1.

Definition of fermentation
2.Difference between two fermentation techniques
3. Fermentation products
4. Role of fermentation in industrial microbiology
- Largescale production of products like important solvents – alcohol
- Production of biomass , exnzymes, antibiotics, acids , etc
- Recombinant products , metabolites

- 5. What factors influence industrial fermentation ?

• Temperature
• pH
• Nature and composition of medium
• Dissolved oxygen
• Dissolved carbon dioxide
• Operation system(batch, fed-batch, continuous)
• Feeding with precursors
• Mixing and shear rates in fermenter
5. About primary and secondary metabolites

Secondary metabolites are products that are non-essential to

survival but serve various useful functions

Synthesis of primary metabolites is usually directly related

with the rate of biomass growth
6. Role of recombinant product in fermentation

Genes from higher organisms may be introduced into microbial cells

capable of synthesizing foreign/ desired protein
IFNs, insuline ,
7. Why is transformation carried out
8. Component of submerged fermenter

Typical Submerged Culture Fermenter

Components of a Fermenter
Substrate
Microbial Inoculum

Product Outlet
9.Compare the three types of fermenter

Typically, continuous fermentations are started as batch cultures

10.Well mixed Continuous
Fermenters
11. Describe the inoculum generation process

Pre/Seed culture > seed fermenter > Production fermenter

Inoculum must be 5-10% of fermenter vessel
This minimize batch time in the vessel and increase productivity

12. Upon what factors does the lag phase depends on

13. Fermentation improvements
14. What are the characteristics of a well designed fermentation medium
-Inexpensive, readily available, consistent quality, good source of C, N,O
O- aeration, C- sugar, N – inorg salt or org material, Vitamin + Mineral

15. How are air, fermentation and media commonly sterilized?

-Air is sterilized using , hydrophobic membrane filter with pore
size 0.1 um . The culture media is sterilized along with
fermenter by heating at 121C for 20 min . For Heat sensitive
media, sterilization is carried out using hydrophilic membrane
filter.

16. How large volume of media is sterilized ?

- Using continuous sterilization process, se batch heating will
be time consuming for heating and cooling
- Preheated above room temp , then flow through a heater
and temp increase to 150C and is held at so as it passes
through the holding coil. Then it moves passing heat to the
preheater which can heat more incoming medium. . Then it
is cooled and then directly enters the fermenter
16. What happens if these factors are altered
Variations in these factors may affect:
(1) the rate of fermentation;
(2) the product spectrum and yield;
(3) the organoleptic properties of the product (appearance,
taste, smell, and texture);
(4) the generation of toxins; nutritional quality and other
physico - chemical properties.
17. Which two groups of chemical can be used as substrate for microbial growth give
example ?
-catabolic- energy source, ammonia,glucose etc,
Anabolic substrate- used in biogenesis,incorporated in cell component so is
conserved

18. Why batch culture cannot maintain high growth rate ?

1. -accumulation of waste product, depletion of
substrate/nutrients

19. What are the parameters of growth kinetics?

1. Yield, maximum specific growth rate, specific mainainance,
saturation constant

20. In a bacterial growth rate equation, dx/dt=ux, when will the

specific growth rate u remain constant and when will it
change ? The specific growth rate μ remains constant only for
limited time and narrow environmental conditions
21. At what condition rate of change in biomass dx/dt = 0 ,
what does it mean?
At steady state, the biomass concentration in the fermenter
does not change,
that is, dx/dt=0

22. In continuous fermenter, what does dilution rate depends on/ D is ratio of?

- The ratio of feed flow rate Q and the volume of the broth VL in the fermenter is known as the dilution rate, D

- When the biomass concentration in feed Xo = 0 , Dilution becomes equal to specific growth rate

- But if the dilution rate exceeds the maximum specific growth rate for the microorganism, the culture will be washed out
23. Well mixed VS Plug flow
- Well mixed- biomass conc does not change at steady state dx/dt=0,
- Plug flow - biomass decreases depends on dilution rate , if D> u, microbes washout.
- Well mixed- Only need to inoculate at the starting
- Plugflow- need to be inoculated continuously

25. Is the optimum temperature for enzyme production and growth same?
No. production 24C, growth 28C

26. When does the enzyme yield reaches its maximum production?
-during the negative acceleration growth phase which occurs just prior to stationary growth,at 24C

27. Which process batch or continuous is better for l asparaginase production ? Why?
A continuous fermentation has advantages of productivity, ease of control, uniformity, and low labor costs. However, the
difficulties of maintenance of sterility and stability in continuous process must be carefully considered. If these problems
are resolved, a continuous process is favored.
24. For asparaginase production , what is yeast extract used for?
Using yeast extract as a growth-limiting substrate

Effect of yeast extract on growth rate.

