although none of the water which they sampled contained either
Escherichia coli, faecal streptococci or aeromonads. Leclerc and Moreau (2002)
examined the microbial ecology of natural mineral water and did find the water
to have contained some groups of bacteria which are pathogenic in mammals.
8 Options for Providing Microbiologically Safe Drinking Water 223

Saleh et al. (2008) examined commercial bottled water from 35 sources, including
purified drinking water and natural spring water. Nine of the 35 sources examined by
Saleh et al. (2008) contained bacteria and among those the authors found:
Acidovorax delafieldii, Acidovorax temperans, (some species of Acidovorax can
cause sepsis in human), Agrobacterium rhizogenes, Burkholderia glumae
(is pathogenic in human), Moraxella caviae (pathogenic in guinea pig),
Parageobacillus thermoglucosidasius (formerly Bacillus thermoglucosidasius)
which produces spores that are used as a challenge for sterilization, Ralstonia
pickettii (formerly Klebsiella terrigena which is pathogenic in human causing
mainly respiratory infections), and Raoultella terrigena (causes sepsis in human).
Totaro et al. (2018) examined spring water and bottled mineral water by polymerase
chain reaction (PCR) and found that, even in the absence of cultivable bacteria, the
bottled water contained evidence of naturally free living human pathogenic amoeba
and bacteria. The organisms detected by Totaro et al. (2018) were Acanthamoeba
polyphaga, Vermamoeba vermiformis, the amoeboid genus Vahlkampfia of which at
least some species are pathogenic for humans, the bacterial genus Legionella, and
non-tuberculous mycobacteria.
Amenu (2014) performed a review of literature on the subject of bottled water and
found a lack of extensive quality standards for packaged water, as compared to the
type of standards which exist for municipal water supplies. The United States
Government Publishing Office (2018) did publish regulations which indicate that
the water which is bottled must be from an approved source, and that publication
included regulations regarding the containers used for the water, the water
processing equipment, and the facilities where the bottling is performed.

8.3.2 Water Vending Machines

Water vending machines purify tap water on site and vend it. Such machines are a
common approach for purchasing drinking water in a form which is less expensive
than is purchasing commercially distributed prepackaged bottled water. The typical
series of processes used in those machines would be activated carbon filtration,
followed by particle filtration, reverse osmosis, a second carbon filter, then ultravi-
olet light. Figure 8.26 shows the appearance of a water vending machine. Some
states in the United States have both performance and inspection requirements for
these machines. The Florida Department of Agriculture and Consumer Services
(2017) has inspection requirements for vending machines that sell either water or
packaged ice. The Florida requirements are for disinfection of the water with either
ultraviolet, or ozone, or some equivalent means, and there also is a requirement of
testing the product water for total and fecal coliforms with specified maximum
allowed bacterial limits. The California Department of Public Health (2014) simi-
larly requires inspection of water vending machines and disinfection of the water
with either ultraviolet, or ozone, or some equivalent, but microbiological testing is
not required by California. Figure 8.27 shows a row of water vending machines for
224 C. J. Hurst

Fig. 8.26 This image

shows a water vending
machine which processes
tap water and then dispenses
the product. Such machines
typically are coin operated.
Water processing done
within that machine
involves a sequence
including initial activated
carbon filtration to remove
organic compounds from the
water, small pore size
filtration to remove
microorganisms including
those which grow on
organic compounds that
accumulate in the activated
carbon filter, reverse
osmosis, a second carbon
filtration to improve taste
following passage through
the reverse osmosis filter,
then ultraviolet light
immediately prior to
dispensing

sale in Thailand, where there is a concern that such coin operated vending machines
frequently distribute water which fails to meet microbial safety regulations
(Fredrickson 2017).

8.4 Community Education Helps with Providing

Background Knowledge of Waterborne Disease
and Acceptance of the Need for Drinking Water
Treatment

Public education can be a critically important aspect of planning water treatment for
very small communities of a few families, and for individual households. Reasons
for removing turbidity are easily understood because doing so improves both the
visual appeal and taste of the water. Understanding the existence and nature of
pathogenic microorganisms requires that people learn to believe in dangers
8 Options for Providing Microbiologically Safe Drinking Water 225

Fig. 8.27 This image is titled “E8661-Pattaya-water-vending-machines” by Vmenkov and shows

water vending machines for sale in Pattaya, Thailand. It is being used under the Creative Commons
Attribution 1.0 Generic license

associated with things that cannot easily be seen since most of the microbes in water
are invisible without some form of technical assistance such as magnified viewing.
The goal of public health microbiology requires that we overcome the invisibility
of pathogenic microorganisms. Achieving that goal includes convincing people of
there being some benefit in filtering and disinfecting water before they drink it, even
though reducing the risk of microbially associated illness may not have been a part of
their earlier education. Understanding and utilizing options for destroying infectivity
associated with those microorganisms that remain in the water after we have applied
turbidity removal processes does sometimes nearly require a leap of faith.
Another issue is that people cannot be helped in ways that they either do not want
or cannot afford. The people that we would help need to feel comfortable with the
technology. Their use of that technology also must be both affordable and reliably
continue to be available.
A community needs to feel ownership of its drinking water treatment processes
and understand responsibility for maintenance of shared equipment. If people sense
that they have a personal involvement with the water treatment techniques then they
are more likely to use those techniques. Instilling a sense of personal involvement
can be even more critical when trying to sustain the use of water treatment technol-
ogies that are being applied to small communities as compared to household
226 C. J. Hurst

treatments, because if no individual person or group of people within a community

feels a sense of personal ownership for the responsibility of participating in main-
tenance of such structures as sand filters, rainwater cisterns, and chlorinators, then
the maintenance will never get done. Brikké and Bredero (2003) have written a good
general background document regarding the technologies of drinking water treat-
ment including technical requirements for design, along with discussing the impor-
tance of assigned maintenance and helping people to sense participatory ownership
of a project so that those people will feel they should care for the equipment. If you
just simply give the information and water processing equipment such as sand filters
to people without those people sensing ownership and evolvement, then no one in
the community will take care of the equipment (Brikké and Bredero 2003).

8.5 Adapting Water Treatment Technologies for Usage by

Very Small Communities

8.5.1 Turbidity Removal by Sedimentation, Coagulation

and Flocculation

Source water may contain sand and other material that contributes to high turbidity.
Turbidity makes the water unpleasant for drinking and the material that contributes
to turbidity often will bind chlorine which complicates the task of chemical disin-
fection. The process of plain sedimentation is used to make water less turbid by
allowing sand, silt, and clay as well as other particulates to settle by gravity.
Sedimentation can be achieved either by constructing a small community sedimen-
tation basin (United States Agency for International Development et al. 1982) or by
allowing the water to settle in buckets. If gravitational sedimentation is not sufficient
to lower the turbidity then it may be necessary to add compounds that will assist in
achieving coagulation and flocculation.
The techniques used for adapting coagulation and flocculation, as well as filtra-
tion and disinfection, downward in scale from the volumes of water required at
municipal levels to the volumes that will provide an adequate amount of microbio-
logically safe drinking water to small communities and households will vary
depending upon the number of people being served. And, importantly it should be
noted that some water treatment techniques which serve well at the household level
will not work at larger scale. For example, the use of boiling as a water disinfection
technique works at the level of an individual household but cannot practicably be
done in a central location to supply the needs for a million people.
8 Options for Providing Microbiologically Safe Drinking Water 227

8.5.2 Adaptation of Slow Sand Filtration for Very Small

Communities

Rapid sand filtration typically is used at facilities that treat surface freshwater for
large communities. Rapid sand filtration occurs as the water flows through a layer of
natural sand. Alternatively, sand may be the major component of a mixed media
filtration bed. The sand or mixed media is underlain with a layer of gravel which
provides for easy drainage of the filtered water. When this technique is used at large
scale water treatment facilities, rapid sand filtration is done in combination with
preliminary steps of coagulation, flocculation and sedimentation. Rapid sand filters
typically will achieve turbidity removal but can accomplish only minimal removal of
bacteria, and so the water processed by rapid sand filtration usually is disinfected
using either chlorine or ozone prior to the water being distributed. Rapid sand
filtration units can be adapted for use even at the household level and that concept
will be presented at a later point in this chapter.
Slow sand filters serve as the standard for successfully achieving bacterial
removal by sand filtration. Slow sand filtration relies upon importance of a hypogeal
biological layer which forms in the uppermost section of the sand because that layer
effects most of the bacterial removal. While rapid sand filters can be allowed to run
dry, slow sand filters cannot be allowed to run dry because that would cause the sand
bed to develop cracks and also risk damaging integrity of the biological layer. If a
slow sand filter does run dry then it must go through a restart process and the
turbidity of the output water should be monitored so as to determine when turbidity
removal has again reached a satisfactory level, indicating that the feedwater is being
filtered effectively, at which point the filter is said to be mature.
Slow sand filtration is one of the treatment technologies which might have
seemed difficult to adapt for very small scale usage, but indeed success has been
found in a form that commonly is termed a biosand filter. Biosand filters are very
small community scale slow sand filtration drinking water treatment systems which
can be used to provide intermittent flow, and they can work effectively after
maturation of the filter (Nair and Ahammed 2014). Figure 8.28 illustrates the design
of these filtration units. There is mention of these filters being used for individual
households, but such filters often would be relatively expensive for household use.
The containers used for housing these filters are about 90 cm meters tall and 30 cm
square (Centers for Disease Control and Prevention 2014b). The two most important
aspects to be noted are firstly that the outlet for the filter is above the top level of the
sand because the filter must be kept submerged to prevent need for a technical
restarting of the filter. The height of the outlet pipe is set to maintain 5–6 cm of water
above the sand (Centers for Disease Control and Prevention 2014b). Secondly, a
diffuser plate is present so that addition of water will not disturb the top layers of the
sand. Water to be filtered is just simply poured on top of the diffuser plate.
Figure 8.29 shows four of these filters being used to supply drinking water for a
small community consisting of a few families in Guatemala. The New England
Water Treatment Technology Assistance Center (2018) has recommended that the
volume of each charge of raw water applied to a biosand filter not exceed 80% of the
228 C. J. Hurst

Fig. 8.28 This is a diagram of a small volume slow sand filter, also called a biosand filter. Use of
this image is courtesy of the author

Fig. 8.29 This image shows biosand filters, they are small volume slow sand filters used to provide
water for drinking and washing. The image is titled “Biosand Filters in Guatemala” by Nora.
jeanine530 and used under the Creative Commons Attribution 3.0 Generic license

filter’s void volume. Allowing turbid source water to settle as a pretreatment before
that water is poured into the filter tank can help to reduce clogging of the filter and
thus help to maintain the filters flow rate. When flow through the biosand filter begins
to slow severely, the diffuser plate can be removed and the biological layer either
agitated or scraped away. The diffuser plate then needs to be reinstalled. Eventually,
as with all slow sand filters, it does become necessary to replace the sand.
8 Options for Providing Microbiologically Safe Drinking Water 229

Clark et al. (2012) has created effective slow sand filters using polyvinyl chloride
buckets and summarized information on both the biofilm layer and fineness of the
sand. Even though most of the filtration effectiveness for slow sand filters occurs in
the biofilm layer, the depth of the filter still is an important consideration because a
filter with greater initial sand depth can be scraped and cleaned more times before
additional sand is needed. The size and uniformity of the sand particles importantly
alters both the flow speed and effectiveness of a slow-sand water filter. Water can
filter faster through coarser sand, and yet fine sand with uniform grain size will
provide more effective although slower filtration. Having finer grain size unfortu-
nately also means that a filters media will clog more easily. Uniformity in size of the
sand particles is helpful for maximizing effectiveness of the filter because having
great variability in grain sizes can result in the smaller sand particles filling in the
spaces between the larger particles and thus contributing to clogging of the filter.
Two negative aspects of biosand filters are that flow limitations can require
excessive waiting, and that inadequate water purification occurs when high flow
rates are imposed. Tellen et al. (2010) examined both increasing the sand’s effective
size and adding zero-valent iron into the media as a disinfectant. Those researchers
found that after 65 days of filter operation, the percent reductions in total coliform,
fecal coliform, and fecal streptococci averaged 98.9% for traditional versus 99% for
their improved biosand filter. Both of those modifications proved to be statistically
significant filter improvements. Therefore, the suggestions of Tellen et al. (2010)
were that increased sand size and addition of zero-valent iron may counter some
drawbacks of traditional biosand filters.
Mwabi et al. (2012) examined biosand filters including those supplemented with
zeolite, versus the use of a sand and gravel bucket filter, a ceramic candle filter, and
silver-impregnated porous pot filters that had been manufactured using plastic
buckets with removable lids. The silver-impregnated porous pot was superior in
effectiveness and consistently produced bacterially acceptable drinking water
regardless of the quality of the source water treated, given sufficient contact time
after filtration so that the silver could take effect against the bacteria. Second best
were the biosand filters, which were similar and sometimes slightly better than the
ceramic candle filter. The bucket filter, which is a rapid filtration technique, dem-
onstrated the worst efficiency at bacterial removal.

8.6 Water Treatment for Individual Households

There are many options for preparing microbiologically safe drinking water at the
household level, and the choices vary based upon the location where the techniques
might be used. Summaries of information on household drinking water treatment
techniques have been published by Agrawal and Bhalwar (2009), Laurent (2005),
Skinner and Shaw (2019c), and the World Health Organization (2011a).
The household level technologies for removing suspended solids include natural
settling and filtering, plus the possibility of assisting sedimentation by using
230 C. J. Hurst

chemically induced coagulation and flocculation with subsequent precipitation

(Oxfam 2012). Filtration can be accomplished by many techniques, with some of
those options such as ion exchange and reverse osmosis requiring a pressurized
source of water and thus having been designed primarily for households which
receive community supplied tapwater. Other filtration technologies such as cloth
filters, granular media filtration including sand, and ceramic filters, have been
developed for emergency situations and for households that do not have community
supplied tapwater, and correspondingly those techniques do not need a pressurized
water supply. Disinfection of the water can be done by using any one of many
techniques including chlorination, heating to pasteurize the water using either fuel or
solar energy, boiling and distillation, as well as by the use of ultraviolet lamps.
General information on performing filtration at the household level and on
performing chlorine disinfection of typical household water volumes can be found
in the reference by Centers for Disease Control and Prevention (2009). Disinfection
of water with ozone is not practical at the household level.
Many of these methods for accomplishing successful drinking water treatment at
the household level will be further explained later in this chapter. Using the methods
in combinations performed either simultaneously or sequentially creates a multi-
barrier approach, of which an example would be coagulation combined with disin-
fection. The reference pathogens cited by the World Health Organization (2011a) for
drinking water treatment were: for bacteria Campylobacter jejuni, for viruses Rota-
virus, and for protozoan Cryptosporidium. The targets which the World Health
Organization has stated for microbial reduction to be considered “protective” were
2log10 reductions in bacteria and protozoa, and a 3log10 reduction in virus (World
Health Organization 2011a). The targets which have been stated for microbial
reduction to be considered “highly protective” were 4log10 reductions in bacteria
and protozoa, and a 5log10 reduction in virus (World Health Organization 2011a).
Having accomplished successful microbial removal it then becomes necessary to
store the water safely to prevent recontamination. Safe storage requires keeping the
water covered. And, because it is not safe to store water in a way that will allow
people to wash their hands in the water that someone else subsequently will be
drinking, drinking water storage containers must have narrow openings (Interna-
tional Network to Promote Household Water Treatment and Safe Storage 2007).
People also must be taught not to drink water from those containers with their
mouths directly on the containers opening!

8.6.1 Coagulation and Flocculation

Using the processes of coagulation and flocculation can be simply done by individ-
ual households as suggested in Fig. 8.30. Aluminium sulfate, commonly known as
alum, and powder from Moringa seeds are two commonly used compounds for
aiding the settling of suspended solids (Oxfam 2012) which then allows a clearer
water to be decanted. Whenever possible, the decanted water should be filtered
8 Options for Providing Microbiologically Safe Drinking Water 231

Fig. 8.30 This image suggests the addition of coagulant to a bowl of water. Use of this image is
courtesy of the author

through a piece of clean cloth to help remove the suspended solids because micro-
organisms associated with those solids likely will remain infectious and represent a
possible source for recontamination of the treated water.
Moringa treatment achieves water flocculation by using a powder prepared from
the seeds of Moringa oleifera (Doerr and ECHO Staff 2005). Sánchez-Martín et al.
(2012) evaluated optimizing the pH, stirring rate and stirring time for that technique.
There have been comparative studies in which the effectiveness of Moringa was
evaluated against aluminum sulfate (Alo et al. 2012; Tunggolou and Payus 2017)
and it has been suggested that seeds of Moringa stenopetala may be more effective
than is Moringa oleifera for purifying water (Abiyu et al. 2018). Habtemariam
(2017) has described the process by which Moringa extracts succeed at water
purification. It is important to note that Moringa treatment does not remove 100%
of all microbial contaminants.
Alum also is used as part of a combined treatment that replies upon disinfection of
the water by either heating or chlorination (Crump et al. 2004; Wrigley 2007). Ferric
sulfate similarly is used as part of a combined treatment with chlorination, as will be
described below.

8.6.2 Filtration

Household water filters are available in different capacities designed for either whole
house use or only kitchen use. Commercially available wholehouse filters can be
installed in the water supply line near to where the water line has entered the house.
A whole house inline paper filter for removing sediments is shown in Fig. 8.13.
232 C. J. Hurst

Some kitchen usage filtration units are designed to be faucet mounted, meaning that
they attach at the end of a standard kitchen faucet and typically those filtration units
include a bypass valve which allows for filtering only water that will be used for
cooking and drinking. Faucet mounted units have small filters that because of their
reduced size must be replaced frequently. Sink mounted kitchen water filters are
connected into the kitchen plumbing. Some of the smaller sink mounted kitchen
filtration units are designed to sit on the counter next to a sink. There are larger
kitchen units designed for installation under the counter and many of those will
include a reservoir tank that holds filtered water. The more common household
filtration options include woven string, pleated paper, spun polypropylene, and
activated carbon. Reverse osmosis filtration units which include several of these
techniques are available for household kitchen use, they typically are installed under
the counter and I briefly will discuss them later in this chapter.
For a general review of household filter units I would suggest the articles by
Agrawal and Bhalwar (2009), and by Johnson and Scherer (2011). Most of the
fabric, polypropylene and paper filters have effective pore sizes greater than the
diameters of viruses and bacteria, and thus any effectiveness which such filters have
against those groups of microorganisms would depend upon removing microorgan-
isms that are associated with solids. Those filters may, however, serve to remove
protozoa and metazoa. I will discuss later in this chapter the use of cloth filters,
including folded sari cloth, to reduce the risk of cholera and dracunculiasis by
removing copepods from water.

8.6.2.1 Household Rapid Sand Filtration

Rapid sand filters can easily be designed for household use. Figure 8.15 shows a
rapid sand filter that was constructed using a 5 US gallon plastic bucket, and these
often are described as being ‘bucket filters’. Both the United States Agency for
International Development et al. (1982) as well as Skinner and Shaw (2019c) have
published designs for constructing rapid sand filters to serve the needs of house-
holds. Such filters are effective at removing turbidity but their capability for remov-
ing microorganisms is considered poor, and so achieving microbiological safety of
the treated water would require that the filtered water be disinfected.

8.6.2.2 Porous Pot Filters

Porous pot filters function by allowing water to permeate through the sides and
bottoms of either stone or ceramic pots into which the water has been placed. The
filtered water then is retained in a less porous container located beneath the porous
pot. If the water to be filtered has a high level of turbidity then you should first
prefilter the water through a clean piece of fine cloth (Oxfam 2012) which will
reduce clogging of the porous pot. That prefiltration easily can be done by placing a
8 Options for Providing Microbiologically Safe Drinking Water 233

Fig. 8.31 These images show a stone water filter at the Santa Catalina monastery in Arequipa,
Peru. The left image is titled “Water filtration stone 2, Santa Catalina monastery, Arequipa, Peru”,
the right image is titled “Water filtration stone, Santa Catalina monastery, Arequipa, Peru”, both are
by Pethrus and being used under a Creative Commons Attribution-share alike 1.0 license. The
ceramic pot beneath the filter serves as a receptacle for the filtered water

piece of clean cloth across the top of the porous pot filter and pouring the dirty water
though that cloth into the pot.

8.6.2.2.1 Introduction to Porous Pot Filters

Porous pot filters also are called porous jar filters. They consist of a permeable pot
which serves as the filter through which water slowly permeates, and there will be a
less permeable container called a receptacle that sits below the filter and into which
the filtered water collects. Historically these types of filters have been made of either
carved stone or ceramic. There still is some usage of the stone porous pot filters
Fig. 8.31 (Ramos 2018) although perhaps that mostly is done for a sense of nostalgic
curiosity, and apparently new stone filters are fashionable as decorative items in
Latin America. Figure 8.32 shows the historical development of ceramic porous pot
filters, which today are manufactured in a more modern style that uses a plastic
bucket as the container into which filtered water collects. A small spigot is fitted to
the receptacle so that filtered water can be withdrawn without inserting either dirty
hands or potentially contaminated implements into the receptacle.
The composition of the mixture used for creating modern ceramic filter pots
consists of kaolin, sawdust and grog. In this sense, grog will be either ground
ceramic or a ground mineral. The formulation and sintering temperature will
together determine the removal efficiency that subsequently can be achieved by
the filtration pot. Lower porosity ceramic will achieve a greater removal of bacteria
234 C. J. Hurst

Fig. 8.32 These images show colonial, Victorian era and modern ceramic filters. The creation of
household ceramic water filters with collection reservoirs for holding the processed water has been a
developing concept for several centuries. The ceramic material used for the filter has a higher
permeability than does the receptacle or reservoir into which the water collects. The upper left
image shows a Colonial era Colombian ceramic filter courtesy of Benjamin Villegas, Villegas
Editores, Bogota, Colombia. The upper right image is of a “germ proof” Victorian era ceramic water
filter with a brass tap, titled “CeramicWaterFilter” by ClarkMills and used under the Creative
8 Options for Providing Microbiologically Safe Drinking Water 235

and turbidity, albeit the filtration rate of lower porosity ceramic correspondingly is
slower. van der Laan et al. (2014) found that the burnt material content of the
ceramic did not affect Escherichia coli removal efficiency. Zereffa and Bekalo
(2017) have examined how the composition and sintering temperature influenced
removal of Ca2+, Mg2+, iron, nitrite, and conductivity, as well as affecting pH of the
filtered water.
Properly caring for a ceramic pot filtration unit should include pouring the raw
water through clean cloth as a prefiltration to remove suspended particulates prior to
placing that water into the filter pot. Prefiltration treatment will remove some
suspended particulates from the water and thereby reduce clogging of the pores
that are in the ceramic body. This prefiltration can easily be done by placing cloth
over top of the ceramic filter pot, then pouring water through the cloth into the filter
pot (Filtron 2018). Silver often is added to the pots and silver is effective as a
disinfectant against some bacteria, fungi and virus, but there are toxicological limits
for ingestion of silver (World Health Organization 2018a). Addition of the silver
may be done by either applying colloidal silver to the ceramic pot or incorporating
AgNO3 into the clay, although incorporating AgNO3 can result in the initial water
which passes through the filter containing too high a level of silver (Mwabi et al.
2012). Silver is commonly used both in other types of domestic ceramic water filters
and in powdered activated carbon filters for the stated purposes of reducing biofilm
growth and potentially providing an additional level of water treatment. Copper and
silver ionization often is used in swimming pool disinfection, also is used to prevent
Legionella bacterial colonization of hot water plumbing systems, and may serve as a
secondary disinfectant of drinking water supplies (World Health Organization
2018a).

8.6.2.2.2 Specific Details About Performance of Porous Pot Filters

Ceramic filters typically can remove most of the larger protozoan and bacterial
organisms from water, but are ineffective at removing the much smaller viral
organisms and the filtration effectiveness varies depending upon the production
quality of the ceramic. Ceramic pot filters now often are coated with silver which
leaches into the water that is being filtered, and if sufficient contact time is allowed
between filtering of the water and the time when that water is consumed, then silver
in the filtered water will show good effectiveness in achieving bacterial inactivation
(Innovación para el Desarrollo y la Cooperación Sur-Sur 2018). Using silver as part
of the pot construction unfortunately seems to decrease the effectiveness with which
the filter removes viruses. Studies have also shown significant bacterial contamina-
tion of the filtered water either when poor-quality locally produced filters are used, or

⁄


Fig. 8.32 (continued) Commons Attribution-Share Alike 3.0 Unported license. The lower images
are a photograph and cutaway drawing of a Filtrón ceramic water filter courtesy of Potters for Peace,
Boulder, Colorado
236 C. J. Hurst

when the receptacle is contaminated at the household level (Centers for Disease
Control and Prevention 2012). Because there is no residual protection for the filtered
water other than the potential presence of silver, it is important that users be trained
to properly care for and maintain both the ceramic filter and receptacle. Given these
precautions, an impressive 60–70% reduction in diarrheal disease incidence has been
documented in users of ceramic pot filters (Centers for Disease Control and Preven-
tion 2012).
van Halem et al. (2007) tested for 12 weeks colloidally silvered and unsilvered
ceramic filter pots from three production locations, Cambodia, Ghana and Nicara-
gua, for removal of microorganisms from canal water and for presence of metals in
the filtrate. They also studied the microstructure of the filter material using mercury
intrusion porosimetry and bubble-point tests. The effective pore size of the ceramic
was measured to have been a mean of 40 mm, which they noted is larger than many
pathogenic microorganisms and that also indicated microbial removal by the filters
to be dependent upon high tortuosities in the filter material facilitating a higher
chance of water contaminants being removed by adsorption, diffusion and
sedimentation.
van Halem et al. (2007) found that turbidity in the raw canal water was well
removed by the filters, but turbidity removal corresponded with clogging of the filter
material. Scrubbing the insides of the pots to remove accumulated layers of solids
that had been retained on the surface of the ceramic was beneficial but did not stop
the effect of long term clogging which occurs within the ceramic body. The filters
without silver did not show different water flow reductions compared to filters with a
silver coating. Notably, those authors do not seem to have prefiltered the water by
any means such as perhaps passing it through a cloth to remove suspended partic-
ulates prior to placing that water into the ceramic filters.
van Halem et al. (2007) found that several metals leached from the ceramic pots,
particularly in the first few weeks, and those were aluminium, antimony, arsenic,
barium, copper, manganese, silicon and silver. For most of those compounds the
effluent measurements eventually decreased to concentrations below World Health
Organization guidelines. Arsenic was the only compound found to leach in concen-
trations above the acceptable level of 10 mgL. Although the arsenic levels did reduce
over time, arsenic levels for the Cambodian filters during even week 12 still were
high. The suggestion was that filter material should be tested for leaching of arsenic,
especially if the ceramic was made in regions where high arsenic concentrations are
known to be present in the soil.
van Halem et al. (2007) compared the filter pots for removal efficiency of total
coliforms naturally present in the canal water, sulphite reducing Clostridium spores,
Escherichia coli K12 and MS2 bacteriophage. Their summary microbiological
results are that bacteria and bacterial spore removal was higher with the silvered
pots, but contrastingly bacteriophage removal was lower with the silvered pots. The
specific microbiological results are that silver impregnated ceramic pot filters suc-
cessfully removed total coliforms, sulphite reducing Clostridium spores, and
Escherichia coli K12. Filters manufactured in Nicaragua without application of
silver showed the highest concentrations of Escherichia coli in the filtered water.
8 Options for Providing Microbiologically Safe Drinking Water 237

The Nicaraguan filters with silver usually did achieve the bacterial guideline level of
no detectable coliforms in any 100 mL sample of filtrate. Water quality produced by
the filters from Ghana and Cambodia was good but did not meet the bacterial
guideline level. MS2 bacteriophage were only partially removed from the water,
and viral removal was significantly better for the filters that lacked colloidal silver
impregnation, a finding that was noted to be in contrast with previously reported
studies. van Halem et al. (2007) concluded that the removal of Escherichia coli
proved better by filters with a layer of silver but that impregnation of colloidal silver
was not needed to remove significant concentrations of Escherichia coli as filters
without silver also reached high log10 reduction values. They also concluded that
colloidal silver was unnecessary for achieving efficient removal of Clostridium
spores by the filter pots.
van der Laan et al. (2014) subsequently found that storage time of the filtered
water in the receptacle, and not contact time with silver during the filtration phase,
was the dominant parameter in achieving Escherichia coli inactivation by silver. The
removal effectiveness for viruses is still of major concern for ceramic pot filtration.
The initial hypothesis of van der Laan et al. (2014) that the absence of silver would
enhance virus removal due to biofilm formation on the ceramic filter element could
not be confirmed.

