BIOPROCESS ASSIGNMENT ( END SEM)

2 marks
1. What happens if excess of carbon is provided to the medium?
A) Microorganisms that utilize carbon as a carbon source will experience a rapid increase in growth
rate when provided with excess carbon. This is because carbon availability is often a limiting factor for
microbial growth, and an abundance of carbon enables cells to undergo faster replication. If the
medium is suitable for microbial growth, providing excess carbon can stimulate
the proliferation of carbon-utilizing microorganisms. This can lead to increased
biomass production and potentially alter the microbial community composition.
2. What do you mean by idle time of a batch bioreactor? Why do you think it might
effect the performance of the reactor?
A) The idle time of a batch bioreactor refers to periods when the reactor is not
actively involved in the cultivation of microorganisms or the production of desired
substances, often due to maintenance, cleaning, preparation, or transition
between batches.
when the reactor is idle, there's a risk of microbial contamination, the nutrients may get
depleted, the pH and temperature of the reactor may shift and the bioproducts that were
produced may degrade over time when left idle.
3. You have been given with two bacterial cell culture and asked to do sub-
culturing and plot the growth curve. You prepared LB broth and inoculated from
the sample that was provided to you. While plotting the curve you observed that
one of the bacterial sample had a prolonged Lag phase than the other.
Accordance to you what can be the potent reasons for your observation.
A) The prolonged lag phase observed in one bacterial sample compared to the other
could be attributed to several potential reasons:

i.The bacterial sample with the prolonged lag phase may have a lower inoculum size
which would require more time for the bacteria to adapt to the new environment,
initiate growth, and reach the exponential phase.

ii. The viability and health of the bacterial cells in the sample could vary between the
two cultures.

iii. Differences in nutrient composition in the LB broth or residual nutrients from the
previous growth medium could impact the lag phase duration.

iv. Limited nutrient availability or imbalances in essential nutrients may delay bacterial
growth and prolong the lag phase.
 v. Factors such as temperature, pH, oxygen availability, and presence of inhibitors or
competitors in the growth medium can affect bacterial growth kinetics and the duration
of the lag phase.

4. What do you mean by mixed growth associated product formation? Can you
justify that it deal with the two other growth associated product formations?
A) Mixed growth associated product formation" refers to the production of multiple products
at different stages of the growth curve, resulting in a mixed profile of product formation over
time.
Growth-Associated Products: These products are directly linked to cell growth. As cells
divide and proliferate, they simultaneously produce the desired compounds. The product
concentration correlates with the growth rate

5. State any two advantages of CSTR?

A) two advantages of continuous stirred tank reactor (CSTR) are
i. CSTRs keep mixing and processing materials without stopping. This helps maintain a
steady flow of products, making them great for making things in big amounts without
interruptions.
ii. Good temperature control
iii. Excellent oxygen transfer
iv. Simple to Adjust: It's easy to change the size of a CSTR to fit different needs. So, whether
you need to make a little or a lot, you can adjust the reactor without much trouble
6. A metallic ion is an activator of an enzyme used in metabolism of substrate for a
particular bacterial specie. Suddenly there is depletion of that particular metallic
ion acting as the activator of the enzyme. According to you which phase of
growth kinetics would the cells continue and why?
A) If there's a sudden depletion of the metallic ion that acts as an activator for an enzyme
crucial in the metabolism of a substrate for a bacterial species, the bacterial growth
kinetics would likely be affected, possibly entering a stationary phase.
Initially, there might be a lag phase as the bacteria adapt to the absence of the
activator ion and adjust their metabolic processes accordingly. During this phase,
bacterial growth is slow as they acclimate to the new conditions.
 If the bacteria can compensate for the absence of the activator ion by activating
alternative metabolic pathways or adjusting enzyme activity, they may continue
growing at a reduced rate during the log phase. However, the growth rate is likely to be
slower compared to optimal conditions due to the metabolic adjustments required.
Eventually, as the bacterial population continues to grow, they may reach a point
where the lack of the activator ion significantly impairs their ability to metabolize the
substrate efficiently. This could lead to a stationary phase, where the rate of cell
growth equals the rate of cell death.
7. How can you minimize the duration of Lag phase of cells within a culture?
A) To minimize the duration by following procedures:
i. Ensure that the inoculum used to start the culture contains actively growing and
healthy cells.
ii.Provide optimal growth conditions such as appropriate temperature, pH,
oxygenation, and nutrient availability.
iii.Supplement the culture medium with growth factors, vitamins, or other supplements
that promote cell proliferation and metabolic activity. These additives can help
stimulate cell growth and shorten the lag phase.
8. What do you mean by diauxic growth?
A) Diauxic growth refers to cellular growth in two distinct phases.It occurs when an
organism encounters two different sugars in its growth medium.One of these sugars
is preferentially metabolized by the organism, leading to rapid growth initially.After
the first sugar is exhausted, the organism switches to metabolizing the second sugar,
resulting in a second phase of growth.
Diauxic growth is a classic example of metabolic regulation in response to changing
environmental conditions, where microorganisms prioritize the utilization of the
preferred carbon source before switching to alternative sources to sustain growth
9. How can you differentiate between balanced and unbalanced growth?

