Industrial Biotechnology

(BT3103)

Sanjeet Mehariya (PhD)

Department of Biosciences
School of Basic Sciences
Manipal University Jaipur
Jaipur-303007, India
Email: [email protected]
Syllabus
• Principles of fermentation technology: Screening and isolation of microorganisms,
maintenance of strains, strain improvement.
• Fermentation media: Natural and synthetic media, sterilization techniques.
• Construction of a Fermenter, Factors affecting Fermentation process.
• Types of fermenters: Solid state, submerged, continuous fermentation, immobilized
enzyme and cell bioreactors.
• Enzyme immobilization: Adsorption, Cross linking, Encapsulation, Entrapment, Covalent
Binding.
• Process of aeration, agitation, temperature regulation and foam control.
• Production of microbial products: Brief account of the following products obtained by
industrial microbiological fermentation, alcoholic beverage-beer, amino acids-glutamic
acid, enzyme-amylase and single cell protein (SCP).
• Biotechnology in specific medical & industrial applications: Retting of jute, microbial
process for immunization (Production of monoclonal antibodies).
 Maintenance of strains
• Maintaining strains, whether they are bacterial, fungal, or other
microorganisms' maintenance always differs.
• Bacteria
• Yeasts
• Fungi
• Microalgae

These microorganisms are integral to various biotechnological processes,

from industrial production and environmental management to
pharmaceuticals and food processing.
Their diverse capabilities make them valuable in the field of biotechnology,
therefor maintenance is very important for any industries.
 Maintenance of strains
• Different steps involves in the maintenance of various type of
industrially important microbes
1. Culture conditions (Temperature, pH, Medium, oxygen
requirements and others)
2. Storage
3. Subculturing
4. Contamination Control
5. Documentation
6. Quality Control
7. Reconstitution of Cultures
Maintenance of bacterial strains (Culture conditions)
• Maintaining bacterial strains effectively is crucial for research,
industrial applications, and diagnostics. Optimal conditions needs to
be ensured for their viability, purity, and performance:
1. Culture Conditions:
• Temperature: Most bacteria grow optimally at 37°C, but some strains
require different temperatures. Psychrophiles grow best at lower
temperatures, while thermophiles require higher temperatures.
• Psychrophiles
• Mesophiles
• Thermophiles
Maintenance of bacterial strains (Culture conditions)
• Psychrophiles
Psychrophiles, or cryophiles, are organisms that can grow and reproduce in
low temperatures, ranging from -20°C to 20°C. The optimal temperature for
growth in psychrophiles is around 15°C or lower, with a maximum
temperature of around 20°C. The minimum temperature for growth is 0°C or
lower.
Psychrophiles are extremophilic bacteria or archaea that have evolved to
thrive in cold conditions. They are found in places that are permanently cold,
such as the polar regions and the deep sea. The word "psychrophile" comes
from the Ancient Greek word, which means "cold, frozen".
• Mesophiles
These organisms are adapted to moderate temperatures, with optimal
growth temperatures ranging from room temperature (about 20°C) to about
45°C. Examples of mesophiles include normal human microbiota and
pathogens like E. coli, Salmonella spp., and Lactobacillus spp. (optimal
temperature for growth 37°C)
Maintenance of bacterial strains (Culture conditions)
• Thermophiles
Thermophiles are microorganisms that are adapted to high temperatures
and can grow between 45 and 90 °C, but their optimal growth temperature
depends on the type of thermophile:
• Moderate thermophiles: Grow best between 45–65 °C
• Extreme thermophiles: Grow best between 65–79 °C
• Hyperthermophiles: Grow best at temperatures above 80 °C
Thermophiles are found in geothermally heated regions like hot springs and
deep sea hydrothermal vents, as well as in decaying plant matter like peat
bogs and compost. They can survive at high temperatures where other
bacteria or archaea would be damaged or killed. Thermophiles also have
enzymes that function at high temperatures, and some of these enzymes are
used in molecular biology. For example, the Taq polymerase used in PCR is a
thermophile enzyme.
Maintenance of bacterial strains (Culture conditions)

Growth rate of bacteria as a function of temperature.