(1)The studies on nutrient requirements of E. aroideae have shown that yeast
extract is essential for cell growth as well as L-asparaginase production.
(2)No yield of L-asparaginase was observed when the cells are grown in the
medium containing 0.05% yeast extract.
(3)A level of 3% yeast extract is inhibitory to both cell growth and enzyme
synthesis as compared to 1.5% yeast extract. However, 3% yeast extract can
give yields of cells and enzyme, respectively, of 3.55 g/liter and 3.41 IU/ml.
NOTE - L-asparaginase activity is detected by amount of ammonia formed

28. Production of L.asparaginase in batch vs continuous fermentation.

Steps of batch fermentation
- Stock culture preparation – e.aroideae is grown in
agar slant with buffer,yeast extract, tryptone
- Inoculum preparation - e. a is transferred from Steps of continuous fermentation
-At first, the fermenter is run batch wise and then shifted to
slant culture to a conical flast with media
continuous
containing buffer,yeast extract, tryptone, and - The feed rate is maintained using a sigmamotor pump
incubated for 12hr at 24C - - the feeding rate anf harvesting rate is kept constant to
- Fermentation – Then it is inoculated in the batch
prevent dilution and maintain constant vol
fermenter in media with lactose yeast extract and - Antifoam is added
buffer - -Cells are separated from broth by centrifugation
- Antifoaming agent is also added , temperature
maintained at 24C , aeration and agitation is also - a linear relationship between specific production rate and
maintained
specific growth rate is observed in continuous fermentation.
- an absence of a linear relationship between the
specific production rate and the specific growth
rate
-
Enzyme formation in either batch or continuous process is found to be greater when E. aroideae

1) Growth Yield
Y = −ds/dt ∼ −Dx/Ds (here –ve means
forward reaction , +ve means inhibition)
• To find the relationship of growth rate (u)
and substrate consumption (q), divide both
part by xdt,
2) Total energy source uptake is used in Growth
and Maintenance
• Y= growth yield
• Dx = increase in biomass
• Ds = utilization / amount of
substrate
• dt = time taken
• U = specific growth rate
• q = substrate consumption rate
• m = maintenance coefficient
m is the maintenance coefficient
Specific rate of catabolic
substrate consumption by non-
growing cells would give m = q
when μ = 0).
Kinetics of Batch Fermentation • X= biomass
concentration
3) Increase in biomass concentration (in exponential phase in batch fermenter). This eqn • U = growth rate
actually says how much is the rate into how much already present hence ux • Xo = biomass conc at
the beginning

4) Increase in biomass concentration at anytime is calculated by integration the equation

Integration

5) For exponential growth, the time to double the biomass is find out by –
td –doubling time, depends on u- specific growth rate
Here as the time to double biomass is
calculated, the biomass X/Xo would give 2.
As final biomass will be double of initial ,so
answer would be 2, hence we use In 2
Population Density • r= birthrate
• a= mortality rate
6) Rate of change in no. of organism / density is find out by – • N = number of
r= birthrate , a= mortality rate
organism
dN / dt = rN − aN2 = rN(1 − N/K), K = r/a •

7) Bacterial growth rate is proportional to instant cell mass (x) and quotient u (growth rate)
remaining constant
dx/dt = μx

8) Integration of the above equation gives the next equation,

x = x0 at time t = 0, gives the exponential eqn
x = x0eμt or ln x = ln x0 + μt
Kinetics of continuous fermentation

1) Eqn for biomass balance or change in biomass

2) At steady state, the biomass concentration in the fermenter does not change,
that is, dx/dt = 0, which gives us

3) Dilution rate is determined by dividing flowrate Q and volume VL, when Xo = 0 (no inflow of
biomass), dilution rate is

, in any well - mixed continuous culture operating at steady state with a finite biomass concentration
in the fermenter, the dilution rate necessarily equals the specific growth rate.
4) biomass concentration Xo at the exit of the fermenter
depends on dilution rate D, as follows:
Clearly, a plug flow fermenter needs to be inoculated
continuously (i.e., X o > 0), or no biomass will be produced.