BIOPROCESS

PRINCIPLES

Isolation, preservation and improvement of

industrial strains
Stages for an Industrial microbial
Fermentation Process

 Strain selection
 Laboratory process development
 Pilot Scale up
 Industrial Scale up
 Fermentation Media Preperation
 Downstream process development
 Product packaging techniques
 Other Commercial consideration
 Waste Disposal
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May 21, 2019 Stages in an Industrial Bioprocess
An Ideal STRAIN
 Genetic Stability
 Efficient production of target product
 Limited need for vitamins and growth factors
 Utilization of wide range of low cost substrates
 Safety, GRAS
 Ready harvesting
 Limited by products production
 Easy genetic amenability

May 21, 2019 Stages in an Industrial Bioprocess

Strain Selection

How?
 Purchase from Culture Collections
 Screening of nature circumstances
 Genetic engineering
 Mutations
 Cell biology techniques
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International Culture Collections

Strains can be purchased from Culture Center

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Screening of nature circumstances

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Isolation and Screening of
Industrial Strain

• Isolation of from the environment is by:

• Collecting samples of free living microorganism from
anthropogenic or natural habitats.
• These isolates are then screened for desirable traits.
• Or by sampling from specific sites:
• Mos with desired characteristics are found among the natural
microflora
• After sampling of the organism the next step is of
enrichment.
Enrichment

 Enrichment in batch or continuous system on a defined

growth media and cultivation conditions are performed to
encourage the growth of the organism with desired trait.
 T h i s will increase the quantity of the desired organism
prior to isolation and screening.
Screening

• Subsequent isolation as pure cultures on solid growth media

involves choosing or developing the appropriate selective
media and growth conditions.
• Next step to enrichment and isolation is Screening.
• The pure cultures must be screened for the desired property;
production of a specific enzyme, inhibitory compound, etc.
• Selected isolates must also be screened for other important
features, such as stability and, where necessary, non-toxicity.
Screening

• These isolation and screening procedures are more easily,

applied to the search for a single microorganism.
• The industrial microorganism should ideally exhibit:
• 1. genetic stability
• 2. efficient production of the target product, whose, route of
biosynthesis, should preferably be well characterized.
Screening
• 3. limited or no need for vitamins and additional growth
factors.
• 4. utilization of a wide range of low-cost and readily
available carbon sources
• 5. amenability to genetic manipulation;
• 6. safety, non-pathogenic and should not produce toxic
agents, unless there is the target product;
• 7. ready harvesting from the fermentation; .
• 8. production of limited byproducts to ease subsequent
purification problems.
Culture
Preservation
 Streptomyces aureofaciens NRRL 2209 was the
first microorganism deposited in a culture
collection in support of a microbially based patent
application.
 Preservation of microbial cultures was critical
for all individuals and firms engaged in the search
for patentable products from and patentable
processes by microorganisms.
Culture Preservation

• Preservation of cultures by freezing, drying, or a

combination of the two processes is highly influenced by
resistance of the culture to the damage caused by rapid
freezing, the dehydrating effects of slow freezing, or damage
caused during recovery.
• To minimize damage, agents have been used that protect
against ice formation by causing the formation of glasses
upon cooling.
Culture Preservation

