BIOPROCESS TECHNOLOGY

What are industrial microorganisms?

Industrial microbiology includes the use
of microorganisms to manufacture food
or industrial products in large quantities.

Numerous microorganisms are used

within industrial microbiology; these include
naturally occurring organisms,
laboratory selected mutants, or even
genetically modified organisms (GMOs)
Microorganisms and its uses
⚫ Production of dairy products: Bacteria are the key players
here.
⚫ Bread Baking: A species of Streptococcus is added to
the dough before making bread to bring about the
required fermentation.
⚫ Alcoholic Drinks
⚫ Organic acids
⚫ Enzymes
⚫ Steroid production
⚫ Help in sewage treatment
⚫ Used as insecticides
WHAT IS A CULTURE?
Population of microorganisms grown under well defined conditions.
WHAT IS MIXED CULTURE?
When a particular species of microbe is present in a very
small number in comparison to the total number of
microorganisms , such culture is called as mixed culture.
WHAT IS PURE CULTURE?
A culture containing only one species of microbe is called
pure culture.
SPECIES- a collection of bacterial cells which share an overall similar
pattern of traits in contrast to other bacteria whose pattern differs
significantly.
STRAIN- A strain is a subset of a bacterial species differing from
other bacteria of the same species by some minor but
identifiable difference.
Strain selection
Important characteristics for strain

• The selection of strains resistant to infection

• The selection of non-foaming strains
• The Selection of strains which are resistant to
components in the medium
• The selection of morphologically favourable strains
• The selection of strains which are tolerant of low oxygen
tension
• The elimination of undesirable Products from a
production strain
• The development of strains producing New fermentation
products
Choosing microorganisms for Industrial
microbiology and Biotechnology
The characteristics of microbes that are
desirable to the industrial microbiologist
are:
⚫ genetic stability,
⚫ easy maintenance and growth, and
⚫ amenability to procedures for extraction
and purification of desired product
Finding microorganisms in nature
⚫ major sources of microorganisms for use
in industrial processes are soil, water,
and spoiled bread and fruits;
⚫ only a minor portion of microbial species
in most environments have been
identified
COMMON METHODS OF ISOLATION OF PURE
CULTURE
•The process of screening a pure culture by
separating one type of microbes from a mixture
is
called Isolation.
Some common isolation methods are;
Streak plate method
Pour plate method-
Loop dilution technique
Serial Dilution technique
Spread plate method
Micromanipulator method
Streak plate method
⚫ Prepare nutrient agar or any required medium
and poured into the petri plates.
⚫ Allow the plates to solidify.
⚫ Sterilize the inoculation loop using flaming
technique.
⚫ Transfer microbial mixture from a tube to the edge
of an agar plate with an inoculation loop as per
illustration.
⚫ Incubate plates at 370C for 24hours.
Streak plate method
⚫ TYPES
Different types of streaking techniques are available. They are T - streak,
Quadrant streak, simple streak, radiant streak and continuous streak.
These techniques are named based on the types of line of streak drawn on
the surface of the medium.
Pour plate method

To isolate and get pure culture of microorganisms

⚫ Prepare nutrient agar and PDA potato Dextrose Agar medium (one plate needs approximately
15 - 20 ml) and sterilize at 1210 C for 15 minutes.
⚫ Dilute the sample up to 1: 10000000 (10-7) using diluents.

⚫ Add 1 ml of samples from 1:1000 (10-3).

⚫ Pour the medium into sample added petriplates.

⚫ Rotate the petriplates clockwise and anticlockwise direction.

⚫ Allow the plates to solidify.

⚫ Similarly perform pour plating for other dilutions like 10-4, 10-5 and 10-6.
⚫ Incubate all nutrient agar plates at 370C for 24 hours and PDA plates at 25 – 300C for 48 hours
and record the results.
Pour plate method
Spread plate method
The method is performed for the assay of chemicals
like antibiotics, vitamins etc.
This method is called spread because L rod or
cotton swab is used to spread the sample.
This techniques also used for the isolation and
enumeration of microorganisms from samples with lower
populations of bacteria and other microorganisms.
Spread plate method
⚫ Prepare nutrient agar medium, sterilize at 1210 C and pour in to the petri plates.

