molecules

Review
Deconstruction of Lignin: From Enzymes to Microorganisms
Jéssica P. Silva 1 , Alonso R. P. Ticona 1 , Pedro R. V. Hamann 1 , Betania F. Quirino 2 and Eliane F. Noronha 1, *

1 Enzymology Laboratory, Cell Biology Department, University of Brasilia, 70910-900 Brasília, Brazil;
wpinheiro.jessica@gmail.com (J.P.S.); alonso.poma.t@gmail.com (A.R.P.T.);
pedror_hamann@hotmail.com (P.R.V.H.)
2 Genetics and Biotechnology Laboratory, Embrapa-Agroenergy, 70770-901 Brasília, Brazil;
betania.quirino@embrapa.br
* Correspondence: enoronha@unb.br; Tel.: +55-61-3307-2152

Abstract: Lignocellulosic residues are low-cost abundant feedstocks that can be used for industrial
applications. However, their recalcitrance currently makes lignocellulose use limited. In natural
environments, microbial communities can completely deconstruct lignocellulose by synergistic action
of a set of enzymes and proteins. Microbial degradation of lignin by fungi, important lignin degraders
in nature, has been intensively studied. More recently, bacteria have also been described as able
to break down lignin, and to have a central role in recycling this plant polymer. Nevertheless,
bacterial deconstruction of lignin has not been fully elucidated yet. Direct analysis of environmental
samples using metagenomics, metatranscriptomics, and metaproteomics approaches is a powerful
strategy to describe/discover enzymes, metabolic pathways, and microorganisms involved in lignin
breakdown. Indeed, the use of these complementary techniques leads to a better understanding of
the composition, function, and dynamics of microbial communities involved in lignin deconstruction.



We focus on omics approaches and their contribution to the discovery of new enzymes and reactions


that impact the development of lignin-based bioprocesses.
Citation: Silva, J.P.; Ticona, A.R.P.;
Hamann, P.R.V.; Quirino, B.F.;
Keywords: lignin; bacteria; biodegradation; auxiliary activities; metagenomics; metaproteomics;
Noronha, E.F. Deconstruction of
metatranscriptomics
Lignin: From Enzymes to
Microorganisms. Molecules 2021, 26,
2299. https://doi.org/10.3390/
molecules26082299
1. Introduction
Academic Editor: Claudio Gioia The conversion of lignocellulosic biomass into biofuels and chemicals has gained inter-
est because of its potential application in biorefineries as a green platform. Lignocellulosic
Received: 26 February 2021 biomass is mainly composed of lignin and polysaccharides (i.e., cellulose, hemicellulose,
Accepted: 26 March 2021 and pectin), arranged in plant cell walls. Cellulose is a homopolymer, composed of D-
Published: 15 April 2021 glucose monomers joined by linear β (1–4) linkages. Hemicellulose is also a sugar polymer,
but it is composed of different monosaccharide molecules mainly joined by β-1,4 glyco-
Publisher’s Note: MDPI stays neutral sidic linkages. Among the sugars that compose hemicellulose, D-xylose, D-mannose, and
with regard to jurisdictional claims in arabinose are present. Hemicellulose polymers are strongly interlinked through covalent
published maps and institutional affil- and non-covalent bonds, and also linked to lignin, which together with cellulose will form
iations.
the recalcitrant lignocellulosic matrix [1,2]. In the plant cell wall, lignin is linked to the
carbohydrate moiety via the ester linkage. This association gives the plant cell wall greater
strength and impermeability [3]. Lignin is formed by radical coupling reactions involving
the three main phenylpropane units: p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S),
Copyright: © 2021 by the authors. linked by C–C and C–O linkages [4,5].
Licensee MDPI, Basel, Switzerland. Lignin is the main plant cell wall component responsible for recalcitrance [6]. Thus,
This article is an open access article pretreatment is an essential step for removing lignin in the process of lignocellulosic
distributed under the terms and biomass conversion into biofuels [7,8]. The high carbon/oxygen ratio and the natural
conditions of the Creative Commons
abundance of lignin make it a promising feedstock material for biological conversion into
Attribution (CC BY) license (https://
value-added products [9]. In addition to the biofuel industry, lignin may also be found as
creativecommons.org/licenses/by/
a by-product from wood-biomass in industrial processes for paper and pulp production.
4.0/).

Molecules 2021, 26, 2299. https://doi.org/10.3390/molecules26082299 https://www.mdpi.com/journal/molecules

 Molecules 2021, 26, x FOR PEER REVIEW 2 of 22
Molecules 2021, 26, 2299 2 of 21

At least 75,000 tons of kraft-lignin were commercialized in 2018, and for the year 2025,
At 250,000
least 75,000 tonsexpected
tons are of kraft-lignin
[10]. were commercialized in 2018, and for the year 2025,
250,000 Thetonsefficient
are expected [10]. of lignin by white-rot basidiomycete fungi has been exten-
degradation
The efficient
sively degradation
studied [11,12]. of lignin
Bacteria by white-rot
are also capable ofbasidiomycete
deconstructing fungi has[5,13],
lignin been exten-
but they
sively studied [11,12]. Bacteria are also capable of deconstructing lignin
are less studied in comparison to fungi [12,14]. Recently, bacteria have been attracting [5,13], but they are
lessgreat
studied in comparison to fungi [12,14]. Recently, bacteria have been
attention due to their high adaptability and biochemical versatility. Furthermore, attracting great
attention
bacteriadue to their
have high adaptability
metabolic pathways that andconvert
biochemical
ligninversatility. Furthermore,
and its derivates bacteria of
into products
have metabolic pathways that convert lignin and its derivates into products
biotechnological interest such as lactic acid, pyruvate, vanillin, lipids, polyhydroxyalka- of biotechno-
logical
noatesinterest
(PHA), such
andascis-muconic
lactic acid, pyruvate, vanillin, [15,16].
acid (cis, cis-MA) lipids, polyhydroxyalkanoates (PHA),
and cis-muconic
In nature,acid (cis,iscis-MA)
lignin [15,16].by the concerted efforts of microbial communities,
deconstructed
In nature, lignin is deconstructed
rather than isolate microorganisms. Therefore, by the concerted effortsofof
the strategy microbial
studying thecommunities,
microbial com-
rather than isolate microorganisms. Therefore, the strategy of studying the microbial
munity may provide a broader comprehension of the process of lignin deconstruction. In
community may provide a broader comprehension of the process of lignin deconstruction.
natural environments, different microorganisms in the microbial community operate syn-
In natural environments, different microorganisms in the microbial community operate
ergistically through the secretion of a variety of biocatalysts. Although in nature coopera-
synergistically through the secretion of a variety of biocatalysts. Although in nature coop-
tion between different microorganisms is common, in the laboratory, not all organisms
eration between different microorganisms is common, in the laboratory, not all organisms
are easily cultured, which is an obstacle to adapting the natural synergistic lignin decon-
are easily cultured, which is an obstacle to adapting the natural synergistic lignin decon-
struction to its bioconversion in industrial processes. However, investigation of natural
struction to its bioconversion in industrial processes. However, investigation of natural
microbial communities using culture-independent approaches (e.g., metagenomics, meta-
microbial communities using culture-independent approaches (e.g., metagenomics, meta-
transcriptomics, and metaproteomics) may deliver a detailed description of the processes
transcriptomics, and metaproteomics) may deliver a detailed description of the processes
and enzymes involved. Thus, the study of lignin-degrading microbiomes (e.g., forests,
and enzymes involved. Thus, the study of lignin-degrading microbiomes (e.g., forests,
animals’ digestive tract, and sewage [17,18]) may be crucial to the development of efficient
animals’ digestive tract, and sewage [17,18]) may be crucial to the development of efficient
industrial
industrial processes
processes forforthethe
useuse of ligninolytic
of ligninolytic feedstocks.
feedstocks.
In the present review, we explore the contribution
In the present review, we explore the contribution of omics of omics approaches
approaches in in discover-
discover-
ing/describing new enzymes and microorganisms in microbiomes adapted to degradelig-
ing/describing new enzymes and microorganisms in microbiomes adapted to degrade
nin. Primarily,
lignin. Primarily, we wefocus
focuson onligninolytic
ligninolyticenzymes
enzymesbelonging
belonging totothethe
auxiliary activities
auxiliary (AA)
activities
family, with emphasis on their impact on lignin valorization in industrial
(AA) family, with emphasis on their impact on lignin valorization in industrial processes. processes.