10. State any three important phenomenon that take place during stationary
phase?
A)
 i. Metabolic Adaptation:Microorganisms undergo metabolic shifts to adjust to the changing
environment and limited nutrients.Metabolic pathways may be reorganized to optimize
energy production and conserve resources.
ii. Secondary Metabolite Production:Many microorganisms produce secondary metabolites
during the stationary phase.Secondary metabolites can include antibiotics, pigments, toxins,
and enzymes that are not essential for growth but contribute to survival and ecological
interactions.Production of secondary metabolites often increases during nutrient limitation
as a strategy for competition or defense
iii. Stress Response Activation:Cells in the stationary phase activate stress response
pathways to cope with environmental stresses.Stress response mechanisms may include the
synthesis of protective proteins, chaperones, and antioxidants to combat oxidative stress
and maintain cellular integrity.

11. Define synchronous culture.

A) Synchronous culture is a technique of growing microorganisms at the same stage of
their growth cycle under controlled conditions. All individuals are in the same phase of
the cell cycle or exhibit a coordinated pattern of growth and division. Synchronous
cultures are typically achieved through methods that temporarily arrest the cell cycle
progression (addition of cytostatic agents), such as Cell cycle synchronization
techniques.
12. If one starts with 10,000 x10 4 cells in a culture that has a generation time
of 2 h, how many cells will be in the culture after 4, 24, and 48 h?

13. What do you mean by fermentation media?

A) Fermentation media, also known as growth media or culture media, are nutrient-rich
solutions used to support the growth and proliferation of microorganisms during
fermentation processes. These media contain essential nutrients required for microbial
metabolism and are tailored to meet the specific nutritional requirements of the target
microorganism or fermentation process.
14. What are the four categories in which fermentation processes can be
categorized?
A) categories are:
i. Alcoholic Fermentation:In alcoholic fermentation, microorganisms such as yeast
metabolize sugars (e.g., glucose) to produce ethanol (alcohol) and carbon dioxide.
ii. Lactic Acid Fermentation:Lactic acid fermentation involves the conversion of sugars (e.g.,
glucose) into lactic acid by lactic acid bacteria.
iii. Acetic Acid Fermentation:Acetic acid fermentation, also known as acetification, involves
the conversion of ethanol into acetic acid by acetic acid bacteria.
 iv. Butyric Acid Fermentation:This type of fermentation is characteristic of obligate anaerobic
bacteria (such as Clostridium species).

15. What do you mean by Homolactic and Heterolactic fermentation?

A) Homolactic fermentation: Converts sugars into lactic acid as the sole end product. It's
common in yogurt and cheese production.

Heterolactic fermentation: Produces a mix of lactic acid, ethanol, carbon dioxide, and other
compounds. It's less common and involves additional metabolic pathways.

16. Name two bacteria that participate in propionic acid fermentation?

A) Propionibacterium freudenreichii and Propionibacterium acidipropionic
17. Why do you think idle time is a negative quality of batch bioreactor?
A) Idle time in a batch bioreactor can be considered a negative quality for several reasons:

i) Efficiency: Idle time represents periods when the bioreactor is not actively producing
desired products. This results in reduced overall productivity and efficiency, as resources are
not fully utilized.

ii) Wastage: During idle time, resources such as energy, utilities, and raw materials may
continue to be consumed without contributing to product generation. This can lead to
increased costs and wastage of resources.

iii) Production Delays: Idle time can prolong the overall production time, leading to delays in
meeting production targets and deadlines. This can have downstream effects on supply
chain logistics and customer satisfaction.

iv) Environmental Impact: Extended idle time may result in unnecessary energy consumption
and emissions, contributing to environmental pollution and carbon footprint.

18. You are taking data of growth kinetics and you observed that the time of
Lag phase appears to be very long,suggest what are the probable reasons for this
condition
A) i.Suboptimal Growth Conditions:Temperature,pH, Oxygen Availability, Nutrient
Concentrations
ii.Low Inoculum Density
 iii. Nutrient Limitation:Carbon, Nitrogen,Phosphorus,Trace Elements
iv. Stressful Conditions:High Osmolarity,Toxic Substances,Antimicrobial Agents
v. Genetic Factors:Mutations,Genetic Variations
vi. Previous Growth History:Subculture Intervals,Storage Conditions,Handling Practices

