Biotechnology

Lecture 2

Dr. Eman Farouk

 Fermentation processes
• Fermentation is an industrial process utilizing living cells for the
production of commercially valuable products either aerobically or
anaerobically.

• In fermentation, living cells are allowed to grow under defined

conditions in a fermenter (bioreactor). The defined conditions
include the use of the proper substrate (medium) and the proper
environmental parameters (e.g. temperature, agitation, pH and
aeration).

• All must be optimized to achieve the highest yield and quality at the
lowest cost possible.
 Bioreactors
• Bioreactors vary from very simple vessel with few controls to highly
complicated ones in which the whole process is under computer
control. Before construction of a fermenter, it must be decided
whether the fermenter is to be used for a special process with certain
organism or for a variety of processes with different microorganisms.

• Simple stirred, aerated fermenters are the ones that are usually used
in industry especially in the pharmaceutical industry. Different
products can be produced in the same apparatus with some
modification.
Bioreactors
• Small fermenter vessels up to 20 liters are usually made of glass,
however, Larger volume vessels are usually made of stainless steel. It
is necessary that fermenter and substrate solutions be sterilized
(mostly inside the fermenter), before the addition of the inoculum
(microbial strain). Small fermenters are sterilized in autoclave while
large fermenters are sterilized by steam generated from distant boiler.

• In aerobic fermentations it is important to achieve the optimal oxygen

concentration.

• It is supplied from compressed, filtered air to the bioreactor and

forced through a sparger to facilitate the dispersion of oxygen to
liquid medium.
• The medium as well as air bubbles are mixed by impellers arranged
on an agitator shaft attached to bottom or top sealed motor. Careful
design is required to achieve optimal oxygen concentration
throughout the fermentation medium since oxygen transfer in large
volume fermenters is difficult.

• Large fermenters are also supplied with internal baffles that help in
mixing. Aeration and mixing are relatively expensive due to the high-
energy costs needed.

• Fermenters may be supplied with pumps to supply acid or alkali in

order to adjust the pH during the fermentation course. Also pumps
to supply nutrients or antifoams may be also required.
Bioreactor
Methods of fermentation:
1. Batch fermentation: the fermenter containing the sterilized culture
medium is inoculated with the microorganism and incubation is
allowed to proceed under optimal conditions for the required period of
time. In batch fermentation, nothing is added to or removed from the
fermenter during the entire fermentation process except air, an
antifoam agent, acid or alkali to control the pH. At the end of the
fermentation cycle, the fermenter is shut off and the contents are
collected and the product is recovered (closed system).

2. Fed-batch process: nutrients are added at intervals as the

fermentation progresses (open system).
3. Continuous Fermentation: sterile nutrients are added continuously
to the fermenter and equivalent amount of product with
microorganism are simultaneously harvested out of the fermenter
(open system).
Scale-up:
• It is the transfer of small scale laboratory fermentation to an
industrial large scale.
• Fermentation processes are usually developed in three stages namely
laboratory scale (flasks, laboratory fermenters), pilot plant scale
(usually 50-200 liters) and production scale (usually in cubic meter
scale depending on the product).
• It should be noted that laboratory-scale process may not work or
work poorly when first attempted at large-scale. The aim of the
scientists carrying out this development stages is to find out the
optimal fermentation conditions required to maintain the highest
product yield.
 Uses of computers in fermentation technology
• Data including pH, O2 , temperature, pressure,
viscosity aeration rate, exhausted O2 and CO2
could be directly fed into computers through
sensors and probes.
• The whole fermentation process could be done
automatically through special programs, which
also alarm when any deviation from the set-up to
inform the production team and sometimes
correct it or stop the fermentation process.
• The data obtained during the fermentation
process can be stored, analyzed and used for
further optimizing the process and can be
compared with other batches.
 Categories of products:
1. Biomass: The product may be the cells themselves e.g. baker’s yeast or single cell
protein (SCP).
2. Biotransformation product.
3. Energy: alcohol, methane (biogas).
4. Intra or extracellular accumulation of metals.
5. Food: preservation and processing.
6. Biodegradation product: degradation of xenobiotics (insecticides and petroleum oil).
7. Enzyme: e.g. amylase, penicillin acylase.
8. Metabolites: either primary metabolite such as citric acid which are usually
produced during the logarithmic phase of growth (trophophase) or secondary
metabolites such as antibiotics, alkaloids or glycosides which are produced during
the stationary phase (idiophase).
9. Immunological product: vaccines and monoclonal antibodies (MCA).
10. Genetically engineered therapeutic protein: Insulin, growth hormone and interferon.
11. Plant tissue culture.
 I. Microbial transformation
Microbial transformation or biotransformation is the ability of m.o. to
modify a wide variety of organic compounds to produce useful products.
 Microbial transformation is a very helpful tool for:

1. Production of more useful & sensitive products.

2. Maintains the original carbon skeleton after obtaining the product.

• These transformations can be oxidation, reduction, hydrolysis,

isomerization, condensation, formation of new carbon bonds and
introduction of functional groups.