2. Lignin
2. Lignin
2.1.2.1.
Structure andand
Structure Composition of Natural
Composition Lignin
of Natural Lignin
Lignin is aisphenolic
Lignin macromolecule
a phenolic macromoleculeof high
of molecular weight,weight,
high molecular composed of three of
composed main
three
units of phenylpropane
main (monolignols):
units of phenylpropane coniferyl alcohol,
(monolignols): coniferylsinapyl alcohol,
alcohol, sinapyl p-coumaryl
andalcohol, and p-
alcohol (Figure
coumaryl 1). When
alcohol incorporated
(Figure into the lignininto
1). When incorporated structure, these
the lignin monomers
structure, thesearemonomers
termed
p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units, respectively [19–21].
are termed p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units, respectively [19– In addition
to these
21]. Inthree maintounits,
addition theseother
three subunits areother
main units, present in theare
subunits lignin structure.
present in the Among them
lignin structure.
areAmong
ferulic acid, ferulates, coniferaldehyde, synapaldehyde, 5-hydroxyiconiferyl
them are ferulic acid, ferulates, coniferaldehyde, synapaldehyde, 5-hydroxy- alcohol,
and acylatedalcohol,
iconiferyl monolignols that contain
and acylated acetate, p-coumarate
monolignols that contain [21–23].
acetate, p-coumarate [21–23].

Figure
Figure 1. Chemical
1. Chemical structuresof
structures ofthe
the three
three monomeric
monomericprecursors
precursorsof of
thethe
lignin macromolecule.
lignin macromolecule.
Molecules 2021, 26, x FOR PEER REVIEW 3 of 22
Molecules 2021, 26, 2299 3 of 21

Depending on its origin, lignin can be classified into three main groups: softwood,
hardwood, and grass
Depending on itslignins. Softwood
origin, lignin can lignins are mainly
be classified composed
into three of guaiacyl
main groups: units,
softwood,
and thus are classified as G-type lignin. Hardwood lignins are mainly
hardwood, and grass lignins. Softwood lignins are mainly composed of guaiacyl units, and composed of dif-
ferent proportions of guaiacyl-syringil (GS-type) [24,25]. Grass lignins
thus are classified as G-type lignin. Hardwood lignins are mainly composed of different have a mixture of
the three aromatic units (HGS), and contain a higher proportion of p-coumaryl
proportions of guaiacyl-syringil (GS-type) [24,25]. Grass lignins have a mixture of the three alcohol
(H)-derived
aromatic units units thanand
(HGS), other ligninatypes
contain higher[26,27]. The highest
proportion proportion
of p-coumaryl of lignin
alcohol is found
(H)-derived
in softwoods
units (24–33%),
than other while
lignin types hardwoods
[26,27]. and grasses
The highest proportionhaveofalignin
smaller amount
is found (~15–28%)
in softwoods
[4,28].
(24–33%), while hardwoods and grasses have a smaller amount (~15–28%) [4,28].
The
The linkages
linkagesbetween
betweenphenylpropane
phenylpropaneunits unitscancanbebe
ether bonds
ether bonds(β-O-4′,
(β-O-44-O-5′)
0 , 4-O-5or0 car-
) or
bon–carbon
carbon–carbon bonds (5–5 , β-5 , β-β , β-1 ) usually called condensed bonds (FigureThe
bonds (5–5′, β-5′,
0 β-β′,
0 β-1′)
0 usually
0 called condensed bonds (Figure 2). 2).
relative
The abundance
relative of these
abundance linkages
of these depends
linkages on theon
depends type
theof monomer
type of monomerpresent in the lignin
present in the
structure. For example,
lignin structure. G-type
For example, lignins
G-type contain
lignins moremore
contain resistant linkages
resistant linkages (β-5, 5-5)5-5)
(β-5, thanthanS-
rich
S-richlignins that
lignins are
that less
are condensed-bonded
less condensed-bonded through
through ether
etherbonds
bonds[23].
[23].

Figure 2. Typical
Figure 2. linkages found
Typical linkages found in in the
the structure
structure of
of natural
natural lignin;
lignin; ether
ether bonds
bonds (β-O-4′,
(β-O-40 , 4-O-5′)
4-O-50 )and
and
condensed bonds (5–5′,
0 β-5′,
0 β-β′,0 β-1′).
condensed bonds (5–5 , β-5 , β-β , β-1 ). 0

In the plant cell wall, lignin is covalently linked to the hemicellulose matrix, forming
the lignin-carbohydrate
lignin-carbohydrate complex
complex(LCC).
(LCC).Among
Amongthe thetypes
typesofoflinkages
linkagesthat areare
that formed
formedin
the LCC,
in the there
LCC, areare
there phenyl glycosides,
phenyl benzyl
glycosides, ester,
benzyl andand
ester, ferulate or deferulate
ferulate esters
or deferulate that
esters
thatlinked
are are linked to lignin
to lignin in 4-OH
in 4-OH andpositions
and 4-O 4-O positions [29,30].
[29,30]. Phenyl-glycoside
Phenyl-glycoside bonds bonds
are oneare
of
one of the most common linkages in the LCC complex and are established between the
carbohydrate reducing ends and the phenolic terminal groups of the lignin macromolecule.
The benzyl ester linkages connect the lignin and carbohydrate moieties through the uronic
Molecules 2021, 26, 2299 4 of 21

acid of the carbohydrates and the hydroxyl group of the lignin. Ferulate or deferulate esters
form most of the LCC linkages in grasses [31]. Ferulate mediates the crosslinks between
polysaccharide–polysaccharide and polysaccharide–lignin. Crosslinks make the lignin
physically close to the cell wall polysaccharides in such a way that lignin blocks the access
of enzymes to the polysaccharides [19,26].

