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