⚫ Dilute the sample up to 1:100000 (10-5).

⚫ Add 1 ml of sample from 10-3 on to the centre of an agar medium using sterile pipette.

⚫ Perform similar procedure for 10-4 and 10-5 dilutions.

⚫ Dip the glass spreader (L- rod) in to a beaker of ethanol/spirit.

⚫ Briefly flame ethanol soaked spreader on Bunsen burner and allowed it to cool.

⚫ Spread the sample evenly over the agar surface.

⚫ Incubate all the plates at appropriate temperature (for bacteria 370C and for fungus 25 – 300 C)
for 24 to 48 hours.
SERIAL DILUTION
MICROMANIPULATOR METHOD
⚫ Micromanipulators
⚫ • Micromanipulators have been built, which permit one to pick out
⚫ a single cell from a mixed culture. This instrument is used in
⚫ conjunction with a microscope to pick a single cell (particularly
⚫ bacterial cell) from a hanging drop preparation.
ADVANTAGES OF MICROMANIPULATOR METHOD
⚫ • The advantages of this method are that one can be reasonably
⚫ sure that the cultures come from a single cell and one can obtain
⚫ strains with in the species.
DISADVANTAGES
⚫ • Disadvantages are that the equipment is expensive,
⚫ • Its manipulation is very tedious, and it requires a skilled operator
ROLL TUBE METHOD
Prepare ten-fold dilution and add 0.1
ml of diluted culture to molten agar
cooled to 500C poured in test tube.
Now tilt the tube and roll so
that medium is formed as a thin
film around the wall of tube.
 Incubate and count the no of
colonies
on next day.
Enrichment methods for isolation of microorganisms
Enrichment methods are useful for quick isolation of specific types
of organisms.
Types of organisms Enrichment method
Thermophiles High temp (42-1000C)
Psychrotrophs low temp (5-150C)
Acidophiles Low pH (2-4)
Halophiles High NaCl concentration
Anaerobes N2 atmosphere
Actinoplanes Pollen grains
Myxobacteria Wood bark
Strains of microorganisms from unusual environments
The unusual environments such as cold habitats, high altitudes, deserts,
deep
sea and petroleum fields are constantly being tried for this purpose.
Such strains may be capable of producing new products of industrial
importance
Screening of metabolites for isolation of microorganisms
The microorganisms can be tested directly for the product
formation, and isolated.
For example – if the product is an antibiotic, then the test
system consists of the strains of organisms which inhibit
zones, on the agar plates.
Another example is the isolation of microorganisms producing
amylases. When grown on agar plates containing starch, and
then stained with iodine, amylase producing organisms can
be identified and isolated.
Screening of microbes
⚫ Although there are many screening techniques, all of
them are generally grouped into two broad
categories.
⚫ They are:
⚫ 1. Primary screening, and
⚫ 2. Secondary screening.
Primary Screening of Microorganisms:
Primary screening may be defined as detection and isolation of the
desired microorganism based on its qualitative ability to produce the desired
product like antibiotic or amino acid or an enzyme etc.
The following are some of the important primary screening
techniques:
Indicator dye technique
The crowded plate
technique Enrichment culture
technique Auxanographic
technique
PRIMARY SCREENING OF ORGANIC ACID PRODUCING
MICROORGANISMS
 The ph indicating dyes may be used for detecting microorganism that
are capable of producing organic acids.

 These dyes undergo colour changes according to its ph.

 Dyes such as Neutral red, Bromothymol blue are added to

the poorly buffered nutrient agar media .

 Colonies are sub cultured to make stock culture.

 Further testing is needed since inorganic acids, bases

are also metabolic products of microbial growth
⚫ Incorporation of CaCO3 in medium is also used to screen
⚫ organic acid producing microbes on basis of formation of clear zone
of dissolved CaCO3 around the colony.
PRIMARY SCREENING OF ANTIBIOTIC PRODUCING
MICROORGANISMS
 Crowded plate technique is used for screening of antibiotic
producing microorganisms.

 Does not give information about the sensitivity of

antibiotics towards other microorganisms.