2.2. Technical Lignins

Technical lignins are produced as by-products in the pretreatment of lignocellulose
procedures to obtain the carbohydrate moiety. These procedures include physical, chemical,
and physicochemical methods, which result in a liquid residue known as black liquor,
mainly composed of lignin, carbohydrates, and ash. Processing methods such as kraft,
lignosulfonate, soda, organosolv, and hydrolyzed lignin are widely used, and each lignin
produced is unique in terms of chemical structure, impurity profile, polydispersity, and
molecular weight [32–34].
Technical lignin has been used for the development of products such as binders,
dispersants or emulsifiers, and sequestrants. Almost 90% of commercial lignin products in
the world are lignosulfonates, often used as polymeric anionic dispersants. Technical lignin
has also been used for civil engineering applications such as soil stabilization, asphalt
stabilization, and cement additives [35–37].
The kraft procedure is the primary pulping process and uses a mixture of sodium
hydroxide (NaOH) and sodium sulfide (Na2 S) solution at high temperatures (150–180 ◦ C),
which breaks ether bonds in lignin through an episulfide intermediate. During cooking,
lignin is degraded into fragments of different molecular weights and is dissolved into
the pulping liquor. After separating cellulose by filtration, the lignin and remaining
hemicellulose in the liquid phase (black liquor) can be used in industrial processes. In
modern pulp mills, the lignin present in black liquor is incinerated to supply energy for
the mill. Isolation of lignin from black liquor for conversion into valuable products such
as biofuel or a chemical compound might be an interesting alternative. Indeed, a lignin
isolation procedure from black liquor known as lignoboost has been introduced. This
procedure begins with adjusting the pH (up to 9.5) of the black liquor with the addition of
carbon dioxide (CO2 ). In the next step, the recovered lignin is re-slurried using sulfuric
acid (H2 SO4 ) to pH 2, and finally, the lignin is isolated by filtration [38,39].
Kraft lignin shows different properties, distinguishing it from native lignin and other
technical lignins. For instance, it has a significant increase of phenolic hydroxyl groups due
to extensive cleavage of β-aryl bonds during the cooking process; furthermore, condensed
structures and biphenyl are formed as a result of the severe procedure conditions. Lignin
condensation usually occurs by the formation of new intermolecular C–C bonds between
lignin fragments. Indeed, C–C bonds such as β–β and β–5 linkages are more difficult
to disrupt than C–O bonds (β-aryl ether linkages). The degree of lignin condensation is
important as it affects lignin recovery after biomass pretreatment. Preventing C–C bond
formation during pretreatment reaction is an important positive variable for downstream
utilization of lignin [30,34,40].
Lignosulfonates are obtained as by-products of sulfite cooking, in which delignifica-
tion of biomass occurs by the addition of sulfite (SO3 2− ) and bisulfite (HSO3 − ). In this
procedure, lignin is sulfonated, degraded, and solubilized. Lignosulfonates contain a large
number of functional groups such as carboxylic, sulfur, and phenolic hydroxyl groups.
Moreover, it presents distinctive physical and chemical characteristics such as solubility in
water, high ash content, and relatively high molecular weight [40].
In the organosolv process, a mixture of organic solvent/solvents and water are added
to the biomass, and these are essentially cooked at high temperatures (190 ◦ C). Solvents
such as ethanol, formic acid, acetic acid, and peroxiorganic acids are commonly used in this
process. After this procedure, lignin is separated via solubilization. The lignin obtained by
this process is less modified. Therefore, the main features of organosolv lignins are low
 this process. After this procedure, lignin is separated via solubilization. The lignin ob-
tained by this process is less modified. Therefore, the main features of organosolv lignins
are low molecular weight, high chemical purity, and the presence of many reactive side
chains available for further chemical reactions, however, it shows poor solubility in water
Molecules 2021, 26, 2299 5 of 21
[38,39].
Depending on the conditions, and nature of the procedure that the lignocellulose bi-
omass is subjected to, the resulting lignin might undergo irreversible repolymerization.
Inmolecular
this case,weight,
lignin’shigh
innatechemical purity, and
recalcitrance the presence
is enhanced, of many
making reactive
it more side chains
challenging to pro-
available for further chemical reactions, however, it shows poor solubility
duce value-added chemicals from it. In some cases, significant amounts of inhibitory in water [38,39].
products Depending
such ason the conditions,
phenolic and nature
acids, furfurals, of the procedure that theand
5-hydroxymethylfurfural, lignocellulose
aldehydes are
biomass is subjected to, the resulting lignin might undergo irreversible
generated, and these can negatively impact downstream processes such as cellulose hy- repolymerization.
In this case, lignin’s innate recalcitrance is enhanced, making it more challenging to produce
drolysis and fermentation [41].
value-added chemicals from it. In some cases, significant amounts of inhibitory products
Ionic liquids (ILs) are green solvents that can also be used for biomass pretreatment.
such as phenolic acids, furfurals, 5-hydroxymethylfurfural, and aldehydes are generated,
Inand
thisthese
case,can
dissolution
negativelyof lignindownstream
impact contained in lignocellulosic
processes biomasses
such as cellulose is accomplished
hydrolysis and
by using organic
fermentation [41]. salts (i.e., alkylbenzenesulfonate, N-methylimidizole, and dimethyl-
sulfoxide)
Ionicthat remain
liquids (ILs) asareliquids at relatively
green solvents lowalso
that can temperatures
be used for(100 °C). pretreatment.
biomass Although ILs are
not yet available
In this at an industrial
case, dissolution scale, there
of lignin contained is growing interest
in lignocellulosic in the
biomasses utilization of by
is accomplished ILs for
the processing
using of biomass.
organic salts IL pretreatments tend
(i.e., alkylbenzenesulfonate, to preserve β-O-4
N-methylimidizole, linkages in the lignin
and dimethylsulfoxide)
that remain
structure, as liquids at
maintaining relatively
similar low temperatures
properties to organosolv (100 ◦lignin.
C). Although
LigninILs canare
benot yet
recovered
available
from the ILsat byan precipitation
industrial scale, viathere is growing
the addition of interest in the such
non-solvents utilization
as waterof ILs for the
or acetonitrile
processing of biomass. IL pretreatments tend to preserve β-O-4 linkages in the lignin
[30,33].
structure, maintaining similar properties to organosolv lignin. Lignin can be recovered from
the ILs by precipitation via the addition of non-solvents such as water or acetonitrile [30,33].
3. Bacterial Enzymes for Lignin Deconstruction
Degradation
3. Bacterial andfor
Enzymes modification of lignin by bacteria involve a repertoire of redox-ac-
Lignin Deconstruction
tive enzymes
Degradation and modification of lignin by(AA)
classified in auxiliary activity families,
bacteria involve according to the
a repertoire Carbohydrate-
of redox-active
Active enZYmes (CAZy) Database [42]. These auxiliary activities involved in
enzymes classified in auxiliary activity (AA) families, according to the Carbohydrate-Activelignin break-
down primarily
enZYmes (CAZy)include the[42].
Database AA1 family,
These which
auxiliary contains
activities laccases;
involved and the
in lignin AA2 family,
breakdown
primarily
which include
includes the AA1 family,
lignin-active which
class-II contains These
peroxidases. laccases; and thealongside
enzymes AA2 family, which
glycoside-hy-
includes lignin-active class-II peroxidases. These enzymes alongside glycoside-hydrolases
drolases work in synergism to degrade lignocellulose (Figure 3). Additionally, other fam-
work
ilies in synergism
show to degrade
a correlation lignocellulose
with oxidation (Figureof3).
or reduction Additionally,compounds
lignin-derived other families
such as
show a correlation with oxidation or reduction of lignin-derived compounds
the AA4 family, which includes vanillyl-alcohol oxidases; the AA5 family, which contains such as
the AA4 family, which includes vanillyl-alcohol oxidases; the AA5 family, which contains
glyoxal oxidases/alcohol oxidases; and the AA6 family, which includes 1,4-benzoquinone
glyoxal oxidases/alcohol oxidases; and the AA6 family, which includes 1,4-benzoquinone
reductases. Families AA3 and AA7 also show members potentially involved in lignin deg-
reductases. Families AA3 and AA7 also show members potentially involved in lignin
radation and and
degradation modification through
modification throughFenton
Fenton reactions
reactions[43].
[43].

Figure 3. Cooperative
Figure model
3. Cooperative modelof of
lignin active
lignin activeenzymes
enzymesandandholocelullases
holocelullasesinin lignocellulosic
lignocellulosic biomass deconstruction. (a)
biomass deconstruction.
Macro organization
(a) Macro (sugarcane
organization bagasse)
(sugarcane and
bagasse) and(b)(b)micro
microorganization (scanningelectron
organization (scanning electron microscopy)
microscopy) images
images are courtesy
are courtesy
of Hamann P.R.V. (c) Carbohydrates and lignin organization in the plant cell wall. (d) cooperative activity of hemicellulases,
cellulases, and ligninases to deconstruct lignocellulose.
Molecules 2021, 26, 2299 6 of 21