8.6.2.3 Ceramic Candle Filters

Ceramic candle filters typically are hollow cylinders with one end closed and
rounded, and the other end having a sealed connection to an outflow drain. The
ceramic usually is made from sintered diatomite (diatomaceous earth) which is the
fossilized remains of diatoms. Water flows from outside the candle to the inside.
These filters can use either gravity or another source of hydrostatic pressure to push

Fig. 8.33 Diagram of a ceramic candle filter that works by gravity. In this drawing there are two
hollow ceramic candles which fasten to the bottom of the upper basin and they extend into the
unfiltered water of the upper basin. The ceramic candles typically are made of sintered diatoma-
ceous earth. Some filter candles now have an activated carbon core, contain exchange resins, and
they may be silvered. The effective pore size of the ceramic candles is graded and seems to range
from 0.5 to 0.9 microns. Use of this image is courtesy of the author
238 C. J. Hurst

water through the ceramic material. Figure 8.33 shows how ceramic candle filters
commonly are used to create a household water filtration device which uses gravity
as the source of hydrostatic pressure. The ceramic candle filters are similar in
concept to the ceramic porous pot filters, except that for the porous pot filters the
upper basin is itself the filter (Oxfam 2012). The filter housing for ceramic candle
filters can have a sealed basin and the system be designed to operate inline connected
to the plumbing of a home water supply. There also are large volume commercial
scale applications for ceramic candle filters. In terms of maintenance, it periodically
is necessary to scrub away large solids that have collected in a layer on the outer side
of the filter candles. Ceramic candle filters typically have effective pore sizes ranging
from 0.5 to 0.9 microns and are able to remove most bacteria and protozoa.
Importantly, not all bacteria will be removed by these ceramic filters as some
bacteria have a diameter smaller than 0.5 microns. There are newer composition
ceramic filters that can achieve partial removal of particulates down to 0.2 microns.
The most modern designs for ceramic candle filters incorporate a first stage which
consists of the ceramic body, a second stage anti-bacterial matrix integrated into the
ceramic mix, a third stage which is an inner core of activated carbon block that can
retain organic chemicals from the water that is being filtered, a fourth stage which is
an ion exchange resin intended to reduce lead and other heavy metals but potentially
also reducing any silver that was added for bacteriocidal quality, and a fifth stage
which imparts silver to the water. Indeed, often now the ceramic filter candles are
silvered as is done with ceramic porous pot filters to provide some antibacterial
disinfectant capability for the filtered water.
The housing containers in which ceramic candle filters are installed generally
now are made of either stainless steel or plastic. A common problem is that people
often clean the outside surfaces of the ceramic candles and then clean the inside of
the bottom container with the same cloth. The outside of the candle is likely to be
contaminated and this approach to cleaning the unit will then cause contamination of
the clean water in the bottom container (Oxfam 2012).
The quality and effectiveness of many locally produced types of ceramic candle
filters may be doubtful, as often is the case for ceramic filter pots. It should not be
surprising that ceramic filters are ineffective for removal of mycoplasma and viruses.
Indeed, viruses initially were defined as “filterable viruses” because, before their
biological nature was understood, they were recognized as being infectious and
capable of passing through a ceramic candle filter. The pores in the ceramic material
of filter candles eventually will clog just as will the pores in a porous pot filter, at
which point the ceramic filter candles will need to be replaced. Oxfam (2012) has
suggested that when deciding whether to distribute candle filters, consideration
should also be given to the availability of replacement ceramic candle filters and
taps. It is important to understand that significant training and follow up will be
needed if people are not familiar with the filters. Another important understanding is
that turbid water may need to be prefiltered through a clean cloth before using the
ceramic candle filter. One means of using cloth to prefilter the water would be to
place the cloth over top of the basin before adding water to the basin, then pour water
into the basin through the cloth.
8 Options for Providing Microbiologically Safe Drinking Water 239

Ceramic filtered water lacks the residual protection that a disinfectant such as
chlorine would provide, and so it is important that users of ceramic filters be trained
to properly care for and maintain the ceramic filters and to not contaminate the
filtered water receptacle.

8.6.2.4 Cloth Filtration

The natural ecology of Vibrio cholerae is its existence as a commensal on the

chitinous shell of aquatic crustaceans including copepods. Ingesting some strains
of that bacterial species will produce the disease cholera, and filtering copepods out
of the source water before drinking that water can reduce the risk of cholera disease
(see Hurst Chap. 7, “Briefly Summarizing Our Understanding of Vibrio cholerae
and the Disease Cholera” pp. 173–184). Colwell et al. (2003) suggested the use of
multiply folded Sari cloth as a filter method applicable at the household level for the
purpose of removing copepods from intended drinking water so as to reduce the
incidence rate of cholera. Household use of cloth filtration is present in Fig. 8.14.
Colwell et al. (2003) also presented alternative suggestions for reducing the inci-
dence of cholera, those being either the use of nylon netting for removing copepods
or boiling of the water, although for some households boiling drinking water
represents the use of expensive firewood. Regarding the Colwell et al. (2003)
reference, there are notes on its first page about boiling plus instructions at the top
of its page 1053 for using the cloth filtration technique. Huq et al. (2010) discovered
by analysis of data that several years after initiation of a cloth filtering project 31% of
the community’s women still used filtration for household water, of which 60% used
sari cloth filtration. Those results showed that sari cloth filtration not only was
accepted and sustained by the villagers and benefited them in reducing the incidence
of cholera, but that use of filtration also benefitted their neighbors who did not filter
water. The benefit to neighbors was unexpected and presumably relates to filtration
having reducing primary transmission via ingestion of contaminated water, and by
having prevented some cases of primary transmission there would have been a
subsequent benefit of reducing the chance that neighbors would acquire cholera by
person-to-person secondary transmission. The reference by Hurst (2018) discusses
primary and secondary transmission routes.
The two types of cloth suggested for this water filtration technique have a large
effective pore size and cannot be expected to remove either bacteria or viruses,
unless those categories of microorganisms are attached either to large suspended
solids or to other particulates such as copepods. Cloth filtration is useful for straining
out suspended natural solids to prevent clogging of ceramic filters. The technique of
cloth filtration possibly can increase the effectiveness of solar disinfection and would
do that mostly by eliminating solids that either can block or absorb ultraviolet
radiation. Removal of solids additionally may reduce the chance of microbial
regrowth following solar disinfection. Cloth filtration also can remove solids that
are produced by the coagulation and flocculation processes which result from using
240 C. J. Hurst

Fig. 8.34 This image is

titled “A typical home RO
system” including
(1) particle filter, (2) reverse
osmosis membrane unit,
(3) pressurized treated-water
storage container, (4) carbon
adsorption post-filter and
(5) separate treated-water
tap. It is by North Dakota
State University, authors
Roxanne Johnson and Tom
Scherer, and being used
under a Creative Commons
Attribution-share alike 3.0
license. https://www.ag.
ndsu.edu/pubs/h2oqual/
watsys/wq1047.pdf

such compounds as alum, ferric sulfate, and Moringa extracts to help clarify water
prior to disinfection of the water.

8.6.2.5 Reverse Osmosis

Reverse osmosis filtration also is available as a household technology Fig. 8.34

(Johnson and Scherer 2013). Yari et al. (2018) studied the microbiological quality
associated with household water desalination devices that included reverse osmosis
membranes. Those devices were being purchased and used by community members
because of the high levels of total dissolved solids in their community provided tap
water. Yari et al. (2018) determined that the heterotrophic bacterial level in the
output water of those household water desalination devices was greater than the
bacterial level found in the input water, and presumably that increase was due at least
in part to development of a biofilm in the filtration units.
8 Options for Providing Microbiologically Safe Drinking Water 241

8.6.3 Disinfection

There are several techniques that can be used to achieve disinfection of drinking
water for individual households. Those most commonly suggested are chlorination,
solar disinfection which is a thermal pasteurization, and boiling. Ion exchange
disinfection also can be done (Agrawal and Bhalwar 2009) and that primarily uses
iodine in the form of either tri-iodide or penta-iodide exchange resins. Portable and
point-of-use devices that use iodine exchange resin have been developed and
extensively evaluated for the inactivation of waterborne pathogens, primarily in
developed countries. Most of these iodide exchange resin devices are designed as
pour through cups, pitchers, and columns through which water is passed so that
microbes come into contact with iodine on the resin. That technology is too
expensive and complex for much of the world’s household needs, although when
cost is not an issue as with the water recycling that takes place on the International
Space Station, iodine resins can become very important and that will be discussed
later in this chapter.

8.6.3.1 Chlorine Disinfection

Chlorine disinfection is an important technique that, given sufficient contact time

and disinfectant concentration, seems effective against all microorganisms although
destruction of protozoan cysts and oocysts can require lengthy periods of chlorine
exposure. It is important to leave a residual level of chlorine in treated water to
protect against accidental recontamination of the water. The available options for
household disinfection using chlorine are sodium hypochlorite which often is
obtained as liquid chlorine laundry bleach (Centers for Disease Control and Preven-
tion 2017; United States Environmental Protection Agency 2017), calcium hypo-
chlorite which is a power and can be purchased for household usage as powdered
bleach, and sodium dichloroisocyanurate which most commonly is available as a
tablet (Clasen and Edmondson 2006).
Sodium dichloroisocyanurate is considered to be more stable that is the sodium
hypochlorite in household bleach (Clasen and Edmondson 2006). Sodium
dichloroisocyanurate is available as effervescent (self-dissolving) tablets which
will disinfect two quarts of water per tablet. It is recommended that turbid water
should first be allowed to settle and then filtered through either a clean cloth, paper
towel, or a coffee filter, if such filters are available, prior to treating the water with
sodium dichloroisocyanurate and then drinking the water (Federal Emergency
Management Agency 2006). There is a general note of caution which must be
considered when treating groundwater that contains reduced forms of arsenic, iron,
manganese, and sulfur, which can react with chlorine-based disinfectants and effec-
tively increase the waters chlorine demand. Naser et al. (2018) is a reference for this
reaction, and in their study they noted that iron present in groundwater did affect the
level of residual chlorine which remained after water was treated with sodium
242 C. J. Hurst

dichloroisocyanurate. Chlorine treatment (Laurent 2005; International Network to

Promote Household Water Treatment and Safe Storage 2007) often is used as a
disinfection technique in combination with coagulants and flocculants, and those
combination techniques will be presented later in this chapter.

8.6.3.2 Pasteurization, Boiling, and Distillation

Boiling is a standard for effectiveness in destroying the infectivity of microorgan-

isms in water. The World Health Organization’s 2015 publication provides a
summary table of microbial inactivation rates by temperature and time for bacteria,
protozoa and virus and clearly shows that a good amount of safety can be accom-
plished by heating water to less than the boiling point. Heating liquid to a specified
temperature that is below the boiling point is termed pasteurization, and the exact
combinations of temperature and time that are used for achieving pasteurization vary
quite a bit. It is better to store the thermally treated water within the same container in
which it was either pasteurized or boiled because thermal treatment will not leave
any residual disinfection capacity in the water as would an alternative treatment such
as chlorination.

8.6.3.2.1 Solar Water Disinfection

Using sunlight to pasteurize water has been suggested and researched by many
people. Pasteurization with sunlight, which is called solar water disinfection and
known by the acronym SODIS, has been done by placing water into clear polyeth-
ylene terephthalate (PET) plastic bottles and then exposing those bottles of water to
sunlight. A good general reference on solar water disinfection would be that by Luzi
et al. (2016). Figure 8.35 shows examples of solar water disinfection. As seen in
Fig. 8.35, the outside of PET bottles may be painted black to increase the rate at
which the contained water can be heated by exposure to sunlight. Ciochetti and
Metcalf (1984) experimented with using a solar box cooker to help heat glass
containers of river water for achieving disinfection. The technique of solar disinfec-
tion offers advantages in that it is inexpensive, easily understood, and easy to apply
at either zero or very low cost. Solar disinfection is independent of energy sources
other than sunlight, and is independent of the supply chains needed for chemical
disinfectants. Solar disinfection is dependent upon access to PET bottles, although
those are available as discarded items virtually everywhere. Solar disinfection does
involve a relatively high labor demand and also this technique requires a long
treatment time. Treatment by SODIS may take 6 h in sunlight and 2 days if the
sun is obscured by clouds. Two days may well be a prohibitive length of time for
people to await a drink of water. Also, solar water disinfection can have limited
aspirational appeal in that this is perceived to be a ‘poor people’s method’. A very
serious detriment is that the water is warm following treatment and people do not
find it refreshing to drink warm water. Another drawback to solar disinfection is that
8 Options for Providing Microbiologically Safe Drinking Water 243

Fig. 8.35 Solar pasteurization of water also is called solar disinfection and abbreviated SODIS.
The upper image is titled “Indonesia-sodis-gross” by SODIS Eawag and is being used under the
Creative Commons Attribution 3.0 Generic license. The lower image shows water in a clear plastic
244 C. J. Hurst

the heated water has no residual disinfectant capacity, and unfortunately some
components of the microbial population that survive thermal treatment can experi-
ence regrowth in the treated water.
There are thermal indicators which consist of small sealed tubes containing a low
melting wax that can be inserted into the bottles and those indicators will show when
the water has reached a safe temperature because the wax will have liquified
(Safapour and Metcalf 1999). Safapour and Metcalf (1999) used a cardboard reflec-
tor to redirect sunlight onto water contained in a black painted jar, and inside the jar
those researchers had placed the reusable water pasteurization indicators (WAPI),
which were a clear polycarbonate tube partially filled with a soybean wax that melts
at about 70
C. It was suggested that if the WAPI tube wax melts and falls to the
bottom of the tube, then it would indicate that pasteurization conditions had been
reached.
Information from Keogh et al. (2017) has indicated that ideally SODIS treatment
requires the water to have a turbidity of less than 30 NTU (nephelometric turbidity
units), although turbidities of natural water can exceed 200 NTU. Those authors
suggested that reducing the turbidity of water would assist in making certain solar
disinfection is effective (Keogh et al. 2017). Another point to notice is that, if the
water initially has a high turbidity, then biofilms as well as sludge layers can develop
within those bottles that contain the treated water. Bacterial regrowth can occur in
those sludge layers. Keogh et al. (2017) indicated that reducing turbidity prior to
solar disinfection may reduce the potential regrowth of bacteria (Keogh et al. 2017).
Keogh et al. (2017) suggested improving microbial stability of highly turbid water
by first using Moringa seed powder as a flocculation agent, subsequently decanting
the cleared water, and then using SODIS to pasteurize the decanted water.
It has been mentioned several times in the literature that the solar disinfection
process will receive benefit from the sun’s ultraviolet light radiation, but it is not
clear how much ultraviolet light actually would be transmitted into the water
containers (Agrawal and Bhalwar 2009).

8.6.3.2.2 Boiling

Boiling water for a sufficient length of time will make the water safe to drink and the
recommendations for duration of boiling range from 1 min to 5 min (Doerr and
ECHO Staff 2005), Federal Emergency Management Agency (2006), United States
Environmental Protection Agency (2017). The length of boiling time required is
dependent upon the altitude at which the boiling occurs (United States Environmen-
tal Protection Agency 2017). Boiling is a simple and very effective way of killing all
classes of microorganisms although the needed fuel can be expensive. Boiling water

Fig. 8.35 (continued) bottle versus a bottle whose exterior has been painted black to increase solar
heating and usage of the image is courtesy of the author
8 Options for Providing Microbiologically Safe Drinking Water 245

Fig. 8.36 The upper image

shows an installed electric
water boiling apparatus. The
image is titled
“Kochendwassergerät” by
Tetris L and is being used
under the Creative
Commons Attribution 1.0
Generic license. The lower
image shows a designated
pot with a lid for boiling and
then storing water, a
translation of the lettering on
this pot is ‘Boiled Water’.
Usage of the lower image is
courtesy of the author

involves the possibility of scalding accidents due to the very hot water temperatures,
and that is a particular hazard if small children are assigned responsibility for
accomplishing the task of boiling the water. Boiled water easily can become
recontaminated once it becomes cooled because the boiled water lacks any disin-
fectant capability (World Health Organization 2017) and for that reason boiled water
should be stored carefully, kept covered, and preferably be kept in the same
container that was used for the boiling. In at least Colombia, it is possible to buy
pots marked “Aqua Hervida” [boiled water] made just for the purpose of boiling
water and then storing the boiled water covered in the same container. There also are
commercial kitchen appliances which will boil water automatically and then allow
easy dispensing of the water (Fig. 8.36).
246 C. J. Hurst

Fig. 8.37 These images show the components and assembly of a solar still designed for using heat
from sunlight to distill water. Usage of these images is courtesy of the author

8.6.3.2.3 Distillation

Distillation will eliminate all microbial contaminants, salts, and turbidity. Effective
distillation can be done using either solar energy or fuel as a source of heat (Skinner
and Shaw 2019c; Federal Emergency Management Agency 2006). Figure 8.37
shows a solar distillation apparatus for which the design is based upon using a
small collecting bucket placed inside of a larger bucket. The larger bucket will hold
the water that is to be distilled. All of this is covered over with a sheet of clear plastic
8 Options for Providing Microbiologically Safe Drinking Water 247

that is sealed to the outer sides of the large bucket using tape. A rock sits as a weight
atop the center of the plastic, directly above the center of the collecting bucket, so
that the condensed water will drip into the collecting bucket. A section of relatively
small diameter plastic tubing, one end of which is inserted into the collecting bucket
and the other end of which extends beyond the outside of the solar still, is used to
obtain distilled water from the collecting bucket using either suction or siphoning. A
section of large diameter plastic tubing with a funnel attached to one end of the
tubing is used for supplying the outer bucket, that funnel is on the end of the tube
which extends outside of the still. The outside of the large bucket can either be
painted black or covered with black plastic to increase the effectiveness of having
sunlight heat the water.

8.6.3.3 Combination Treatments that Are Designed to Both Remove

Turbidity and Disinfect the Water

Combined flocculation and disinfection can be done using commercially produced

products that are available as tablets and powders. The use of ferric sulfate as a
coagulant with sodium dichloroisocyanurate as a disinfectant has been evaluated by
Légaré-Julien et al. (2018) whose studies used commercially marketed double-
layered tablets for which a 5 min manual stirring period is recommended, followed
by 55 min of settling, and then cloth filtration. Allowing a settling period of twice
that time was suggested for cold water temperatures (Légaré-Julien et al. 2018). The
use of ferric sulfate as a coagulant in combination with calcium hypochlorite as a
disinfectant has been evaluated by several researchers and that combination is
available as a commercial powdered product called “PUR Water Purification
Sachets” that are sold in sealed packets. The procedure used for that product is to
add the contents of a sealed packet to an open container holding 10 L of water, stir
for 5 min, let the solids settle to the bottom of the bucket and then filter the water
through a cotton cloth into a second container. After that filtration, there is a waiting
period of 20 min for the hypochlorite to further inactivate microorganisms (Centers
for Disease Control and Prevention 2014a; Laurent 2005; United Nations Develop-
ment Programme 2007). It has been reported that prevalence of diarrhea was
approximately 40% lower in households which used that combination of ferric
sulfate as a coagulant in combination with calcium hypochlorite as disinfectant
(Chiller et al. 2006). Crump et al. (2004) compared the combination of ferric sulfate
and calcium hypochlorite versus using either alum alone, sodium hypochlorite alone,
and alum plus disinfection with sodium hypochlorite. The findings of Crump et al.
(2004) were that: the combined flocculant-disinfectant product would be useful in
settings where source waters are both highly microbially contaminated and turbid,
and that all high turbidity samples treated with alum alone failed to reach the World
Health Organizations bacteriologic potability standard.
248 C. J. Hurst

8.7 The Water Recycling Unit Used on the International

Space Station

When humans began sealing ourselves into living quarters that kept us isolated from
the natural life support systems of the terrestrial surface on which we had evolved, it
became necessary to develop artificial life support systems which could sustain us
for at least a short while. Efforts at developing artificial life support systems began in
1896 with the Holland submarine “Plunger” and have progressed to the closed
ecological systems that are being used onboard the International Space Station
(Hurst et al. 1997).
Figure 8.38 shows the designs of several earth orbital “Space stations” as they
have been imagined and of one which exists in our present reality. The lower left part
of that image is titled “Space Wheel” by Syd Mead and is a futuristic suggestion
from the 1950’s of what an earth orbital station eventually might look like. The
central part of that image is a drawing from the beginning of this century titled
“Global Economy Saipan” by Andrew Au that hangs on my office wall, and for me it
depicts construction of biomechanical orbital stations. The upper right part of that
image is a photograph of the International Space Station.
The prospect of prolonged residence in space with either minimal or no resupply
from earth has presented an enormous technical challenge. One of the most techni-
cally challenging tasks has been the successful adaptation of water treatment tech-
nology for use by crew members onboard the International Space Station, which
represents a very isolated household. The water recycling unit on the International
Space Station was designed with the goal of providing 10 L of drinkable water per
day for each person in the station. All possible sources of water available to the
International Space Station need to be collected and recycled. The only supplemental
water is that which comes on resupply flights from earth. I was fortunate to have
participated in testing the viral removal efficiency for that recycling unit (Roman and
Hurst 1998). Combining treatment technologies into a multistage process allows for
increased assurance of health safety. My guess would be that successfully removing
chemicals was more challenging than the demands required for microbial removal.
The sources of water used for testing the recycling unit came from facilities at the
NASA (National Aeronautics and Space Administration) Marshall Space Flight
Center in Huntsville, Alabama where the testing was conducted. Figure 8.39
shows some of those water sources. The upper left image shows the closed entry
door to the testing room, which was a temporary building housed inside of a larger
building. Much of the source water would be collected from peoples usage of that
testing room. The person seen in the upper left image has walked across a tacky sheet
on the floor to remove particulates from the bottoms of his shoes, and he is shown
using a powered shoe brush required to eliminate particles from the sides of his shoes
prior to his entering that room. Inside of that room was a shower shown in the upper
right image, from which the drainage water was collected. The room also contained
toilet facilities shown in the lower left image, from which the urine provided an
additional source of water. The lower right image shows a laundry washing machine
8 Options for Providing Microbiologically Safe Drinking Water 249

Fig. 8.38 This image shows the designs of earth orbital space stations and is a composite created
by Christon J. Hurst, used with his permission. The upper right image is a photograph of the
International Space Station, is titled “International Space Station after undocking of STS 132” by
NASA/Crew of STS-132 and it is a public domain image. The central image is titled Macromeme:
Global Economy Saipan by Andrew Au, imagined as depicting construction of biomechanical
orbital stations and is used here with permission of the owner. The lower left image is titled “Space
Wheel”, Copyright© Syd Mead, Inc. www.sydmead.com and used with permission

which supplied spent water and a clothing dryer from which escaping humidity was
collected. That laundry equipment was not in the testing room. Figure 8.40 shows
the other areas inside of the testing room. The upper image shows a sink from which
water used for hand washing, wet shaving and tooth brushing was collected, and a
microwave oven which would have released some steam to also be recycled. The
lower image shows exercise equipment plus shirts which had been worn during
250 C. J. Hurst

Fig. 8.39 These images show sources of water that were used for evaluating the water reclamation
unit developed at NASA in Huntsville, Alabama for the International Space Station. Most of the
water was collected from sources located within in a small temporary structure contained inside of a
much larger building. Clockwise from the upper left these photographs show: a person using a
powered shoe brush to clean their shoes before entering the temporary structure; a shower bath; a
laundering machine and clothes dryer; and a toilet with urine and fecal collection separated. The
laundering machine and dryer were housed separately. Usage of these images is courtesy of the
author
8 Options for Providing Microbiologically Safe Drinking Water 251

Fig. 8.40 These images show additional sources of water that were used for evaluating the water
reclamation unit developed at NASA in Huntsville, Alabama for the International Space Station.
The upper image shows a sink used for hand washing and dental hygiene plus a microwave oven for
heating meals. The lower image shows exercise equipment and t-shirts hung to dry. In the bottom
center area of the lower photograph condensate from a room dehumidifier can be seen collecting
into glass storage containers. Usage of these images is courtesy of the author
252 C. J. Hurst

exercising and then been hung up to collect the moisture which would evaporate
from those shirts. The water collected from all of those sources was registered by weight.
When the quantity of water collected from those sources was not sufficient, it was
supplemented with ‘ersatz’ water which contained a defined list and concentrations of
compounds.
Figure 8.41 shows the water recycling unit. The wastewater recycled by this unit
included a combination of water containing human metabolic waste from humidity
condensate, showering, hand washing, urine distillate, tooth cleaning, and facial wet
shaving plus water from laundering. A laboratory prepared solution also was
processed which represented the equivalent of water from fuel cells and a mixture
analogous to animal humidity condensate plus equipment off-gassing contaminants.
Processing of the water begins with the upper left image in which can be seen two
large stainless steel pressure cans that hold the water mixture to be treated. The upper
left image also shows a thin vertical white cartridge filter that removes particulates
from the water. The step of particulate filtration is followed by the water passing
through resin columns packed in stainless steel tubes which are called Unibeds,
shown in the upper right image. The Unibeds include ion exchange resin, organic
adsorption resin, inorganic adsorption resin, and iodine release resin. The iodine
subsequently is removed by passage of the water through an iodine removal resin.
Catalytic oxidation is the next stage of treatment, shown by equipment in the lower
right image, and that treatment consists of oxygenation, high temperature
catalyzation, and de-gassing. The water next passes through an ion exchange column
that releases iodine. Finally, the product water is stored in containers behind the
white panels that are shown on the bottom of a rack in the lower left image and the
water will be filtered yet once more prior to use. That recycling system was
repackaged to fit into a double wide rack for mounting in the International Space
Station. Figure 8.42 shows a display which demonstrates what a double rack looks
like from the front and how that rack unit fits into the curved sides of the space
station. The water recycling processor shown in Fig. 8.41 needed to be reconfigured
to fit into a double rack.
8 Options for Providing Microbiologically Safe Drinking Water 253

Fig. 8.41 These images show the International Space Station Water Processor. Clockwise from the
upper left these images show the sequence of water processing: the stainless steel tanks which store
wastewater that is to be processed and visible in the lower left quadrant of the metal rack is a
particulate filter with 0.5 micron porosity; stainless steel tubes called unibeds which contain ion
exchange and adsorbent resins; a catalytic oxidation reactor plus a process control monitor; and two
enclosed storage tanks on the bottom half of a rack for containing the processed water. Usage of
these images is courtesy of the author
254 C. J. Hurst

Fig. 8.42 These images demonstrate what a double width equipment rack looks like for the
International Space Station and how those racks fit against the interior curved sides of the space
station. The water recycling unit was reconfigured to fit into one double width equipment rack.
Usage of these images is courtesy of the author

Compliance with Ethical Standards

Conflict of Interest Christon J. Hurst declares that he has no conflict of interest.