Maintenance of bacterial strains (Culture conditions)
• Medium: Use the appropriate medium based on the bacterial strain's
needs:
• General Purpose: Luria-Bertani (LB) broth/agar, nutrient broth/agar.
• Selective Media: Media with specific nutrients or inhibitors to select for or
against certain bacteria (e.g., MacConkey agar for Gram-negative bacteria).
• Enrichment Media: To enhance the growth of specific types of bacteria (e.g.,
blood agar).
• Oxygen Requirements:
• Aerobes: Require oxygen (e.g., Pseudomonas).
• Anaerobes: Grow without oxygen (e.g., Clostridium). Use anaerobic
chambers or sealed jars.
• Facultative Anaerobes: Can grow with or without oxygen (e.g., E. coli).
Maintenance of Fungal strains (Culture conditions)
• Fungi:
• Temperature: Most fungi grow well at temperatures between 25-30°C.
Some fungi require lower (psychrophiles) or higher (thermophiles)
temperatures.
• Medium: Use specific media based on fungal needs:
• General Purpose: Potato dextrose agar (PDA), Sabouraud dextrose agar
(SDA).
• Selective Media: Media with specific nutrients or inhibitors to select for or
against certain fungi (e.g., Sabouraud dextrose agar with antibiotics).
• Enrichment Media: Used to enhance the growth of specific types of fungi.

Humidity: Fungi often require high humidity for optimal growth, especially
when dealing with sporulation.
Maintenance of Yeasts strains (Culture conditions)
• Maintaining yeast strains involves specific practices to ensure their viability,
purity, and performance. Here's a detailed guide to help you effectively manage
yeast cultures:
Culture Conditions:
• Temperature: Yeasts generally grow well at temperatures between 25-30°C.
Some strains may have specific temperature requirements, so always check the
strain's optimal growth temperature.
• Medium: Use the appropriate growth medium based on the yeast strain’s needs:
• General Purpose: Yeast extract-peptone-dextrose (YPD) medium or Sabouraud dextrose
agar (SDA).
• Selective Media: Media with specific nutrients or inhibitors to select for or against certain
yeasts (e.g., YPD with antibiotics if necessary).
• Oxygen Levels: Yeasts are often facultative anaerobes, meaning they can grow
with or without oxygen. However, some yeasts may have specific requirements:
• Aerobic Conditions: Generally favorable for yeast growth and for processes like
fermentation.
• Anaerobic Conditions: Required for specific strains, especially those used in fermentation
processes.
Maintenance of microalgal strains (Culture
conditions)
Maintaining microalgal strains requires specific techniques to ensure their
health, viability, and genetic stability. Microalgae are used in a range of
applications from biofuel production to environmental monitoring, so
proper maintenance is crucial. Here’s a comprehensive guide for
maintaining microalgal strains:
• Light: Microalgae typically require light for photosynthesis. The light
intensity and duration depend on the species. Generally, a light cycle of
12-16 hours of light and 8-12 hours of darkness is used.
• Temperature: Most microalgae grow well at temperatures between 20-
30°C. Some species may have more specific temperature requirements,
so check the strain’s optimal conditions.
 Maintenance of microalgal strains (Culture
conditions)
• Medium: Use appropriate growth media based on the species’ needs:
• General Purpose: Bold's Basal Medium (BBM), F/2 medium, or other suitable
nutrient-rich media.
• Selective Media: For specific strains or to enhance certain growth conditions
(e.g., media with specific nutrients or inhibitors).
• pH: Maintain the pH at a level suitable for the microalgae strain. Many
microalgae thrive at pH values between 6-8, but this can vary.
• Carbon Source: Some microalgae may need additional carbon sources
like CO2 or bicarbonates to support growth, especially in closed
systems.
 Maintenance of strains (Storage)
2. Storage:
• Storage of microbes is crucial for preserving their viability and genetic
integrity over time. Different types of microbes—bacteria, fungi, yeasts,
algae, and archaea—may require specific storage methods due to their
unique characteristics. Here’s a comprehensive guide to the various
methods used for storing different types of microbes:
• 1. Bacteria:
• Short-term Storage:
• Refrigeration: Store bacterial cultures at 4°C for short-term needs. This method is
suitable for up to a few weeks.
• Long-term Storage:
• Cryopreservation: Mix bacterial cultures with a cryoprotectant (e.g., 15-20%
glycerol or dimethyl sulfoxide) and store in cryovials at -80°C or in liquid nitrogen (-
196°C). This method preserves cultures for several years.
• Lyophilization (Freeze-Drying): Freeze-dry the bacterial culture to remove
moisture, allowing for long-term storage. The lyophilized bacteria are stable at room
temperature.
 Maintenance of strains (Storage)
• 2. Fungi:
• Short-term Storage:
• Refrigeration: Store fungal cultures at 4°C for short-term maintenance. This slows down growth
without killing the fungi.
• Long-term Storage:
• Cryopreservation: Mix fungal cultures with a cryoprotectant (e.g., glycerol or dimethyl
sulfoxide) and store at -80°C or in liquid nitrogen. This method ensures viability for several years.
• Lyophilization (Freeze-Drying): A method used for long-term preservation by removing moisture.
Freeze-dried fungal cultures can be stored at room temperature.
• 3. Yeasts:
• Short-term Storage:
• Refrigeration: Store yeast cultures at 4°C for up to a few weeks. This helps slow down their
metabolism and extend viability.
• Long-term Storage:
• Cryopreservation: Mix yeast cultures with a cryoprotectant (e.g., 15-20% glycerol) and store at -
80°C or in liquid nitrogen. This preserves yeast strains for long-term use.
• Lyophilization (Freeze-Drying): Yeast cultures can also be freeze-dried for long-term storage. The
lyophilized yeast is stable at room temperature.
 Maintenance of strains (Storage)
• 4. Microalgae:
• Short-term Storage:
• Refrigeration: Store microalgal cultures at 4°C for short-term maintenance. This slows down
growth and metabolism without killing the algae.
• Long-term Storage:
• Cryopreservation: Mix microalgal cultures with a cryoprotectant (e.g., glycerol or dimethyl
sulfoxide) and store in cryovials at -80°C or in liquid nitrogen. This ensures long-term preservation.
• Lyophilization (Freeze-Drying): A less common method for microalgae but can be used to
preserve cultures for extended periods. The process involves removing moisture from the culture.
• 5. Archaea:
• Short-term Storage:
• Refrigeration: Store archaeal cultures at their optimal growth temperature for short-term
maintenance. For some extremophiles, this may involve storing at high temperatures.
• Long-term Storage:
• Cryopreservation: Mix archaeal cultures with a cryoprotectant (e.g., glycerol) and store at -80°C
or in liquid nitrogen. This method preserves the strains for long-term storage.
• Lyophilization (Freeze-Drying): Less commonly used but effective for preserving extremophiles
by removing moisture.
 Maintenance of strains (Storage)
• General Considerations for All Microbes:
• Cryoprotectants: Use appropriate cryoprotectants to prevent ice
crystal formation during freezing, which can damage microbial cells.
Common cryoprotectants include glycerol and dimethyl sulfoxide
(DMSO).
• Container Types: Use appropriate storage containers such as
cryovials for freezing and airtight containers for lyophilized samples.
• Labeling and Documentation: Clearly label all storage containers
with relevant information such as strain name, date, storage
conditions, and any additional details. Maintain comprehensive
records of storage practices for accurate tracking and retrieval.
 Maintenance of strains
3. Subculturing:
• Regularly subculture strains to prevent them from becoming
overgrown or mutated.
• This involves transferring a small amount of the culture to fresh
medium to renew growth.
• Follow good aseptic techniques to prevent contamination.
4. Contamination Control:
• Always work in a sterile environment using sterile equipment.
• Regularly inspect cultures for signs of contamination (e.g., unusual
growth patterns, discoloration).
 Maintenance of strains
5. Documentation:
• Keep detailed records of strain source, growth conditions, subculturing
dates, and any observations related to the strain’s behavior or health.
6. Quality Control:
• Regularly check the identity and purity of the strain, especially if it’s used
for critical applications. Techniques such as PCR, microscopy, and
biochemical tests can be useful.
7. Reconstitution of Cultures:
• When reviving a stored strain, use proper techniques to reconstitute it. For
cryopreserved strains, gradually thaw and transfer to fresh growth medium
under sterile conditions.
• By following these practices, you can maintain the viability and purity of
your strains for ongoing research or industrial applications.
 Strain improvement
• Strain improvement is one element of fermentation process management. It is the process of
increasing the productivity of a microorganism by improving or selecting for a more productive
phenotype. In other words, it is the act of making a strain of microorganisms produce more of what we
want, such as metabolites or enzymes.
Ideal Characteristics of Strain
• Rapid growth
• Genetic stability
• Non-toxicity to humans
• Ability to use cheaper substrates
• Elimination of the production of compounds that may interfere with downstream processing
• To improve the use of carbon and nitrogen sources.
• Reduction of cultivation cost
• Shorter fermentation time.
 Purpose of Strain Improvement
• Increase the productivities