The basic mechanism of bacterial lignin deconstruction is analogous to that previously

described for filamentous fungi, in which enzymes with oxidative activity present a cen-
tral role. Enzymes are commonly described as being involved in lignin degradation in
fungi such as laccases (E.C 1.10.3.2) and lignin peroxidases (E.C 1.11.1.7), and manganese
peroxidases (E.C 1.11.1.13) are also present in bacteria [44,45]. Following Cazy database
categorization, based on protein sequence and functional validation, most of the enzymes
involved in lignin and aromatic compound oxidation are grouped in auxiliary activity (AA)
families I and II. The AAI group comprises laccase/di-phenol:oxireductases/ferroxidases
(EC 1.10.3.2), laccase-like multicopper oxidases (EC 1.10.3.-), and ferroxidases (EC 1.10.3.2).
Group AA2 includes ascorbate peroxidases (EC 1.11.1.11), versatile peroxidases (EC
1.11.1.16), lignin peroxidases (EC 1.11.1.14), peroxidases (EC 1.11.1-), cytochrome-c peroxi-
dases (EC 1.11.1.5), and manganese peroxidases (EC 1.11.1.13).
Laccases are multi-copper oxidases displaying activity against monophenols and
compounds containing p-diphenol structures [46,47]. One of the first evidences of lac-
case activity produced by bacteria was observed for Azospirillum lipoferum, an alpha-
proteobacteria obtained from rice rhizosphere. These enzymes displayed oxidative activity
against p-phenylenediamine, hydroquinone, L-DOPA (L-3,4-dihydroxyphenylalanine),
syringaldazine (4-hydroxy-3,5-dimethoxy-benzaldehyde azine), and ABTS (2,20 -azino-
bis(3-ethylbenzothiazoline-6-sulfonic acid)) [48]. The enzyme responsible for the laccase
activity was later purified and characterized as a multimeric protein. Similar findings have
been described for fungal laccases, which can be monomeric, dimeric, or tetrameric [46].
Extremophile bacteria can also be laccase producers. Rezaei et al. (2017), purified
a monomeric laccase (75 kDa) produced by the halotolerant bacterium Aquisalibacillus
elongatus [49]. The purified enzyme showed activity against a variety of substrates in-
cluding polyphenols such as tannic acid and monomeric phenols such as catechol, gal-
lic acid, and guaicol [49]. In another report, Yu Chen et al. (2013) obtained a trimeric
thermostable laccase from the thermophilic actinomycete Thermobifida fusca, produced
using sugarcane biomass as the substrate. The purified enzyme had maximum activ-
ity at 60 ◦ C, and exhibited oxidizing activity against the dyes p-phenylenediamine, and
2,6-dimethylphenylalanine [50].
Manganese peroxidases (MnP) contain a heme structure with a central manganese
ion. These enzymes catalyze the reduction of substrates using H2 O2 as an electron donor,
catalyzing the oxidation of lignin [51,52]. Manganese peroxidases have been obtained
from a great variety of bacteria. Oliveira et al. Oliveira (2009) reported the purification of
MnPs from Bacillus pumilus, and Paenibacillus sp., isolated from wood-decaying material
and paper mill effluent, respectively [51]. MnPs have also been identified in the alpha-
proteobacteria Aurantimonas manganoxydans and Erythrobacter sp. [52]. An MnP produced
by Bacillus subtilis from decomposing natural rubber in soil was reported by Nayanashree
and Thippeswarny (2015) [53]. Although there are a number of reports on bacterial MnPs,
they are much less characterized in comparison to laccases.
Lignin peroxidases are a widely studied class of proteins from the fungus Phanerochaete
chrysosporium. Enzymes from this class can catalyze the oxidation of lignin and phenolic
related compounds using peroxide [4]. Their counterpart in bacteria has been reported in
a variety of genera including those typically found dwelling in lignin-rich environments.
For example, a Bacillus sp. isolate (CSA105) from a paper mill effluent was able to produce
classic plant cell wall degrading enzymes (e.g., cellulases) as well as lignin-active enzymes
including a lignin peroxidase [54]. Still, regarding the phylum firmicutes, Gomare et al.
(2008) reported the production and purification of a lignin peroxidase from Brevibacillus
laterosporus MTCC 2298. The reported LiP displayed activity against the industrially-
relevant dyes (i.e., methyl orange, and Blue-2B) [55].
Bacteria isolated from natural environments including actinobacteria have also been
described as important players in lignin degradation mediated by LiP. Yee and Wood
(1997) reported a LiP produced by Streptomyces viridosporum T7A with activity against 2,4-
dichlorophenol, a compound usually found in pesticides and herbicides, which can be an
Molecules 2021, 26, 2299 7 of 21

important environmental contaminant [56]. Similar results were reported for a 49.7 kDa per-
oxidase from Streptomyces sp. AD001 that showed activity against 2,4-dichlorophenol [57].
In addition to the classical oxidases reported as presenting a central role in lignin degra-
dation by bacteria, recent studies show other activities such as β-etherases enzymes, which
are involved in lignin/phenolic compounds deconstruction/consumption. β-etherases
cleave β-aryl ether bonds found in lignin and are receiving attention because they can
degrade this kind of linkage in high-molecular-weight lignin. β-Etherase activity has been
described for Sphingomonas paucimobilis SYK-6, and thus far it is one of the few enzymes
with a non-oxidative mechanism against lignin [58].
One of the early studies on β-etherases was a report of the genes ligE and ligF coding
for two β-etherases with similarities to the glutathione-S-transferase protein family in
Sphingomonas paucimobilis SYK-6 [58]. Later, these β-etherases from S. paucimobilis were
expressed in E. coli and shown to degrade high-molecular-weight lignin [58,59].
With the advent of new techniques to prospect new putative genes encoding lignin-
modifying and deconstruction enzymes, and the development of more accurate protocols
to identify products of lignin degradation and/or modification, it is expected that a more
diversified group of proteins will be categorized as being involved in lignin biodegradation.
Many reports of putative enzymes involved in lignin breakdown are based on the presence
of proteins or sometimes gene expression when a microorganism is cultured in the presence
of lignin or lignin-related compounds. However, more direct experimental data are still
needed to validate the role of specific enzymes in lignin deconstruction. Examples include
dioxygenases and veratryl alcohol oxidases, which do not have a widespread role in lignin
and aromatic compound degradation in bacteria and require further validation of their role
in lignin deconstruction.

4. Approaches to Access Bacterial Community Structure and Functionality

Metagenomics is a culture-independent approach that can be used to describe a
microbiome in two different levels: structural and functional. At a structural level, a mi-
crobial community can be described regarding the microorganisms present, from phylum-
to species-level, in addition, ecological interactions and evolutionary aspects can be ex-
plored [60,61]. In contrast, functional metagenomics explores the bacterial community
genomic diversity of a sample, allowing the identification of genes and biochemical path-
ways prevalent in a microbiome (Figure 4). The choice of the target environment for omics
studies is essential for mining biocatalysts. As shown in Table 1, in natural or modified
microbiomes where lignin degradation occurs naturally, a repertoire of bacteria and genes
that are involved in lignin degradation has been identified.
Different experimental approaches can be used to access the structural and functional
profiles of microbial communities. Metataxonomics, for example, is based on the sequenc-
ing of phylogenetic marker genes such as 16S rRNA, providing information about the
structural composition of a bacterial microbiome, sometimes reaching the hierarchical
level of species [62–64]. On the other hand, shotgun metagenomics is an undirected se-
quencing of all the microbial DNA in a sample, allowing access to both the structural
and functional community profile. Functional information can also be accessed through
phenotypic screening, which includes the construction of metagenomic libraries, and the
use of different techniques to identify genes with the desired function such as activity-based
screening [65–67]. In addition to direct analysis of environmental samples using DNA se-
quencing techniques, culture-enrichment is a powerful tool to establish microbial consortia
with desired properties. This technique basically consists of cultivating environmental
communities on desired substrates such as lignin and/or lignocellulosic materials under
specific conditions, thus providing the enrichment of microorganisms with the desired
function. This selected microbial community is then subjected to the culture-independent
methods previously described or the traditional culture-based ones.
 Molecules 2021, 26, 2299 8 of 21
Molecules 2021, 26, x FOR PEER REVIEW 8 of 22