Ethical Approval This article does not contain any studies with human participants or animals.

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Chapter 9
Microbiome of Drinking Water Distribution
Systems

Laurence Mathieu, Tony Paris, and Jean-Claude Block

Abstract Drinking water distribution systems appear as vulnerable engineered

systems constantly sown and colonized by microorganisms representing several
hundreds of species. Such microorganisms originate firstly from finished potable
drinking water, secondly from growth in the distribution systems in the bulk water
and on the surface of pipes and reservoirs as biofilms, and thirdly from accidental or
continuously low introduction of pathogens.
This biomass is a source of technological problems (microbiologically influenced
corrosion, red water, odor, and taste of tap water) caused by some specific microbes.
Investigation and control of the microbial population community and its behavior in
drinking water distribution systems should be the cornerstone of efforts to protect
distribution system integrity, water quality, and public health. This silent contami-
nation could also be responsible for endemic gastrointestinal illnesses attributable to
tap water meeting current standards.
As shown throughout this literature review (mainly limited to papers published
from 1990 to 2014), distribution systems must be considered as bioreactors. First, we
present bugs systematically found in drinking water distribution systems all over the
world (bacteria, viruses, yeasts, fungi, protozoa, microcrustaceans, rotifers, and
oligochaete worms). Then, we analyze and discuss several items related to biofilms
grown under conditions relevant to drinking water environments including mecha-
nisms of biofilm formation, structure, cohesiveness, biodiversity, and pathogen

L. Mathieu (*)
EPHE-PSL Research University, LCPME, Nancy, France
Laboratoire de Chimie Physique et Microbiologie pour les Matériaux et l’Environnement,
CNRS-Lorraine University, Villers-lès-Nancy, France
e-mail: laurence.mathieu@univ-lorraine.fr
T. Paris
Eurofins – IPL Est, Nancy, France
J.-C. Block
Laboratoire de Chimie Physique et Microbiologie pour les Matériaux et l’Environnement,
CNRS-Lorraine University, Villers-lès-Nancy, France

© Springer Nature Switzerland AG 2019 261

C. J. Hurst (ed.), The Structure and Function of Aquatic Microbial Communities,
Advances in Environmental Microbiology 7,
https://doi.org/10.1007/978-3-030-16775-2_9
262 L. Mathieu et al.

reservoirs. Finally, the chapter concludes with a review of some of the parameters
governing biofilm accumulation in drinking water distribution systems.

9.1 Introduction

Drinking water distribution systems have been in use for several centuries, but the
first modern ones meant for transporting drinking water in every part of a city were
built at the beginning of the eighteenth century. Such public drinking water supplies
have a substantial impact on public health as it gives access to safe, potable drinking
water and de facto prevents the epidemiological transmission of waterborne patho-
gens. Most drinking waters are transported (after more or less complex treatments
depending on the quality of the resource) to the consumers thanks to underground
distribution networks, which represent huge and costly, engineered systems
consisting of a combination of reservoirs and pipes.
Distribution systems are challenging habitats for micro- and macroorganisms.
However, bacteria, viruses, yeasts, fungi, protozoa, microcrustaceans, rotifers, and
oligochaete worms occur systematically all over the world in reservoirs and pipes
that transport drinking water through municipal water distribution systems. Those
types of organisms also exit in private areas (i.e., after the water meter) representing
household supply infrastructures where high times of retention, small diameter pipes
with high area/volume ratios, and generally higher temperatures favor microorgan-
ism growth that resultantly exceeds guideline values for microbiological parameters
(Völker et al. 2010; Proctor and Hammes 2015). Indeed, many waterborne micro-
organisms become autochthonous in distribution systems despite the relatively low
concentrations of organic and inorganic nutrients needed for their carbon and energy
requirements and addition of chemical disinfectants (generally oxidants such as
chlorine or chloramine).
Live organisms (saprophytic and pathogenic) are everywhere even in the very
recently built drinking water distribution systems. They originate firstly from fin-
ished potable drinking water, secondly from growth in the distribution systems in the
bulk water and on the surface of pipes and reservoirs as biofilms, and thirdly from
accidental or continuously low introduction of pathogens. In the first instance,
distribution systems are constantly seeded with indigenous bacteria escaping from
or produced in drinking water treatment plants (especially from sandfilters or
biofilters) (Servais et al. 1991; Pinto et al. 2012; Bichai et al. 2014; Lautenschlager
et al. 2014). This biomass shapes the downstream microbial water and biofilm
community structure (Prévost et al. 1998; Velten et al. 2011; Pinto et al. 2012;
Wang et al. 2013b). Second, the growth of both saprophytic bacteria, some of which
being of sanitary interest (e.g., Legionella), and microinvertebrates occurs in the
bulk water as well as within biofilms and also loose deposits, all of which contrib-
uting to increasing biomass throughout the network. Third, there occur accidental
rare massive intrusions of pathogens variously caused by ineffective treatments,
treatment breakthroughs, or leakages associated with negative transient pressure
9 Microbiome of Drinking Water Distribution Systems 263

(Besner et al. 2011), and all of which can be the cause of major waterborne outbreaks
(Hrudey and Hrudey 2004; Craun 2012; Ashbolt 2015). There additionally are low
continuous intrusions of fecal pathogens (e.g., rotavirus, Campylobacter jejuni) and
fecal indicators (Westrell et al. 2003; Miles et al. 2009). This silent contamination
could be responsible for endemic gastrointestinal illness rates as described by
Payment et al. (1997), who reported that 14–40% of gastrointestinal illnesses were
attributable to tap water meeting current standards and that the water distribution
systems appeared partly responsible for these illnesses.
Drinking water distribution systems thus appear as vulnerable engineered sys-
tems constantly sown and colonized by microorganisms representing several hun-
dreds of species. Walls of pipes, reservoirs, taps, valves, seals, and fitting accessories
appear as the critical key areas for survival and growth of many of these microor-
ganisms in the form of biofilms. This sessile biomass is also the source of techno-
logical problems (microbiologically influenced corrosion, red water, odor, and taste
of tap water). Investigation and control of the microbial population community and
its behavior in drinking water distribution systems should be the cornerstone of
efforts to protect distribution system integrity, water quality, and public health.
In this chapter, after a brief description of distribution systems that must be
considered as bioreactors, the most relevant scientific publications (mainly based
on those published from 1990 to 2014) about microorganisms and
microinvertebrates in drinking waters will be presented in an overview. Then, we
will analyze and discuss several items related to biofilms grown under conditions
relevant to drinking water environments including mechanisms of formation, struc-
ture and cohesiveness, biodiversity, and pathogen reservoirs. Finally, the chapter
will conclude with a review of some of the parameters governing biofilm accumu-
lation in drinking water distribution systems.

9.2 Drinking Water Distribution Systems: Multiphasic

Unsteady-State Bioreactors

Drinking water distribution networks represent huge systems combining reservoirs

and pipes (as an example, public networks in France equal to approximately
900,000 km in total; the length of private networks could be equivalent or greater).
Pipes of 2 m to 2 cm in diameter are made of very different materials depending on
the year of laying in the past century (Malm et al. 2012). Materials used in networks
are highly diverse: lined or unlined cast iron, ductile iron, galvanized stainless steel
(GS), stainless steel (SS), linings (asphaltic, cement, epoxy), polyvinyl chloride
(PVC), plasticized polyvinyl chloride (PVCp), high-density polyethylene (HDPE),
polyethylene cross-linked with aluminum foil between both layers (PEx), polypro-
pylene (PP), ethylene propylene diene monomer (EPDM), and copper especially in
private houses. All these materials are colonized by microorganisms, whatever the
topographical and physicochemical characteristics of their surface.
264 L. Mathieu et al.

Biofilm 40% Loose deposits 56% W+P 4%

Fig. 9.1 Comparison of viable biomass in percentage (%) (as inferred from ATP results) of
different phases within 1 m-long water pipes (PVC, 110 mm in diameter): from left to right pipe
wall biofilm, loose deposits, and water + particles (W+P) (adapted from Liu et al. 2014a)

9.2.1 Where Is Most of the Biomass Located in the Network?

Drinking water distribution systems have been engineered for transporting water but
work de facto as slow bioreactors producing biomass in different phases of the
system: in the bulk water, on particles in suspension, on pipe walls in biofilm, and in
loose deposits which represent material easily removed from the network by water
flushing. The distribution of the biomass between these phases can differ greatly
from one network to another, but it is generally admitted that most of the biomass is
not found in the bulk water but either attached to surfaces (biofilm) or accumulated
into loose deposits (also called deposits, soft deposits, sediments, or hydraulically
mobile sediments). Indeed, a mass balance carried out by Kjellerup et al. (2006)
showed that 77% of the bacteria were located at surfaces and 23% in the bulk water.
In another work, it was shown that soft deposits easily recovered from the pipes by
water flushing represent the first reservoir of bacteria (in combination with other
bugs), ahead of biofilm, water, and suspended particles (Fig. 9.1). Interestingly, in
the study of Liu et al. (2014a), bacteria from the loose deposit phase (dominated by
Sphingomonas spp.) were clearly different from the bacteria of the bulk water
(dominated by Polaromonas spp.).
In addition, Gauthier et al. (1999a) counted up to 109 CFU/g of dry deposits and
Zacheus et al. (2001) up to 109 bacterial cells and 104 fungi/g. Van Lieverloo et al.
(2012) found that aquatic sow bugs (Asellidae) and oligochaete worms, both known
to have caused rare though embarrassing consumer complaints, represented 98% of
the biomass in deposits flushed from mains. Loret and Greub (2010) underlined that
the concentration of free-living amoebae (FLA) was especially high in sediments,
which constitute ecological niches where they can feed bacteria. Last, Lipponen
et al. (2002) reported high numbers of nitrifying bacteria and nitrification potentials
in the sediments.
Exposure of consumers’ drinking water to particle-associated bacteria appears
low (25–30 cells attached on a single particle leading to 1–3.5
103 cells/mL)
(Stoodley et al. 2001; Liu et al. 2013a). However, the presence of such bacterial
aggregates should not be underestimated as they clearly affect QMRA (quantitative
microbial risk assessment) results especially in the presence of a threshold in the
dose-response relationship (Maul 2014).
9 Microbiome of Drinking Water Distribution Systems 265

Fig. 9.2 Diurnal changes in bacterial parameters (cells and ATP) of water treatment plant (WTP)
and distribution system (DN) at one single point of use on the network. The figure is by Nescerecka
et al. (2014) and used under the Creative Commons Attribution 2.0 Generic license

9.2.2 Water Distribution Systems Work as Unsteady-State

Bioreactors

Steady state in distribution systems could be defined by a constant concentration of

all elements (bacteria, organic matter, chlorine). It cannot be reached in distribution
systems because the water flow determined by consumer use is highly variable (high
during the day; low at night in large pipes or even stagnant in house plumbing) with
some peaks of consumption in the early morning, around noon, and later in the
afternoon (Digiano and Zhang 2004). Flow rates vary from 0 (water stagnation) to
1.5 m/s (during peak hours) leading to variable water residence times from several
days (up to 1 month at worst due to bad hydraulic conditions) to a few hours,
respectively. Water stagnation is known to seriously affect bacterial water quality.
As an example, overnight stagnation at household temperatures results in a signif-
icant increase in bacterial cell concentration in the first samples flushed from the taps
(Lautenschlager et al. 2010).
As a result the microbial characteristics of distributed waters highly vary on a
daily basis due to bacterial growth especially during stagnation episodes and bio-
mass shearing from biofilm and mobilization of loose deposits during high flow rate
events (Fig. 9.2).
266 L. Mathieu et al.

9.2.3 Flowing to the Tap: A Long Way

From treatment to tap, finished drinking waters may travel several days through
miles of pipes. Then, many microorganisms take advantage of this long distance and
period of time to grow (inaccurately called regrowth by some practitioners). The
environmental conditions found in the drinking water distribution systems support
microbial life. Indeed, the so-called oligotrophic drinking waters carry hundreds of
μg/L of assimilable organic matter (AOM), which represent feast for bacteria whose
individual mass is around 0.2 pg (in other words, AOM can support the growth of
millions of bacteria/L). pH is often near the neutrality (ranging between 6 and 8, on
average), and water temperature typically varies in cold waters from 4  C to 25  C
depending on the season and the geographical location. As a result bacterial numbers
increase considerably all along the transport chain (up to one log in some cases)
being favored by concomitant disinfectant decrease (see some field studies, e.g.,
Prévost et al. 1998; Power and Nagy 1999; Niquette et al. 2001; Kormas et al. 2010;
Bal Krishna et al. 2013; Lautenschlager et al. 2013; Nescerecka et al. 2014).
Interestingly, the bacterial community structure may also change along the way.
For example, Lautenschlager et al. (2013) reported that during transport through the
network, the proportion of Proteobacteria phylum varied with an increase in
Betaproteobacteria at the end of the section studied in the network where hydraulic
transport time was greater than 48 h (Fig. 9.3).
In some systems, analysis of the bacterial community and bacterial counts suggest
that temporal variation patterns are stronger than spatial patterns. As an example,
Pinto et al. (2014) showed that the patterns in spatial dynamics were weaker than
those observed for the temporal trends, which exhibited seasonal cycling correlating
with temperature and source patterns and also demonstrated reproducibility on an
annual time scale. McCoy and Van Briesen (2014) analyzing the variance of Alpha-,
Beta-, and Gammaproteobacteria and bacterial concentrations in different sites of a
distribution system suggested that temporal patterns are stronger than spatial
patterns.

0h 44 h 49 h
0.95 105 cells… 1.05 105 cells… 1.3 105 cells…

Alphaproteobacteria Betaproteobacteria Gammaproteobacteria Deltaproteobacteria

Fig. 9.3 Pyrosequencing analysis of drinking water sampled on taps located at different distances
from the plant defined by the transport time of the water from 0 to 49 h for the most distant point
(adapted from Lautenschlager et al. 2013)
9 Microbiome of Drinking Water Distribution Systems 267

9.3 Bacteria and Bugs in Drinking Waters

The numerous and biologically diverse microorganisms and macroorganisms within

distribution systems form complex ecosystems.

9.3.1 A Few Million Bacteria per Liter of Drinking Water

What the consumers and water authorities know on a routine analysis basis about
microbial characteristics of drinking water is very limited and exclusively based on
mandatory controls and, to make it short, on analysis of fecal indicators such as
Escherichia coli along with assessment of the heterotrophic plate count (HPC). HPC
method has been used for decades but is time-consuming (72-h incubation at 20  C)
and provides very different information depending on the physiological state of the
bacteria, the availability of nutrients, and the disinfectant concentration. Moreover,
the use of culture-dependent methods has long been recognized to underestimate the
number of live bacteria (LeChevallier et al. 1980; Martiny et al. 2005). Thus, HPC
numbers found in the literature vary from 0 CFU/mL (chlorinated waters just at the
intake of the distribution system) to 10,000 CFU/mL in distribution systems
(Mathieu et al. 1995; Zacheus et al. 2001; Chen et al. 2013; Vaz-Moreira et al. 2013).
In truth, potable waters (defined by zero cultivable Escherichia coli/100 mL and
zero cultivable enterococci/100 mL) carry millions of bacterial cells per liter.
Throughout many distribution systems all over the world, most of the bacterial
cell numbers as counted either by microscopy after staining with specific fluoro-
chromes or using fast flow cytometry method (FCM) are around 108 cells/L (with
variations ranging from 107 to 5
108 cells/L). Many of those cells are indirectly
defined as active due do their high nucleic acid content (HNA bacteria) (Phe et al.
2005; Berney et al. 2008; Ramseier et al. 2011).
The concentrations of microorganisms could even be higher than indicated by the
cited data because some very small-sized bacteria could have been missed by these
analytical methods. According to Rinta-Kanto et al. (2004) and Wang et al. (2007),
ultramicrobacteria passing through 0.02 or 0.01 μm porosity filters represent 0.1% of
all cells in drinking water. Cultured ultramicrocells isolated from drinking waters
and identified by their 16S ribosomal DNA sequences were closely related to
members of the Proteobacteria, Actinobacteria, and Firmicutes (Silbaq 2009).
Estimation of the viable bacterial biomass in water by assessment of ATP
concentration (a culture-independent method) appears successful, quite easy, and
rapid (Berney et al. 2008; Hammes et al. 2008, 2010a). Good correlation was
observed between ATP concentration not with the total number of cells (Liu et al.
2013b), but with the number of cells with undamaged membranes (Hammes and Egli
2010; Hammes et al. 2010a). Undamaged bacteria in tap waters could represent half
of the total number of cells (Kahlisch et al. 2012). To sum up such the promising
ATP technique could substitute for the currently mandatory HPC method.
268 L. Mathieu et al.

9.3.2 Bacterial Diversity in Drinking Waters: Dominance

of Proteobacteria

Like all natural aquatic environments, water distribution systems are home to
millions of bacteria/L and hundreds of species of microorganisms. Additionally,
each combination of water resource and treatment plant produces drinking water
with a unique bacterial composition.
Different methods have been used to study the structure of bacterial communities
in distribution systems. Culture-dependent methods have long been, and still are,
used for isolating specific groups or species, often those of either pathogenic or
metabolic interest such as Actinobacteria, Actinomycetes, Aeromonas, Helicobacter,
Legionella, Mycobacteria, and nitrifiers depending on the technical or health-related
issues to be addressed. More recently, less than 10 years ago, our understanding of
the microbial ecology of distribution systems greatly improved thanks to molecular
biology investigation, fingerprint determination, and sequencing-based approaches.
These techniques are still being upgraded, and next-generation techniques will
provide less expensive and time-consuming methodologies as well as new software,
tools for analyzing gene sequences, and new data banks (Douterelo et al. 2014a; Liu
et al. 2016).
As shown in Table 9.1, bacteria phyla are very diverse in drinking water (11 phyla
and surely more than 100 species). Interestingly, the 17 publications reported in
Table 9.1 show a large dominance of Proteobacteria whatever the geographical
location, water resource origin, or season. However, each water distribution system
has a unique bacterial composition (especially at the level of genus and species).

9.3.3 Other Bugs in Drinking Water

Drinking water distribution systems harbor a relatively wide-ranging ecosystem

including fungi, amoebae, and microinvertebrates, which form with heterotrophic
bacteria a trophic food chain.

9.3.3.1 Fungi

Fungi are diverse in drinking water and composed of many genera and species
(Hageskal et al. 2009). Aspergillus, Fusarium, Trichoderma, Mucor, Rhizopus, etc.
have been found sometimes in high concentrations (1–2200 CFU/mL) in drinking
water distribution systems (Hageskal et al. 2006; Pereira et al. 2009, 2010; Poitelon
et al. 2009a; Siqueira et al. 2011; Oliveira et al. 2013; Van der Wielen and Van der
Kooij 2013; Al-gabr et al. 2014). Some opportunistically pathogenic species (e.g.,
Fusarium oxysporum and F. dimerum) which are capable of causing life-threatening
9 Microbiome of Drinking Water Distribution Systems 269

Table 9.1 Some examples of phyla and genera identified in drinking water by culture-independent
techniques
Phyla/class and genera
Authors Water sampling Methods (dominants)
Douterelo Pilot made of recirculating 16S rRNA gene PCR Dominance of
et al. loops (HDPE) fed with followed by Proteobacteria (Alpha-
(2013) chlorinated drinking water pyrosequencing > Beta-class) followed by
+ HDPE coupons Firmicutes (Clostridia and
Bacilli classes)
At the genus level:
Methylocystis,
Methylocella,
Sphingopyxis,
Polaromonas,
Hydrogenophaga,
Hyphomicrobium, Clos-
tridium, Bosea, Nevskia,
Mesorhizobium
Douterelo Two sections of a DWDS 16S rRNA gene PCR Dominance of
et al. in the Northwest of followed by conven- Proteobacteria (mostly
(2014b) England composed of tional cloning and Alpha- > Delta-
HDPE or cast iron and fed sequencing > Gamma-class)
with treated surface water > Firmicutes (Clostridia,
Bacilli) > Actinobacteria
> Spirochaeta
At the genus level:
Spirochaeta,
Methylobacterium, Clos-
tridium,
Desulfobacterium,
Flavobacterium,
Lysinibacillus, Pseudo-
monas, Flavobacterium,
Clostridium, Bacillus
Note: Differences in the
structure of the bacterial
community between sam-
ples from polyethylene
and cast iron pipes
Eichler DWDS of the City of 16S rRNA and 16S Bacteroides 
et al. Braunschweig (Germany) rRNA gene-based sin- Proteobacteria (Beta-
(2006) fed with treated surface gle-strand confirmation > Alpha-class)
water + chlorine polymorphism (SSCP) > Actinobacteria
fingerprint Note: DNA-based and
RNA-based fingerprints
differed in their major
taxonomic groups
(continued)
270 L. Mathieu et al.

Table 9.1 (continued)

Phyla/class and genera
Authors Water sampling Methods (dominants)
Henne DWDS of the City of 16S rRNA and 16S Proteobacteria (Alpha-
et al. Braunschweig (Germany) rRNA gene-based sin- + Beta-class)
(2012, fed with treated surface gle-strand confirmation > Actinobacteria,
2013) water + chlorine polymorphism (SSCP) Bacteroidetes,
fingerprint Planctomycetes
Note:
Gammaproteobacteria
proportion increases in
RNA-based fingerprints
Holinger 17 DWDS in cities along 16S rRNA gene PCR Proteobacteria
et al. Arkansas and Mississippi followed by > Cyanobacteria (includ-
(2014) river basins (USA) pyrosequencing ing chloroplasts)
> Actinobacteria (with
Mycobacteria)
 Firmicutes,
Bacteroidetes
Kahlisch DWDS—City of Braun- 16S rRNA and 16S 16S rRNA genes domi-
et al. schweig (Germany) fed rRNA gene-based sin- nated by
(2012) with treated surface water gle-strand confirmation Betaproteobacteria,
+ chlorine polymorphism (SSCP) Bacteroidetes, and
fingerprint Actinobacteria
16S rRNA dominated by
Alpha-, Beta-,
Gammaproteobacteria,
Bacteroides, and
Cyanobacteria
Li et al. Four end points (red water 16S rRNA gene-based Dominance of
(2010) events) of the Beijing followed by conven- Proteobacteria (Alpha-,
(China) DWDS fed with tional cloning and Beta-, Gamma-, Delta-,
treated surface water sequencing Epsilon-class),
+ chloramination Bacteroidetes,
Actinobacteria
At the genus level:
Caulobacter,
Brevundimonas,
Sphingomonas,
Rhodobacter, Afipia,
Hyphomicrobium,
Rhodocyclus,
Ferribacterium,
Propionivibrio,
Gallionella,
Comamonadaceae,
Legionella, Pseudomonas,
Nevskia, Sulfuricurvum,
Flavobacteriaceae,
Sphingobacterium, Acti-
nomyces, Arthrobacter,
Microbacterium
(continued)
9 Microbiome of Drinking Water Distribution Systems 271

Table 9.1 (continued)

Phyla/class and genera
Authors Water sampling Methods (dominants)
Lin et al. Reservoir tanks above 16S rRNA gene PCR Proteobacteria (85%)
(2014) DWDS (Hubei province, followed by  Bacteroidetes (14%)
China), fed with treated pyrosequencing  Actinobacteria (1%)
surface waters from At the genus level:
branches of Yangtze River Enhydrobacter,
+ chlorine dioxide Acinetobacter,
Rheinheimera, Delftia,
Acidovorax, Comamonas,
Blastomonas,
Brevundimonas,
Porphyrobacter,
Sphingobacterium,
Chryseobacterium,
Flavobacterium, Nubsella
Liu et al. PVC pipe sections cut from 16S rRNA gene PCR Dominance of
(2014b) a DWDS located in the followed by Proteobacteria (Beta-
northern part of the Neth- pyrosequencing > Alpha- > Delta-,
erlands and fed with Gamma-class),
unchlorinated drinking  Firmicutes
water > Actinobacteria,
Chloroflexi,
Bacteroidetes,
Nitrospirae,
Acidobacteria
At the genus level:
Dominance of
Polaromonas (69%),
Sphingomonas (13%),
Acidovorax (5%),
Janthinobacterium (4%)
Martiny Loop-shaped reactor 16S rRNA gene-based Dominance of Nitrospirae
et al. equipped with stainless followed by conven-  Proteobacteria (Alpha-
(2005) steel coupons. Aerated fil- tional cloning and > Beta- ¼ Gamma-
tered and groundwater sequencing ¼ Delta-class > Epsilon-
(unchlorinated) class) > Acidobacteria
> Planctomycetes,
Bacteroidetes
Note: Richness showed a
strong seasonal trend
Pinto et al. Full-scale DWDS of Ann 16S rRNA gene PCR Dominance of
(2014) Arbor (Michigan, USA) followed by Proteobacteria (Beta-
carrying a combination of pyrosequencing > Alpha- > Gamma-
treated surface and class), Betaproteobacteria
groundwater (especially Acidovorax
and Georgfuchsia) domi-
nated in summer when
Alphaproteobacteria
(especially
Brevundimonas) domi-
nated in winter
Note: Richness showed a
strong seasonal trend
(continued)
272 L. Mathieu et al.

