large scale microbial fermentation, problems of large scale microbial fermentation, microbial fermentation.

1. 1
Large scale microbial fermentation
principle and its problems
Numair Ahmad
24
Indus International Institute

2. 2
What is fermentation?
• Pasteur’s definition: “life without air”, anaerobe
red ox reactions in organisms
• New definition: a form of metabolism in which the
end products could be further oxidized
For example: a yeast cell obtains 2 molecules of
ATP per molecule of glucose when it ferments it
to ethanol

3. 3
What is fermentation techniques (1)?
Techniques for large-scale production of microbial products.
It must both provide an optimum environment for the
microbial synthesis of the desired product and be
economically feasible on a large scale. They can be divided
into surface (emersion) and submersion techniques. The latter
may be run in batch, fed batch, continuous reactors
In the surface techniques, the microorganisms are cultivated
on the surface of a liquid or solid substrate. These techniques
are very complicated and rarely used in industry

4. 4
What is fermentation techniques (2)?
In the submersion processes, the microorganisms grow in a
liquid medium. Except in traditional beer and wine
fermentation, the medium is held in fermenters and stirred to
obtain a homogeneous distribution of cells and medium. Most
processes are aerobic, and for these the medium must be
vigorously aerated. All important industrial processes
(production of biomass and protein, antibiotics, enzymes and
sewage treatment) are carried out by submersion processes.

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

6. 6
Some important fermentation products

7. 7
Some important fermentation products

8. 8
Winemaking fermenter

9. 9
General Aspects of Fermentation
Processes

10. 10
Fermenter and its types
The heart of the fermentation process is the fermenter.
Simple fermenters (batch and continuous)
Fed batch fermenter
Air-lift or bubble fermenter
Cyclone column fermenter
Tower fermenter
Other more advanced systems, etc
The size is few liters (laboratory use) - >500 m3
(industrial applications)

11. 11
Cross section of a fermenter for Penicillin production

12. 12
In the manufacture of penicillin,
fermentation is the preferred route. The
fermentation substrate consists mainly of the
sucrose found in corn steep liquor and
lactose together with minerals and
phenylacetic acid. The preferred mold is
Penicillium chrysogenum and the first stage
of the process is the transfer of the mold to
the fermenter from the test tube where it has
been stored.

13. 13
The whole process is aerobic (it
requires air). The fermentation is
carried out over 4 or 5 days at 25-
27°C. This gives a yield of about 0.5%
penicillin. The mycelium, the mold
cells plus insoluble metabolites, is
filtered off in a coated drum filter.

14. 14
The filtrate is adjusted the pH 2
with sulphuric acid. At this pH, the
penicillin exists as an undissociated
acid; consequently, it is soluble in
organic solvents. After extraction
the solution is decolourised and
impurities are removed by activated
carbon in a second drum filter.

15. 15
The penicillin is then precipitated
by addition of a solution of
potassium acetate. This converts
the penicillin into a negative ion,
which makes it insoluble in the
organic solvent. Inn the final steps
the penicillin is crystallized and
dried.

16. 16
Flow sheet of a multipurpose fermenter and its
auxiliary equipment

17. 17
Fermentation medium
• Define medium  nutritional, hormonal, and
substratum requirement of cells
• In most cases, the medium is independent of the
bioreactor design and process parameters
• The type: complex and synthetic medium (mineral
medium)
• Even small modifications in the medium could
change cell line stability, product quality, yield,
operational parameters, and downstream processing.

18. 18
Medium composition
Fermentation medium consists of:
• Macronutrients (C, H, N, S, P, Mg sources  water,
sugars, lipid, amino acids, salt minerals)
• Micronutrients (trace elements/ metals, vitamins)
• Additional factors: growth factors, attachment
proteins, transport proteins, etc)
For aerobic culture, oxygen is sparged

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Inoculums
Incoculum is the substance/ cell culture that is
introduced to the medium. The cell then grow in the
medium, conducting metabolisms.
Inoculum is prepared for the inoculation before the
fermentation starts.
It needs to be optimized for better performance:
• Adaptation in the medium
• Mutation (DNA recombinant, radiation, chemical
addition)

20. Problems faced in
microbial fermentation
20

21. Bacterial contamination
21

22. 22
This is the most common type of
contaminant in bacterial cultures.
Depending on the type of bacterium
contaminating the process, the effects
brought about are different. In all cases,
one may expect reduced yield of product,
and possible contamination by
metabolites of contaminating bacteria. In
some cases toxins may be produced by
contaminants.

23. 23
The probability of occurrence of a particular
type of contaminations caused by bacteria
may differ, depending on the process itself
and the organism used in the process. One of
the most frequently occurring contaminants
are lactic acid bacteria and sporulating
bacteria. Clean-up and re-occurrence
prevention of especially the latter presents a
considerable challenge.

24. Virus contamination
24

25. 25
Viruses are the most dangerous contaminant
in this type of fermentation. Due to small
sizes, they are the toughest enemy to prevent
entering the process, and their occurrence
may result in very long, lasting even several
months, paralysis of whole facility
productivity. Due to the fact that eukaryotic
cell cultures are usually used for production
of highly valuable products, a risk of
running fermentation without optimal
protection is something what can cost you a
lot.

26. Fungal infection
26

27. 27
Fungal cultures can be contaminated
also by other fungi. As this type of
contamination may be relatively hard
to detect, good measures of precaution
should be undertaken to prevent it.

28. Dairy contamination
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29. 29
In dairy industry, controlling contamination
issues is very important, not only due to
possible health hazards. Milk is a raw
material, which cannot always be sterilized,
and almost every batch contains
bacteriophages infectious to bacteria used in
production process.

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Moreover, some phages attacking
lactic acid bacteria are resistant to
pasteurization, and thus, they are
much harder to eliminate. As it is not
always possible to prevent
bacteriophages from entering
production process, there is a
possibility to prevent re-infections,
which are the ones to blame for vast
majority of production failures.