Figure 4. General scheme of the metagenomic strategies for the identification of lignin-degrading
Figure 4. General scheme of the metagenomic strategies for the identification of lignin-degrading
enzymes from different bacterial communities.
enzymes from different bacterial communities.
Table 1. Auxiliary activity families (AAs) present in different bacterial communities as surveyed from metaomics studies.
Different experimental approaches can be used to access the structural and functional
profiles of microbial
Bacteria Associatedcommunities.
with Lignin Metataxonomics, for example, is based on the sequenc-
DNA Source AAs Families References
Degradation
ing of phylogenetic marker genes such as 16S rRNA, providing information about the
Brazilian Caatinga soil structural composition
Proteobacteria, Actinobacteria,ofAcidobacteria
a bacterial microbiome,
AA1, AA3, AA7 sometimes reaching the [68] hierarchical
Proteobacteria,
level Acidobacteria,
of species [62–64]. Onand the other hand, shotgun metagenomics is an undirected se-
Forest soil AA3 [69]
Actinobacteria
quencing of all the microbial DNA in a sample, allowing access to both the structural and
Caulobacteraceae, Acidobacteria,
functional community profile. Functional information
AA1, can also
AA3, AA4, AA5, AA6,beandaccessed through phe-
Soil Solirubrobacterales, Elusimicrobia, [70]
notypic screening, which includes the AA7
construction of metagenomic libraries, and the use
Nevskiales, and Cystobacteraceae
of different techniques
Geodermatophilus, to identify
Thermobispora, and genes with the desired function such as activity-based
Antarctic soil AA3, AA4, and AA7 [18]
screening
Amycolatopsis[65–67]. In addition to direct analysis of environmental samples using DNA se-
Agricultural soil Proteobacteria
quencing techniques, culture-enrichment AA3,
is aand AA6
powerful [71] consor-
tool to establish microbial
Legionella, Acinetobacter, and Pseudomonas,
tia with desired properties. This technique basically consists of cultivating environmental
Termite gut microbiome Myxococcus, Streptomyces, and AA1, AA3, AA4, AA5, and AA6 [72]
communities
Actinoplanes on desired substrates such as lignin and/or lignocellulosic materials under
Arion gut microbiome specific
n.s. conditions, thus providing theAA2, enrichment
AA3, AA4,ofand
microorganisms
AA6 with [73]
the desired
Folsomia gut microbiome function. This
Proteobacteria selected
and microbial community
Actinobacteria is then
AA3, AA6, andsubjected
AA7 to the culture-independent
[74]
Prevotella, Bacteroides,
methods previouslyClostridium,
described or the traditional
Bovine rumen AA6, AA5,culture-based
AA4, AA7, and ones.
AA3 [75]
Fibrobacter, and Ruminococcus
In the next topic of this review, bacterial communities that use lignin as a carbon
Firmicutes, Bacteroidetes, Spirochaetaes,
Camel rumen source and genes encoding lignin-modifying AA3, AA4, AA6, and
auxiliary AA7 (AAs) present in
activity [76]
natural or
Fibrobacteres, and Proteobacteria
Elephant feces enriched
n.s. microbiomes will be described, with a
AA4 and AA6special focus on omics techniques.
[77]
n.s. = data not shown.
4.1. Microbiomes Characterized Using Metagenomic Approaches
4.1.1. Soil
As previously discussed, environmental samples are an interesting source of micro-
organisms specialized in lignin deconstruction. One such example is soil bacteria, which
Molecules 2021, 26, 2299 9 of 21

In the next topic of this review, bacterial communities that use lignin as a carbon
source and genes encoding lignin-modifying auxiliary activity (AAs) present in natural or
enriched microbiomes will be described, with a special focus on omics techniques.

4.1. Microbiomes Characterized Using Metagenomic Approaches

4.1.1. Soil
As previously discussed, environmental samples are an interesting source of microor-
ganisms specialized in lignin deconstruction. One such example is soil bacteria, which play
an active role in deconstructing the litter deposited in the Brazilian Caatinga soil. In a study
that analyzed metagenomic libraries sequences from this microenvironment, considerable
genetic potential for the deconstruction of lignin was observed in which members of the
phyla Proteobacteria, Actinobacteria, and Acidobacteria acted mainly through enzymes
belonging to auxifamilies AA3, AA7, and AA1 [68].
In the topsoil of coniferous forests, the phyla Proteobacteria, Acidobacteria, and
Actinobacteria with the potential to encode genes for auxiliary enzyme activities, especially
those belonging to AA3 families, have also been identified [69]. Interestingly, in another
study, Wilhelm et al. (2019) [70], also characterized this microbiome in North America
through the association of stable isotope probing (SIP), 16S rRNA gene amplicon, and
shotgun metagenomics. The SIP technique was used to label microorganisms capable of
assimilating the 13 C-labeled substrates (hemicellulose, cellulose, or lignin) in the different
soil layers. The authors demonstrated that bacterial deconstruction of lignin seems to occur
throughout the entire soil column, and not just in the upper layer. Different bacteria with
ligninolytic potential were identified both in the organic layer and in deeper mineral layers
(Table 1). Among them are new lignin degraders from mineral soils belonging to the non-
cultivable clades of Caulobacteraceae, Acidobacteria, Solirubrobacterales, Elusimicrobia,
Nevskiales, and Cystobacteraceae. Likewise, the genes of the most abundant AA families
(AA3, AA4, and AA6) were mainly from bacteria.
Antarctic soil microbial communities represent a genetic reservoir of cold-active
ligninolytic enzymes. Bacteria belonging to the genera Geodermatophilus, Thermobispora,
and Amycolatopsis exhibited potential for deconstructing lignin, with enzymes belonging to
families AA7, AA3, and AA4, respectively, being the most prevalent [18].
Functional metagenomic analysis of a microbial community present in hydrocarbon-
contaminated agricultural soils has shown that these environments also harbor bacteria
specialized in lignin deconstruction. Members of the phylum Proteobacteria were identified
as the main group, having genes for enzymes with auxiliary activity belonging to families
AA3 and AA6 [71]. In these different microbiomes, bacteria belonging mostly to the phyla
Proteobacteria, Acidobacteria, and Actinobacteria are central lignin decomposers. These
bacteria use a different set of enzymes to deconstruct the lignin structure (Table 1), and it is
worth mentioning that AA3 probably plays a role in lignin deconstruction in the soil as it is
constantly part of the group of the most abundant enzymes.

4.1.2. Invertebrate Digestive Tract

Bacterial communities inhabit a diversity of environments, and numerous bacteria
live in association with invertebrate hosts such as termites, wood wasps, beetles, and wood-
feeding roaches [78,79]. Insects play an important ecological role in the carbon cycle, and
the breakdown of lignocellulosic fibers (e.g., wood, grass, and litter) [80]. Bacteria express
and secrete carbohydrate-active enzymes in the invertebrate gut system, thus contributing
to the digestion of lignocellulosic substrates [81].
To date, little is known about lignin degradation in the termite gut, whether it is per-
formed by the termites themselves or mediated by unidentified gut bacterial communities
involved in lignin degradation [82]. Although the majority of metagenomic studies of
termite luminal fluid have shown no evidence for lignin degradation, some bacteria have
been isolated and identified from termite gut such as Trabulsiella sp., Rhodococcus erythropolis,
and Streptomyces, which have exhibited peroxidase and laccase activities [83–85].
Molecules 2021, 26, 2299 10 of 21