Table 9.1 (continued)

Phyla/class and genera
Authors Water sampling Methods (dominants)
Poitelon Three DWDS of Paris V6 ribosomal sequence Dominance of
et al. (France) fed with treated tag (SARST-V6) Proteobacteria (Beta-
(2009a, b) surface water + chlorine method  Gamma- > Alpha- and
Water sampled at the outlet Delta-class)
of the plants  Acidobacteria,
Planctomycetes, Chla-
mydiae, Nitrospirae
Prest et al. Full-scale DWDS of 16S rRNA gene PCR Proteobacteria (Beta-
(2014) Kralingen (Rotterdam area, followed by class mainly)
Netherlands) fed with pyrosequencing > Planctomycetes
treated surface water > Bacteroides
+ chlorine dioxide > Chloroflexi,
Acidobacteria,
Firmicutes,
Actinobacteria
Note: Combined
pyrosequencing with flow
cytometry data
Revetta Cincinnati DWDS (Ohio, 16S rRNA (RT PCR) Proteobacteria
et al. USA)—treated surface followed by conven- > Cyanobacteria,
(2010) water tional cloning and > Actinobacteria,
Water sampled at the tap sequencing Bacteroidetes,
Planctomycetes,
Firmicutes,
Actinobacteria
Note: Dominance of
unclassified bacteria
(>50%)
Vaz- Full-scale DWDS fed with 16S rRNA gene- Proteobacteria (Alpha-
Moreira chlorinated-treated water denaturing gradient gel ¼ Beta-  Gamma-class)
et al. (Portugal) electrophoresis (DGGE) >>> Actinobacteria
(2013) profiling > Bacteroides
> Cyanobacteria
> Planctomycetes
> Aquificae,
Acidobacteria, Firmicutes
Wang DWDS Pinellas county qPCR + amplicon Proteobacteria dominate
et al. (Florida, USA). Annual sequencing of the 16S (26.8–99.7%) followed by
(2014a) chlorine burn performed rRNA gene Bacteroides,
during late summer to mit- Actinobacteria,
igate nitrification problems Acidobacteria,
in chloraminated DWDS Firmicutes, and
Verrucomicrobia
At the class level:
Alpha-, Beta-,
Gammaproteobacteria
(48%) followed by
Betaproteobacteria
(continued)
9 Microbiome of Drinking Water Distribution Systems 273

Table 9.1 (continued)

Phyla/class and genera
Authors Water sampling Methods (dominants)
(16.9%) and
Alphaproteobacteria
(12.7%)
Note: Burn stages shift
dominance to
Bacteroidetes, Firmicutes,
etc.
Williams Cincinnati DWDS (Ohio, 16S rRNA gene-based Alphaproteobacteria
et al. USA)—treated surface followed by conven- dominate in both chlori-
(2004) water—ductile iron pipe tional cloning and nated and chloraminated
loops + chlorine or sequencing DWDS. Other phyla
chloramine detected Cyanobacteria,
Firmicutes,
Betaproteobacteria,
Gammaproteobacteria
At the genus level:
Sphingomonas,
Brevundimonas,
Caulobacter,
Sphingobium,
Acidovorax, Pseudomo-
nas, Xanthomonas,
Legionella, Nitrospira,
Mycobacterium, etc.
DWDS drinking water distribution system, HDPE high-density polyethylene, PVC polyvinyl
chloride

infections in immunocompromised patients should be controlled in healthcare envi-

ronments (Sautour et al. 2012), which means understanding the potential contribu-
tion of drinking water as a source of infectious disease within healthcare facilities.
The disinfection of water systems contaminated with fungi appeared difficult as
many strains are definitively unaffected by chloramine (Ma et al. 2015).

9.3.3.2 Free-Living Amoebae and Small Eukaryotes

Free-living amoebae (FLA) (Naegleria, Acanthamoeba spp., Vermamoeba

vermiformis, etc.) are widespread in environmental waters and distribution systems
[see the review by Loret and Greub (2010) and Thomas and Ashbolt (2011)]. While
many FLA are ubiquitous and harmless to humans, several genera are pathogenic,
particularly Acanthamoeba (keratitis, encephalitis) and Naegleria (encephalitis).
Amoebae were found in 79% of household water samples from Ohio (USA)
particularly in showerheads (52%) and kitchen sprayers (50%) (Stockman et al.
274 L. Mathieu et al.

2011). Their prevalence and concentration correlated with the level of organic matter
and the season (Valster et al. 2009; Marciano-Cabral et al. 2010). Wang et al.
(2012a, c) found the occurrence of Vermamoeba vermiformis to be greater than
that of Acanthamoeba spp., but the opposite was also reported in other distribution
systems (Valster et al. 2009; Lin et al. 2014). Additionally, chlorinated disinfectant
efficiency (bleach, chlorine dioxide, chloramines) is of course dose-dependent, and
cysts are highly more resistant to disinfection than trophozoites (Dupuy et al. 2011,
2014).
FLA can act as hosts for amoeba-resisting bacteria, which represent a major
concern in terms of public health as they can gain virulence by intracellular growth
in amoebae and chlorine cannot easily disinfect intracellular pathogens. FLA diver-
sity and intra-amoebal composition show significant differences among drinking
water distribution systems. Many bacterial genera can be found in amoebae, includ-
ing Stenotrophomonas, Bradyrhizobium, Sphingomonas, Pseudoxanthomonas,
Acidovorax, Legionella, and Chlamydiae (Corsaro and Venditti 2010; Delafont
et al. 2013, 2014).
Besides amoebae, populations of other small eukaryotes can be detected in
drinking waters, such as dinoflagellates, ciliates, and metazoan (Poitelon et al.
2009a; Otterholt and Charnock 2011). Reported cases of diseases traceable to free-
living indigenous eukaryotes in drinking waters seem however very rare.

9.3.3.3 Invertebrates

Invertebrates such as worms and crustacea are likely to be found in drinking water
distribution systems. A large survey of invertebrates in drinking water was carried
out in the Netherlands and showed systematic occurrence but large variations in their
abundance (Van Lieverloo et al. 2012). Aquatic sow bugs (Asellidae, 1–12 mm up to
1500 per m3) and oligochaete worms (Oligochaeta, 1–100 mm, up to 9900 per m3)
were found associated with loose deposits. Smaller crustaceans (0.5–2 mm) domi-
nated in water from mains (Cladocera and Copepoda up to 14,000 per m3). Common
invertebrates in tap water were Rotifera (<1 mm) and nematode worms (Nematoda,
<2 mm). A parallel study done in Denmark showed a nationwide occurrence of
Asellus aquaticus (<2 mm) (Christensen et al. 2011). Annelida (worms),
Platyhelminthes, and mollusks (snails) were also found (Fig. 9.4). In all the systems
studied, the microbial quality of drinking water was high, and some biotic relation-
ships were shown with corrosion, organic matter, and biofilm (Christensen et al.
2011; Van Lieverloo et al. 2012). Apart from the esthetic problems and some
consumer complaints caused by larger animals like A. aquaticus, no hygienic
problems seem to be associated with such bugs.
9 Microbiome of Drinking Water Distribution Systems 275

Fig. 9.4 Microinvertebrates sampled from a non-chlorinated drinking water supply. (a) Adult and
juvenile Asellus aquaticus (Malacostraca), (b) seed shrimp (Ostracoda), (c) flatworm (Turbellaria),
(d) land slug from a clean water tank, (e) Cyclops sp. (Maxillopoda), (f) Tubifex sp. (Clitellata), and
(g) springtail (Entognatha). Reprinted from publication by Christensen et al. (2011), with permis-
sion from Elsevier

9.4 Drinking Water Biofilm Development

Drinking water biofilms result from a dynamic process of assembly of microorgan-

isms, on surfaces, which is controlled by both physical and biological mechanisms.
The outcome is a constantly renewed organo-mineral layer on the pipe surface,
densely populated with many bacteria and bugs. Its high cohesiveness and biodi-
versity limit its easy cleaning by mechanical and chemical methods and prevent its
complete eradication.

9.4.1 Stages of Biofilm Formation Under Conditions

Relevant to Drinking Water Environments

Colonization of surfaces by bacteria begins through a series of recruitment processes

that are well identified but not so easily depicted and understood in the case of
drinking water. We now know that the typical models of disease-associated patho-
genic bacterial biofilms (i.e., Pseudomonas or staphylococci), which have been
illustrated at the molecular level (regulation pathways, signaling, genetics, etc.),
must have parallels in drinking water systems. But equivalent detailed knowledge is
276 L. Mathieu et al.

missing in the field of drinking water biofilms, which to date have been more
characterized by their structural, mechanical, and phenotypic aspects.
Several stages can be distinguished in the bacterial biofilm development process.
The very first ones are clearly governed by physicochemical processes (convective
transport, diffusion, and reversible adhesion), and then other stages are controlled by
live bacteria (phenotypic differentiation, growth). Finally, the process of detachment
results from both physical shear forces and cell migration under bacterial control.
Understanding these different mechanisms better represents a real challenge
especially for efforts at limiting development of biofilms in drinking water distribu-
tion systems (e.g., by selecting an appropriate material which should be inert,
non-adherent, robust, and inexpensive!). Here, we briefly describe the different stages
of drinking water biofilm development on the surface of new pipes newly installed in
the network, which can be summarized as follows: formation of a conditioning film,
adhesion and growth of bacteria, grazing, and detachment.

9.4.1.1 Preconditioning of Surfaces

Whatever the primary mechanism for biofilm formation may be, it begins with the
attachment of a set of organic polymers recruited from the bulk water and forming
patchy spots upon the substrate onto or besides which bacteria will land. This
so-called conditioning film is formed within a few minutes or hours, essentially as
soon as a material is exposed to natural waters, i.e., waters containing minerals and
polymerlike organic molecules. This preliminary step in biofilm development is
clearly under-explored, and only a few studies have attempted to characterize this
tiny organic deposit. Some of these polymers have been described as gel colloidal
particles consisting of acidic polysaccharides (approximately 0.2 μg/L of xanthan
gum equivalent) (Van Nevel et al. 2012), but glycoproteins could represent the
dominant polymers in conditioning films formed in drinking water (Francius et al.
2017). These first events certainly modify the characteristics of the surface, making it
appropriate (or inappropriate) for further colonization by pioneer microorganisms
(Dang and Lowell 2000).

9.4.1.2 Adhesion of Pioneer Bacteria

Bacterial adhesion in drinking water distribution systems is controlled by the

physicochemical characteristics of both the cells and the pipe surfaces. On the one
hand, negative charges (Walker et al. 2005; Ojeda et al. 2008) are distributed on the
material surface and in the bio-interphase (i.e., the volume formed by the cell
envelopes) of bacteria described as hydrodynamic permeable soft colloids
(Gaboriaud et al. 2008; Gosselin et al. 2011). On the other hand, hydrophobic
polymers in the bacterial envelopes (e.g., as mycolic acids in Mycobacteria enve-
lopes) confer to the cells some hydrophobic properties, which will play a key role in
“locking” the bacteria on the substratum more efficiently than can hydrophilic cells
9 Microbiome of Drinking Water Distribution Systems 277

(Van Loosdrecht et al. 1987; Tatchou-Nyamsi-König et al. 2008). As a result,

depending on the surface characteristics of the pipe material, the polymers recruited
for the conditioning film, and the hydrodynamic shear stress that governs the mass
transfer at the wall, at least 103 bacterial cells/cm2, will adhere onto the surface in a
few hours.

9.4.1.3 Growth of Attached Drinking Water Bacteria

Development of a biofilm on the surface results from both continuous deposition of

cells from the bulk water and bacterial growth. Attachment of bacteria to the pipe
surface via electrostatic and hydrophobic interactions is not harmful for most
bacteria. Live bacteria stay metabolically active and multiply, and some of them
produce exopolymers (EPS), which will reinforce the adhesion of cells and cohesion
of the growing multilayer biofilm (Percival et al. 1998; Flemming and Wingender
2010). The average apparent growth rate (μ) of drinking water biofilms varies from
0.041 per day to 0.2 per day (Block et al. 1993; Boe-Hansen et al. 2002; Manuel
et al. 2007; Park et al. 2012; Pedersen 1990). This dynamic process is seemingly
limited to microorganisms in the upper layer of the biofilm due to diffusion limita-
tion phenomena in the deeper layers. Thus, biofilm activity potential drops rapidly
within the first few days of biofilm development (Kalmbach et al. 1997). Addition-
ally, there is a shift in the bacterial community structure along with an increase in cell
surface area coverage, bacterial richness, and overall community diversity
(Kalmbach et al. 1997; Martiny et al. 2003). For example, Douterelo et al. (2014c)
reported that pioneer species (i.e., Pseudomonas spp. and Janthinobacterium spp.),
which form the initial drinking water biofilm on polymeric material, were partly
replaced by a multispecies biofilm within 3 weeks. Under drinking water conditions,
it seems that cell coverage of the substratum is relatively slow (due to the fact that
nutrients are limited, bacteria produce not only cells but also EPS, and the structure is
continuously subjected to grazing and hydraulic shear stress—see below). Predicting
how long it takes for biofilm to cover 80% of a new pipe surface by using data from
Paris et al. (2007) gives a time of 900 days, which is in agreement with direct
observations from Martiny et al. (2003).

9.4.1.4 Protozoa Grazing “in and on” Drinking Water Biofilms

Free-living amoebae use the biomass of biofilms as food. During the first period of
drinking water biofilm formation, grazing favors large clumps by single cell or small
aggregate consumption. Grazing may be very effective and was estimated equally to
a consumption rate of 1–2
104 cells/cm2 h (Sibille et al. 1998; Paris et al. 2007). It
impacts biofilm construction by removing 80% of the attached cells in the first weeks
of flowing drinking water into the systems (Paris et al. 2007; Declerck et al. 2009) or
slowing down the average apparent biofilm generation time by up to 47 days
(Pedersen 1990). As shown by Derlon et al. (2012), predation had a significant
278 L. Mathieu et al.

influence on both the total amount and the structure of biofilm developed on
ultrafiltration membranes.

9.4.1.5 Drinking Water Biofilm Detachment

Detachment of bacterial cells or clumps from biofilms is of paramount importance to

contamination of drinking water. It explains that the diversity of the bacteria flowing
in a network is always a combination (in an unknown ratio) of cells originating from
the treatment plant and those detached from the biofilm. Stoodley et al. (2001) using
a drinking water lab reactor showed detachment of single cells and aggregates,
which could contain from 10 to 1.7
103 cells (small aggregates were numerous).
Such a detachment rate estimated by using the mathematical relations proposed by
Van Der Wende et al. (1989) is de facto equal to the growth rate. Values of kshear
given in the literature are of the same order of magnitude, i.e., 0.039 per hour (Bois
et al. 1997).

9.4.2 Biofilm Structure and Cohesiveness

The five stages briefly described above lead to the slow formation of mature
biofilms, which are de facto very heterogeneous. The term heterogeneity refers to
the extent of the nonuniform distribution of any selected constituent in any of the
compartments of the biofilm system, such as the distribution of the biomass, the
microscale chemical gradient, the microorganism species and their spatial organiza-
tion, as well as the genetic expression and physiological state of the bacteria (Stewart
and Franklin 2008; Lewandowski and Boltz 2011).
Drinking water biofilms formed under turbulent conditions are thin systems (from
a few micrometers to more than 100 μm) composed of 105–107 cells/cm2 in patchy
distribution. This limited number of attached cells is clearly the result of both
nutrient diffusion limitations, which prohibit the growth of many members of the
consortium, and continuous detachment and erosion of the biofilm canopy. Under
laminar flow or stagnation conditions, pipe surfaces are also partly covered with
bigger soft, brown deposits composed of cells, organic matter, and iron oxides (a few
millimeters high). Indeed, drinking water biofilms are far from being continuous
films but rather dispersed populations of single cells and aggregates partly covering
the surface area (Fig. 9.5), with sometimes the formation of streamer-like aggregates
(up to 200 μm in length for around 1
106 bacterial cells).
The cohesion of biofilms is related to an exopolymer matrix (EPS) (Flemming
2016), whose composition in biofilms grown under drinking water conditions is not
very well documented due to sensitivity limitations of most analytical techniques
and interferences of metals trapped in the biofilms. Bacteria can produce different
appendages such as curli fibrils (0.1–10 μm with a width of 4–12 nm). The fibrils
consist mainly of CsgA protein, which is assembled in a fibrillar tertiary structure
9 Microbiome of Drinking Water Distribution Systems 279

Fig. 9.5 Examples of biofilms on HDPE coupons colonized by 3-month-old drinking water
biofilms without chlorination and under turbulent flow conditions at 20  C. Microscopic observa-
tions after in situ staining with Sybr-II (left, magnitude
200) and DAPI (right, magnitude
1000)

(Larsen et al. 2007). The presence of adhesins with similar amyloid-like structure
was also described for some Gammaproteobacteria. The tendency to produce
amyloid adhesins seemed more pronounced in oligotrophic environments such as
drinking water distribution systems (Larsen et al. 2007). In brief, by analogy with
laboratory experiments or studies on thick environmental biofilms, it can be assumed
that EPS are composed of polymers and heteropolymers such as polysaccharides,
proteins, glycoproteins, lipids, and eDNA (Flemming and Wingender 2010).
The entanglement of the EPS matrix brings cohesion and viscoelasticity to the
biofilm by forming a net, whose mesh size could vary from 5 nm for the most
cohesive clusters (i.e., those with the lowest detachment shear stress applied with
atomic force microscopy) to 11 nm for the least cohesive clusters (Mathieu et al.
2014; Abe et al. 2012). Entanglement is related to the nature and quantity of the EPS,
and the degree of reticulation of the matrix depends on cation bridging and hydro-
phobic “glue.” By referring both to the radius of gyration exhibited by polysaccha-
rides in the range of 10–100 nm and the long polysaccharides detected in drinking
water biofilms with lengths ranging from 1 to 10 μm (Abe et al. 2011), we can
conclude that the macromolecules within the biofilm curl up, thus leading to higher
entanglement. Mathieu et al. (2014) determined that each macromolecule could
probably generate from 10 to 103 self-contact points and a 1 μm3 biofilm could
contain up to 104 macromolecules. They deduced that the maximum concentration
of contact points due to the macromolecules of the network ranges from 104 to 107
per μm3.
Such a huge network of exopolymers firmly attached to cells and to the substra-
tum makes cleaning of water pipes very difficult. The cohesiveness of small biofilm
aggregates is very high (i.e., their detachment requires high hydrodynamic strength).
Flushing the pipes by rapid circulation of water (generating in this example a
hydrodynamic shear stress of about 10 Pa) removes only large clusters and leaves
on the surface small aggregates of bacteria which will start to multiply. Water
flushing drinking water pipes appears effective in loose deposit mobilization and
shearing of fragile scales but is quite ineffective in the removal of viscoelastic
structures such as biofilms. Effective cleaning procedures should break up the matrix
280 L. Mathieu et al.

and change the elastic properties of bacterial biofilms. Only treatments such as
oxidation induce changes in the mechanical properties of drinking water biofilms
that destabilize the biofilm cohesiveness by creating breaks in the matrix structure
(Mathieu et al. 2014; Tachikawa and Yamanaka 2014).

9.4.3 Bacterial Diversity in Biofilms

Understanding the microbial ecology in distribution systems is still a challenging

task. Without a detailed inventory of the microorganisms growing in distribution
systems, water utilities are forced, in a sense, to “fly blind” when making treatment
decisions (Ingerson-Mahar and Reid 2012). The common questions arising when
trying to study microorganisms in drinking water distribution systems irrespective of
their life style are what types of microorganisms are present, how abundant they are,
how their activities shape the environment or influence other organisms, and how the
environment influences the structure and the function of the microorganisms present.
Different methods have been used in an attempt to address some of these questions
from culture-dependent to culture-independent techniques. The latter (genetic fin-
gerprints, metagenomics, pyrosequencing, and so on) (see the review by Douterelo
et al. 2014a) cast new light and provide exciting information. Still, the role of most of
the bacteria detected in drinking water biofilms remains uncertain and the synergistic
interactions unknown, just like the effect of protozoan grazing and the survival of
intracellular bacteria. However, these new analytical methods in just a few years
have profoundly changed our understanding of drinking water biofilms.
First, we do know that the biodiversity in biofilms is high, as a result of
environmental parameters driven from both the bulk water (nutrients, electron
acceptors, temperature, ions, pH) and the substratum (nutrients or toxic leaching
from the material, physicochemical properties) and because of the heterogeneity of
the organo-mineral deposits, which creates a mosaic of microenvironments. Second,
the persistence of most of the attached bacteria and the development of a metastable
ecosystem generate richness and diversity largely different from that of planktonic
bacteria (Martiny et al. 2005; Henne et al. 2012; Douterelo et al. 2013; Liu et al.
2014a, b; Lürhig et al. 2015).
Bacterial biofilm communities have been for the most part explored in a descrip-
tive way over the last decade. As shown in Table 9.2, three classes of Proteobacteria
(α-, β-, γ-) are systematically present and predominate, and only their relative
abundance varies. Besides Proteobacteria, other more or less dominant phyla are
also detected such as Firmicutes, Verrucomicrobia, Planctomycetes, and
Bacteroidetes. It could also be suspected that in specific situations (e.g., corroded
materials, phosphate or disinfectant treatment, electron donors) some distinctive
populations settle in (Norton and LeChevallier 2000; Martiny et al. 2005). At the
class and genus levels, the inhabiting bacterial flora of drinking water biofilms
clearly shows an extraordinary richness but does not allow us to draw, for the
moment, any consensual scheme of colonization or any understanding and sense
Table 9.2 Some examples of community composition identified in drinking water distribution system biofilms by culture-independent techniques
Dominant phyla and classes
Authors Biofilm sampling Methods (major genera) Comments
Chen et al. Unlined cast iron pipe sections 16S rRNA gene-based Firmicutes, Actinobacteria, and The major bacterial members in
(2013) from a treated surface water from PCR-DGGE followed Proteobacteria (Alpha-, Beta-, iron tubercle community were
China—pipe tubercles scraped re-amplification and conven- Gamma-classes) identical to those in the stagnant
and grounded tional cloning and sequencing of At the genus level: tap water
the major DGGE bands Rhizobium, Pseudomonas, Alpha-, Beta-, and
Lactococcus, Brevundimonas, Gammaproteobacteria shared
Rheinheimera, Arthrobacter, comparable dominance within
Bacillus, Herbaspirillum tubercles and stagnant tap water
Douterelo Pilot made of recirculating loops 16S rRNA gene PCR followed Proteobacteria are dominating Bacterial community composi-
et al. (2013) of high-density polyethylene by pyrosequencing (Gamma- > Beta- > Alpha-clas- tion differed between biofilms
pipes equipped with HDPE cou- ses) followed by Firmicutes and bulk water samples and
pons and fed with chlorinated (Clostridia and Bacilli classes) before and after flushing
drinking water At the genus level: The bacterial composition and
Pseudomonas, Zooglea, community structure of biofilms
Janthinobacterium, and changed between the three dif-
9 Microbiome of Drinking Water Distribution Systems

Sphingomonas ferent hydraulic regimes

Species richness and diversity
tended to be higher at highly
varied flow
Douterelo Pilot made of recirculating loops 16S rRNA gene PCR followed Dominance of Proteobacteria Sequencing analysis of the clone
et al. (HDPE) + HDPE coupons Chlo- by conventional cloning and (Beta- > Gamma- > Alpha-clas- libraries showed changes in the
(2014a, b) rinated drinking water sequencing ses) biofilm bacterial community
At the genus level: composition from day 7 to day 28
Pseudomonas, Gradual increase in species rich-
Janthinobacterium, ness and diversity over time dur-
Methylophilus, ing the 28 days of biofilm
Stenotrophomonas, accumulation
Undibacterium, Dechloromonas, Identify bacterial groups
Acidovorax, Bacteroidetes, involved in initial attachment to
281

(continued)
Table 9.2 (continued)
282

Dominant phyla and classes

Authors Biofilm sampling Methods (major genera) Comments
Flavobacterium, Curvibacter, HDPE pipes and subsequent
Porphyrobacter, Sphingomonas, adhesion
Nevskia, Sphingopyxis,
Novosphingobium,
Methylobacterium, Acinetobacter
Feazel et al. Showerheads from nine cities in 16S rRNA gene PCR followed Actinobacteria, Proteobacteria, Showerhead biofilms enriched in
(2009) the USA by conventional cloning and Firmicutes Mycobacteria, 100-fold above
sequencing At the genus level: the water content
Mycobacterium gordonae, Myco-
bacterium avium, Pseudomonas,
Sphingomonas, Staphylococcus,
Streptococcus, Burkholderia,
Neisseria, Acinetobacter,
Legionella
Henne et al. Different sampling locations of 16S rRNA and 16S rRNA gene- Dominance of Proteobacteria Large differences in biofilm fin-
(2012) the Braunschweig (G) DWDS fed based single-strand confirma- (Alpha-  Gamma- > Beta-clas- gerprints, indicating a high spa-
with treated surface water tion polymorphism (SSCP) ses) > Chlamydiales tial variability in DWDS
+ chlorine fingerprint >>> Acidobacteria, Reduced richness compared to
Planctomycetes, Firmicutes, bulk water
Actinobacteria, Chloroflexi, Higher relative abundances of
Bacteroidetes, Nitrospira single phylotypes in biofilms
Relatedness of RNA-DNA
within the biofilm fingerprints
Hong et al. Two water meters (WMa and 16S rRNA gene PCR followed Dominance of Proteobacteria The two water meters had differ-
(2010) WMb) from Urbana Champaign by pyrosequencing (Beta- > Alpha- > Gamma-clas- ent bacterial community compo-
DWDS (Illinois, USA) ses)  Firmicutes, Deinococcus- sitions
Thermus, Bacteroidetes, Presence of few methanotrophs
Actinobacteria belonging to the
L. Mathieu et al.
 At genus level: Methylococcaceae
Acidovorax, Sphingomonas, (Gammaproteobacteria) due to
Methylophilus, Lysobacter, methane in the groundwater
Porphyrobacter, Bosea, resource
Methylobacterium, Methylocystis,
Bradyrhizobium, Sphingopyxis
Keinänen- Polycarbonate slides in annular 16S rRNA and 16S rRNA gene- Gammaproteobacteria (Nevskia Sequences corresponded to sev-
Toivola reactor connected to Cincinnati based followed by conventional ramosa) and Mycobacterium eral genera containing patho-
et al. (2006) DWDS (Ohio, USA). Fluoridated cloning and sequencing genic species
and chlorinated drinking water Presence of iron-oxidizing,
nitrite-oxidizing genus
Kelly et al. Ductile iron pipe sections from Tag pyrosequencing of bacterial Proteobacteria (Gamma-, Alpha- High seasonal variability
(2014) DWDS, Pinellas county (Florida, 16S rRNA genes of the classes), Actinobacteria Good relationship between
USA) fed with chlorinated drink- extracted DNA At the genus level: diversity and bacterial abundance
ing waters Methylomonas (41% of the
sequences), Acinetobacter, Myco-
bacterium, Pseudomonas,
Methylobacterium
Lin et al. Reservoir tanks of the DWDS in 16S rRNA gene PCR followed Preponderance of Proteobacteria Differences in the bacterial com-
9 Microbiome of Drinking Water Distribution Systems

(2014) Hubei province (China), fed with by pyrosequencing (44.6%) (Alpha-  Beta- munity composition between
treated surface waters from > Gamma-classes) biofilm and water samples
branches of Yangtze River  Gemmatimonadetes Biofilms more diverse microbial
+ chlorine dioxide > Chloroflexi > Bacteroidetes communities than water
Biofilm removed by scraping > Nitrospirae
At the genus level:
Sphingobium, Porphyrobacter,
Hyphomicrobium,
Brevundimonas,
Phenylobacterium,
Nitrosomonadaceae,
Hydrogenophaga, Mycobacte-
rium, Clostridium,
Planctomycetes, Nitrospira
283