• Biotransformation applies for steroids, antibiotics, hydrocarbons &

terpenes. The transformation reaction is considered to be a
detoxification reaction by the microorganism using induced
enzymes.
Biocatalysts used in biotransformation:
A variety of substances such as growing cultures, immobilized cells and enzymes may be
involved in microbial biotransformation.
A- Growing cultures:
Biotransformations can be catalysed by growing cultures, as cells are grown on a suitable
liquid medium in a shake-flask, and after a period of growth, the substrate for
transformation is added directly to the cell culture.
B- Isolated enzymes:
In this case, the enzyme required for biotranformation is liberated from the cells by
cell disruption and then purified to the required degree using a range of
chromatographic technique.
C- Immobilized cells:
Immobilization of cells refers to the technique of anchoring the cells on an inert
support for their stability and functional reuse. By employing this technique, cells
are made more efficient and cost-effective for their industrial use.

 A number of transformation processes employ immobilized cells, offering the

advantage that the process can be carried out continuously and the cells can be
used over and over again. Immobilized bacterial cells, which catalyse one-stage
or multi-stage reactions, are presently used commercially in the production of
aspartic acid, L-alanine and malic acid.
 Steroid biotransformation
• Steroids are group of lipid compounds having the same basic
cyclopentano-phenanthrene nucleus (4-fused rings).
• Steroids such as estrogen, progesterone and androgens are used as therapeutics.
Progesterone and estrogens derivatives are used as contraceptives. cortisone
derivatives as anti-inflammatory agents in rheumatoid arthritis.

• One of the major biotechnological aspects in this area is the application of a

wide range of the microorganisms including bacteria and fungi in converting
steroid substances into the pharmacologically active compounds or other useful
intermediates.

• Actinomycetes including Rhodococcus, Mycobacterium, Corynebacterium and

Nocardia species, have become widely recognized as bacteria able to rapidly
degrade sterols, producing steroid pathway intermediates that find application
as precursors in drug synthesis.

• Aspergillus and Rhizopus nigricans are example of fungi used in

biotransformation of steroids.
 • Low cost sterols of animal origin such as cholesterol & of plant origin
such as diosgenin are converted chemically to progesterone which in
turn is used as a substrate for biotransformation.

Cortisones:
By Rhizopus nigricans (bread mold) and stereo-specific hydroxylation (several step
synthesis), over 800 tons/year of major corticosteroids are produced.
Corynebacterium simplex is used to transform cortisol (hydrocortisone) to
prednisolone by incorporatign 1,2 double bond in ring A of the cortisol
molecule to produce prednisone which have the property of markedly
increased anti inflammatory effect.
• Steroids can be produced by chemical synthesis, but the process is
laborious & expensive.

• As example, chemical conversion of deoxycholic acid (bile acid) to

cortisone required 37 steps & yielded 1 g cortisone from 615 g
deoxycholic acid.

• The price of 1 g cortisone was reduced from 200$ to only one $ or

less when the process was applied by Rhizopus nigricans.
Steps of the fermentation process:
1. Microorganism is allowed to grow for about 17-48 h under controlled
conditions (pH, oxygen, temp.).

2. The steroid substrate is added in suitable solvent.

3. Conditions are readjusted for steroid transformation.

4. Product recovery.

5. Removal of the mycelium by filtration or centrifugation.

6. Extraction of the steroid product with organic solvent & crystallization

of the product.
 Steroid transformation by fungal spores:

Steroid transformation by fungal spores provides the following advantages:

1. Simple medium.
2. No fear of contamination.
3. After transformation spores are separated & recycled.
4. Ease of product recovery.
5. Cost is much reduced.
Fungal spores are often as active as vegetative cells in transformation of steroids. The
microorganism is allowed to grow in presence of the steroid as inducer for long period
to produce enough spores. Spores are then collected in buffer, purified & freed from
any vegetative debris. Spores are then mixed with the steroid substrate in sugar
medium & transformation is carried out as under vegetative cells.
Transformation of non-steroidal compounds:
• Gluconobacter melanogenus is used to transform glycerol to dihydroxyacetone
which is used in lotions and cosmetics.
• Cryptococcus neoformans is used to transform unsaturated fatty acids to
prostaglandins which have wide medical applications as contraceptives, pain
alleviators, treatment of cardiovascular and digestive disorders.
Bioransformation of antibiotics:
• The microbial transformation of existing antibiotics has been done with the
objective of developing new, modified and improved antibiotics which contain
many qualities like reduced toxicity, broad antimicrobial spectrum, enhanced oral
adsorption, less resistant or allergic effects.
Biotransformation of natural pencillin G to several
semi-synthetic penicillns:
• Penicillin G acylase is the key enzyme used in the industrial production of β-
lactam antibiotics. This enzyme hydrolyzes the side chain of penicillin G and
related β-lactam antibiotics releasing 6-amino penicillanic acid (6-APA), which
is the building block in the manufacture of semisynthetic penicillins.
• Penicillin acylase from Escherichia coli strain ATCC 11105, Bacillus
megaterium strain ATCC 14945 and mutants of these two strains is currently
used in industry.
• Penicillin acylase produced by Saccharomyces cerevisiae is also used in
biotransformation of penicillin G to semi-synthetic penicillins.
Natural penicillin Ampicillin