Nevertheless, recent taxonomic and functional metagenomic analysis of gut microbiota

of seven species of termites (subfamilies Syntermitinae, Nasutitermitinae, Apicotermitinae,
and Termitinae) has revealed the presence of bacterial genes that encode enzymes that play
a role in lignin deconstruction [72]. As a result, a set of CAZymes (CAZy database [86]),
AA1, AA3, AA4, AA5, and AA6 families involved in the degradation and modification of
aromatic compounds were detected in almost all termite species investigated. The most
common bacterial genera harboring genes encoding for the AA1 family were Legionella,
Acinetobacter, and Pseudomonas, for the AA5 family these were Myxococcus and Streptomyces,
and for the AA7 family, it was Actinoplanes.
In another study of termites, Su et al. (2016), using 454 pyrosequencing of 16S rRNA,
analyzed the microbiota gut of four species of termites (three species of wood-feeding
termites and one species of a fungus-feeding termite) and Spirochaetes (11–55%), Firmicutes
(7–18%), Bacteroidetes (7–31%), and Proteobacteria (8–14%) were detected as the main phyla
for all four termites. Furthermore, based upon the automatically taxonomy-to-phenotype
mapping, bacterial metabolic activities related to lignin degradation and modification of
lignin-derived aromatic compounds were identified [87].
These findings show that the termite gut system contains bacteria whose genes encode
enzymes for lignin degradation or modification, although their function in the termite
gut remains unclear. Moreover, the bacterial communities differ between the midgut and
hindgut segments (P1 to P5) of termites [88]. These differences may be related to oxygen
availability. For instance, oxygen is low in the P3 segment, which may hinder oxidative
degradation of lignin, while gut segments with aerobic conditions may harbor several
species capable of lignin degradation [81].
In another study aiming to investigate the microbiota of the Asian long-horned beetle
midgut, several bacterial reads with copper oxidase (Cu-oxidase) domains were searched
in the protein family database (Pfam [89]), and many of these reads had a similarity to
laccases, multicopper oxidases, and polyphenol oxidases. Additionally, a considerable
number of annotated sequences had conserved domains with similarity to hypothetical
proteins, which could represent uncharacterized laccase-type enzymes for lignin degra-
dation. Furthermore, bacterial dye-decolorizing peroxidases that can cleave the β-aryl
ether linkages in both syringyl and guaiacyl lignin in the presence of hydrogen-peroxide
were detected. Genes for β-aryl ether degrading enzymes, classified as β-etherases or
glutathione-S-transferases, were also identified [17].
A repertoire of lignocellulose-degrading enzymes has also been identified in the
gut microbiome of the common black slug Arion. This study revealed more than 3383
CAZymes including multiple AAs families, associated with lignin degradation including
members of the AA2, AA3, and AA4 families, which are involved in oxidative degradation,
and the AA6 family, which catalyze the biodegradation of aromatic compounds such as
monolignols [73].
Using shotgun sequencing and bioinformatics approaches, Agamennone et al. (2019)
analyzed the gut microbiome associated with the soil invertebrate Folsomia candida. A
total of 2004 genes encoding enzymes that degrade cellulose, starch, and lignin were
detected. Among these, 1988 (99.2%) were of bacterial origin and mainly originated from
Proteobacteria (43%) and Actinobacteria (36%). Among the CAZyme families, 81 genes
were identified as auxiliary activities and the most abundant were the family members
of AA3 family (32% of the total), AA6 (27%), and AA7 (17%). The repertoire of auxiliary
activities identified suggested that the bacterial microbiome plays a role in the degradation
or modification of lignin contributing to the adaptation of the invertebrate to life in the
soil [74].

4.1.3. Vertebrate Digestive Tract

Bacterial CAZymes are essential to ruminants as they hydrolyze fibrous plant mate-
rials, which are utilized by host animals as energy sources [90]. Studies of the ruminal
microbiome at the domain level established that 97.5% of sequences belong to bacteria, 1.3%
Molecules 2021, 26, 2299 11 of 21

to archaea, and 0.9% to eukaryotes [75]. The phyla Bacteroidetes, Firmicutes, Proteobacteria,
and Fibrobacteres were identified as being involved in plant biomass degradation [75,91,92].
According to Jose et al. (2017), approximately 0.46% of AA families of the total number
of CAZyme families are involved in bovine rumen plant biomass deconstruction. It should
be noted that AAs, which are involved in lignin degradation and act in conjunction with
other hydrolytic enzymes, were observed with lower incidence (Table 1). Indeed, these
results corroborate early reports that showed limited lignin degradation in the ruminal
environment, mostly because of its natural microaerobic condition [93]. Five enzyme
families essential for degradation or modification of lignin were detected in the rumen (i.e.,
AA6, AA5, AA4, AA7, and AA3). Phylogenetic analysis showed that species of bacteria
belonging to genera Prevotella, Bacteroides, Clostridium, Fibrobacter, and Ruminococcus are
key contributors of CAZymes inhabiting the cow’s rumen [75].
Beloqui et al. (2006) reported RL5, a gene coding for a polyphenol oxidase with laccase
activity from a bovine rumen metagenomic library. Characterization of the recombinant
laccase produced in Escherichia coli revealed its ability to oxidize different substrates such
as syringaldazine, 2,6-dimethoxyphenol, veratryl alcohol, guaiacol, tetramethylbenzidine,
4-methoxybenzyl alcohol, 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS),
and phenol red, over a wide pH range. Phylogenetic analysis assigned this laccase sequence
to the genus Bacteroides [94].
Similarly, Ufarté et al. (2018) found in the bovine rumen microbiome producers of en-
zymes able to oxidize phenolic compounds such as ABTS (2,20 -azino-bis(3-ethylbenzothiazoline-
6-sulfonic acid)), syringaldehyde, 1-hydroxybenzotriazole, acetosyringone, and 2,20 -azino-
bis without the addition of mediators such as copper or manganese, presenting activity in
a wide range of temperatures (45 to 60 ◦ C) and pH (4.5 up to 5.5) [95].
Work focusing on the rumen microbiome of a more exotic ungulate, the camel, iden-
tified bacteria associated with lignin degradation. These bacteria were able to produce
enzymes with auxiliary activities categorized into four families (AA3, AA4, AA6, and
AA7), of which a 1,4-benzoquinone reductase belonging to AA6 accounted for >90% of the
1319 sequences. The auxiliary activity enzyme (AA6) was also abundant in the cow rumen
biogas reactor microbiome [76]. Still, regarding the camel’s rumen microbiome, Firmicutes
species contributed with 48% of the AAs found, Bacteroidetes species with 35%, Spirochaetaes
with 8%, and Fibrobacteres and Proteobacteria species contributed with 4% [76].
Families of AAs represented 0.13% of the total number of CAZyme families found in
the fecal samples of Asian elephants (Table 1). Among these, AA4 was the most abundant
(50%) followed by AA6 members (20%) [77]. These numbers showed a different prevalence
in comparison to the biogas fermenter, camel, and cow rumen where AA6 was the most
abundant family [75,76].

4.2. Culture Enrichment

Culture enrichment using lignin as the main carbon source facilitates the identification
of key microorganisms for its deconstruction and biotransformation. For example, the
taxonomic and functional analysis of bacterial consortia derived from soil and chicken
feces enriched in wood chips or filter paper, and subcultured in alkali lignin containing
medium showed that Pseudomonas has a central role in the deconstruction of aromatic
compounds. In addition to their high abundance in the consortia, they also had most of the
genes involved in the ortho-cleavage and degradation pathways of benzoate and catechol.
Other genera such as Klebsiella, Variovorax, Leclercia, and Enterobacter were also associated
with lignin degradation [96].
Moraes et al. (2018) [97], used 16S rRNA gene sequencing and shotgun metagenomics
to characterize a ligninolytic consortium obtained by cultivation of a sample of sugarcane
soil in a medium containing low molecular weight soluble lignin as a carbon source.
The authors reported an increase in the abundance of the families Alcaligenaceae and
Micrococcaceae and reported for the first time the involvement of the genus Pseudomaniobacter
in the degradation of aromatic compounds derived from lignin. Functional analysis of this
Molecules 2021, 26, 2299 12 of 21

consortium revealed the presence of different families of auxiliary activities (AA3, AA4,
AA6, and AA7) and genes of the main pathways related to the degradation of aromatic
compounds such as benzoate and phthalate (Table 2).

Table 2. Auxiliary activity families (AAs) and metabolic pathways of aromatic compounds consumption present in different
bacterial consortium identified in metagenomic studies.

