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'.

FERMENTATION BIOTECHNOLOGY

MICRO-ORGANISMS

• Several species belonging to the following categories of micro-organisms are used in fermentation processes:

• PROKARYOTICUnicellular :   Bacteria, CyanobacteriaMulticellular :   Cyanobacteria

• EUKARYOTIC  Unicellular :   Yeasts, algaeMulticellular :   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 sourceProtein                                    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 metabolitesSecondary 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)

Spore or vegetative cell suspension as inoculum

Slants or shake flask

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

Fermentation Production

Down Stream Processing/Recovery

Quality Control/QA

Store/Warehouse

Effluent Treatment/Incineration

API