(continued)
Table 9.2 (continued)
284

Dominant phyla and classes

Authors Biofilm sampling Methods (major genera) Comments
Liu et al. Particle-associated bacteria sam- 16S rRNA gene PCR followed Dominance of Proteobacteria
(2013a) pled at the entry of three by pyrosequencing (Beta- and Gamma-class  Alpha
unchlorinated DWDS and Delta-class) > Nitrospirae
> Planctomycetes,
Cyanobacteria, Euryarchaeota,
Acidobacteria > Actinobacteria,
Bacteroidetes, Crenarchaeota,
Chloroflexi, Gemmatimonadetes
At the genus level:
Legionella, Nitrospira,
Gallionella, Planctomycetes,
Caulobacter, Hyphomicrobium,
Aquabacterium,
Comamonadaceae,
Nitrosomonas, Crenothrix,
Caldilinea, Rhodopirellula,
Rhodospirillaceae, Leptolyngbya
Liu et al. PVC pipe sections cut from a 16S rRNA gene PCR followed Dominance of Proteobacteria Stable bacterial communities in
(2014a) DWDS located in the northern by pyrosequencing (Alpha-  Beta-, Gamma-class), bulk water, pipe wall biofilm, and
part of the Netherlands and fed >> Actinobacteria (3%), suspended solids throughout the
with unchlorinated drinking water Chloroflexi (2%), Bacteroidetes distribution system
(2%), Nitrospirae (1%), Bulk water bacteria (dominated
Firmicutes (1%), and by Polaromonas spp.) were
Acidobacteria (1%) clearly different from the biofilm
At the genus level: bacteria (dominated by
Dominance of Sphingomonas Sphingomonas spp.)
 Janthinobacterium,
Pseudomonas
L. Mathieu et al.
Liu et al. PVC and cast iron faucets located 16S rRNA gene PCR followed Dominance of Proteobacteria Difference in bacterial composi-
(2014b) in a DWDS of Hubei province, by pyrosequencing (Alpha-  Beta-, Gamma-class), tion of the PVC biofilms (pre-
China Actinobacteria dominance of Hyphomicrobium)
At the genus level: and cast iron biofilms (corrosion-
Hyphomicrobium, associated bacteria)
Aquabacterium, Acinetobacter, Bacterial communities in the
Limnobacter, Mycobacterium, bulk water and biofilm samples
Nevskia, Porphyrobacter, Pseu- were significantly different
domonas, Rhodobacter
Martiny Loop-shaped reactor equipped 16S rRNA gene-based followed Dominance of Nitrospirae Attached and planktonic com-
et al. (2005) with stainless steel coupons and by conventional cloning and  Proteobacteria (Gamma- and munities form separate clusters
fed with aerated filtered and sequencing Alpha-class > Beta- and Delta- Increase in the biofilm richness
unchlorinated groundwater class) > Acidobacteria over long period of time
> Planctomycetes, Firmicutes,
Bacteroidetes
Revetta Glass and PVC coupons from 16S rRNA gene-based followed Proteobacteria (Gamma-, Beta-, No significant difference in
et al. (2013) semi-closed pipe loop system or by conventional cloning and Alpha-class), Actinobacteria, community structures between
annual reactors fed with sequencing Bacteroidetes, Nitrospirae, materials used
9 Microbiome of Drinking Water Distribution Systems

monochlorinated drinking water Firmicutes Decrease in

from Cincinnati DWDS (Ohio, At the genus level: Gammaproteobacteria and
USA)—treated surface water Early stages of biofilm were increase in Actinobacteria with
dominated by Serratia, biofilm aging
Cloacibacterium, Increasing diversity with sam-
Diaphorobacter, and Pseudomo- pling time
nas Phylogenetic structure of biofilm
Older biofilms dominated by communities varied with biofilm
Mycobacterium, Flavobacterium, aging
Phenylobacterium, Acidovorax
(continued)
285
Table 9.2 (continued)
286

Dominant phyla and classes

Authors Biofilm sampling Methods (major genera) Comments
Schmeisser EPDM-coated valves in DWDS 16S rRNA gene-based followed Dominance of Proteobacteria
et al. (2003) (northwest of Germany) by conventional cloning and (Alpha- ¼ Gamma- > Beta-class)
sequencing > Actinobacteria > Firmicutes
At the family level:
Rhizobiales, Pseudomonas,
Enterobacteriales,
Burkholderiales,
Caulobacterales, Actinomycetales
Williams Polycarbonate coupons in annular 16S rRNA gene-based followed Dominance of Proteobacteria Disinfectant practice changed the
et al. (2005) reactor connected to DWDS by conventional cloning and (Alpha-, Beta-, and Gamma- biofilm bacterial communities
+ chloramine or chlorine sequencing class), Actinobacteria, Reduction in species richness in
Planctomycetes the chloraminated biofilm
At the genus level:
– In the presence of chloramine:
Mycobacterium and
Dechloromonas
– In the presence of chlorine:
Porphyrobacter,
Hyphomicrobium, Bosea,
Sphingomonas, Bradyrhizobium
DWDS drinking water distribution system, EPDM ethylene-propylene-diene monomer, PVC polyvinyl chloride
L. Mathieu et al.
9 Microbiome of Drinking Water Distribution Systems 287

of the interactions between phyla and between genera. Indeed, each drinking water
biofilm population has a unique bacterial population structure. Metabolomics could
be an interesting way to document the biofilm activity by analyzing the chemical
compounds (metabolites) that are either consumed or excreted by the microorgan-
isms as a result of their metabolic activity. These metabolites can be used to infer the
type of microorganisms present in the biofilm, as well as the nature of their activity
(Beale et al. 2013).

9.5 Accumulation of Pathogens and Microorganisms

of Public Health Interest in Drinking Water Biofilms

Whatever their structure and thickness, biofilms shape a new soft hydrated surface
on the bare pipe material providing new specific properties. Thus, the hydrophilic-
hydrophobic balance as well as the topography of the surface of the naked material
may be greatly changed. The colonization of the drinking water biofilm by water-
borne pathogens requires at least two things: (1) effective collisions with and
physicochemical interactions between the biofilm surface and the pathogens and
(2) the capacity for these microorganisms to adapt to this new environment despite
being in competition for nutrients and space with the bacteria already attached.
First, predicting effective adhesion of pathogens onto biofilms is rather difficult
due to the lack of information on the surface properties of microorganisms including
waterborne pathogens despite growing efforts in this field (Gaboriaud et al. 2008;
Ojeda et al. 2008). Additionally, characterization of biofilm is also limited and
complex. Janjaroen et al. (2013) showed, for example, that the roughness of biofilms
increases with age, which for us could be explained by higher accumulation of
hydrophobic material within the EPS matrix. This makes sense as the authors found
more Escherichia coli cells attached to “old” biofilms. Shen et al. (2015) also
reported that L. pneumophila adhesion was enhanced by biofilm roughness. Indeed,
as reported by Van Loosdrecht et al. (1987) and Tatchou-Nyamsi-König et al.
(2008), hydrophobic bacteria do stick more efficiently despite their surface-negative
charges, which determine a repulsive barrier. However, even few variations in
envelope polymeric composition may greatly change the surface properties of
bacteria as shown for Legionella (Gosselin et al. 2011) and the sorption of
Escherichia coli shown to be growth phase dependent (Walker et al. 2005). The
same was noted for nanoparticle-like bacteriophages GA and Qβ for which small
surface property variations significantly affect the rate at which adsorption occurs
(Fig. 9.6).
Second, many microorganisms survive for a relatively long period of time within
distribution systems and partly colonize drinking water biofilms despite a compet-
itive environment. For instance, some waterborne opportunistic pathogens occurring
in premise plumbing systems, including strains of Legionella spp., non-tuberculosis
Mycobacteria, Pseudomonas, fungi, and Acanthamoeba, are easily detected in
288 L. Mathieu et al.

Fig. 9.6 Modeling of the adsorption of bacteriophages GA (filled triangle and full line) and Qβ
(filled square and dashed line) under static conditions as a function of time. The full and dashed
lines are, respectively, representative of the equations y ¼ (1.25
105t)0.5 and y ¼ 0.0552exp
(2.95
103t) with t being time expressed in min (Hébrant et al. 2014).

biofilms where they multiply. It means that both the pre-existing autochthonous
biofilm population and antagonistic competition do not prevent biofilm colonization
by allochthonous species. From this point of view, pre-colonization of new pipes
with healthy microbial consortia capable of repelling opportunistic pathogens is still
a faraway “silver bullet” solution (Wang et al. 2013a). In some cases, better survival
of heterotrophic pathogens could even be suspected in the biofilm, as it would
represent a necrotrophic organic matter resource (Temmerman et al. 2006; Valster
et al. 2011) and a reactive barrier to disinfectant diffusion (see Sect. 9.6). Intracel-
lular persistence or growth of pathogenic microbes within amoebae and other
eukaryotic invertebrates in drinking water (Bichai et al. 2008) could explain the
capacity of some microorganisms to colonize effectively drinking water biofilms
(Garcia et al. 2013; Ovrutsky et al. 2013).
Biofilms can act as either temporary or long-term reservoirs and habitats for
bacterial pathogens. As summarized in Fig. 9.7, some of them do find in biofilms
almost systematically a favorable ecological environment (Legionella, Mycobacteria,
and Pseudomonas aeruginosa), while for others (depending on temperature, avail-
ability of organic matter, and chlorine), occupancy is transient (E. coli, Klebsiella,
Salmonella, Campylobacter, and Helicobacter). Last, viruses, Giardia, and Crypto-
sporidium oocysts (i.e., bugs unable to multiply outside hosts or under drinking water
environmental conditions) will likely have a reduced persistence and wash out more
or less rapidly (Helmi et al. 2008).
9 Microbiome of Drinking Water Distribution Systems 289

Fig. 9.7 Behavior of indigenous bacteria, waterborne pathogens, and microorganisms of sanitary
interest in drinking water biofilms: ① zone with microbial growth rate higher or just slightly lower
than the dilution rate: indigenous saprophytic bacteria (Legionella, Mycobacteria, P. aeruginosa,
free-living amoebae); ② zone of transient colonization: E. coli, Salmonella, Campylobacter, and
Helicobacter; and ③ zone of fast washout of microorganisms unable to grow in drinking water
biofilms: virus, Cryptosporidium, Giardia (adapted from Batté et al. 2003)

Many of these microorganisms can also be internalized (e.g., Pseudomonas,

Mycobacteria, Candida yeasts) or even multiply (e.g., Legionella) inside free-living
amoebae which may be found in relatively high density (up to 103 FLA/cm2 of
biofilm) (Sibille et al. 1998).

9.5.1 Escherichia coli

Escherichia coli have rarely been detected in drinking water biofilms (Singh et al.
2003; Juhna et al. 2007; Feazel et al. 2009), which makes sense as best efforts are
made to inactivate this fecal indicator in drinking waters. However, recent publica-
tions, for the most part, did not describe the community structure identified in the
phylum Gammaproteobacteria at the genus level, and either noncultivable or dead
E. coli could have been present (Schmeisser et al. 2003). Their growth was demon-
strated (Camper et al. 1991; Szewzyk et al. 1994; Fass et al. 1996; Li et al. 2006) and
allowed only transient stays in biofilms. Variations between the results from these
rare assay reports could be explained by factors such as strain influence, local
electrochemical gradients, and organic matter availability. Indeed, as shown in
planktonic culture by Vital et al. (2010), growth of Escherichia coli and its effective
competition with autochthonous bacterial communities depend on the concentration
of organic matter. Their adhesion is also dependent on the ionic environment as
E. coli adhesion rates increased with ionic strength on new PVC surfaces and very
young biofilms, but seems independent of the solution chemistry for older biofilms.
290 L. Mathieu et al.

This suggests that the physical structure of biofilms could play a role in facilitating
the adhesion of E. coli cells (Janjaroen et al. 2013).

9.5.2 Helicobacter

Helicobacter were also detected and could persist for relatively long periods of time
in drinking water biofilms (Mackay et al. 1998; Azevedo et al. 2006; Giao et al.
2008; Linke et al. 2010).

9.5.3 Legionella pneumophila

Legionella pneumophila (the most studied pathogenic species—causative agent of

Legionnaires’ disease) and nonpathogenic species Legionella have been found in
water supplies at temperatures below 18  C (Rodriguez-Martinez et al. 2015). They
were recovered from 16% to 19% of samples collected in cold water systems of
within-building distribution systems (Völker et al. 2010; Donohue et al. 2014) and
detected in drinking water biofilms with molecular analytical tools (Williams et al.
2005; Wang et al. 2012a; Buse et al. 2014; Lin et al. 2014). The lowest concentration
and the greatest diversity of Legionella were observed in the water supply with low
organic matter concentration and high protozoan richness, and biofilms were found
to harbor up to 100 Legionella/cm2 (Wullings et al. 2011). The role of intracellular
replication in amoebae appears essential for Legionella proliferation. As an example,
Declerck et al. (2009) showed that biofilm-associated Legionella only increased after
intracellular replication in Acanthamoeba castellanii. Many experimental systems
built to test Legionella behavior in drinking water biofilms confirm these observa-
tions, i.e., that Legionella exhibit effective adhesion, long persistence and growth
despite autochthonous communities, and chlorination cycles (Rogers et al. 1994;
Van Der Kooij et al. 2005; Vervaeren et al. 2006; Langmark et al. 2007; Lehtola
et al. 2007; Cooper et al. 2008; Hindre et al. 2008; Moritz et al. 2010; Stewart et al.
2012).

9.5.4 Mycobacteria

Environmental Mycobacteria (M. avium, M. gordonae, M. intracellulare,

M. lentiflavum, M. tiuscia, etc.) were very frequently reported in distribution sys-
tem biofilms at densities from 100 to 5
103 per cm2 and can be considered as
common inhabitants of public distribution systems (Falkinham et al. 2001;
Schmeisser et al. 2003; September et al. 2004; Torvinen et al. 2004; Vaerewijck
et al. 2005; Williams et al. 2005; Keinänen-Toivola et al. 2006; Feazel et al. 2009;
9 Microbiome of Drinking Water Distribution Systems 291

Wang et al. 2012a, c; Kelly et al. 2014). Slow but significant mycobacterial growth
was expected or demonstrated in drinking water distribution systems and biofilms:
(1) the number of Mycobacteria found in biofilms was higher than in the bulk water
(Feazel et al. 2009), (2) growth was observed in laboratory assays under conditions
relevant to drinking waters (Steed and Falkinham 2006), and (3) the highest numbers
of Mycobacteria were found at the distal sites of the distribution systems (Falkinham
et al. 2001; Torvinen et al. 2004).

9.5.5 Pseudomonas

Pseudomonas spp. occurrence in drinking water distribution systems was reported to

be fairly high from 2% (Völker et al. 2010) to 13% (Wang et al. 2012a) in cold water
systems of within-building distribution systems. They are frequently identified by
culture-independent molecular techniques in drinking water biofilms (Schmeisser
et al. 2003; Feazel et al. 2009; Chen et al. 2013; Douterelo et al. 2013, 2014b; Kelly
et al. 2014; Lin et al. 2014; Liu et al. 2014a).
Pseudomonas aeruginosa, which is responsible for opportunistic infections, has
become a major cause of nosocomial infections worldwide especially in intensive
care units and neonatal units (Walker et al. 2014). The bacteria adopt a sedentary
community lifestyle by forming a biofilm through various adhesive systems (Giraud
et al. 2010) and are able to colonize water system biofilms (taps, u-bends, etc.)
especially when some biodegradable organics are leached from the pipe material
(Moritz et al. 2010).

9.6 Factors Controlling Biofilm Accumulation in Drinking

Water Distribution Systems

Biomass variations in drinking water distribution systems occur due to the combined
influence of several parameters (eight parameters and their effects are listed in
Table 9.3), which themselves vary with water resources, treatments, seasons,
and drinking water system construction materials (Langmark et al. 2007; Pinto
et al. 2012; Henne et al. 2013; Wang et al. 2012a, b, 2013b, 2014a). These
multiparametric environmental effects are not easily quantifiable at the level of
species, and only few models have attempted to predict biomass variations (total
number of cells or HPC). Standard correlation analysis techniques have indicated
that biomass variations in drinking water are due to the combined influence of
organic matter, chlorine, and temperature (Niquette et al. 2001; Zhang and Digiano
2002).
Among all the parameters listed in Table 9.3, some have been well studied as they
represent operational parameters that practitioners either may control (i.e.,
292 L. Mathieu et al.

Table 9.3 Some parameters (beneficial and detrimental effects) governing biomass accumulation
in drinking water distribution systems and biofilms
Parameters Major effects References
Biodegradable or Substrate saturation constants Van Der Kooij and Hijnen (1988);
assimilable organic (Ks) are shown to be as low as a LeChevallier et al. (1991); Servais
carbon (BDOC or few μg/L et al. (1995); Van Der Kooij et al.
AOC) In the bulk water, consumption of (1995); Sibille et al. (1997);
10 μg/L organic carbon would Niquette et al. (2001); Hammes
result in the formation of as many et al. (2010b); Sack et al. (2014)
as 104–105 cells/mL (the yield
factor should be less in biofilm as
some of the matter is used for EPS
production)
For limiting growth in drinking
water distribution systems, AOC
should be limited to 50 μg/L or
BDOC to 100 μg/L
Disinfectants: chlo- Drinking water biofilms are shown Chen and Stewart (1996);
rine or chloramine to be systematically more difficult Gauthier et al. (1999b); Lu et al.
to inactivate than planktonic bio- (1999); Hallam et al. (2001);
mass due to the strong depletion of Lehtola et al. (2005); Steed and
chlorine at the surface and within Falkinham III 2006; Szabo et al.
the biofilm (consumption by EPS) (2007); Morrow et al. (2008);
Monochloramine penetration Berry et al. (2009); Lee et al.
within biofilm is better than that of (2011); Hwang et al. (2012);
chlorine (in spite of similar diffu- Wang et al. (2012b, c, 2014a);
sion coefficients). This penetration Xue et al. (2012); Xue and Seo
does not necessarily result in (2013); Mathieu et al. (2014)
immediate bacterial viability loss
In drinking water distribution sys-
tems, chlorine (>1 mg/L Cl2) is
unable to eradicate biofilms
Di-oxygen Coliforms adhere quite well in Farkas et al. (2013); Wang et al.
oxygen-depleted environments (2014b)
Di-oxygen appears to be a major
driver for eukaryote persistence
Hydrodynamics Accumulation of biomass on the Lautenschlager et al. (2010);
surface is more rapid under turbu- Manuel et al. (2010); Simoes et al.
lent conditions than under laminar (2010)
flow
Increasing shear stress resulted in
lower cell number of cells per unit
surface area of PVC and stainless
steel material
Water stagnation favors growth in
bulk water (the total cell balance
biofilm + water was constant).
Growth rate under stagnation con-
ditions is as high as 0.22 per hour
(continued)
9 Microbiome of Drinking Water Distribution Systems 293

Table 9.3 (continued)

Parameters Major effects References
Phosphorus Phosphorus can originate from Miettinen et al. (1997); Sathasivan
water resource and water treatment and Ohgaki (1999); Appenzeller
(anticorrosion as phosphate) and et al. (2001, 2002); Lehtola et al.
also be released from some pipe (2002); Morton et al. (2005);
materials Polanska et al. (2005); Gouider
Microbially available phosphorus et al. (2009); Li et al. (2010); Jiang
(MAP) can show a wide concen- et al. (2011); Inkinen et al. (2014)
tration range in drinking waters
(0.3–30 μg/L)
Phosphorus can be sometimes the
limiting factor for bacterial growth
Temperature A slight temperature increase Bagh et al. (2004); Norton et al.
(from 15  C to 35  C) induces a (2004); Silhan et al. (2006);
higher number of HPC in biofilms Inkinen et al. (2014)
High temperature reduces biomass
and biodiversity in biofilms (hot
water systems at 60  C or more)
Temperatures >35  C are required
to control the occurrence of Myco-
bacterium avium in pipelines
Pipe materials Some elastomeric surfaces have Rogers et al. (1994); Kerr et al.
more abundant biofilms due to (1999); Lu et al. (1999);
organic leaching than other mate- Kalmbach et al. (2000); Niquette
rials (except corroded iron sur- et al. (2000); Kilb et al. (2003);
faces). Leaching can be as high as Schwartz et al. (2003); Lehtola
0.15–30 μg TOC/cm2 day and 0.06 et al. (2004, 2005); Gagnon et al.
to 30 μg AOC/cm2 day (2005); Moritz et al. (2010);
The effect of pipe construction Pavissich et al. (2010); Yu et al.
material is measurable on prokary- (2010); Allion et al. (2011);
ote biomass not on eukaryotes Bucheli-Witschel et al. (2012);
Copper limits biofilm accumula- Wang et al. (2012c, 2014a); Buse
tion during the first months of pipe et al. (2014); Douterelo et al.
usage. In long-term biofilms grown (2014b, c); Liu et al. (2014a)
on copper, the number of cells can
be the same, but diversity and
physiology may be affected by
toxic Cu ions
No differences in biofilm accumu-
lation on stainless steel of different
grades were found
(continued)
294 L. Mathieu et al.

Table 9.3 (continued)

Parameters Major effects References
Corrosion of iron- Corrosion and corrosion products Norton and LeChevallier (2000);
bearing materials as well as iron (Fe II and Fe III) Appenzeller et al. (2001, 2005);
increase bacterial survival and cul- Butterfield et al. (2002);
tivability and stimulate the rate of Grandjean et al. (2005, 2006);
biofilm development Lehtola et al. (2005); Szabo et al.
Corrosion of iron or copper pipes (2007, 2012); Hindre et al. (2008);
induces a rapid consumption of Wang et al. (2012b); Szabo and
chlorine at the pipe surface and a Minamyer (2014)
low efficacy of the disinfectant
Growth of some species
(Legionella pneumophila) can be
inhibited under iron-rich
conditions
AOC assimilable organic carbon, BDOC biodegradable dissolved organic carbon, EPS
exopolymers, TOC total organic carbon

disinfectant type and concentration, concentration of biodegradable organic matter,

nature of pipe materials) or that vary during cleaning procedures (i.e., water flush-
ing). None of these procedures allow biofilm prevention nor its eradication. We
further discuss two of these major parameters (i.e., disinfectants and nutrients)
below.

9.6.1 Chlorine and Other Chlorinated Oxidants

Chlorine and other chlorinated oxidants have been widely used for more than a
century. They both injure and inactivate bacterial cells by oxidation and substitution
reactions with envelope polymers (as shown by increases in membrane permeability)
and affect key intracellular polymers such as nucleic acids (Saby et al. 1997; Phe
et al. 2005; Ramseier et al. 2011). They also injure all living microorganisms
including protozoa (Mogoa et al. 2010). However, complete biofilm disinfection is
never achieved as chlorine penetration is limited by a reaction-diffusion interaction
with all the organic material accumulated on the pipe wall surface (De Beer et al.
1994; Chen and Stewart 1996; Lee et al. 2011), and, as a consequence, some species
escape the treatment depending on the nature and concentration of the disinfectant.
First, it is important to understand that the dominant bacterial species surviving
after either chlorination or chloramination are different from those which
predominated before treatment. As an example, Ling and Liu (2013) showed by
16S rRNA gene pyrosequencing analysis that chloramination selected Acinetobacter
and Acidobacteria as dominant populations, while natural biofilm development
leads to the selection of members of Nitrospira and Bacteroides. Roeder et al.
(2010) also observed a shift in the bacterial composition of biofilms (compared by
DGGE) and low similarities in treated versus untreated biofilms (especially with
9 Microbiome of Drinking Water Distribution Systems 295

Alpha-Proteobacteria Beta-Proteobacteria Gamma-Proteobacteria Other EUB

Fig. 9.8 Reversible shift in the Proteobacteria populations of drinking water biofilms when
subjected to discontinuous chlorination (adapted from Mathieu et al. 2009)

chlorine dioxide). Changing disinfection regimes from chlorine to monochloramine

in drinking water biofilms leads to population shifts, and the emergence of
Legionella species in chlorinated biofilms and Mycobacteria in chloraminated
ones (Santo Domingo et al. 2003; Pryor et al. 2004; Williams et al. 2005). Codony
et al. (2005) showed that discontinuous chlorination reduced the susceptibility of
biofilms to the disinfectant. Mathieu et al. (2009) reported that Beta- and
Gammaproteobacteria survived better to increasing chlorination than did
Alphaproteobacteria in drinking water biofilms. Interestingly, as soon as the selec-
tion pressure was over (by decreasing chlorine), the proportions of Alpha-, Beta-,
and Gammaproteobacteria phyla returned in a few weeks to the initial equilibrium
showing resilience of the bacterial community (Fig. 9.8).
Second, a physiological response of the bacteria remaining after disinfection
could be noticed at several levels:
– On the one hand, either overproduction or synthesis of different EPS may be
speculated from reports showing how discontinuous chloramination affects
drinking water biofilm architecture (Milferstedt et al. 2013). The same is expected
from the work of Schwering et al. (2014) who reported also novel characteriza-
tion of morphologically distinct microcolonies after chlorination.
– On the other hand, resistance to chlorine is induced in many species after
sublethal exposure. Knowing the key importance of chlorine for antimicrobial
strategies, it is however surprising to observe that only limited knowledge is
currently available regarding the ways in which bacteria sense and respond to
reactive chlorine species. Most demonstrations of responses to either chlorine or
chloramine have been largely done in planktonic cultures: according to Dukan
and Touati (1996) dps, katE, and katG genes, as well as oxyR regulons, were
involved in protective functions against chlorine. Du et al. (2015) showed that
proteins involved in stress regulation and stress responses were among those
upregulated under both starvation and chlorine or monochloramine disinfection.
Glutathione appears as a key regulator in intracellular redox protection against
chlorine (Chesney et al. 1996; Saby et al. 1999). Escherichia coli responded to
monochloramine by activating not just one single antioxidant system but diverse
296 L. Mathieu et al.

defense response including oxidative stress, DNA repair, and genes involved in
attachment (fimbriae, curli) (Holder et al. 2013). Exposure of Legionella
pneumophila to chlorine induced, among other things, the expression of cellular
antioxidant proteins, stress proteins, and transcriptional regulators (Bodet et al.
2012). Although information about bacterial responses to reactive oxygen species
(ROS) is vast, work addressing bacterial responses to reactive chlorine species
has begun only recently. Transcriptomic and proteomic studies provide new
insights on the bacteria defense strategies against these important antimicrobial
compounds (see the review by Gray et al. 2013).
Most of these observations could be extrapolated to biofilm communities. Indeed,
chlorine induced soxS (transcriptional activator of the superoxide response) to a
greater extent in the dormant cells than in the active cells of the biofilm. In addition,
chlorine-dependent induction of soxS was more prominent in aerobically grown cells
(Kim et al. 2009). Comparative transcriptomic analysis revealed that planktonic
Escherichia coli exposed to monochloramine shares a transcriptional fingerprint
with cells grown under biofilm conditions that are known to decrease
monochloramine sensitivity characterized by general metabolic inhibition, redox
and oxidoreductase response, and cell envelope integrity response (Berry et al.
2010). In another situation (biofilm grown on Cu), the authors showed that UV
irradiation intensified the recA-mediated SOS response (the main mechanism to
repair DNA injuries and other damages) and suggested it could be responsible for
differences in the taxonomic composition of biofilms (Jungfer et al. 2013).
To sum up, biofilms and the noncellular organic matter that accumulate on pipe
walls have a high reducing potential, which leads to high chlorine consumption at
the surface of the material as well as to chlorine having limited diffusion into the
biofilm, thus reducing the exposure of biofilm-associated bacteria to chlorine and
also to chloramine. As a consequence many biofilm bacteria do survive low disin-
fectant exposures and induce physiological protective reactions (e.g., oxidative
stress resistance), which lead to higher resistance to further disinfection. As a result,
continuous addition of disinfectants into drinking water distribution systems
(0.1–0.5 mg/L Cl2) limits growth of bacteria in the bulk water but cannot eradicate
biofilms (Mathieu et al. 2009, 2014).

