1. FERMENTATION TECHNOLOGY

2. CONTENTS Fermentation technology: 1. Definition
2. Principle of fermentation
3. Types of fermentation
4. Surface culture
5. Solid state fermentations,
6. Microbial transformation,
7. Preparation of
Alcohol
Penicillin,
Streptomycin and
Riboflavin.

3. What is Fermentation?

5. Some important fermentation products
Organism
Use
Ethanol
Saccharomyces cerevisiae
Industrial solvents, beverages
Glycerol
Production of explosives
Lactic acid
Lactobacillus bulgaricus
Food and pharmaceutical
Acetone and butanol
Clostridium acetobutylicum
Solvents
-amylase
Bacillus subtilis
Starch hydrolysis

6. Industrial fermentation processes may be divided into two main types, with various combinations and modifications. These are
1. Batch fermentations and
2. Continuous fermentations.
Batch fermentations
A tank of fermenter is filled with the prepared mash of raw mate­rials to be fermented. The temperature and pH for microbial fermen­tation is properly adjusted, and occasionally nutritive supplements are added to the prepared mash. The mash is steam-sterilized in a pure culture process. The inoculum of a pure culture is added to the fermenter, from a separate pure culture vessel. Fermentation proceeds, and after the proper time the contents of the fermenter, are taken out for further processing. The fermenter is cleaned and the process is repeated. Thus each fermentation is a discontinuous process divided into batches.

7. Continuous fermentation
Growth of microorganisms during batch fermentation confirms to the characteristic growth curve, with a lag phase followed by a loga­rithmic phase. This, in turn, is terminated by progressive decrements in the rate of growth until the stationary phase is reached. This is because of limitation of one or more of the essential nutrients. In continuous fermentation, the substrate is added to the fermneter continously at a fixed rate. This maintains the organisms in the logari­thmic growth phase. The fermentation products are taken out conti­nuously. The design and arrangements for continuous fermentation are somewhat complex.
FED-BATCH FERMENTATION
Substrate increments as the fermentation progresses.
started as batchwise with a small substrate concentration.
Initial substrate is consumed, addition of fermentation medium

8. Aerobic fermentations
A number of industrial processes, although called 'fermentations',
Fig. 1. Aerobic fermenter.
are carried on by microorganisms under aerobic conditions. In older aerobic processes it was necessary to furnish a large surface area by exposing fermentation media to air. In modern fermentation processes aerobic conditions are maintained in a closed fermenter with submerged cultures. The contents of the fermenter are agitated with au impeller and aerated by forcing sterilized air (Fig 1).

9. Anaerobic fermentations
Basically a fermenter designed to operate under micro-aerophilic or anaerobic conditions will be the same as that designed to operate under aerobic conditions, except that arrangements for intense agitation and aeration are unnecessary. Many anaerobic fermentations do, how­ever, require mild aeration for the initial growth phase, and sufficient N agitation for mixing and maintenance of temperature.

10. Bacterial growth

11. lag phase (adapt to their surroundings)
exponential growth (grow in numbers)
stationary phase (stop growing)
death phase
During lag phase, bacteria adapt themselves to growth conditions. It is the period where the individual bacteria are maturing and not yet able to divide. During the lag phase of the bacterial growth cycle, synthesis of RNA, enzymes and other molecules occurs. The log phase (sometimes called the logarithmic phase or the exponential phase) is a period characterized by cell doubling.[3] The number of new bacteria appearing per unit time is proportional to the present population. If growth is not limited, doubling will continue at a constant rate so both the number of cells and the rate of population increase doubles with each consecutive time period. The stationary phase is often due to a growth-limiting factor such as the depletion of an essential nutrient, and/or the formation of an inhibitory product such as an organic acid. Stationary phase results from a situation in which growth rate and death rate are equal. The number of new cells created is limited by the growth factor and as a result the rate of cell growth matches the rate of cell death. The result is a “smooth,” horizontal linear part of the curve during the stationary phase. At death phase,(Decline phase) bacteria run out of nutrients and die.

13. Fermentation techniques
surface (solid state) submersion techniques.
microorganisms cultivated on the surface of a liquid or solid substrate.
complicated and rarely used in industry.
Mushroom, bread, cocoa, tempeh
microorganisms grow in a liquid medium.
(biomass, protein, antibiotics, enzymes and sewage treatment) are carried out by submersion processes.

