Bacterias are fermented in optimal growth conditions in labs and at commercial places to extract various drugs and useful proteins which are genetically engineered or are present inside the bacterias. to maximize the process certain things are to be considered, which are described in the slide

1. B Y A B H I S H E K G I R I
MAXIMIZING THE EFFICIENCY
OF FERMENTATION PROCESS
M.sc-II
SEM-
III P-I

2. WHY TO MAXIMISE?
• In any type of fermentation process that is used to grow
cells, it is necessary to monitor & control culture
parameters, such as
I. Dissolved oxygen concentration.
II. pH, Temperature &
III. Degree of mixing.
• Changes in any of these parameters can have a
dramatic effect on the yield of cells & the stability of the
protein product.

3. OPTIMAL GROWTH
(OXYGEN)
• The maximal oxygen demand in a fermentation,
Qmax = Xμmax/Yo2
• Where, X = cell mass
μmax = maximal specific growth rate.
Yo2 = growth yield based on oxygen consumed.
• Oxygen supplied in the form of sterilized air.
• However, introducing air produces bubbles, & thus the rate of
transfer of oxygen to the cells is insufficient.
• Thus, fermenter design should monitor these changes in the
culture.

4. OPTIMAL GROWTH
(pH)
• Most microorganisms grow optimally between 5.5 & 8.5.
• However, cellular metabolites are released into the growth
medium.
• Therefore, the pH must be monitored & either acid or base
must be added as needed to maintain the pH.
• After adding fermentation broth should be mixed
throughout so that the pH is same in entire reaction vessel.

5. OPTIMAL GROWTH
(TEMPERATURE)
• Microorganisms grown at a temperature below the optimum
grow slowly & have a reduced rate of cellular production.
• Whereas if grown at a temperature above the optimum
growth there may be premature induction of the expression
of the target protein.
• If it is under control of temperature-sensitive repressor, or
induction of a heat shock response, will produce proteases
that lower the yield of the protein product.

6. OPTIMAL GROWTH
(MIXING)
• Adequate mixing of a microbial culture is essential to
assure an adequate supply of nutrients to the cells &
prevention of the accumulation of any toxic metabolic by-
product in local, poorly mixed regions.
• Effective mixing is easily attained with small-scale
cultures, but it is one of the major problem with the scale
of fermentation is increased.

7. OPTIMAL GROWTH
(AGITATION)
• Agitation of the fermentation broth affects other factors,
such as
1. The rate of transfer of oxygen from the gas bubbles to the
liquid medium & then from the medium to the cells,
2. Efficient heat transfer,
3. Accurate measurement of specific metabolites in the
culture fluid, &
4. Efficient dispersion of added solutions such as acids,
bases, & antifoaming agents.
• On these grounds, it might be concluded that the more
mixing there is, the better the growth.

8. • However, excessive agitation of a fermentation broth can
cause hydomechanical stress (shear)
• Thus damages larger microbial or mammalian cells, & a
temperature increase, which may also decrease cell
viability.
• Thus a balance must be struck between the need to
provide through mixing & the need to avoid damage to the
cells.

9. ADDITIONAL CONSIDERATION FOR
GEM
• For scaled-up fermentations that has nothing to do with the
technical aspects of the process but depends instead on
whether a GeM is being used.
• Although most recombinant microorganisms are not
hazardous, it is nevertheless important to ensure they are
not inadvertently released into the environment.
• Therefore, fail-safe system are used to prevent accidental
spills of the live recombinant organisms & to contain them if
they occur.
• Furthermore, all organisms must render them nonviable
before they are discharged from the production facility

10. HOW TO MAXIMIZE?
1. 1.HIGH-DENSITY CELL CULTURES

11. HIGH-DENSITY CELL CULTURES
• A major objective of fermentation is to maximise the
volumetric production.
• In practice, cell concentration of more than 50 gm. cells/liter
of culture have been obtained with fed-batch cultures of
recombinant E-coli.
• Some nutrients like carbon & nitrogen can inhibit cell growth
if they are present at too high concentration.
• In addition, fermentations that use complex media
containing peptone or yeast extract, can vary from one
media to another & are not always reproducible.

