FERMENTATION BIOTECHNOLOGY
Biotechnology
The integrated use of biochemistry, microbiology and chemical engineering to
exploit plant materials and genetic resources for the production of specific
products and services
Fermentation
Fermentation is considered as 'any process for the production of a product by
means of mass culture of micro-organisms'.
MICRO-ORGANISMS
• Several species belonging to the following categories of micro-
organisms are used in fermentation processes:
• PROKARYOTIC
Unicellular : Bacteria, Cyanobacteria
Multicellular : Cyanobacteria
• EUKARYOTIC
Unicellular : Yeasts, algae
Multicellular : Fungi, algae
REQUIREMENTS FOR ARTIFICIAL CULTURE
Growth of Microorganisms under artificial culture requires Growth
(Nutrient) Medium
Microbial Growth is influenced by:
1) Kind and concentration of the ingredients of the medium.
2) pH
3) Temperature
4) Purity of the cultured organism
NUTRIENT SOURCES FOR INDUSTRIAL FERMENTATION
Carbon source
Glucose Corn sugar, Starch, Cellulose
Sucrose Sugarcane, Sugar beet molasses
Lactose Milk whey
Fats Vegetable oils
Hydrocarbons Petroleum fractions
Nitrogen source
Protein Soybean meal, Corn steep liquor, Distillers' soluble
Ammonia Pure ammonia or ammonium salts
Nitrate Nitrate salts
Phosphorous source Phosphate salts
PHASES OF MICROBIAL GROWTH
Lag phase
Period of adaptation
Log phase (Tophophase or growth phase in case of Fungi)
Rate of growth of the organism steadily increases.
Stationary phase (Idiophase or production phase in case of Fungi)
Growth ceases
FERMENTERS AND BIOREACTORS
A large growth vessel used to culture microorganisms on a large scale, frequently
for the production of some commercially valuable product
A bioreactor differs from a fermenter in that the former is used for the
mass culture of plant or animal cells, instead of micro-organisms.
Fermenters/bioreactors are equipped with an aerator to supply oxygen in
aerobic processes, a stirrer to keep the concentration of the medium
uniform, and a thermostat to regulate temperature, a pH detector and
similar control devices.
DESIGN OF INDUSTRIAL FERMENTATION PROCESS
• The fermentation process requires the following:
a) a pure culture of the chosen organism, in sufficient quantity and in the correct
physiological state;
b) sterilised, carefully composed medium for growth of the organism;
c) a seed fermenter, a mini-model of production fermenter to develop an inoculum to
initiate the process in the main fermenter;
d) a production fermenter, the functional large model; and
e) equipment for i) drawing the culture medium in steady state, ii) cell separation, iii)
collection of cell free supernatant, iv) product purification, and v) effluent treatment.
Items a) to c) above constitute the upstream and e) constitutes the downstream, of the
fermentation process
FERMENTORS
FERMENTOR
TYPES OF CULTURE SYSTEMS
BATCH CULTURE
Culture is stopped at stationary phase for recovery.
Advantage- optimum levels of product recovery.
Disadvantages- wastage of unused nutrients, the peaked input of labour
and the time lost between batches.
FED-BATCH CULTURE
A fresh aliquot of the medium is continuously or periodically added,
without the removal of the culture fluid.
A low but constantly replenished medium has the following advantages:
maintaining conditions in the culture within the aeration capacity of the
fermenter;
removing the repressive effects of medium components such as rapidly used
carbon and nitrogen sources and phosphate;
avoiding the toxic effects of a medium component; and
providing limiting level of a required nutrient for an auxotrophic strain.
CONTINUOUS CULTURE
Growth of the organism is controlled by the availability of growth limiting
chemical component of the medium
Continuous processing may suffer from contamination, both from within and
outside
Continuous culturing is highly selective and favours the propagation of the
best-adapted organism in culture
PRODUCTS OF FERMENTATION PROCESSES
Fermentation processes aim at one or more of the following:
Production of cells (biomass) such as yeasts;
Single cell protein
Extraction of metabolic products such amino acids, proteins (including enzymes),
vitamins, alcohol, etc., for human and/or animal consumption or industrial use such as
fertiliser production;
Primary metabolites
Secondary metabolites
Modification of compounds (through the mediation of elicitors or through
biotransformation); and
Production of recombinant products.
Genetically manipulated Escherichia coli, Saccharomyces cerevisiae, other
yeasts and even filamentous fungi are now being used to produce interferon,
insulin, human serum albumin, and several other products.
GENETIC IMPROVEMENT OF FERMENTATION PROCESSES
MUTATION
Exposing a culture of a micro-organism to UV light, ionising radiation
or certain chemicals
RECOMBINATION
Recombination is defined as any process that brings together genes
from different sources
DNA MANIPULATION
INDUSTRIAL FERMENTATION
Microbiology lab
Cell maintenance and storage
Preparation of master and working cell banks
Inoculum Development
Sterility and purity checking of fermenter samples
Fermentation
Media Preparation tanks
Seed Tanks
Fermentors
Dosing/Feed tanks
Online control – DCS system
Down Stream Processing
Centrifuges/Filters
solvent solvent extraction vessel and layer separators
Chromatographic columns
Dryers
Sieve's
QC/QA and Analytical Development Lab
Analysis of in process samples
Analysis of raw materials
Finished good analysis
Impurity profiling
Analytical method development
Microbiological testing of finished goods and process water.
Environmental monitoring of the plant
QA functions
Engineering
Supply of utilities like Steam, instrument air, cooling water, chilled water etc.
Maintenance works
Validation and calibration of equipments/instruments
Store/Warehouse
Storage of raw materials
Storage of finished goods
Storage of engineering spares
Storage of solvents and gas cylinders
Microbiology lab - Inoculum Development
Master cell bank
(Lyophil vials)
Working cell bank
(slants or glycerol stocks)
Slants or shake flask
Spore or vegetative cell
suspension as inoculum
Fermentation
Seed
Seed media preparation and sterilization.
Inoculation of seed tanks with laboratory inoculum aseptically.
Growing seed culture (15-48 hrs). Control of pH, air and agitation.
Sampling and microbiological testing
Production
Production media preparation and sterilization.
Inoculation of production fermenter with appropriately grown seed aseptically.
Running of fermentation under controlled conditions ( pH, dissolved oxygen, agitation
and feed rate).
Sampling and microbiological testing
Run time (1 day to 15 days)
Harvest
Down Stream Processing
Fermentation broth storage
Cell disruption- Sonication, Beads, French press, Microfluidizer ect.
Cell/cell debries separation- Press filters, RVFs, Nutch Filters, Centrifuges etc.
Purification of product by solvent solvent extraction or chromatography
Precipitation/Lyophilization
Drying
Packaging
OPERATIONAL DIAGRAM OF LARGE-SCALE FUNGAL
BATCH FERMENTATION SYSTEM
Preculture Preparation of Fermentation Recovery of enzyme-
inoculum containing medium
FERMENTATION PLANT OPERATIONS
Microbiology Laboratory
Quality Control/QA
Fermentation Production
Down Stream Processing/Recovery
Store/Warehouse
Effluent Treatment/Incineration
API