 Dilutions are made and then pouring and spreading of

soil samples that give 300 to 400 or more colonies per
plate.

 Colonies showing antibiotic activity are indicated by

zone of
inhibition around the colony .

 Such colonies are sub cultured and purified by streak

Antibiotic producing Amylase producing
microbe microbes
Antibiotic activity against
microbe
AUXANOGRAPHY TECHNIQUE
This technique is largely employed for detecting microorganisms
are able to produce growth factors (e.g. Amino acid and vitamins) extracellular.

ENRICHMENT CULTURE TECHNIQUE

This technique was used to isolate the desired microorganisms form
a heterogeneous microbial population present in the sample.

Either medium or incubation conditions are adjusted so as to favor

the growth of the desired microorganism.
SECONDARY SCREENING
⚫ It’s a systematic screening programme intended to isolate industrially important or
useful microorganisms .

SOME IMPORTANT POINTS ASSOCIATED WITH SECONDARY SCREENING ARE

 It is useful in sorting of microorganisms that have real commercial value. The

microorganisms having poor applicability in fermentation process are discarded.

 Provides the information whether the product formed by microorganisms is new

or
not.

 This may be accomplished by paper , thin layer, chromatographic technique.

Screening for new metabolites, and isolation of microorganisms
Research work is particularly directed for identifying
chemotherapeutically important products for the
treatment of tumors,
bacterial diseases (newer antibiotics against resistant
strains) and
viral diseases,
besides several other substances (e.g. hormones, enzyme
inhibitors).
In addition, isolation of microorganisms for improvement of
food industry, and for efficient degradation of the environmental
pollutants and hazardous chemicals also assumes significance.
An isolated producer strain should possess the following
characters:
⚫ 1. It should be able to grow on relatively cheaper substrates.
⚫ 2. It should grow well in an ambient temperature preferably at 30-40°C.
This reduces the cooling costs.
⚫ 3. It should yield high quantity of the end product.
⚫ 4. It should possess minimum reaction time with the equipment used in
a fermentation process.
⚫ 5. It should possess stable biochemical characteristics.
⚫ 6. It should yield only the desired substance without producing
undesirable
substances.
⚫ 7. It should possess optimum growth rate so that it can be easily cultivated on a
large scale.
Microbes in Industrial Products
⚫ Beverages.
Yeasts are the widely used microorganism for the production of beverages
like beer, brandy, rum, wine, whiskey, etc.

⚫ Organic acids.
Microbes are also used for the industrial production of certain organic
acids.

⚫ Enzymes.
⚫ Antibiotic.
⚫ Vitamins.
A List of important microorganisms and their
products
Microorganism Product
Algae
Chlorella sorokiniana Single cell protein
Spirulina maxima Single cell protein
Bacteria
Acetobacter aceti Acetic acid
Bacillus subtilis Bacitracin
Actinomycetes
Streptomyces aureofaciens Tetracycline
Fungi
Aspergillus niger Citric acid
Strategies for improvement of
industrially important strains
-
What are strains?
• A strain is a genetic variant or subtype of a microorganism (e.g., a virus,
bacterium or fungus).
• Microbial strains can also be differentiated by their genetic makeup using
metagenomic methods to maximize resolution within species.

What are industrial strains?

• Strains which synthesize one component as the main product are
preferable, since they make possible a simplified process for
product recovery.

Why is strain development important in industrial microbes?

• Prerequisite for efficient biotechnological processes at industrial scale is
the use of microbial strains which produce high titre of the
desired product.
• The process of enhancing the biosynthetic capabilities of microbes to
produce desired product in higher quantities is defined as microbial
strain improvement.
From where we can find industrial
strains?
• The first step in developing
producer strains is the
isolation of concerned
microorganisms from the
natural habitats.
• What we are looking?
• • Fromprocedure
The where we canofget?
isolation,
detection, and separation of
microorganisms of our interest
from a mixed population by using
highly selective procedures is
called Screening.
Proper strain used in industry genetically regarded as safe
(GRAS)
 Methods of strain improvement
• Mutation and mutant selection
• Recombination
– Transduction
– Transformation
– Conjugation
– Protoplast fusion
– Parasexual recombination
• Recombinant DNA technology
 Mutation and mutant selection
• A mutation is a sudden and heritable change in the
traits of an organism.
• Mutations occurring without any specific
treatment are called “spontaneous mutation”.
• Mutation are resulting due to a treatment
with certain agents are known as “induced
mutation”.
• Application of mutagens to induce mutation is called
mutagenesis.
• Agents capable to induce mutations are
called mutagens.
 Mutagens