12. • Acetate, is inhibitory to cell growth, is produced by E.coli
both when the cells are grown under oxygen-limited
conditions & in the presence of excess glucose.
• Using glycerol instead of glucose as the carbon source,
lowering the culture temperature or using Gem to shunt
acetate into less toxic compounds.
• Oxygen may become limited in such cultures. To overcome
this problem, the rate of introduction of air, the agitation
rate, or both can be increased.
• Alternatively, expression of the gene Vitreoscilla hemoglobin
can increase uptake of oxygen & improve enzyme
production in Bacillus subtilis, increases erythromycin
production by Saccharopolyspora erythraea.

13. • High density cell cultures are most readily attained in fed-
batch cultures.
• The addition of nutrients following depletion of some of the
original nutrients may be constant, stepwise, or exponential.
• Addition of nutrients can be automated.
NUTRIENT
TIME
CONSTANT
Specific growth rate
NUTRIENT
TIME
STEPWISE
Specific growth
rate

14. HOW TO MAXIMIZE?
1. 2. INCREASING PLASMID STABILITY

15. INCREASING PLASMID STABILITY
• Loss of plasmid is the major industrial problem in large-scale
growth of recombinant E. coli cells.
• Plasmid loss often limits the yield of plasmid-encoded
recombinant proteins.
• Plasmid instability in bacterial cultures is typically due to
unequal distribution of plasmids to daughter cells during
growth & cell division.
• Once lost, cells grow faster, with the result that cells lacking
plasmid eventually dominating the culture.
• One approach to avoid this problem is to include antibiotic
resistance gene & then add to the culture.

16. • In addition to the obvious economic cost of the antibiotic,
disposal of spent growth medium is a potential
environmental hazard.
• One way to deal with this problem is to delete an essential
gene from the chromosomal DNA of the host bacterium & at
the same time place this gene on the plasmid that is being
stabilised.
• As a result , only plasmid-carrying cells can grow, making
the bacterial strain totally dependent upon maintenance of
the plasmid.
• With this system, selection that utilizes antibiotics is no
longer necessary thereby decreasing both cost &
environmental risk.

17. HOW TO MAXIMIZE?
1. 3. QUIESCENT E. COLI CELLS

18. QUIESCENT E. COLI CELLS
• It is difficult to engineer recombinant bacteria to produce
large amounts of foreign protein & at the same, time to grow
to a high cell density.
• It would be advantageous to be able to grow cells to a high
density & then shift the allocation of available resources
from growth to foreign-protein production.
• With this in mind, workers engineered a quiescent cell
expression system in which a plasmid-encoded protein is
expressed in non-growing but metabolically active cells.
• Understanding the commercial potential of this unique
system, scientist who developed this approach have applied
for patent to protect their intellectual property rights

19. QUIESCENT STATE IN E. COLI
• Quiescent stage is established by overexpression of Rcd, a
regulatory protein, in an hns mutant E. coli.
• Hns gene codes for histone like nucleoid-structuring protein.
• E.coli gradually cease synthesizing host protein but continue
synthesizing plasmid-encoded foreign proteins.
• In both batch & fed-batch modes, the quiescent cells
produce less biomass & secrete considerably more scFv
protein than control E.coli cells engineered to express scFv
under the control of the pL promoter.

20. Rcd gene is placed under
the transcriptional control of
the pR promoter while the
recombinant protein gene
(encoding a single chain
antibody variable fragment
scFv) was controlled by the
pL promoter.
The activities of both pR &
pL was repressed by a
temperature-sentitive cI
repressor protein

21. HOW TO MAXIMIZE?
1. 4. PROTEIN SECRETION

22. PROTEIN SECRETION
• High-level cytoplasmic expression in E.coli of different
foreign proteins results in the formation of inclusion bodies
consisting of insoluble improperly folded proteins.
• Even if soluble purifying it from a cytoplasmic extract is
major undertaking.