9.6.2 Organic Matter from Treated Water

Organic matter [measured either as dissolved organic carbon (DOC) or assimilable

organic carbon (AOC) or biodegradable dissolved organic carbon (BDOC)] repre-
sents a key parameter for heterotrophic biomass growth in drinking water distribu-
tion systems (autotrophic bacteria develop much more slowly). Bacteria favor
growth in biofilm when waterborne organic matter is limited to less than a few
hundreds of μg/L, while the opposite situation is observed (preferred growth in the
bulk water) when AOC > 500 μg/L (Tsai et al. 2004). Thus, threshold values have
9 Microbiome of Drinking Water Distribution Systems 297

been proposed for AOC (<50 μg/L) and BDOC (<100 μg/L) to achieve a limitation
of bacterial growth and result in the drinking water distribution system being defined
as biostable.
The impact of more recalcitrant macromolecules has been explored on biofilm
activity (Camper 2004; Rodrigues et al. 2010). Such macromolecules, i.e., humic
substances or polysaccharides and proteins of phytoplanktonic and bacterial origin,
cannot be assimilated directly due to their high molecular weight. Therefore, their
biodegradation should occur first in the biofilms as extracellular enzymes are needed
before their assimilation. Indeed, Sack et al. (2014) showed that biopolymers
promote drinking water biofilm formation at microgram-per-liter levels. These poly-
mers are bound to and hydrolyzed by cell-attached enzymes to be degraded into
growth-promoting compounds (i.e., <700 Da). Furthermore, the biofilms grown
with carbohydrates or proteins clearly differed in bacterial community composition.
The protein-adapted biofilm communities were more diverse than those of the
polysaccharide-adapted biofilm suggesting that proteins in distribution systems are
more utilized and are therefore more likely to promote biofilm formation. The
community structure also changed according to the nature of the polymers:
Cytophagia, Flavobacteriia, Gammaproteobacteria, and Sphingobacteriia grew
during polysaccharide addition, while Alpha-, Beta-, Gammaproteobacteria,
Cytophagia, Flavobacteriia, and Sphingobacteriia grew during protein addition.

9.6.3 Organic Matter Leached from Pipes

Since the 1990s, plastic pipes have been recognized to support biofilm growth (Kilb
et al. 2003; Schwartz et al. 2003; Moritz et al. 2010; Wang et al. 2012c). Biomass
increase and population selection are material dependent (Rogers et al. 1994). As
shown by Kalmbach et al. (2000), the bacterial community on soft PVC material
differed significantly from those on other materials; the dominant species
Aquabacterium commune was replaced by other Betaproteobacteria hybridizing to
an amount of 66% with the Aquabacterium citratiphilum-specific probe beta 4. Such
a selective effect could be related to plasticizers contained in soft PVC such as
sebacate, azelate, and adipate, which are metabolizable by Aquabacterium. In
another study, Liu et al. (2014b) showed that PVC biofilms were dominated by
Hyphomicrobium-like phylotypes (66%), whereas the latter represented only 2–7%
of the microbial community found on cast iron. Finally, by flushing pipes, Douterelo
et al. (2014b) found that the highest species richness and diversity were in the
samples of material mobilized from plastic pipes (as compared to cast iron) with a
high relative abundance of Alphaproteobacteria (23%), Clostridia (10%), and
Actinobacteria (10%) coming from the plastic pipe biofilm communities.
298 L. Mathieu et al.

9.6.4 Other Nutrients Leached from Pipe Materials

Iron from corroded pipes (Norton and LeChevallier 2000; Grandjean et al. 2005;
Wang et al. 2012b, 2015) and phosphorus from both some plastic materials and
corroding cast iron (Lehtola et al. 2004; Morton et al. 2005) do contribute to the
biofilm growth and diversity. Iron corrosion has been extensively studied, and many
specialized species take advantage of this environment. As an example, Wang et al.
(2012b, c) showed that iron-reducing bacteria Shewanella, iron-oxidizing bacteria
Sediminibacterium, and sulfur-oxidizing bacteria Limnobacter thiooxidans strains
promoted iron corrosion by synergistic interactions in the primary period (see also
Tables 9.2 and 9.3).
To sum up, nutrients (quantity and quality) govern biofilm dynamics and speci-
ation. Due to high affinity of most bacteria for nutrients on the order of a few μg/L
(Van Der Kooij and Hijnen 1988; Van Der Kooij et al. 1995), it appears illusory to
eradicate heterotrophic biofilms from drinking water distribution systems. Reducing
organic matter thanks to the best available technology and limiting corrosion are the
best ways to limit biofilm activity and diversity.

9.7 Conclusion

Drinking water distribution systems have been engineered for transporting water but
work de facto as slow bioreactors producing biomass in different phases of the
system (in the bulk water, on particles in suspension, in loose deposits, on pipe walls
in biofilm). The microbial characteristics of distributed waters highly vary on a daily
basis due to bacterial growth especially during stagnation episodes and biomass
shearing from biofilm and mobilization of loose deposits during high flow rate
events.
Potable waters (defined by zero cultivable Escherichia coli/100 mL and zero
cultivable enterococci/100 mL) carry millions of bacterial cells per liter. Bacteria
phyla are very diverse in drinking water (up to 48 phyla and in excess of 4000 unique
operational taxonomical units, OTUs—Proctor and Hammes 2015), with a large
dominance of Proteobacteria whatever the geographical location, water resource
origin, or season. However, each water distribution system has a unique bacterial
composition (especially at the level of genus and species). Moreover, drinking water
distribution systems harbor a relatively wide-ranging ecosystem including fungi,
free-living amoebae, small eukaryotes, and microinvertebrates, which form with
heterotrophic bacteria a trophic food chain.
Besides drinking water biofilms formed under turbulent conditions are thin
systems (from a few to more than 100 μm) composed of 105–107 cells/cm2 patchy
distributed. Three classes of Proteobacteria (α-, β-, γ-) are systematically present
and predominate, and only their relative abundance varies in drinking water biofilms.
Besides Proteobacteria, other more or less dominant phyla are also detected such as
9 Microbiome of Drinking Water Distribution Systems 299

Firmicutes, Verrucomicrobia, Planctomycetes, and Bacteroidetes. Biofilms can act

as either temporary or long-term reservoirs and habitats for bacterial pathogens.
Some of these pathogens do find in drinking water biofilms almost systematically a
favorable ecological environment (Legionella, Mycobacteria, and Pseudomonas
aeruginosa), while for others (depending on temperature, availability of organic
matter, and chlorine), occupancy is transient (E. coli, Klebsiella, Salmonella, Cam-
pylobacter, and Helicobacter). Last, viruses, Giardia, and Cryptosporidium oocysts
(i.e., bugs unable to multiply outside hosts or under drinking water environmental
conditions) will likely have reduced persistence and washout more or less rapidly.
Microbiome variations in drinking water distribution systems occur due to the
combined influence of several parameters, which themselves vary with water
resources, treatments, seasons, and drinking water system construction materials.
Only recently, less than 10 years ago, our understanding of the microbial ecology of
distribution systems greatly improved thanks to molecular biology investigation,
fingerprint determination, and sequencing-based approaches. These techniques are
still being improved, and next-generation techniques will provide less expensive and
time-consuming methodologies as well as new software, tools for analyzing the gene
sequences, and new data banks. For the time being, understanding the microbial
ecology in distribution systems is still a challenging task. Without a detailed
inventory of the microorganisms growing in distribution systems and their interac-
tions, water utilities are forced, in a sense, to “fly blind” when making treatment
decisions and managing microbial contamination risks.

Compliance with Ethical Standards

Conflict of Interest Laurence Mathieu declares that she has no conflict of interest. Tony Paris
declares that he has no conflict of interest. Jean-Claude Block declares that he has no conflict of
interest.

Ethical Approval This article does not contain any studies with human participants or animals
performed by any of the authors.

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Chapter 10
Isolation and Cultivation of Bacteria

Martin W. Hahn, Ulrike Koll, and Johanna Schmidt

Abstract Isolation and cultivation of microorganisms enables in combination with

cultivation-independent methods comprehensive research on ecology and function
of microbes. The availability of cultures provides opportunities for ecophysiological
experiments, enables high-quality genome research and represents a mandatory
prerequisite for the description of new taxa of prokaryotes. Unfortunately, access
to microorganism by cultivation methods is still quite limited, and the majority of the
microbial diversity remains uncultured so far. This chapter discusses the potential
reasons for this lack in cultivability and reviews advances in cultivation methods for
prokaryotes. Detailed analysis of the media and methods used by taxonomists for
isolation of bacterial strains used for description of >1000 new species revealed that
the description of new taxa in the ranks of species and genera is currently not
methodically limited. On the other hand, isolation of strains enabling the description
of taxa representing higher ranks is obviously strongly limited by the currently
applied methods. Consequently, different cultivation strategies are required
according to the scientific goals of the respective research. While taxonomists
interested in the description of new species affiliated with any genus or family can
isolate new strains suitable for this task by using standard cultivation methods and
media, ecologists interested in cultivation of model organisms appearing in natural
systems with high cell numbers, as well as taxonomists interested in isolation of
strains representing new higher ranking taxa like orders, classes and phyla, should
rather use high-throughput non-standard methods in combination with optimized
screening protocols.

M. W. Hahn (*) · U. Koll · J. Schmidt

Research Department for Limnology, University of Innsbruck, Mondsee, Austria
e-mail: martin.hahn@uibk.ac.at

© Springer Nature Switzerland AG 2019 313

C. J. Hurst (ed.), The Structure and Function of Aquatic Microbial Communities,
Advances in Environmental Microbiology 7,
https://doi.org/10.1007/978-3-030-16775-2_10
314 M. W. Hahn et al.

10.1 Introduction

Isolation and cultivation of bacteria is an easy task. Take an agar plate of a standard
medium, for instance, Marine Agar (Zobell 1941) for marine or R2A Medium
(Reasoner and Geldreich 1985) for freshwater or soil bacteria, and spread 100 μL
liquid sample or soil suspension on the plate. You will find several colonies on the
plate after incubation at room temperature for a few days. Differences in colony
morphology and coloration usually indicate that different taxa are present on such
plates. Most of the grown colonies could be easily purified and propagated as pure
cultures. So, why is cultivation of bacteria and other microorganisms still a big issue
in microbiology and related disciplines dealing with ecology and diversity of
microorganisms? The answer is that those easy to cultivate bacteria represent only
a minor fraction of the current bacterial diversity on earth (Rappe and Giovannoni
2003; Hugenholtz et al. 1998). The majority of bacteria present in the environment,
frequently estimated as 99% or >99% of cells (Pham and Kim 2012; Amann et al.
1995), is assumed to be difficult to be cultivated or partially even not culturable at all.
Importantly, easy to cultivate microorganisms are assumed to represent taxa growing
in their natural habitats only with low abundance and contributing, due to their low
numbers, probably only marginally to the metabolism of ecosystems (Staley and
Konopka 1985; Pace et al. 1986). The majority of bacteria are resistant to standard
cultivation approaches and thusly have escaped cultivation by microbiologists, a
truth that was overlooked by microbiologists for about a century. Recent advances in
cultivation techniques resulted in isolation and cultivation of many difficult to be
cultured microorganisms representing abundant taxa in, for example, marine or
freshwater systems. Investigation of recently cultured taxa enabled, for instance,
new insights in ecology and diversity of microorganisms abundant under natural
conditions (Giovannoni et al. 2005a, b; Hahn et al. 2012b). However, despite these
successes, the vast majority of microbial diversity is still not represented by culti-
vated organisms, which limits research in many scientific disciplines. This includes,
for instance, such important fields of research as the development of new antibiotics,
which is required due to the emerging problem of antibiotic resistance in bacterial
pathogens. The development of new antibiotics is steadily decreasing since a couple
of decades (Braine 2011), which has several reasons where one reason is the limited
access to antibiotic substances encoded by so far uncultured bacteria.

10.2 What Is Isolation and Cultivation?

Microorganisms were cultivated by humans as fermentation starters since ancient

times. Traditional fermentation starters were used for production or conservation of
various food (e.g. sourdough, soybeans, milk products) and beverage products
(e.g. beer). The scientific concept of purposeful establishment and maintenance of
cultures and especially of pure cultures of microorganisms was mainly developed in
10 Isolation and Cultivation of Bacteria 315

the second half of the eighteenth century. One of the leading scientists was Robert
Koch (Brock 1999). In the 1880s, mainly Koch and coworkers developed the
concept of pure cultures, i.e. clonal cultures derived from a single cell, and the
methodology for establishment and maintenance of such cultures (Blevins and
Bronze 2010). Further key innovations established by Robert Koch, Walther
Hesse, Fanny Hesse and Julius Richard Petri were the introduction of agar for
solidification of liquid media (e.g. broth) (Hesse 1992) and the development of
glass-made Petri dishes (Petri 1887). This methodology based on dishes filled with
sterile solidified media is still the basic microbiological technique for isolation,
cultivation and maintenance of prokaryotes. Nowadays, this direct plating of sam-
ples from the environment (e.g. aquatic samples or soil suspensions) onto agar plates
is circumscribed by the term standard methods (or “traditional methods” or “culti-
vation with standard media”) for isolation and cultivation of prokaryotes
(Vartoukian et al. 2010; Hazan et al. 2012; Stewart 2012). Microorganisms
responding to such treatments with macroscopic growth on agar plates are termed
readily culturable microorganisms (Kell et al. 1998; Barer and Harwood 1999;
Handelsman 2004).
Cultivation of microorganisms always includes the process of isolation of par-
ticular microbes from other members of their community. In many cases, the
physical separation of the targeted microbes is done in one or two subsequent
steps, for instance, by serial dilution of samples, plating of the dilution on agar
plates and subsequent transfer of single colonies to another plate. The dilution-to-
extinction method (Button et al. 1993; Connon and Giovannoni 2002) also uses
dilution for isolation of bacteria. Precision of the dilution procedure can be increased
by using a MicroDrop microdispenser system (Bruns et al. 2003a; Gich et al. 2005).
Other methods for physical separation, i.e. isolation, of cells are filtration through or
onto filters with a certain pore size (Hahn et al. 2004) and fluorescence-activated cell
sorting (FACS), which is frequently used for isolation of picocyanobacteria
(e.g. Prochlorococcus and Synechococcus). The latter method makes use of the
autofluorescent traits of these phototrophic bacteria (Moore et al. 1998; Crosbie et al.
2003). Laser microscopes (optical tweezers) were used for isolation of archaeal cells
from mixed cultures (Huber et al. 1995, 2000).
Frequently, microbiologists are interested in isolation or detection of specific
prokaryotes, e.g. strains of a particular species or genus (e.g. pathogenic bacteria) or
strains with a specific physiology (e.g. nitrifiers). Such targeted cultivation is usually
performed by using selective cultivation media in combination with preferably
selective incubation conditions and may also include a specific pretreatment of
samples (e.g. fractionation by filtration). A high number of media suitable for
specific isolation and cultivation of various microorganisms as well as many
media with a broader applicability were developed during the past 130 years. A
very broad collection of recipes of microbiological cultivation media is provided by
the Handbook of Microbiological Media (Atlas 2010). All available media are more
or less selective for certain groups of organisms sharing specific physiological traits.
Consequently, there is no unselective medium suitable for cultivation of all readily
culturable microorganisms. When considering the broad spectrum of microbial
316 M. W. Hahn et al.

physiologies ranging from chemoorganoheterotrophs (e.g. E. coli and yeasts) and

photolithoautotrophs (e.g. Cyanobacteria) to chemolithoautotrophs (e.g. nitrifiers),
it is obvious that a universal medium meeting the demands of all existing microor-
ganisms is impossible to be created.
In many cases, targeted isolation of specific readily culturable microorganisms is
hampered by either their low abundance (cell numbers) under natural conditions or
by the lack of a highly specific microbiological medium or the lack of selective
cultivation conditions. For such microorganisms, enrichment cultures are employed,
which increase the relative abundance of the targeted microorganisms by using semi-
selective conditions. Frequently, liquid media are used for such enrichments, and
samples from enrichments are subsequently spread on agar plates providing suitable
growth conditions. One example for such a protocol is enrichment of Vibrio cholera
in alkaline peptone water (Lesmana et al. 1985) for detection of this pathogen in
stool samples of patients. In other cases, enrichment of target organisms can be
achieved by sample fractionation, e.g. by filtration using filters with certain pore
sizes (Watanabe et al. 2009). Sample fractions penetrating filters, e.g. 0.2 μm filters
[isolation of spirochaetes, Polynucleobacter or other bacteria (Hahn 2004; Johnson
1977)], or sample fractions retained by filters [e.g. isolation of filamentous
cyanobacteria and other filamentous bacteria (Rippka 1988)] may be of interest.
The success of enrichment treatments may be influenced by various factors including
sample transport and cultivation conditions (Alam et al. 2006) and the presence of
superior competitors or specific phage (Muniesa et al. 2005).
Successful cultivation of a microorganism includes subcultivation and potential
maintenance of the culture. The first subcultivation is in many cases unsuccessful,
and a strain initially growing on an agar plate or in a liquid culture is lost (Kenters
et al. 2011). Some researchers do not distinguish between temporary cultivation
without successful subcultivation (Zengler et al. 2002, 2005) and maintenance and
sustainable cultivation; however, this difference is crucial when it comes to physi-
ological or taxonomic investigations of cultivated microorganisms.
As illustrated in Fig. 10.1, standard methods for isolation and cultivation of
prokaryotes employ various adjustment “screws” enabling an increase of specificity
of the method for cultivation of the targeted organisms. In other cases, a low
specificity is wanted in order to isolate new taxa. Interestingly, a large fraction of
newly described species is still isolated and cultivated by using low-specificity
standard methods (see below). An example is direct plating of water samples or
soil suspensions on R2A Agar, which yielded 11% of all newly described species in
the years 2009 and 2010. All so-called standard methods have in common that they
employ solidified media with high nutrient concentrations and aim on cultivation of
microorganisms as macroscopically visible colonies.
Prokaryotes previously not cultivated by using standard methods, e.g. all bacteria
and archaea not ready to grow on standard agar plates with high nutrient concentra-
tion, are considered to be difficult to cultivate prokaryotes. This may include strains,
which are basically able to grow under such conditions but for which suitable media
were not developed yet. It is obvious that it is difficult to draw a line separating
readily culturable and difficult to be cultured taxa. On the other hand, it is easier to
10 Isolation and Cultivation of Bacteria 317

Fig. 10.1 General workflow of isolation and cultivation experiments. Steps highlighted by the grey
area encompass what is usually meant by the term cultivation method

distinguish between previously cultivated and “uncultured” (or not yet cultured)
taxa. This separation is usually done by performing comparisons of small subunit
(SSU) rRNA gene sequences [also known as 16S rRNA (Prokaryotes) and 18S
rRNA genes (Eukaryotes)] of organisms of interest with sequences deposited in
public nucleic acid sequence databases (GenBank, EMBL-ENA, DDBJ). These
databases contain very large collections of SSU rRNA gene sequences representing
at least type strains of all prokaryotic species described so far, as well as large
numbers of ribosomal sequences of other cultured and environmental sequences of
uncultured microorganisms. Environmental sequences were retrieved by molecular
methods from environmental DNA samples without cultivation of organisms
(Amann et al. 1995). Such sequences are frequently labelled as “uncultured” or
“environmental” or “cloned” sequence. Sequence comparisons are frequently
performed by using the BLAST (Basic Local Alignment Search Tool) algorithm
318 M. W. Hahn et al.

(Altschul et al. 1990) provided, for instance, by NCBI (http://blast.ncbi.nlm.nih.gov/

Blast.cgi). This algorithm searches databases for sequences similar to the query
sequence (i.e. the SSU rRNA gene sequence of interest), aligns the similar sequences
and calculates the sequence similarity (also termed sequence identity), which is the
percentage of identical nucleotides of the aligned sequences. Usually, prokaryotes
sharing >97%, >98% or >99% SSU rRNA gene sequence similarity with previ-
ously cultivated strains are considered to represent already cultivated organisms.
However, a really diagnostic similarity threshold cannot be defined because of the
low phylogenetic resolution of the SSU rRNA sequences. Even organisms sharing
identical SSU rRNA genes may represent distinct species (Stackebrandt and Ebers
2006) differing in physiological and ecological traits (Jaspers and Overmann 2004).
Various methods aiming on cultivation of either “difficult to culture” or so far
“uncultured” microorganisms have been developed. Many of these methods avoid
utilization of media with high nutrient concentrations, and some of these methods
yield cultures exclusively growing in liquid media (Rappe et al. 2002). While the
majority of so far cultivated prokaryotes are maintained on solidified medium, the
trend in cultivation of eukaryotic microorganisms is rather the opposite. Besides
cultures of yeasts and a minor fraction of protists, eukaryotic microorganisms are
frequently cultivated in liquid media. Furthermore, the majority of such cultures do
not represent pure cultures consisting only of a clone of a single organism. Fre-
quently such liquid cultures consist of a single eukaryote or a few eukaryotes mixed
with a not characterized diversity of prokaryotes. Especially phagotrophic protists
like ciliates and many other heterotrophic protists (protozoa) are maintained in
cultures regularly fed with prey organisms like algae. A typical ciliate culture usually
consists of the ciliate; its food, e.g. an algae; and an unknown number of diverse
bacterial taxa. Such mixed cultures are termed xenic cultures, while pure cultures
free of other organisms are termed axenic cultures.

10.3 Quantification of the Uncultured Fraction of Microbial

Diversity

The scientific literature is full of quantitative statements on culturability of environ-

mental bacteria (or microorganisms) which consider proportional estimation of the
“not yet cultured” or “not culturable” fraction of bacterial (or microbial) diversity.
Many papers state that of natural bacterial communities, only 1% or <1% of cells
enumerated microscopically can be grown on standard media (i.e. represent readily
culturable cells). Other papers state that only 1% or <1% of cells of such commu-
nities are culturable. Such statements usually do not mention methods considered for
cultivation, for instance, standard or non-standard methods. In addition, some
publications applied this 1% or <1% figure to the so far cultivated fraction of the
global bacterial diversity. There are papers which use this 1% figure for bacterial cell
numbers or diversity, and other papers use it for all microorganisms. The almost
10 Isolation and Cultivation of Bacteria 319

universal application of the (<) 1% figure seems to suggest that quantification of

cultivation success and estimation of the fraction of so far uncultured taxa are both
trivial issues but this is not the case. While efficiency of particular cultivation
experiments can be measured, the latter issue can currently only be addressed by
unfirm estimations and speculations.
The success of cultivation experiments can be quantified as cultivation efficiency
in different ways by referring to parameters characterizing a particular sample used
for a cultivation experiment. Those parameters are usually cell numbers, but taxa
numbers or community fractions could also be used (Fig. 10.2). Nonquantitative
measures of cultivation success could be presentation of new taxa not represented by
previously described genera, families or even taxa of higher rank (Tamaki et al.
2011; Mori et al. 2009; Zhang et al. 2003).
The traditional measure of cultivation efficiency is the comparison of the number
of cells present in a sample with the number of cultures obtained from the sample
(Jannasch and Jones 1959). The number of cells in the sample is nowadays usually
enumerated either by epifluorescence microscopy (Bruns et al. 2003b) or by flow
cytometry (Button and Robertson 2001) of stained samples; however, application of
these methods to samples from soil, sediments or faeces is difficult. The determina-
tion of the number of cultivated cells depends on the kind of cultivation methods
used in the experiment. If solidified media are employed, the number of colonies
(CFU, colony-forming units) is used as a measure of cultivated cells (Jannasch and
Jones 1959). If exclusively liquid media are used, the number of cultivated cells is
determined as most probable number (MPN) by dilution experiments (Bruns et al.
2002). The cultivation efficiency (CEc) is expressed as percentage of total number of
counted cells represented by cultures. In standard cultivation experiments,
i.e. spreading of samples on agar plates, the cultivation efficiency equals the ratio
of cultivable cells to the total number of countable cells expressed as a percentage
{[(number of colonies obtained per sample volume)/(number of cells counted per
sample volume)]
100} (Fig. 10.2). The determined efficiencies usually depend on
the kind of investigated sample and the kind of cultivation method used for the
experiment (Jannasch and Jones 1959; Bruns et al. 2002; Janssen et al. 2002; Davis
et al. 2005; Sait et al. 2006). Some review papers provided overviews on quantitative
results of cultivation experiments by categorization of various types of environmen-
tal sample (Amann et al. 1995; Schleifer 2004; Puspita et al. 2012). The data
presented by Amann and colleagues, as well as by Schleifer represent exclusively
quantifications comparing microscopical counts with the number of colony-forming
units (CFU) on agar plates, while the review by Puspita and colleagues also included
most probable numbers obtained from cultivation experiments using liquid media.
The reported cultivation efficiencies ranged from 0.0007 to 0.2% for desert soil
(Connon et al. 2007) to 58% for human faeces (Wilson and Blitchington 1996). For
some habitat types, for instance, for soil, the three reviews presented diverging
figures. In the oldest review paper, a cultivation efficiency of 0.3% (Torsvik et al.
1990) is mentioned, while the latest review reported a range of 2.4–19% (Sait et al.
2002). This difference may have resulted from the more sophisticated cultivation
methods employed by Sait and colleagues. However, due to the broad variety of
320 M. W. Hahn et al.

Fig. 10.2 Theoretical example on quantification of cultivation success. (a) A sample containing ten
cells representing four taxa is used in a cultivation experiment. The experiment yields three cultures
representing two taxa. (b) The three isolates represent 30% of cells contained in the sample
(cultivation efficiency based on cell numbers, CEC), 50% of taxa contained in the sample (CET)
and 70% of the community contained in the sample (sum of relative abundance of cultured taxa in
sample, i.e. community fraction, CECF). The specific cultivation efficiencies for taxon A, B, C and
D are 33%, 0%, 0% and 100%, respectively. Ce./Sa. cells/sample, Iso./Sa. isolates/sample, Re.Ab.
relative abundance
10 Isolation and Cultivation of Bacteria 321

reported efficiencies and methodological differences between the reviewed studies,

it is not reasonable to calculate an average cultivation efficiency. Importantly, a
rough trend of increasing cultivation efficiencies with increasing trophic status of
habitats or samples is obvious (Puspita et al. 2012). Rather oligotrophic environ-
ments like desert soil, fresh and seawater tend to result in lower efficiencies than
more nutrient-rich (i.e. higher concentrations of organic carbon) environments like
soil, sediments, activated sludge or faeces. On the other hand, due to difficulties in
microscopic enumeration of cell numbers in complex samples like soil, sediment and
activated sludge samples, cultivation efficiencies determined for those samples may
suffer from underestimation of total cell numbers. However, it is possible that such
potential overestimation of cultivation efficiency is at least partially compensated by
another bias. Complex and heterogeneous samples may contain clumped bacterial
cells (e.g. microcolonies) or microscopic objects carrying several attached bacterial
cells (e.g. small soil particles) which could result in underestimation of cultivation
efficiency if such cells are separately counted by microscopy but formed only a joint
colony on an agar plate. Janssen and colleagues determined for untreated soil
samples that the potential cultivation efficiency cannot exceed about 65% of present
prokaryotic cells due to the presence of a high percentage of clumped cells (Janssen
et al. 2002). Sonication of samples was demonstrated to reduce the resulting
underestimation of cultivation efficiency (Janssen et al. 2002). Another factor
potentially resulting in underestimation of cultivation efficiency is formation of
microcolonies overlooked in macroscopical counting of CFUs (Jannasch and
Jones 1959). In a pioneering study, Jannasch and Jones investigated the influence
of different media and cultivation methods on the cultivation efficiency of marine
bacterioplankton (Jannasch and Jones 1959). Both formation of microcolonies on
filter membranes and formation of macrocolonies on standard agar plates were
considered for the determination of cultivation efficiencies. Standard cultivation
(macrocolonies) resulted in an average cultivation efficiency of about 0.7% of
microscopic counts, which was probably a too high figure due to underestimation
of total bacterial numbers caused by microscopic limitations at the time the study
was performed. However, counting of microcolonies resulted in a 20–35 times
higher efficiency (about 13–23%) compared to the standard cultivation. Other
investigations confirmed these trends for other habitat systems (Ferrari et al. 2005,
2008). Furthermore, prolonged incubation of agar plates was demonstrated to
increase visibility of small colonies formed by slowly growing bacteria (Davis
et al. 2005, 2011). Besides consideration of macro- and microcolonies, quantitative
cultivation experiments conducted by using liquid low nutrient media usually result
in increased cultivation efficiencies (Bruns et al. 2002, 2003a, b; Köpke et al. 2005).
It has to be noted that most of these mentioned cultivation efficiencies were only
based on temporary cultivation without any proof for feasibility of sustained
cultivation.
A second but more theoretical perspective on cultivation efficiency is coverage of
microbial diversity present in a sample or system by cultivation. This measure of
cultivation efficiency (CET) compares taxon or species richness of the sampled
community with the taxon richness of the cultivated fraction of the community
322 M. W. Hahn et al.