Consortium Source and Bacteria AA Pathways of Consuming

Substrate References
with Ligninolytic Potential Families Aromatic Compounds
Enrichment from
Catechol ortho-
chicken feces and
cleavage and
soil (Pseudomonas, Alkali lignin - [96]
benzoate degradation
Klebsiella, Variovorax, Leclercia, and
pathways
Enterobacter)
Enrichment from
Benzoate degradation
sugarcane plantation
to catechol, catechol
soil (Proteobacteria, Low-molecular- AA2, AA3,
ortho-cleavage,
Actinobacteria, weight (LW) AA4, AA6, and [97]
catechol meta-
Firmicutes, lignin AA7
cleavage, and
Alcaligenaceae, and
phthalate degradation
Micrococcaceae)
Enrichment from soil
(Brevundimonas,
Wheat straw AA2, AA4,
Caulobacter,
switchgrass and AA6, and AA7 - [98]
Pseudomonas,
corn stover
Citrobacter, and
Aeromonas)
Enrichment from
AA2, AA3,
compost ecosystems
Corn stover AA4, AA6 and - [99]
(Proteobacteria and
AA7
Firmicutes)
Enrichment from
compost ecosystems
(Symbiobacterium
thermophilum, T.
curvata,
Mycobacterium Rice straw AA2 - [100]
xenopi,
Amycolicicoccus
subflavus and
Mycobacterium
thermoresistibile)
Enrichment from
compost ecosystems
(Sphaerobacter, Ortho-cleavage of
Hyphomicrobium, protocatechuate and
Thermus Switchgrass AA2 4- [101]
thermophilus, hydroxyphenylacetate
Sphaerobacter, degradation
Gemmatimonadetes,
Paenibacillus)
Enriched from
AA1, AA2,
compost ecosystems
CMC AA4, AA6, and - [102]
(Thermobacillus
AA7
species, Bacillus)

Three microbial consortia started from soils supplemented with wheat straw (WS1-M),
switchgrass (SG-M), and corn stover (CS-M) were described as presenting a set of genes
belonging to auxiliary activities families. AA6 family members were the most abundant,
followed by members of the AA2, AA7, and AA4 families. The highest rate of lignin
Molecules 2021, 26, 2299 13 of 21

deconstruction, approximately 59%, was recorded for the SG-M consortium, which had
a high abundance of the genera Brevundimonas, Caulobacter, Pseudomonas, Citrobacter, and
Aeromonas [98].
In a microbial consortium generated from the cultivation of compost samples in media
containing corn stover as the carbon source, 22 genes encoding AAs were assigned to
Firmicutes and Proteobacteria. Only four genes encoding members of the AA2 family
have been attributed to Escherichia and Klebsiella from the Proteobacteria phylum [99]. In
contrast, a different straw-adapted compost microbial consortium was enriched for AA2
family genes, distributed among Actinobacteria species, a novelty, since these members
were not previously recognized as active on lignin. Among the species identified were Ther-
momonospora curvata, Mycobacterium xenopi, Amycolicicoccus subflavus, and Mycobacterium
thermoresistibile [100].
In a thermophilic consortium adapted to degrade switchgrass through multiple pas-
sages at 60 ◦ C, several putative genes belonging to the AA1 family were assigned to Thermus
thermophilus, Sphaerobacter, Gemmatimonadetes spp., and Paenibacillus spp. Additionally, genes
for families of glutathione S-transferases have also been found in the genus of Hyphomi-
crobium. This family of enzymes contains members that are involved in the cleavage of
beta-aryl linked lignin dimers [101]. In another thermophilic bacterial consortium enriched
with carboxymethylcellulose (CMC), six bacterial genomes were partially reconstructed
and named as BZ1, BZ2, BZ3, BZ4, BZ5, and BZ6. Taxonomic and phylogenetic analysis
suggest that three of the six reconstructed genomes belong to new bacteria: Thermobacillus
(BZ1); a member of a new genus in the family Paenibacillaceae (BZ3), similar to Paenibacillus
and Cohnella; and a member of a new deep-branching family in the Clostridia (BZ6) class.
Six families of auxiliary activities related to the degradation of lignin (AA1, AA2, AA4,
AA6, and AA7) were found in the six reconstructed genomes [102].

4.3. Metatranscriptomics and Metaproteomics Approaches

4.3.1. Metatranscriptomics
Metatranscriptomics is the study of the rRNA and mRNA of a microbial community,
which provides information on the active functional profile through the evaluation of gene
expression (Figure 5) [103]. In contrast to metagenomics, which describes the composition,
function, and relative abundance of different members of a microbial community, metatran-
scriptomics provides insight into the metabolically active part of the community. Therefore,
metatranscriptomic analysis is restricted to transcribed genes, and the non-transcribed
portions of the genome that are frequent in metagenomic data are not present [104]. How-
ever, technical limitations for effective RNA recovery, low quantities of bacterial mRNA
in samples, and the short lifetime of mRNA can restrict the application of this approach.
The transcriptional profile of auxiliary activity genes involved in lignin degradation is
summarized in Table 3.

Table 3. Families of auxiliary activities (AAs) present in different bacterial communities, as researched from metaomics
studies.

Sample Omics Techniques Applied AAs Families References

Compost ecosystems Metatranscriptomics AA2, AA3, AA4, AA6, and AA7 [105]
Soil microbiota Metatranscriptomics AA2 and AA6 [106]
Forest soil Metatranscriptomics AA1 and AA3 [69]
Termite gut Metatranscriptomics AA2, AA4, and AA6 [107]
Termite gut Metatranscriptomics AA4 and AA6 [108]
Bacterial consortium Metaproteomics AA2 and AA7 [109]
Bacterial consortium Metaproteomics AA2 [99]
Soil microbiota Metatranscriptomics AA2 and AA6 [106]
Forest soil Metatranscriptomics AA1 and AA3 [69]
Termite gut Metatranscriptomics AA2, AA4, and AA6 [107]
Termite gut Metatranscriptomics AA4 and AA6 [108]
Molecules 2021, 26, 2299 14 of 21
Bacterial consortium Metaproteomics AA2 and AA7 [109]
Bacterial consortium Metaproteomics AA2 [99]

Figure 5. General
Figure 5. schemefor
General scheme forfunctional
functionaland
and genetic
genetic profiling
profiling of oxidative
of oxidative enzymes
enzymes exploiting
exploiting
metatranscriptomic sequence data.
metatranscriptomic sequence data.
Active metabolic pathways of the microbial community in a thermophilic compost-
ing operation in the São Paulo Zoo Park have been unraveled through metatranscrip-
tomics [105]. Lignin deconstruction occurred sequentially and synergistically by the action
of ligninases that reached peak abundance at the end of the composting process. The
majority of ligninolytic enzymes belonging to the classes AA2 (7–28% of all AAs) and
AA6 (32–66%), followed by moderate amounts of AA3, 4, and 7, were found in all sam-
ples. Members of Clostridiales, Bacillales, and Actinomycetales were implicated as the major
compost-degrading microbes, particularly in the thermophilic and mature stages of the
composting process [105].
The temporal expression dynamics of a set of plant biomass-degrading enzymes by a
bacterial consortium growing on sugarcane bagasse was studied. Here, Jiménez et al. (2018)
described the importance of the expression of auxiliary activities AA2 and AA6. The highest
expression levels of transcripts encoding AA2 and AA6 members of families derived from
Paenibacillus and Brevundimonas were observed at early stages (at 12 h) of the consortium’s
growth. According to the authors, this could be related to cell protection against oxidative
damage and electron transfer. Finally, the expression of catalase-peroxidases (AA2) was
carried out by Chryseobacterium and Brevundimonas at the final stages (192 h), suggesting
that the degradation of lignin occurred in the final stages of growth of the consortium [106].
Moreover, Žifčáková et al. (2017) investigated the glycoside hydrolases and auxiliary
enzyme contribution of fungi and bacteria in two different stations on top forest soil. A
high diversity of transcription in families of AA3 (mixed activities in lignocellulose) and
AA1 (laccases) was reported. Concerning CAZymes functional groups, those destined for
cellulose were more transcribed, followed by those that act on lignin [69].
Molecules 2021, 26, 2299 15 of 21

Tokuda et al. (2018) identified transcripts encoding three families of auxiliary activities
belonging to AA6, AA4, and AA2 involved in bioconversion of phenolic compounds in
the hindgut compartment from termites (Nasutitermes takasagoensis) [107]. Additionally,
Marynowska et al. (2017), studied the lignocellulolytic potential of the higher termite gut
prokaryotes. They reported transcripts encoding enzymes such as AA4 (vanillyl alcohol
oxidase), and AA6 families (1,4-benzoquinone reductase) [108].