23. Investigator observed that
expression levels were quite low
when they expressed several
different foreign proteins under
transcriptional control of the
strong pm/xyLS
promoter/regulator system.
Use of translocation signal
sequences significantly
stimulated the levels of
expression of these 3 human
proteins.
20-30% of the protein that was
produced was found to be
insoluble form.
A strategy that minimizes the
extent of insoluble protein
formation needs to be
developed.

24. HOW TO MAXIMIZE?
1. 5. REDUCING ACETATE

25. REDUCING ACETATE
• Acetate inhibits both cell growth & protein production & also
wastes carbon & energy resources.
• Removing acetate from the culture during fermentation can
be achieved by several different method including
continuous dialysis & the use of macroporous ion-exchange
resins.
• However, these methods tend to remove nutrients that are
necessary for cell growth along with the acetate.
• Use of fructose & mannose is used as a carbon source thus
lowering levels of acetate & higher yields of protein.

26. • Another method is to reduce the uptake of glucose by cells
by adding methyl α-glucoside, a glucose analogue.
• This effect can also achieved by using an E.coli host cell
that contained a mutation in ptsG, a gene encoding enzyme
II in the glucose phosphotransferase system.
• Ex : In a batch culture both wild-type & ptsG mutant E.coli
expressing β-galactosidase activity,
Wild-type = 10gm/lit
ptsG mutant = 15gm/lit
at the same time, the mutant cells synthesized about
25% more β-galactosidase/gm of cells than wild type cells.
• it is much easier & quicker to alter host cell by genetic
transformation than by mutagenesis & selection, alternative
methods were developed.

27. • One of these method
include introducing a
gene encoding the
enzyme acetolactate
synthase into host cells.
• This enzyme catalyses
the formation of
acetolactate from
pyruvate, thereby
decreasing the flux
through acetyl coA to
acetate.
• The transformed cells
produced 75% less
acetate than the
nontransformed cells.
• Acetoin which is
produced in approx. 50-
fold less toxic than
acetate. The protein yield
was also doubled.

28. • Another way of converting acetate to acetoin is to redirect it
to TCA cycle.
• In one study workers overexpressed the gene for the
enzyme phosphoenol pyruvate carboxylase, which converts
it to oxaloacetate, they obtained 17% increase in the
specific growth rate of E.coli cells & 44% decrease in
acetate production.
• Unfortunately, overexpressing this enzyme also decreases
the amount of glucose uptake by the bacterial cells &
diminishes the growth rate.
• Another group of workers tranformed the host cell with the
gene for the enzyme pyruvate carboxylase isolated from
gram negative bacteria Rhizobium etli.
• Thus acetate levels were decreased & cell yield was
increased & synthesis of foreign proteins was also increased

29. Addition of pyruvate carboxylase
allow s E.coli to use the available
carbon more efficiently, directing it
away from acetate toward
biomass & protein formation.
Similarly, this may also be done
by converting aspartate to
fumarate.
To do this, E.coli were
transformed with the gene for L-
aspartate ammonium lyase
(aspartase) under the control of
strong tac promoter .
Aspartate activity is induced by
addition of IPTG at the mid- to late
log phase of growth.

30. SUMMARY
• To maximise the fermentation process optimal growth
conditions must be maintained.
• In high density cell cultures nutrients like carbon &
nitrogen should not exceed the optimal level.
• Acetate production must be lowered for maximizing the
efficiency.
• Plasmid stability must be increased and also insoluble
protein secretion must be restricted.

31. REFERENCE
BOOKS
• Bernard R. Glick and Jack J. Pasternack, Molecular
Biotechnology – Principles and applications of
recombinant DNA, ASM Press, Washington DC.
• S. S. Purohit, Biotechnology – Fundamentals and
applications, 3rd Edition, Agrobios, India
WEB LINKS
• http://www.massey.ac.nz/~ychisti/FermentInd.PDF
• http://www.rpi.edu/dept/chem-eng/Biotech-
Environ/SeniorLab/Fermentation/