(Fig. 10.2). If a community would consist in total of 1000 taxa and a cultivation
experiment would result in cultivation of 200 of those taxa, CET would be 20%. This
approach requires binning of organisms present in the sample and represented by
cultures in taxa. Because the majority of prokaryotes dwelling in natural environ-
ments represent undescribed species (see below), classification into species catego-
ries makes no sense. An alternative method, which is used in various diversity
studies, is classification in operational taxonomic units (OTU) defined by sequence
similarity or phylogenetic position of organisms (Wang et al. 2007; Schloss and
Handelsman 2005). Usually such OTU classifications are based on sequences of
SSU rRNA genes (Schloss and Westcott 2011). In sequence similarity-based bin-
ning, sequences of cultured or uncultured organisms, respectively, are compared
pairwise and binned according to their similarity values. Threshold values frequently
used for OTU classification are 97% or 99% SSU rRNA gene sequence similarity
(Thompson et al. 2005).
A third taxon-based measure of cultivation efficiency (CECF) refers to the fraction
of the studied community represented by obtained cultures (Fig. 10.2). This measure
includes the relative abundance of taxa in the community and weights abundant taxa
more than rare taxa. This measure is of interest if the aim of the cultivation
experiment is to provide model organisms representing important players of a
community. The probably most impressive example of cultivation of strains
representing a large fraction of a community is the isolation of SAR11 bacteria
(Rappe et al. 2002). SAR11 represents a clade of Alphaproteobacteria comprising
by average 20–30% of marine bacterioplankton in surface waters (Morris et al.
2002). Strains affiliated to this clade were cultivated by a high-throughput technique
based on dilution-to-extinction and cultivation in nutrient-poor media (Rappe et al.
2002; Connon and Giovannoni 2002). The first obtained isolates were described as
Candidatus Pelagibacter ubique (Rappe et al. 2002). Further investigation of the
obtained isolates provided deep insights in their physiology, ecology and evolution
(Giovannoni et al. 2005a, b; Vergin et al. 2007; Tripp et al. 2008). Knowledge
gained by such studies even enabled the development of improved media better
suited for cultivation of members of the SAR11 clade (Carini et al. 2013). Other
examples are the cultivation of Polynucleobacter strains representing at least at the
time of sampling about 60% of the bacterioplankton community in a freshwater pond
(Hahn 2003; Hahn et al. 2005; Jezberova et al. 2010). The contribution of more than
50% of the total cell number of freshwater bacteria by Polynucleobacter bacteria is
not a usual figure; however, a contribution of more than 10% by this taxon are
typical values for many freshwater systems (Jezberova et al. 2010, 2012). By
targeted isolation of Limnohabitans (Kasalicky et al. 2013) and Polynucleobacter
(Hahn et al. 2004; Watanabe et al. 2009), it is possible to cover by average about
20% of freshwater bacterioplankton (Jezbera et al. 2012). Strains affiliated with the
genera Polynucleobacter and Limnohabitans are known to grow on agar plates
solidified with a standard agar concentration of 1.5% (w/v) (Hahn 2003; Hahn
et al. 2009; Kasalicky et al. 2010, 2013), and at least Polynucleobacter strains can
be isolated from lake or pond water by direct plating of samples on modified R2A
(Watanabe et al. 2009, 2012) or NSY agar plates (Hahn et al., unpublished data). But
10 Isolation and Cultivation of Bacteria 323

this does not mean that cultivation experiments with direct plating of samples from
habitats with >10% Polynucleobacter bacteria known to grow efficiently on such
media consistently would result in cultivation efficiencies of >10% (Hahn et al.,
unpublished data).
To our knowledge, large-scale cultivation experiments aiming on cultivation of a
maximum number of taxa from a single environmental sample by using a large
number of media and methods (Fig. 10.3) have not been performed so far. Applica-
tion of a large number of different cultivation methods would be required because of
selectivity of all methods and media. Frequently, taxon-based cultivation studies
compare phylogenetic positions of obtained isolates and their previously cultured
closest relatives (with sequences available from public databases) by construction of
phylogenetic trees with SSU rRNA sequences (Zengler et al. 2002) and claim
cultivation success if at least some of the obtained cultures are only distantly related
(e.g. <94% or <97% SSU rRNA similarity) to previously cultured strains (Zengler
et al. 2002; Hahn et al. 2004; Janssen et al. 1997, 2002; Sait et al. 2002; Connon and
Giovannoni 2002; Page et al. 2004; Cho and Giovannoni 2004). In a much more
frequent kind of study aiming on characterization of the diversity of natural com-
munities of microorganism, SSU rRNA sequences of uncultured organisms obtained
by cultivation-independent methods are also compared with sequences of cultured
organisms but with all previously cultured and sequenced strains independently of
their origin (Britschgi and Giovannoni 1991; Mullins et al. 1995; Hugenholtz et al.
1998; Zwart et al. 2002; Newton et al. 2011; Janssen 2006). Each such study
focusing on a natural system demonstrated that the vast majority of microorganisms
(including eukaryotic microorganisms) is not represented by closely related cultured
relatives. However, this kind of analysis only suggests that the vast majority of
extant microorganisms was not cultured so far, but does not provide a reliable
estimation of the percentage of yet uncultured diversity.
It can be concluded that the cell number-based cultivation efficiency (CEc) of
direct plating on standard bacteriological media is frequently <1% of microscopical
cell counts but may exceed 1% if nutrient-rich environments like activated sludge are
investigated (Amann et al. 1995). Isolation experiments using liquid low nutrient
media tend to result in higher CEc (Bruns et al. 2003a, b). However, presentation of a
general figure for CEc of naturally occurring prokaryotic communities is impossible.
Due to the selectivity of media and methods, the determination of cultivability of
members of a certain community would require a large number of cultivation
experiments employing a broad range of different media (Fig. 10.3). Since different
media may cultivate overlapping fractions of taxa present in the studied community,
the cumulative cultivation efficiency of the tested media has to be determined as
taxon-based efficiency CET. Studies based on taxon richness, i.e. the number of taxa
present in the studied community, are faced with the problem of determination of the
richness of the rare microbial biosphere (Sogin et al. 2006; Kunin et al. 2010). This
problem could be circumvented by exclusion of rare taxa contributing, for instance,
less than 0.1% of the community. The presence of rare taxa culturable only with a
few specific media would also be a problem in a large-scale experiment aiming on
the estimation of the fraction of “unculturable” taxa (compare Fig. 10.3). However,
324 M. W. Hahn et al.

Fig. 10.3 Schematic illustration on hypothetical cumulative cultivation success resulting from an
infinite number of cultivation experiments with different cultivation methods (M1 to M1) applied to
a natural community of prokaryotes in a particular habitat (e.g. water of a hot spring). Each method
is able to cultivate a certain percentage of cells and taxa (e.g. strains sharing >99% SSU rRNA
10 Isolation and Cultivation of Bacteria 325

due to the lack of appropriate experiments employing a large number of different

media, even sound estimates of the fraction of basically cultivable taxa present, for
instance, in marine prokaryotic communities are currently lacking. We can only
speculate on the fraction of taxa resistant to cultivation by the currently available
methods.
Obviously, the determination of CEc for natural microbial communities by using
one or a few standard media does not result in meaningful data. The majority of
microbiologists acknowledge that cultivation methods are not suitable for determi-
nation of diversity of communities or abundance of particular taxa (Amann et al.
1995). This is similar in clinical microbiology; however, in contrast to environmen-
tal microbiology, clinical microbiologists developed a large number of cultivation-
dependent assays for specific detection of the majority of bacterial pathogens of
humans (Delmée et al. 2005; Becker et al. 2013; Gould et al. 2009). Such highly
selective media and methods are largely lacking for the majority of readily culturable
environmental prokaryotes.
A question of great scientific importance is about the global richness of microbial
diversity, and the fraction of this diversity so far cultivated by microbiologists and
described as species by taxonomists. Unfortunately, it is a highly difficult and
currently probably impossible task to address these questions properly. First
attempts to estimate the global number of operational taxonomic units defined by
sequence similarities of SSU rRNA greater than 97% (OTU97%) were done (Curtis
et al. 2002, 2006), but the vast diversity of prokaryotes and again the problem of
accessing and quantification of the rare biosphere (Huse et al. 2010; Kunin et al.
2010; Sogin et al. 2006) limit the results of such efforts. Due to these reasons, and
due to limitations in transforming OTU numbers into species numbers (Stackebrandt
and Ebers 2006), the currently available estimations on the global number of
undescribed prokaryotic species are based on vague estimations and assumptions,
which results in a quite large range of estimates. While the current (March 2015)
number of validly described species of prokaryotes is about 13,000 (www.bacterio.
net), the estimates of prokaryotic species number on earth range from >11,100
(Mora et al. 2011) to <2
106 in the ocean (Curtis et al. 2002) to 109 species of
prokaryotes (Dykhuizen 1998, 2005). The highest of these estimates suggest that
99.999% of the existing prokaryotic species thus far have not been taxonomically
described.

⁄


Fig. 10.3 (continued) sequence similarity). The diagram at the bottom depicts the cumulative
number of taxa cultivated by using the infinite number of methods. Fractions A and B represent
taxa culturable by standard and non-standard methods, respectively. Fraction C represents rare taxa
with medium requirements only met by a few special media. Due to the rareness of the taxa in the
environment it is unlikely that representatives of the taxa are present in inoculi used for the few
experiments with the suitable media. As a result strains represented by this fraction will remain
uncultured. If unculturable taxa really exist, they would be represented by fraction D, but this
fraction could not be distinguished from fraction C
326 M. W. Hahn et al.

10.4 Reasons for Either Lack of or Low Culturability

of Prokaryotic Cells

A large number of papers have tried to explain why many prokaryotic cells are
refractory to cultivation (Vartoukian et al. 2010; Stewart 2012; Rappe and
Giovannoni 2003; Pham and Kim 2012; Alain and Querellou 2009). Most authors
assume that a multitude of reasons are responsible for this phenomenon and that
reasons might be different from taxon to taxon or even from cell to cell. The known
potential reasons for lack of culturability of a certain cell can in principle be
classified to three different categories (Fig. 10.3): (1) cells are nonviable, (2) cells
are not culturable by the applied method, and (3) cells are viable and could basically
be cultured by the applied method but lack at present the appropriate physiological
status for growth under the artificial conditions provided by the method.

10.4.1 Nonviable Cells

The first category covers dead (nonviable) cells; however, the status dead is difficult
to be defined in prokaryotes and other microorganisms (Roszak and Colwell 1987).
Usually the presence of an intact cell membrane is used to distinguish between viable
and nonviable cells (Nocker et al. 2006), because an intact membrane is required to
maintain the proton motive force essential for prokaryotic energy metabolism.
Several cytological methods are used to diagnose the physiological stage of single
cells or populations (Kell et al. 1998). A couple of those methods are based on
testing the presence of an intact permeability barrier (e.g. life/dead stain), while other
methods are based on the detection of messenger RNA, which rapidly disappears in
cells lacking transcription. Due to methodical limitations in detection of nonviable
cells in environmental samples, only little is known about the abundance of
nonviable cells under natural conditions.

10.4.2 Unsuitability of Cultivation Method Employed

The second category covers cases, where the applied cultivation method is
unsuitable for cultivation of a certain taxon. The term method circumscribes here
again the entire cultivation procedure (Fig. 10.1), which includes potential
pretreatment of sample (e.g. dilution or size fractionation), the kind of medium or
media used for the cultivation experiment, as well as the employed cultivation
conditions (e.g. temperature, gas phase, illumination, agitation). This global view
is necessary because a single detail may hinder growth of taxa otherwise able to
grow under the offered conditions.
10 Isolation and Cultivation of Bacteria 327

A crucial component of each cultivation method is the medium (or media)

employed. The medium should cover all requirements of the organism to be culti-
vated. As discussed above, media are always selective, and creation of a universal
medium is most likely impossible. The literature is full of examples of successful
struggle for development of media suitable for cultivation of certain targeted micro-
organisms (e.g. Mycobacterium tuberculosis, Legionella pneumophila). The large
number of described media (Atlas 2010) emphasizes the importance of media
development in cultivation of microorganisms. Media may be unsuitable for culti-
vation of certain organisms because they lack essential growth factors. Legionella
pneumophila, the causative agent of Legionnaires’ disease, for instance, requires L-
cysteine in the medium (Ewann and Hoffman 2006), and the abundant marine
bacterium Candidatus Pelagibacter ubique requires reduced sulphur compounds
(Tripp et al. 2008). Furthermore, media may lack common goods produced under
natural conditions only by a small fraction of a microbial community but utilized by
a wider range of microbes. Known common goods are siderophores (D’Onofrio et al.
2010) required by some bacteria for iron uptake under iron-limited conditions or
enzymes protecting from oxidative stress. Bogosian and colleagues demonstrated
that Vibrio vulnificus in the viable but nonculturable state can be cultivated with an
up to 1000-fold higher efficiency if the cultivation medium is supplemented with
catalase an hydrogen peroxide (H2O2)-decomposing enzyme (Bogosian et al. 2000),
and Morris et al. demonstrated that heterotrophic bacteria (helper bacteria) protect
Prochlorococcus strains against oxidative stress and increase by this protection the
cultivation efficiency for this important marine phototroph (Morris et al. 2008).
Recently, Tanaka and colleagues revealed that autoclaving of agar in the presence
of phosphate, which is a usual procedure in media preparation, resulted in the
formation of H2O2 causing a significant reduction of the number of CFU on plates
inoculated with environmental samples. Furthermore a negative influence on the
diversity of taxa able to grow on the plates was demonstrated (Tanaka et al. 2014).
These findings reemphasize the importance of using compounds like catalase or
pyruvate known to eliminate H2O2 as supplements in microbial media used for
isolation of strains from the environment (Stevenson et al. 2004). On the other hand,
it remains unknown if only the formation of H2O2 caused inferior cultivation
efficiencies of plates solidified with agar compared to other solidifying reagents
like gellan gum (Janssen et al. 2002; Tamaki et al. 2005).
The abovementioned phenomenon that prokaryotes can only be cultured in the
presence of helper cells was repeatedly observed (Kaeberlein et al. 2002; Morris
et al. 2008; Jezbera et al. 2009; Garcia et al. 2014), but in most of the cases it is not
known which shared goods or service are provided by the helpers. One example is
the growth of the freshwater actinobacterium Candidatus Limnoluna rubra enabled
by the betaproteobacterial helper bacterium Polynucleobacter sp. on an agar plated
(Hahn 2009). The Actinobacteria can only grow in the immediate proximity of
colonies of the helper strain (Fig. 10.4), which indicated dependence on a substrate
or service provided by the helper. Interestingly, Polynucleobacter strains were also
found to be efficient helper strains in cultivation of two other but only distantly
related Actinobacteria. These Actinobacteria represent two different lineages within
328 M. W. Hahn et al.

Fig. 10.4 Coculture of the

actinobacterium Candidatus
Limnoluna rubra strain
MWH-EgelM2-3 (orange
pigmentation) and the
betaproteobacterium helper
strain Polynucleobacter
sp. (without pigmentation)
on an NSY agar plate. The
plate was inoculated with a
dense mixed culture of the
two strains grown in liquid
NSY medium. Despite the
sample was evenly spread
on the agar plate, colonies of
the actinobacterium grew
only in the immediate
proximity of
Polynucleobacter colonies.
So far, Candidatus
Limnoluna rubra could only
be grown in the presence of
the helper strain (Hahn
2009)

the acI Actinobacteria clade (Jezbera et al. 2009; Garcia et al. 2014). The Epstein lab
systematically exploited the helper concept for cultivation of previously uncultured
prokaryotes by using diffusion chamber devices (Kaeberlein et al. 2002; Bollmann
et al. 2007). Besides lack of specific growth factors or substrates, other factors may
cause a certain medium to be unsuitable for cultivation of a specific prokaryote. Such
factors could be the adjusted pH value, a too high substrate concentration and the
presence of inhibiting compounds.

10.4.3 Temporary Impairment Due to Physiological Status

The third category includes cases where the organisms are in principle able to grow
on the provided medium and at the provided incubation conditions but currently lack
the physiological status enabling them to start growth under the provided conditions.
Those cells may require an appropriate adjustment of their physiological status
(i.e. gene expression pattern) to the provided cultivation conditions. Initiation of
10 Isolation and Cultivation of Bacteria 329

the adjustment may be triggered either by a specific stimulus or by an appropriate

transition of conditions towards the artificial cultivation conditions. A classic exam-
ple regarding the influence of the physiological status of particular cells on cultiva-
tion success is the viable but nonculturable (VBNC) state of bacteria (Roszak and
Colwell 1987). Several pathogenic and non-pathogenic taxa, e.g. Escherichia coli
and Vibrio spp., are known to be able to enter this dormant physiological state if
environmental conditions turned unsuitable for their growth (Oliver 2010). As
mentioned above cells in this state can be wakened and enabled again for growth
on standard media by specific treatments. However, in the genus Polynucleobacter
representing important freshwater bacteria, the lack of previous cultivation success
cannot be explained by hindering due to VBNC states. This ubiquitous and abundant
freshwater taxon (Jezberova et al. 2010) was not cultivated before 2003 (Hahn
2003). After successful cultivation of a larger number of strains by a novel method,
ten strains were tested for their abilities to grow on plates of ten different standard
media (Hahn 2003). Surprisingly, the tested strains could grow by average on 84%
of the tested complex media. Interestingly, R2A Medium was found to be the best-
performing medium supporting growth of all ten strains with above-average yields
which represents a medium very frequently used for isolation of bacteria from
environmental samples (see below). Given the abundance and ubiquitous presence
of Polynucleobacter bacteria in freshwaters and their ability to grow on a very
frequently used standard medium, the fact of the previous lack of cultivation of
this taxon requires a special explanation. The first isolation of Polynucleobacter
strains was performed by stepwise acclimatization of bacteria to higher substrate
concentrations and exclusion of a large fraction of potential competitors by sample
fractionation (filtration). Control experiments with skipped acclimatization and
direct plating of fractionated samples on agar plates did not result in growth of
Polynucleobacter strains (Hahn et al. 2004), leaving only the acclimatization pro-
cedure as explanation for successful cultivation. The stepwise acclimatization to
higher substrate concentrations may help to avoid substrate-accelerated death known
to result from exposure of growth limited bacteria to increased concentrations of the
growth-limiting substrate (Postgate and Hunter 1963). A mechanistic but unproven
explanation for the effect of the acclimatization procedure could be that the gradual
transition to higher substrate concentrations gives metabolically rather inflexible
bacteria the opportunity to readjust their metabolism to the strongly changing
environmental conditions. It can be assumed that such a gradual transition is
especially of importance in bacteria adapted to rather stable environmental condi-
tions. Polynucleobacter bacteria are characterized by a rather small genome size of
about 2 Mbp resulting in a rather spare metabolic network and only a low number of
signal transduction genes (Hahn et al. 2012b). Signal transduction is of crucial
importance in sensing of environmental changes; therefore Polynucleobacter and
other bacteria with limited signal transduction abilities may simply miss the required
prompt readjustment of their metabolism to suddenly changed environmental con-
ditions. The demonstrated isolation of Polynucleobacter strains by direct plating
(without acclimatization) on an improved R2A Medium (Watanabe et al. 2009,
2012) seems to argue against this hypothesis; however, inoculation of an agar
330 M. W. Hahn et al.

plate with 100 μL of a lake water sample [about 106 bacteria mL1 with a relative
abundance of Polynucleobacter bacteria of 1–10% (Jezberova et al. 2010)] results in
seeding of 103–104 Polynucleobacter cells on each agar plate but only in growth of a
few Polynucleobacter colonies (Watanabe et al. 2012). These few colonies may
represent a small fraction of Polynucleobacter cells possessing by chance a gene
expression pattern suitable for surviving a sudden change from in situ conditions to
artificial cultivation conditions. Acclimatization enabled cultivation of several pre-
viously uncultured bacteria, including, for instance, Limnohabitans spp. (Kasalicky
et al. 2013) representing important freshwater bacteria (Jezbera et al. 2012), as well
as various freshwater Actinobacteria characterized by very small genome sizes. Such
freshwater Actinobacteria, e.g. Rhodoluna lacicola MWH-Ta8, possess genomes
with only 1.4 Mbp (Hahn et al. 2014), which is only slightly larger than the genome
size of Cand. Pelagibacter ubique and other SAR11 strains (Giovannoni et al.
2005b). In contrast to SAR11 strains, R. lacicola and other freshwater
Actinobacteria are able to grow on agar plates with high substrate concentrations.
Despite the Acclimatization Method attempted for enabling the cultivation of a
broad diversity of previously uncultured bacteria, it well may fail in cultivation of
obligate oligotrophs like SAR11 bacteria.
Other examples of in principle cultivable but only very rarely cultured bacteria
are certain Verrucomicrobia and Acidobacteria strains. Especially the latter phylum
represents bacteria highly abundant in soil systems, but despite their environmental
significance, this phylum is extremely underrepresented in culture collections. Some
of the few cultivated strains were obtained by sophisticated isolation strategies
(Koch et al. 2008), while other strains could be cultivated by using simple direct
plating of samples on only slightly modified standard medium (Mannisto et al.
2011). Similar success was reported for novel Verrucomicrobia strains (Sangwan
et al. 2005). As in the case of the Polynucleobacter bacteria, these observations on
Acidobacteria and Verrucomicrobia could be explained by basic culturability of at
least some lineages of these phyla but lack of a physiological state (readily
culturable) enabling survival of a sudden change to artificial cultivation conditions
in the vast majority of cells affiliated with such culturable lineages. Potentially the
percentage of readily culturable cells in populations of some taxa can be positively
influenced by application of signal compounds. Burns and colleagues demonstrated
that the supplementation of media with signal compounds like cAMP and
homoserine lactones resulted in an increase of general cultivation efficiency
(Bruns et al. 2002, 2003b).
10 Isolation and Cultivation of Bacteria 331

10.5 Does Genome Size Play a Role in Culturability

of Prokaryotes?

Genome size of prokaryotes varies from <0.1 Mbp to >10 Mbp. A look on the
distribution of genome size among cultivated genome-sequenced bacteria indicates
underrepresentation of bacteria with small genome size among the sequenced
strains. The average genome size of the 3602 prokaryotic genomes (almost all
representing cultivated strains) contained in the Integrated Microbial Genomes
(IMG) database (Markowitz et al. 2012) and classified with the sequencing status
“finished” is 3.5 Mbs. Only 26% of those genomes possess genome sizes <2 Mbp,
and the vast majority of these small genomes represent host-associated
(e.g. Helicobacter), symbiotic (e.g. Buchnera) or pathogenic strains
(e.g. Borrelia). While small genomes represent about one quarter of all finished
genomes, they represent only one-seventh (14%) of all 604 genomes linked to the
ecosystem types “marine”, “soil” and “freshwater”, and remarkably, the average size
of genomes associated with strains from these ecosystems is 4.2 Mbp, thus larger
than the overall average of all genomes. By contrast, metagenomic investigations
(Yooseph et al. 2010; Biers et al. 2009), single-cell genomic analysis (Swan et al.
2013) and genome sequencing of cultivated strains representing abundant taxa
(Dufresne et al. 2005) suggested that at least the water column of marine and
freshwater systems are dominated by prokaryotes possessing small genome sizes.
Cultures of representatives of abundant free-living marine or freshwater taxa char-
acterized by small genome size (e.g. Cand. Pelagibacter, Prochlorococcus, fresh-
water Actinobacteria, Polynucleobacter) were usually not obtained by standard
methods, and their cultivation is still considered as being difficult. Obviously,
there is a link between small genome size and underrepresentation of respective
isolates in culture collections. These free-living prokaryotes share the trait of small
genome sizes with bacteria possessing an obligate endosymbiotic or parasitic life-
style like Buchnera aphidicola, an obligate endosymbiont of aphids. In both cases,
the free-living pelagic bacteria and the obligately host-associated bacteria, the small
genome sizes resulted from reductive genome evolution (Batut et al. 2014; Mira
et al. 2001; Giovannoni et al. 2014).
The low culturability of obligate symbionts and parasites is assumed to be a
consequence of reductive genome evolution taking place in the evolutionary process
transforming free-living or facultatively host-associated taxa to obligately host-
associated organisms. Genes required for synthesis of essential compounds essential
for free-living stages but provided by host cells to host-associated stages usually lose
functionality due to mutations and lack of stabilizing selection. This results in
transformation of genes to pseudogenes, which usually undergo further gene erosion
and get finally completely lost. Other genes encoding crucial cell functions
(e.g. predation defence) not required by host-associated stages may also undergo
this process of gene loss. This reductive genome evolution results in streamlined
genomes and in physiologies demanding specific compounds and environmental
conditions which are provided by their specific host. The details of these demands
332 M. W. Hahn et al.

are usually unknown, and therefore it is difficult to provide under in vitro conditions
obligately host-associated strains with all their requirements. A typical example for
these difficulties in cultivation is Buchnera aphidicola (Gammaproteobacteria,
Enterobacteriaceae) the endosymbiont of various aphid species. These obligate
endosymbionts were intensively investigated for decades, they are well represented
by complete genome sequences, and detailed knowledge on their physiology and
ecology is available but still pure cultures representing this taxon are lacking.