15. Industrial fermenters
Can hold up to litres of culture
Cells are provided with nutrients and very carefully controlled environment to keep them in desired growth stage
Usually made out of stainless steel as many fermentations produce acid.
Nutrients and other materials are fed in by valve operated pipelines.

16. Industrial fermenters

17. Industrial fermenters
Conditions in the fermenter are carefully monitored to regulate cell growth.
Fermenter and all pipework must be sterile before fermentation begins
This is usually achieved by flushing the whole system with superheated steam before the production begins.

18. Industrial fermenters

19. Industrial fermenters
Interior is monitored by sterilisable probes which record temp., pressure, stirrer speed, pH, oxygen and carbon dioxide levels.
These are all recorded and electronic control systems with automatic valves will regulate them.
E.g. if medium becomes too acidic, bases can be added from a reservoir to correct the pH

20. Industrial fermenters
A fermenter with a gallon capacity

21. Industrial fermenters
The production of microbial products is called fermentation but it is not fermentation in the defined sense of the word.
Process if frequently aerobic so ferementer has to be well aerated.
Incoming air is filtered and pumped into the base of the fermenter – a valve releases the pressure from the top f the tank.

22. Industrial fermenters

23. Industrial fermenters
The aeration will be sufficient to mix many cultures
If the culture is thick or sticky additional stirring is required by a motor driven paddle called an impeller.
While initially the culture may need warming to start of the process – once it has started a cooling system is vital.

24. Industrial fermenters
Control panel for an industrial fermentation operation

25. Industrial fermenters
An antibiotic producing fermentation may use a tonne of sugar a day.
The organisms are likely to raise the temperature of the culture by more than 10C per hour ; more heat will come from the activity of the impeller.
This rise in temperature could quickly kill the microbes if not cooled.
Cooling is achieved by either a water jacket or cooling coils inside the fermenter.

26. Industrial fermenters

27. Industrial fermenters
Fermenters in an American winery

29. Selection of microbes
Screening to find the right microorganism out of many species which can produce potentially valuable product.
Both naturally occurring and genetic variant.

30. Fermentation medium Downstream processing Sl. no. Nutrient
Raw Material 1.
C - source
Corn sugar, molasses, starch, glucose 2.
N - source
Soyabean meal, corn steep liquor, yeast extract 3.
Fat and hydrocarbons
Vegetable oil, petroleum fractions 4.
Ammonia
Pure ammonia or ammonium salts 5.
Nitrate
Nitrate salts 6.
Nitrogen
Air (from nitrogen fixing bacteria) 7.
P - source
Phosphate salts
Downstream processing

31. Precursors improve the yield or quality of products that are incorporated without any major change in the product. E.g., phenyl acetic acid for penicillin, cobalt for vitamin B 12.
Inducers: The majority of the enzymes used in industrial fermentation are inducible and are synthesized in response of inducers: e.g. starch for amylases, maltose for pollulanase, pectin for pectinase.
Chelators: Chelators are the chemicals used to avoid the precipitation of metal ions. Chelators like EDTA, citric acid, polyphosphates are used in low concentrations.
Antifoaming agents(alcohol anf fatty acids)
Phosphates as buffers
Sometimes growth factors and calcium carbonate

32. There are 5 major groups of commercially important fermentation:
1. Microbial cells or biomass as the product, e.g. single cell protein, bakers yeast, lactobacillus, E. coli, etc.
2. Microbial enzymes: catalase, amylase, protease, pectinase, glucose isomerase, cellulase, hemicellulase, lipase, lactase, streptokinase, etc.
3. Microbial metabolites :
Primary metabolites – ethanol, citric acid, glutamic acid, lysine, vitamins, polysaccharides etc.
Secondary metabolites: all antibiotic fermentation
4. Recombinant products: insulin, hepatitis B vaccine, interferon, granulocyte colony-stimulating factor, streptokinase
5. Biotransformations: phenylacetylcarbinol, steroid biotransformation, etc.

33. Penicillin

34. Streptomycin Soybean meal Glucose NaCl 28C pH 7.6-8 10 days 3phases
Rapid growth of microbe
Streptomycin production
C depletion

35. riboflavin pH 4.5 26-28C 96 to 120 hrs Submerged aerated fermentation
Yeild 500 to 600 microgram/ml

36. Alcohol