• Regulating the activity of the enzymes

• Increasing the permeability

• To change un-used co-metabolites

• Introducing new genetic properties into the organism

 Methods of Strain Improvement
• Mutation
• Selection
• Growth selection
• Hybridisation
• Genetic engineering
• Metabolic engineering
 Strain improvement
• Mutation
• The mutation is one method of strain
improvement. Mutation is the process of
randomly changing the genetic code of a
cell. This can cause new genes to be
formed or can change the activity of
existing genes. Mutation can happen
spontaneously or can be induced by
radiation or chemicals. It can also be
passed from one generation to the next.
• Many times, the mutation will not have
any effect on the cell. However,
occasionally a mutation will improve the
cell in some way. For example, it may
make it grow faster or be more resistant to
disease.
 Strain improvement
• Selection
Selection is another method of strain improvement. Selection is the process of
choosing cells or organisms that have certain desirable traits and then breeding them
together.
The advantage of selection is that it can be very precise. It can target specific genes or
traits that are desired. This can result in strains that are very robust and have many
desirable qualities.
Mutation and selection are two of the most common methods of strain improvement.
However, there are many others. Some of these include:
• Growth selection
This is a method where the best-growing cells are selected for breeding. This can be
done by picking the cells that grow the fastest or the largest. This can also be done by
selecting cells that have a high yield.
 Strain improvement
• Hybridisation
• This is the process of mating two different strains together to create a new strain.