10.6 Cultivation and Description of New Prokaryotic Taxa

Since a few decades, a mandatory requirement for the valid description of new
species of prokaryotes is the deposition of cultures of the type strain in at least two
public culture collections located in two different countries (Tindall et al. 2006).
Journals like the International Journal of Systematic and Evolutionary Microbiology
(IJSEM), which is the most important journal for publication of descriptions of new
prokaryotic taxa (see below), do not accept manuscripts proposing new species for
publication before certificates of deposition from two culture collections were
presented. Thus, IJSEM is a good source for data suitable for analyses on which
new taxa were isolated with what method and medium.
According to the List of Prokaryotic Names with Standing in Nomenclature
(LPSN, http://www.bacterio.net) (Parte 2014), the current number of validly
described prokaryotic species is about 13,000. About 600 new species and about
100 new genera were described annually during the past 10 years (Fig. 10.5). The
vast majority of these new taxa is based on new isolates and only a minor fraction of
the new taxa represents revised previously described taxa. Furthermore, most of the
descriptions and revisions are published in IJSEM. We analysed all descriptions of
bacterial taxa published in volumes 2009 and 2010 in the IJSEM. The analysis of
new archaeal taxa was omitted because the number of such descriptions is too small

Fig. 10.5 Total number of

new prokaryotic species and
genera described per year in
the period 1980–2013. The
graph depicts data obtained
from the LPSN webpage
(http://www.bacterio.net)
10 Isolation and Cultivation of Bacteria 333

for revealing significant trends. In 2009 and 2010, a total of 1042 new species and
168 new genera were described in this journal (Table 10.1), which represented 82%
and 77% of all new species and genera, respectively, described in these 2 years
(Fig. 10.5). We analysed the methods and media used for isolation of the type strains
representing the new taxa. We distinguished between “standard” and “non-standard
methods”, whereas the former category includes all isolations performed by direct
plating of environmental samples on agar plates, while the latter comprises all
cultivation methods deviating from this simple approach, for example, by establish-
ment of an enrichment culture preceding the plating on agar plates or application of
advanced cultivation methods.
Examining the information summarized in Table 10.1, it can be seen that 51% of
the 1042 newly described species were isolated using standard methods. Nineteen
percent of the 1042 newly identified species were isolated by using non-standard
methods. The remaining 30% of the newly described species were regarded to have
been obtained using isolation methodology reported in the respective papers too
imprecise for assignment into either of those two methodological categories
(labelled in Table 10.1 as N/A). Comparison of data for standard and non-standard
methods revealed that a larger diversity (measured as Shannon indices) is covered by
the latter methods. The differences in the calculated Shannon and Shannon’s equi-
tability indices (Krebs 1999), respectively, seem to be minor; however, there is a
clear trend suggesting that the new species based on isolation by non-standard
methods are more equally distributed over genera. With other words, the employed
standard methods preferred certain genera and resulted by average in description of
2.1 new species per previously or newly described genus, while the used
non-standard methods resulted in a higher evenness regarding the distribution of
new species across genera (1.5 new species per genus). Consequently, the used
non-standard methods resulted in a more frequent description of new genera
(Table 10.1). While by average only each seventh new species obtained by standard
methods also represented a new genus, each fourth new species obtained by a
non-standard method had to be placed in a new genus. Comparison of sequence
similarity of SSU rRNA genes of the type strains of new species with the sequence of
their closest related species (Table 10.1) furthermore suggested that non-standard
methods obtain by average a bit more distantly related new taxa; however, this
difference in sequence similarity may mainly reflect the more frequent isolation of
strains representing new genera by non-standard methods. Species descriptions
(category “N/A” in Table 10.1) based on type strains isolated by methods neither
classified as standard nor as non-standard methods resemble in most of the analysed
parameters those descriptions based on standard methods (Table 10.1). The observed
differences in trends between standard and non-standard methods (Table 10.1) can at
least partially be attributed to a higher diversity of media used by the analysed
non-standard compared to the standard approaches (Table 10.2).
More pronounced differences between results of isolations performed with stan-
dard and non-standard methods were observed regarding the phylum affiliation of
the described new taxa. Almost 97% of the 1042 new species described belonged to
the “big four” phyla (Hugenholtz 2002), i.e. Proteobacteria, Actinobacteria,
 334

Table 10.1 Statistics on methods and media used for isolation of strains described as new species in papers published in the International Journal of Systematic
and Evolutionary Microbiology (IJSEM) in 2009 and 2010. The two Shannon indices were used to measure the diversity of described new taxa at the genus level
Average SSU rRNA
Genera Shannon’s similarity (closest
New species (new and Species/ Shannon equitability New genera New genera/ species)
Method or medium Number % old) genus Index (H ) (EH) Number % new species %
All methods (total) 1042 100.0 520 2.0 5.8 0.93 168 100.0 0.16 96.6
Standard methods 530 50.9 248 2.1 5.4 0.95 80 47.6 0.15 96.7
Non-standard 198 19.0 130 1.5 4.9 0.98 47 28.0 0.24 96.2
methods
N/Aa 314 30.1 142 2.2 4.9 0.95 41 24.4 0.13 96.9
9 Top media, total 450 43.2 250 1.8 5.2 0.95 61 36.3 0.14 97.1
Marine Agar 139 13.3 106 1.3 4.6 0.98 36 21.4 0.26 96.4
R2A Medium 117 11.2 72 1.6 4.1 0.95 10 6.0 0.09 96.8
Trypticase Soy 42 4.0 37 1.1 3.6 0.99 5 3.0 0.12 96.7
Medium
Nutrient Agar 42 4.0 31 1.4 3.3 0.96 3 1.8 0.07 98.2
Humic Acid 35 3.4 19 1.8 2.7 0.90 2 1.2 0.06 98.6
Vitamin Agar
Luria Broth Agar 35 3.4 25 1.4 3.0 0.95 2 1.2 0.06 97.9
Starch Casein 16 1.5 14 1.1 2.6 0.98 2 1.2 0.13 98.1
Agar
MRS Agar 14 1.3 4 3.5 0.8 0.54 0 0.0 0.00 98.3
Plate Count Agar 10 1.0 10 1.0 2.3 1.00 1 0.6 0.10 96.7
Other media, total 592 56.8 334 1.8 5.5 0.94 107 63.7 0.18 97.1
Note that these taxonomic descriptions represent 81.8% of all descriptions of new prokaryotic species in this 2-year period (Fig. 10.5)
a
N/A, classification in either the category standard or non-standard methods was not be possible due to the lack on specific information on the method
M. W. Hahn et al.
10 Isolation and Cultivation of Bacteria 335

Table 10.2 Comparison of kind of media used for isolation of type strains described as new
species either isolated with methods classified as standard or non-standard methods
Standard Non-standard
methods methods
Total number of described new species 530 198
Total number of different media used 129 119
Shannon index (H) 3.51 4.45
Shannon’s equitability (EH) 0.72 0.93
Most frequently used medium (number of obtained new Marine Agar Marine Agar (11)
species) (110)
2nd most frequently used medium (number of obtained R2A Agar (96) Nutrient Agar (9)
new species)
The data refer to descriptions of new species published in the International Journal of Systematic
and Evolutionary Microbiology (IJSEM) in the volumes 2009 and 2010

Table 10.3 Distribution of the 1042 new species described in 2009 and 2010 in IJSEM across
phyla
All methods Standard methods Non-standard methods
(% of total) (% of total) (% of total)
Proteobacteria 38.0 35.9 43.4
Actinobacteria 27.1 28.7 15.7
Firmicutes 17.2 16.8 24.2
Bacteroidetes 14.1 17.7 8.1
Other phyla 3.6 0.9 8.6

Firmicutes and Bacteroidetes; however, standard and non-standard methods pre-

ferred different phyla. As compared to standard methods, the use of non-standard
methods resulted in isolation of a smaller proportion of strains representing new
species affiliated with the phyla Actinobacteria and Bacteroidetes but a higher
proportion of Proteobacteria, Firmicutes, and representatives of other phyla
(Table 10.3). Standard methods resulted only in the description of five species
(0.9% of new species obtained by standard methods) not belonging to the “big
four” phyla, while non-standard methods resulted in the description of 17 new
species (8.6% of new species obtained by non-standard methods. The 5 type strains
obtained by standard methods represent exclusively Deinococcus species
(Deinococcus-Thermus phylum), but the 17 type strains obtained by non-standard
methods represent 9 different phyla (Aquificae, Chloroflexi, Deferribacteres,
Deinococcus-Thermus, Ignavibacteriae, Planctomycetes, Synergistetes,
Thermodesulfobacteria, Thermotogae) and 1 candidate of a novel phylum (Mori
et al. 2009). Four more phyla (Acidobacteria, Caldiserica, Fusobacteria and Spiro-
chaetes) are represented by strains obtained with unclassified methods (data not
shown).
Analysis of the phylum affiliation of newly described genera even suggests higher
selectivity by standard compared to non-standard methods (Fig. 10.6). While
non-standard methods enabled description of new genera affiliated with nine
336 M. W. Hahn et al.

Fig. 10.6 New genera described in the IJSEM journal in 2009 and 2010. Methods used for
isolation of the type strains were classified as standard or non-standard methods. For 41 (24.4%)
of the new genera, the published information on employed isolation methods were not sufficient for
assignment to either of these two categories. These new genera descriptions were assigned to a
separate category (N/A, not available). Numbers at the top of the columns represent the total
numbers of the described new genera and the percentage of newly described species also
representing newly described genera, i.e. the frequency of new genera among the described new
taxa (species)

different phyla, standard methods enabled proposals of new genera representing only
four different phyla. Furthermore, non-standard methods resulted over-
proportionally and under-proportionally in description of new genera affiliated
with the phyla Firmicutes and Actinobacteria, respectively.
A detailed analysis of the media used for isolation of the 1042 type strains
representing the newly described species revealed that almost 25% of type strains
were obtained by use of only two different media, i.e. Marine Agar (Zobell 1941)
and R2A Agar (Reasoner and Geldreich 1985), and almost 43% of type strains were
obtained by only nine different media (Fig. 10.7, Table 10.1). Note that the same
medium could be employed by standard and non-standard methods (Table 10.2).
Among the nine top media, Marine Agar is outstanding regarding the ratio of the
number of new genera per newly described species (Table 10.1). Thirty-six strains
(25.9% of type strains) of the 139 type strains obtained by Marine Agar represented
new genera, while the success of the other eight media for identifying new genera
ranged only from 0% to 13% (average 8.0%). Among the nine top media, R2A Agar
and Trypticase Soy Medium (TS) resulted in isolation of only four new species not
affiliated with one of the “big four” phyla, respectively.
New species obtained by the three top media, i.e. Marine Agar, R2A Agar and
Trypticase Soy Medium, showed only small taxonomic overlaps with regard to their
genus affiliations (Fig. 10.8). Only three genera received new species from all three
media. These genera were Bacillus (seven new species), Pseudomonas (five species)
and Paracoccus (three new species). The majority of genera (78%) received one or
more new species from only one of the three media. Obviously, these three media
10 Isolation and Cultivation of Bacteria 337

Fig. 10.7 Media used for isolation of the 1442 type strains described as new species in papers
presented in the International Journal of Systematic and Evolutionary Microbiology (IJSEM) in
2009 and 2010. Media used for description of 5–9 species, 2–4 species or only a single species were
lumped together in categories, respectively. Media used for isolation of the type species were not
mentioned for 14.1% of the described new species

Fig. 10.8 Venn diagram depicting taxonomic overlap of newly described species isolated with the
three most frequently used media [Marine Agar, R2A Agar and Trypticase Soy Medium (TS)]
regarding their genus affiliation. For instance, 5% of the 298 type strains isolated with one of these
three media were affiliated with genera covered by all three media, but 41.6% of strains are affiliated
with genera only covered by isolation experiments using Marine Agar. The presented data refer to
descriptions of new species published in 2009 and 2010 in IJSEM

strongly differ in their taxonomic selectivity, which could, at least partially, be

influenced by application of these media for different kinds of environmental
samples. Marine Agar is typically used for processing of samples from marine or
salt lake environments, while the other media typically are used for isolation
experiments on non-marine samples (e.g. freshwater or soil samples).
338 M. W. Hahn et al.

Fig. 10.9 Relationship between percentage of strains obtained by standard isolation methods and
the average SSU rRNA (16S rRNA) sequence similarity with the closest related described species in
newly established taxa of different rank. Only the highest rank established for a certain type strain
was considers. That means that data for type strains representing new orders were not included in
the lower categories although these type strains usually also represent new species, genera and
families. Numbers in brackets depict the number of taxa established with the respective rank

Across all media used for isolation of type strains of new species, the genera
Streptomyces (25 new species), Paenibacillus (21 new species) and Bacillus (20 new
species) were the 3 genera receiving the highest numbers of new species. Interest-
ingly, only 8% of the new Streptomyces species but 43% of the new Paenibacillus
and 65% of the new Bacillus species were obtained by one of the nine top media
(Table 10.1).
A general trend regarding suitability of standard methods for isolation of strains
representing new taxa of higher taxonomic rank is obvious (Fig. 10.9). Strains
resulting in the description of new families or orders were only rarely isolated by
using standard methods. Only 14.3% of strains resulting in description of new
families were obtained with a standard method. All other new families and all new
orders described during the period under review were obtained either by
non-standard methods or methods which could not be classified due to the lack of
sufficient information.
The contribution of advanced cultivation methods (see below) to the description
of new prokaryotic taxa is still quite small. Some of the previously proposed
advanced cultivation methods did not contribute to the isolation of a type strain
representing a new species. The high-throughput cultivation (HTC) technique did
contribute a substantial number of new species and interesting taxa of higher rank
(Table 10.4). High-throughput cultivation combined with dilution-to-extinction and
initial cultivation in low nutrient media (frequently accomplished by using simply
either autoclaved sea or freshwater as the cultivation medium) as developed in the
lab of Stephen J. Giovannoni (Connon and Giovannoni 2002; Stingl et al. 2007)
resulted in the description of more than 100 taxa (Table 10.4). Pure cultures of
10 Isolation and Cultivation of Bacteria 339

Table 10.4 Contribution of selected advanced cultivation methods to isolation of type strains
described in the period 2000–2014 as new species in IJSEM
Taxa
Method Reference for method described
HTC dilution-to-extinction Connon and Giovannoni (2002), Stingl >100
et al. (2007)
Acclimatization Method Hahn et al. (2004, 2005) 17
MicroDrop technique Bruns et al. (2003a), Gich et al. (2005) 5
Encapsulation of cells in gel Zengler et al. (2002, 2005) –
microdroplets
Diffusion growth chamber Kaeberlein et al. (2002), Bollmann et al. –
(2007)

strains initially grown in low nutrient liquid media typically then were transferred
and maintained on high nutrient agar media (usually Marine Agar). Establishment of
pure cultures suitable for maintenance on standard solidified media was the crucial
requirement for deposition of a strain in public culture collections and subsequent
valid description of new taxa. Currently, the research group of Jang-Cheon Cho is
probably most active in isolation of new taxa by HTC techniques and their taxo-
nomic description. Some of the highlights among the >50 taxa described by Cho and
colleagues are the description of the new class Opitutae of the phylum
Verrucomicrobia (Choo et al. 2007) and the establishment of a new family affiliated
with the Gammaproteobacteria (Kim et al. 2007). Other taxa of environmental
significance, e.g. members of the marine SAR11 cluster (Morris et al. 2002), could
be cultivated by HTC techniques (Rappe et al. 2002) as pure cultures in autoclaved
sea water, but due to their obligate oligotrophic metabolism, they could not be
maintained on media suitable for deposition in public culture collections. Due to
this restriction, only the proposal to establish a candidatus species, i.e. Cand.,
Pelagibacter ubique, was possible so far (Rappe et al. 2002).
It should be noted that the HTC technique is not the only method following a
strategy of initial cultivation in sterile seawater, screening of cultures and subsequent
seeking transfer of interesting cultures to solidified standard media. A couple of
other researchers also described new taxa based on type strains isolated with such
strategies; however, these other strategies were not labelled with a brand name like
“HTC technique”, which makes detailed literature analyses of their contribution to
taxonomic advances more difficult. An interesting example for such a cultivation
experiment is the isolation of the type strain of Sphingopyxis alaskensis (Schut et al.
1993) and its subsequent taxonomic description as a new species (Vancanneyt et al.
2001; Godoy et al. 2003).
The MicroDrop technique (Gich et al. 2005; Bruns et al. 2003a) enabled the
description of a new genus affiliated with the phylum Acidobacteria (Koch et al.
2008). This phylum contributes in high percentages to soil bacterial communities
(Foesel et al. 2014), and cultivation-independent investigations have suggested a
huge diversity within this phylum (Eichorst et al. 2007). However, the Acidobacteria
represent one of the most understudied bacterial phyla currently containing only nine
340 M. W. Hahn et al.

validly described genera with a total of 22 species. Furthermore the type species
identifying both a new species and genus representing planktonic freshwater bacteria
was isolated by using the MicroDrop technique (Jogler et al. 2013).
The Acclimatization Method (Hahn et al. 2004, 2005) was used for isolation of
strains enabling description of nine new species, two new genera and six candidatus
species (Table 10.4). This method aims on a gradual transfer of bacteria from their
natural low nutrient concentration environments to the artificial high nutrient con-
centration conditions usually provided by microbiological media. Bacteria present in
inoculi from environmental samples are subjected to stepwise increases in nutrient
concentrations in liquid media, and strains able to grow at high concentrations are
finally transferred to a solidified standard medium (frequently NSY agar plates). This
acclimatization procedure is usually combined with an initial selection step, either a
filtration or dilution of samples, which shall exclude environmentally rare but under
artificial conditions highly competitive taxa able to rapidly overgrow more abundant
but slower-growing taxa.
Interestingly, the selection and the acclimatization steps of the Acclimatization
Method just represent intermediate steps in the cultivation process, which can be
skipped in comparative analyses. Hahn et al. (2004) compared the results of culti-
vation experiments using lake water as inoculum performed with and without those
steps. Thus they compared results of experiments utilizing the Acclimatization
Method with results of direct plating of samples on agar plates (standard plating).
NSY medium was exclusively used in all experiments performed by using those two
methods. The sets of strains (Table 10.5) obtained with these two different methods
from the water columns of freshwater lakes and ponds differed substantially regard-
ing their phylogenetic compositions (Fig. 10.10). The majority (68.8%) of strains
isolated with the Acclimatization Method belonged to the class Betaproteobacteria
(phylum Proteobacteria) and the phylum Actinobacteria (Fig. 10.10), which are
known to usually contribute together to the majority of bacteria dwelling as

Table 10.5 Comparison of strains and taxa isolated by the Filtration-Acclimatization Method
(FAM) and direct plating on NSY agar
FAM Direct plating
Number of strains 73 27
Number of taxa (>97% SSU rRNA simil.) 16 20
Singleton taxa (% all taxa) 62.5 80.0
Taxa closer to uncultured bacteria (% all taxa) 56.3 0
Similarity (%) with closest species (average of all taxa) 94.5 97.6
Similarity (%) range (min. to max.) 86.9–99.5 92.2–100
Taxa with >97% similarity to closest species 12.5 55.6
Both methods employed the same medium (liquid or solidified NSY medium) and resulted in pure
cultures maintained on NSY agar plates and subsequently characterized by sequencing of their SSU
(16S) rRNA genes. For comparison strains sharing >97% SSU rRNA sequence, similarities were
assigned to the same taxon. The relationship to other uncultured and cultured organisms was
estimated by performing BLAST analysis with SSU rRNA sequences against public databases.
The presented data were adopted from Hahn et al. (2004)
10 Isolation and Cultivation of Bacteria 341

Fig. 10.10 Comparison of the phylogenetic affiliation of bacterial strains isolated with the Accli-
matization Method (Hahn et al. 2004) and standard plating. Both methods used the same medium
(NSY medium) and resulted in strains able to grow on agar plates. The graph is based on data
published by Hahn and co-authors previously (Hahn et al. 2004) and depicts the relative contribu-
tion (%) of the respective phylogenetic groups to the total number of strains isolated by the two
methods (Table 10.5)

bacterioplankton in freshwater systems (Glockner et al. 2000; Newton et al. 2011).

These two taxa were also present among the strains obtained by standard plating
(Fig. 10.10) but contributed numerically only 10%; thus they represented only a
minor fraction. Other taxa more typically known to usually contribute only minor
factions to freshwater bacterioplankton (e.g. phylum Deinococcus-Thermus and
Gammaproteobacteria) were represented as unanticipatedly larger proportions
(Fig. 10.10). The presence of a large fraction of spirochetes among the strains
obtained by the Acclimatization Method was a result of the filtration step (Hahn
2004; Canale-Parola et al. 1966) but not basically a result of the acclimatization
procedure [as suggested by control experiments (Hahn et al. 2004)]. The direct
plating resulted in isolation of a higher number of taxa (defined by >97% SSU
rRNA similarity) albeit a smaller total number of strains was obtained by this method
in comparison to the Acclimatization Method (Table 10.5). However, the latter
method resulted by average in isolation of strains and taxa more distantly related
to previously characterized isolates and species. Furthermore, while all taxa obtained
by the standard plating methods were more closely related to previously described
species, about half of the taxa retrieved by the Acclimatization Method were more
closely related to so far uncultured bacteria only characterized by cultivation-
independent methods (Table 10.5; Hahn et al. 2004). Importantly, this method
resulted in isolation of several previously uncultured taxa known to be abundant
freshwater bacteria (e.g. Polynucleobacter spp., Limnohabitans spp., planktonic
freshwater Actinobacteria), while the standard plating did not obtain such taxa. In
general, the Acclimatization Method obtained more candidates for new species and
342 M. W. Hahn et al.

genera than the standard plating method. Some of these candidates were meanwhile
described as new taxa (e.g. Hahn et al. 2010, 2012a; Jezbera et al. 2009). The lower
ratio of taxa discovered per isolated strain (0.2 for the Filtration-Acclimatization
Method versus 0.7 for the standard plating methods) probably resulted from the
Filtration-Acclimatization Method entailing too strong of a selection step,
i.e. filtration through 0.2 mm filters. This filtration might exclude too many taxa
otherwise potentially cultivable by the Acclimatization Method. The use of filters
with larger pore size or replacement of the filtration step by a dilution step [Dilution-
Acclimatization Method (Hahn et al. 2005; Kasalicky et al. 2010, 2013)] may
increase the yield of new taxa per isolated strain. The better performance of the
Filtration-Acclimatization Method compared to the standard plating in isolation of
more distantly related (to previous characterized species) taxa essentially resulted
from the acclimatization procedure. This was demonstrated by control experiments
comparing the effects of the filtration step and the acclimatization procedure on the
cultivation results (Hahn et al. 2004).
Other advanced cultivation methods, for instance, either the cultivation by using
diffusion growth chambers simulating a natural environment and enabling cocultivation
(Kaeberlein et al. 2002; Bollmann et al. 2007) or the encapsulation method (Zengler
et al. 2002, 2005) have not contributed to the description of new prokaryotic species so
far. A potential reason for this lack of contribution might be that these methods, as well
as the HTC method (see above), do not primarily aim on isolation of strains able to grow
on typical (high nutrient) bacteriological media. The ability of strains to grow on high
nutrient media facilitates sustainable maintenance of cultures as well as deposition of
strains in public culture collections, but the lack of this ability does not fundamentally
prevent the description of new taxa as, for instance, demonstrated by the description of
Mariprofundus ferrooxydans (Emerson et al. 2007) and the subsequent description of
the new class Zetaproteobacteria (phylum Proteobacteria), which harbours the genus
Mariprofundus (Makita et al. 2017).
In summary, the analysis of species descriptions published in IJSEM and the
study comparing the Acclimatization Method with standard plating suggested that it
is quite easy to isolate and cultivate bacterial strains representing undescribed
species. For instance, 20% of the analysed 1042 descriptions of new species resulted
from direct plating of environmental samples on either Marine Agar or R2A Agar.
About 50% of the new species and numerous of the additional strains identified were
successfully isolated by standard methods without requiring either sophisticated
pretreatments, enrichment procedures or other laborious non-standard cultivation
methods (Table 10.1). Similar trends were observed in the abovementioned direct
plating of freshwater samples on NSY agar plates (Hahn et al. 2004). Isolation of
27 strains resulted in nine strains, which reliably represented new candidate species
as suggested by the fact of their SSU rRNA sequence values having less than 97%
similarity to type strains of the closest related described species (Stackebrandt and
Goebel 1994, 2006). An impressive study by Joseph and colleagues also demon-
strated that simple cultivation on agar plates (including non-standard media) can
retrieve numerous candidates for new species from soil samples, including possible
genera and families affiliated with so far understudied phyla like Acidobacteria
10 Isolation and Cultivation of Bacteria 343

(Joseph et al. 2003). Obviously, progress in taxonomic description of new bacterial

species is not limited by cultivation and deposition of type strains in public culture
collections but probably rather by efforts, costs and taxonomic expertise required for
successful taxonomic characterization and description of new species (Tindall et al.
2010). New methods and media are currently not required for adding new species
and genera to previously established families; however, application of standard
cultivation methods results only rarely in description of new taxa of higher rank
(Fig. 10.9). Thus, deeper taxonomic exploration of the so far poorly characterized
bacterial diversity strongly requires more sophisticated cultivation experiments.

10.7 Strategies for Cultivation

The selection of an appropriate strategy for cultivation experiments largely depends

on the project goals. Taxonomists interested in obtaining new strains suitable for
description of novel species and genera may use standard methods and media (see
above). The use of non-standard methods may increase the yield of taxonomically
novel groups; however not all obtained cultures may be suitable for deposition in a
public culture collection, which is currently one of several mandatory requirements
for description of new prokaryotic taxa (Tindall et al. 2010). Acclimatization pro-
cedures in combination with various standard media can be expected to even gain
higher yields of novel taxa. Thus far, acclimatization experiments were almost
exclusively performed by using only a single medium (Hahn et al. 2004) leaving
the potential of this strategy largely unexploited.
Microbial ecologists interested in the study of specific environmentally signifi-
cant but so far uncultured taxa may first require an appropriate method for high-
throughput screening of cultures (Rappe et al. 2002). Fluorescent in situ hybridiza-
tion (FISH) or PCR with probes or primers, respectively, specific for the targeted
organisms represent efficient screening methods. Cultivation experiments should be
started with methods and media best mimicking the natural conditions under which
the targeted organisms thrive. Initial use of low substrate concentration media, for
instance, sterilized sea or lake water, is recommended. If cultivation on such low
substrate concentration media is successful, then transfer to standard media can be
tried. Acclimatization procedures may help if a single step transfer to substrate-rich
media is not successful.
If genomic data on so far uncultured target organisms were obtained by either
metagenomics or single-cell genomic methods, then analysis of the available data
may help to better understand the physiology and ecology of the targeted organisms.
Such knowledge could be used to refine the cultivation strategy and the composition
of the employed media. Cocultivation with other organisms thriving in the same
habitat may be considered if initial trials on cultivation in pure culture are found to
fail. Once a stable mixed culture is established, experiments on supplementation of
the used media with substances potentially required by the target organisms could be
performed. Sequencing and subsequent analysis of genomes of the organisms
344 M. W. Hahn et al.

contained in an established mixed culture also could be performed. Genomic insights

could help to improve the medium so as to release the targeted organisms from their
dependence on the helper organisms.

Compliance with Ethical Standards

Funding This study was supported by the Austrian Science Fund (FWF) project 27160-B22
(Mikroevolution eines planktischen Süßwasserbakteriums).

Conflict of Interest Martin W. Hahn declares that he has no conflict of interest. Ulrike Koll
declares that she has no conflict of interest. Johanna Schmidt declares that she has no conflict of
interest.

Ethical Approval This article does not contain any studies with human participants or animals
performed by any of the authors.

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