Physical Chemical Biological

Radiation Base analogs Transposon

Intercalating
Heat Virus
agents

Deaminating
Bacteria
agents

Metals
The practical isolation of mutants
Transduction
 Transformation
• Bacterial transformation is a
process of horizontal
gene transfer by
which some
bacteria take up foreign
genetic material (naked DNA)
from the environment.

• Cells in which transformation

can occur are
‘competent’ cells.
 Conjugation

Bacterial conjugation : is
the transfer of genetic
material between bacterial
cells by direct cell-to-cell
contact or by a bridge-like
connection between two
cells.

Conjugation types:
1) F+ x F- Conjugation
2) Hfr x F- Conjugation
3) F’ x F- conjugation
Protoplast fusion
Parasexual recombination
 Genetic Engineering
• Genetic engineering (GE) is the process of using
recombinant DNA (rDNA) technology to
alter the genetic makeup of an organism.
• Genetic engineering is accomplished in three basic
steps:
– The isolation of DNA fragments from a donor organism
– The insertion of an isolated donor DNA fragment into a
vector genome
– The growth of a recombinant vector in an appropriate
host.
 Improvement of microbial processes by GE
Primary metabolites
• New processes for the production of amino acids and vitamins have
been developed by recombinant DNA technology.
• Escherichia coli strains were constructed with plasmids bearing amino
acid biosynthetic operons.
• Cloning extra copies of threonine export genes into E. coli led to increased
threonine production.
• An engineered strain of Corynebacterium glutamicum producing L -tryptophan
was further modified by cloning in additional copies of its own transketolase gene.
• Biotin has been made traditionally by chemical synthesis but recombinant
microbes have approached a competitive economic position. The cloning of a
biotin operon ( bioABFCD) on a multicopy plasmid allowed E. coli to produce 10000
times more biotin than did the wild-type strain.
• Riboflavin production in Corynebacterium moniagenes - was developed by cloning
and overexpressing the organism's own riboflavin biosynthesis genes and its own
promoter sequences.
• A novel process for vitamin C synthesis involved the use of a genetically
engineered Erwinia herbicola strain containing a gene from Corynebacterium sp.
 Improvement of microbial processes by GE
• Secondary metabolites
• Studies revealed that many antibiotic biosynthesis genes were arranged in
clusters.
• The entire cephamycin C pathway was cloned and expressed from
a cephamycin-producing strain of Streptomyces cattleya into
another cephamycin producer, Streptomyces lactamgens , a two- to
three-fold improvement was obtained.
• Microbial enzymes
• Genes encoding many microbial enzymes have been cloned and the enzymes
expressed at levels hundreds of times higher than those naturally produced.
• Scientists at Novo Nordisk isolated a very desirable lipase for use
in detergents from a species of Humicola.
• For production purposes, the gene was cloned into Aspergillus oryzae , where
it produced 1000-fold more enzyme and is now a commercial product.
• The α-amylase gene from Bacillus amyloliquefaciens was cloned
using multicopy plasmid pUB110 in B. subtilis
 Improvement of microbial processes by GE

Polymers, fuels, foods and beverages

• Recombinant DNA of Xanthomonas
manipulation
campestris increased titers of xanthan by two-fold.
• Alcohol dehydrogenase II and pyruvate decarboxylase genes
from Zymomonas mobilis were inserted in E. coli.
• Beer wort contains barley β-glucans which reduce the filtrability of
beer and lead to precipitates and haze in the final product. The
gene coding for endoglucanase was transferred from Trichoderma
reesei to brewer's yeast and the engineered yeast strain efficiently
hydrolyzed the β-glucans.
Bioconversions
• Recombinant Candida pasteurianum can carry out the conversion of
glycerol to 1,3-propanediol.