 Methods to protect against the negative effects

of dehydration include adaptation to lower
effective water activity by pre-incubation in high
osmotic pressure solutions.
 Damage caused by thawing after freezing can be
minimized by rapid melting and by the
composition of the medium used for growth after
preservation.
Culture Preservation
 There are various preservation methods .
 To date, preservation in liquid nitrogen is still the most
successful long-term method.
Serial Transfer
 Based upon its ease of use, serial transfer is often the first
“preservation” technique used by microbiologists.
 T h e disadvantages of relying upon this method for culture
maintenance include contamination, loss of genetic and
phenotypic characteristics, high labor costs, and loss of
productivity.
Preservation inDistilled
Water
 T h i s method (Castellanimethod, 50 years ago) was
extensively tested on 594 fungal strains:
 6 2 % of the strains growing and maintaining their
original morphology.
 I n another study, 76% of yeasts, filamentous fungi, and
actinomycetes survived storage in distilled water for 10
years.
Preservation inDistilled
Water
 T h e pathogen Sporothrix schencki concluded that even
though long-term survival was good when this procedure
was used, there was a noted loss in virulence.
 Castellani technique should be considered as one of the
options for practical storage of fungal isolates.
Preservation under Oil
• One of the earlier preservation methods was the use of mineral oil
to prolong the utility of stock cultures.
• Mineral oil has been found to prevent evaporation from the
culture and
• Decrease the metabolic rate of the culture by limiting the supply
of oxygen.
• This method is more suitable than lyophilization for the
preservation of non-sporulating strains.
 Lyophilization
 O n e of the best methods for long-term culture preservation of m
yan
microorganisms is freeze-drying (lyophilization).
 T h e commonly used cryoprotective agents are skim milk (15% [wt/vol]
for cultures grown on agar slants and 20% for pelleted broth cultures)
or sucrose (12% [wt/vol] final concentration).
 I t should be noted that some plasmid--containing bacteria
aresuccessfully preserved by this method.
 Storage over Silica Gel
•
Neurospora has successfully been preserved over
•
silica gel.
• Preservation on Paper
• Drying the spores on some inert substrates can preserve spore-forming fungi,
actinomycetes, and unicellular bacteria.
• Fruiting bodies of the myxobacteria, containing myxospores, may be
preserved on pieces of sterile filter paper and stored at room temperature or at
6°C for 5 to 15 years.
• Preservation on Beads
• The method involving preservation on beads (glass, porcelain) , developed by
Lederberg, is successful for many bacteria.
Liquid Drying
• To avoid the damage that freezing can cause, a liquid—drying
preservation process is applied.
• It has effectively preserved organisms such as anaerobes that are
damaged by or fail to survive freezing.
• This procedure was preferred over lyophilization for the maintenance of
the biodegradation capacity of six gram--negative bacteria capable of
degrading toluene.
• Malik’s liquid-drying method was also found to be markedly superior to
lyophilization for the preservation of unicellular algae.
Cryopreservat
ion
 Microorganisms may be preserved at - 5 to - 20°C for 1,
to 2years by freezing broth cultures or cell suspensions in
suitable vials.
 D e e p freezing of microorganisms requires a cryoprotectant
such as glycerol or dimethyl sulfoxide (DMSO) when
stored at -70°C or in the liquid nitrogen at -156 to -196°C.
Cryopreservat
ion
 Broth cultures taken in the mid--logarithmic to
late logarithmic growth phase are mixed with an
equal volume of 10 to 20% (vol/vol) glycerol or 5
to 10% (vol/vol) DMSO.
Alternatively, a 10% glycerol-sterile broth
suspension ofgrowth from agar slants may be
prepared.
Preser vation in Liquid
Nitrogen
 Storage in liquid nitrogen is clearly the preferred method for
preservation of culture viability.
Screening / Isolation
 What is screening?

 Primary Screening
 Secondary Screening

ENRICHMENT : Anearobes in N2 Atmospheres,

Acidophiles in Extreme pH values (2-4)

TEST SYTEMS: For Amylases the test system would

be agar plates with starch, selection of colonies after
staining with iodine

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 Strain Isolation SCHEME
 Soil or water sample is suspended in a definite
amount of sterile water and agitated
 The supernatant is diluted 10-1 –10-10
 Samples from this dilution are placed on culture
media and then incubated
 Single colonies from the plates are picked and
purified by restreaking
 Pure strains are maintained as agar cultures in test
tubes

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 Isolation of from the environment is by: • Collecting
samples of free living microorganism from
anthropogenic or natural habitats. • These isolates
are then screened for desirable traits. • Or by
sampling from specific sites: • Mos with desired
characteristics are found among the natural
microflora • After sampling of the organism the
next step is of enrichment.

May 21, 2019 Stages in an Industrial Bioprocess

 Genetic
Engineering
Incorporation into
artificial plasmids of
genes from a wide
variety of sources has
made possible the
transfer of genetic
material across
virtually any species
barrier.
Various high value
added products have
been produced from
the Genetic engineering
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methods
 Mutation

Via chemical
or physical,
and biological
means

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Cell Biology Techniques
 Protoplast fusion
(promote high frequencies of genetic recombinants)

 Removing the cell wall with lytic enzymes in the

presence of osmotic stabilizers.
 In the presence of fusogenic agent such as
polyethylene glycol (PEG), protoplasts are
induced to fuse and form transient hybrids or
diploids.

May 21, 2019 Stages in an Industrial Bioprocess