4.3.2. Metaproteomics
Metaproteomics allows the assessment of the complete protein content of an envi-
ronmental microbiota (Figure 6). The main challenges of metaproteomics include sample
complexity, low peptide identification, and lack of complete databases [110,111]. Although
there are still only a few studies that have reported the presence of enzymes related to
lignin degradation, the use of this approach in lignocellulolytic bacterial communities has
Molecules 2021, 26, x FOR PEER REVIEW
demonstrated the presence of auxiliary activities involved in the deconstruction of lignin 16 of 22
(Table 3).

Figure 6. General scheme of the metaproteomic analysis for the identification of lignin-degrading
Figure 6. General scheme of the metaproteomic analysis for the identification of lignin-degrading
enzymes from different bacterial communities.
enzymes from different bacterial communities.

Forexample,
For example,the theanalysis
analysisofof thebacterial
the bacterial consortium
consortium secretome
secretome adapted
adapted to using rice
to using
straw
rice as aascarbon
straw source
a carbon showed
source showed thethe
presence
presence ofof
1616domains
domains ofofauxiliary
auxiliaryactivities
activitiesbelong-
ing to three
belonging families
to three (AA2,
families AA7,
(AA2, andand
AA7, AA10).
AA10).Interestingly, in in
Interestingly, addition
additiontotosingle-domain
single-
domain CAZymes, other proteins with multiple domains have been identified,
CAZymes, other proteins with multiple domains have been identified, for example, lig- for example,
ligninolytic proteins containing
ninolytic proteins containing three
three distinct
distinctAA2
AA2domains.
domains.TheThemain
maingroups
groups respon-
responsible
sible
for the deconstruction of this biomass belonged to the phyla Proteobacteriaand
for the deconstruction of this biomass belonged to the phyla Proteobacteria and Bac-
Bacteroidetes [109].
teroidetes [109].
In contrast, for another consortium adapted to corn stover, only one AA2 protein
In contrast, for another consortium adapted to corn stover, only one AA2 protein
(catalase/peroxidase) from Escherichia coli was considered potentially involved in the
(catalase/peroxidase) from Escherichia coli was considered potentially involved in the deg-
degradation of lignin [99]. Likewise, in another bacterial consortium adapted for using
radation of lignin [99].
switchgrass as a carbon source, Likewise, in another
only a putative bacterial
laccase assignedconsortium adapted for using
to the Gemmatimonadetes
switchgrass
was identified as a carbon source, only a putative laccase assigned to the Gemmatimonadetes
[101].
was identified [101].
5. Application of Lignin-Active Enzymes
5. Application of Lignin-Active
Nowadays, with Enzymes
a growing interest in green processes, microbial enzymes are the
major biological players sought after for industrial
Nowadays, with a growing interest in green applications. Lignin-active
processes, microbialenzymes areare the
enzymes
prospected for many applications such as decontamination of industrial dyes [112], general
major biological players sought after for industrial applications. Lignin-active enzymes
are prospected for many applications such as decontamination of industrial dyes [112],
general delignification for diminishing lignocellulose recalcitrance [16], and the produc-
tion of aromatic compounds of industrial relevance [8]. As previously discussed, enzymes
produced by filamentous fungi are the prime source for industrial applications, however,
Molecules 2021, 26, 2299 16 of 21

delignification for diminishing lignocellulose recalcitrance [16], and the production of aro-
matic compounds of industrial relevance [8]. As previously discussed, enzymes produced
by filamentous fungi are the prime source for industrial applications, however, bacteria
can produce enzymes with interesting kinetic parameters that should be further exploited.
The major challenge in using enzymes produced by bacteria instead of those secreted
by classic filamentous fungi such as Trichoderma sp. is related to enzyme production levels.
Filamentous fungi that are prospected for enzyme production are naturally specialized in
secreting enzymes with activity against lignin and holocellulose [113,114]. The utilization of
similar enzymes obtained from bacteria requires recombinant production of these enzymes
in host cells suitable to industrial conditions. In this regard, heterologous expression
and a direct evolution approach to improve catalytic activity, enhance expression levels,
and improve laccase stability have been pursued to convert bacterial laccases into highly
efficient and commercially valuable biocatalysts [13].
Bio-delignification by AA1 family (laccases) provides a clean and efficient treatment
method of lignocellulose without damaging the cellulose, which is important for com-
mercial bioethanol production. Researchers have shown that laccases can work more
efficiently in combination with hemicellulases for lignocellulose saccharification. However,
Rocha-Martín et al. [115], reported that supplementation of laccase in enzymatic hydrolysis
showed contradictory results, depending on pretreatment type and biomass used. The
addition of laccase to softwood hydrolysis resulted in high glucose yield, while a negative
effect was obtained for hardwood hydrolysis.
AA1 families have successfully reported effectiveness for kraft pulps and biobleaching.
Arias et al. (2003) showed that the biobleaching of eucalyptus kraft pulps by lacasse (AA1_1
family) from Streptomyces cyaneus CECT 3335 using ABTS as a mediator resulted in the
significant decrease in the kappa number up to 2.3 U and increased pulp brightness. An
alkaline and halotolerant bacterial laccase (SilA) produced by Streptomyces ipomoea CECT
3341 was also used to enhance the conventional chemical bleaching process of an industrial
eucalyptus kraft pulp [47,116].
Lignin deconstruction and detoxification of aromatic compounds by bacterial enzymes
is a widely investigated topic. However, details on bacterial uptake and metabolization of
the compounds released during lignin breakdown is still a relatively less investigated topic.
Recent studies are not only focusing on how the bacterial enzymatic machinery works in
lignin deconstruction, but also on investigating how these compounds are metabolized.
Although the intention of this review is not to describe the detailed pathways, it is clear
that the bacterial metabolism of lignin can generate interesting biotechnological products
such as bioplastics, vanillin, and lipids that can be further transformed into biofuels [43].

6. Conclusions and Future Perspectives

The use of metagenomic approaches to study lignocellulosic microbial communities
present in different environments has revealed the ligninolytic potential of several bacte-
ria including new non-cultivable clades such as Caulobacteraceae, Solirubrobacterales, and
Cystobacteraceae. It has also allowed access to a set of potential bacterial genes for auxiliary
activities, belonging mainly to families AA2, AA3, AA4, AA5, AA6, and AA7, which are
involved in the degradation of lignin and other components of the plant cell wall. One
limitation of metagenomics is that it does not provide information about active microbial
communities. This is why complementary approaches such as metatranscriptomics and
metaproteomics need to be used. Studies using these two latter approaches have shown
the presence of auxiliary bacterial activities related to lignin degradation (Table 3). To date,
there are still few studies related to bacterial degradation of lignin using omics approaches,
and these mainly use metaproteomics. Future work aimed at the bacterial degradation
of lignin needs to take advantage of the integrative use of the three omics approaches.
Together, these will likely generate a more comprehensive understanding of microbial
degradation and/or modification of lignin.
Molecules 2021, 26, 2299 17 of 21

Author Contributions: J.P.S.: writing—original draft preparation. A.R.P.T.: writing—original draft

preparation. P.R.V.H.: writing—original draft preparation. B.F.Q.: writing—review, and editing.
E.F.N.: writing—review and editing, funding acquisition. All authors have read and agreed to the
published version of the manuscript.
Funding: This research was funded by grants from EMBRAPA, Universidade de Brasília, CAPES,
and CNPq. J.P.S. and P.R.V.H. are recipients of a CAPES doctoral scholarship. E.F.N. and B.F.Q. are
recipients of a CNPq research fellowship.
Conflicts of Interest: The authors declare no conflict of interest.

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