• The advantage of hybridisation is that it can create strains that are very robust and
have many desirable qualities. However, it can also be unpredictable. It is not always
possible to predict the outcome of a cross between two strains.
• Genetic engineering
• Genetic engineering is a method where genes from one organism are inserted into
another organism.
• The advantage of genetic engineering is that it allows you to select specific genes
that you want to improve the strain. You can also insert genes from other organisms
that have desirable qualities. This can result in strains that are very robust and have
many desirable qualities.
 Strain improvement
• Genetic engineering: This
approach involves manipulating the
genetic sequence of a
microorganism to improve its
productivity. One common strategy
is to insert genes from other
organisms that encode enzymes
that catalyze the production of the
desired product.
 Strain improvement
• Metabolic engineering: This approach involves altering the metabolic pathways of a
microorganism to redirect its resources towards the production of the desired product.
• One common strategy is to knock out genes that are not essential for product
formation and replace them with genes from other organisms that encode enzymes
that catalyze the production of the desired product.
•
 Microbial culture collections in India
• Microbial Culture Collection (MCC): Located in Pune, this center is part of the National
Centre for Cell Science (NCCS) on the Savitribai Phule Pune University Campus.

• Microbial Type Culture Collection (MTCC): Located in Chandigarh at the Institute of

Microbial Technology (IMTECH), this center was established in 1986 and is funded by the
Council of Scientific and Industrial Research (CSIR) and the Department of Biotechnology
(DBT). The MTCC's goals include acting as a depository for microbial cultures, supplying
cultures to research institutions, universities, and industries, and conducting research on
microbial diversity and taxonomy.

• National Fungal Culture Collection of India (NFCCI): Located in Pune.

 Microbial culture collections in India
• National Collection of Industrial Microorganisms (NCIM): Located in Pune, this center is
part of the National Chemical Laboratory's Biochemical Science Division.

• National Bureau of Microorganisms: Agriculturally important, Mau (U.P.) India

• National Collection of Dairy Cultures: Located in Karnal, this center is agriculturally

important.
 Microbial culture collections
Microbial Collections
Agricultural Research Service USDA
ALGAE a world catalogue of algal collections
ATCC American Type Culture Collection, USA
BCCM Belgian Co-ordinated Collections of Microorganisms
BGSC Bacillus Genetic Stock Center
BCC BIOTEC Culture Collection, Thailand
CABRI CABRI (Common Access to Biological Resources and Information) by EC
CBS Centraalbureau voor Schimmelcultures
CCAP The Culture Collection of Algae and Protozoa, UK
CCUG University of Goteborg, Sweden (>38000 strains)
CGSC E.coli Genetic Stock Center
Developmental, Cell and Molecular Biology Group Duke University
DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
 Microbial culture collections
Microbial Collections
Fungal Genetics at U. Texas Houston Medical School
MA (Real Jardin Botanico, Madrid) cryptogamic collections
HAMBI University of Helsinki, Finland
IEGM Institute of Ecology and Genetics of Microorganisms
IMI CABI Bioscience Genetic Resource Collection
INVAM The International Culture Collection of Arbuscular and VA Mycorrhizal Fungi
JCM Japan Collection of Microorganisms
JSCC Japan Society for Culture Collections on-line database
MGD Microbial Germplasm Database
Matsushima Mycological Memoirs No.8
Microbial Information Network of China
MICH University of Michigan Fungus Collection
MSDN Microbial Strain Data Network mirrored in Japan
NCCB
 Microbial culture collections
Microbial Collections
NCIMB
NCTC National Collection of Type Cultures (U.K.)
NCPF National Collection of Pathogenic Fungi (U.K.)
NCPV National Collection of Pathogenic Viruses (U.K.)
Quinone Database
RDPII (Ribosomal Database Project II)
The Chlamydomonas Genetics Center
TISTR
UAMH
UKNCC
UNSW University of New South Wales
VKM All Russian Collection of Microorganisms
World Phytophthora Collection, University of California Riverside (US)
HBMMD HARBOR BRANCH MARINE MICROBE DATABASE
Thanks for Today !!