19. What are the changes that take place within a bacterial cell during its
transition from lag to log phase?
A)
● Metabolic Activation: Bacterial cells transition from a state of low
metabolic activity during the lag phase to increased metabolic
activity during the log phase.
● Nutrient Utilization: Bacterial cells begin to actively take up and utilize nutrients
from the surrounding environment to support cell growth and division.
● DNA Replication: As bacterial cells prepare for division, DNA replication
machinery becomes active, leading to the synthesis of new DNA strands.
● increase in Cell Numbers: As cell division proceeds, the bacterial population
rapidly increases in number, leading to exponential growth.
20. What do you mean by doubling time?
A) Doubling time refers to the amount of time it takes for a population of cells or organisms
to double in number through exponential growth. It is a measure of the rate of population
increase during the exponential growth phase.
T(double) =ln2/K
=0.693/K

21. Derive the unit for dilution.

Suppose we have a solution where 1 mL of a solute is diluted with a total volume of 10 mL of

solvent.The dilution ratio can be expressed as
1:10, which means 1 part of solute is diluted with
10 parts of solvent.
In general, the unit for dilution is typically expressed as a ratio (e.g., 1:10, 1:100, etc.) or as a fraction
(e.g., 1/10, 1/100, etc.), indicating the relationship between the volume of solute and the total
volume of the solution.

22. What do you mean by generation time?

A) Generation time, also known as doubling time, is the time it takes for a population of cells
or organisms to double in number through exponential growth. It is a fundamental parameter
used to describe the rate of population increase during the exponential growth phase.

23. Give the mathematical expression for generation time.

24. Name two complex agar.

 Nutrient Agar,Tryptic Soy Agar (TSA), Lysogeny Broth (LB) Agar
25. State two uses of vector.

i. Gene Cloning: Vectors are used to replicate and propagate foreign DNA fragments in host
cells for further analysis.

ii. Recombinant DNA Technology: Vectors serve as carriers to combine DNA sequences from
different sources, enabling applications in biotechnology, medicine, agriculture, and research.

26. You are operating two bioreactors, one batch and another continuous.
According to you which will achieve stationery phase and why?
A) In a batch bioreactor, the growth of microorganisms occurs in a closed system where
nutrients are added at the beginning and the culture is harvested at the end of the run. As
nutrients deplete and waste products accumulate, the growth rate decreases, eventually
leading to the stationary phase.
In a continuous bioreactor, fresh media is continuously added, and an equal volume of spent
media is removed to maintain a constant volume. This constant influx of nutrients and
removal of waste products can potentially sustain exponential growth indefinitely, without
entering the stationary phase.
Therefore, the batch bioreactor is more likely to achieve the stationary phase because it
operates under finite nutrient conditions, while the continuous bioreactor is designed to
maintain exponential growth by continuously supplying nutrients.

27. What are the conditions that result in the death phase within batch
bioreactor?
A)In short, the death phase in a batch bioreactor occurs due to:

● Nutrient depletion.
● Accumulation of waste products and inhibitory metabolites.
● Physiological changes leading to decreased viability and cell death

28. Differentiate between obligate aerobes and obligate anaerobes.

29. What do you mean by photolithotrophs?
A) Photolithotrophs" are organisms that utilize light as their energy source (photo-) and
inorganic compounds as their electron source (-litho-). In simpler terms, they obtain energy
from sunlight and use inorganic molecules (such as minerals or certain chemicals) as
electron donors to drive their metabolic processes.
30. If a microbial population increases from 5 × 10⁵ to 2 × 10⁶ cells/mL in 2
hours, calculate the generation time.
31. What happens if excess of carbon is provided to the medium? repeat
A) Microorganisms that utilize carbon as a carbon source will experience a rapid increase in growth
rate when provided with excess carbon. This is because carbon availability is often a limiting factor for
microbial growth, and an abundance of carbon enables cells to undergo faster replication. If the
medium is suitable for microbial growth, providing excess carbon can stimulate
the proliferation of carbon-utilizing microorganisms. This can lead to increased
biomass production and potentially alter the microbial community composition.
32. Name the two most important components of a medium that should be
present in bacterial culture.
A) The two most important components of a medium for bacterial culture are:

Carbon Source: Bacteria require a carbon source for energy and growth. Common carbon
sources include sugars (e.g., glucose, sucrose), amino acids, organic acids, and alcohols.
Glucose is one of the most commonly used carbon sources in bacterial culture media.

Nitrogen Source: Nitrogen is an essential component of proteins, nucleic acids, and other
cellular components. Bacteria require nitrogen sources to synthesize amino acids and
nucleotides for growth and metabolism.

33. Why Foaming is usually is not a problem in synthetic medium?

A) foaming is usually not a problem in synthetic medium due to its simplified and well-
defined composition, reduced protein content, controlled nutrient complexity, enhanced
foam control with additives, and better process understanding, which collectively minimize
the likelihood of foam formation compared to complex media.
34. Synthetic media have many advantages but then also they are of little use
in industrial fermentation. Why?
A) while synthetic media offer precise control over nutrients and are great for small-scale
experiments, they have limitations for large-scale industrial fermentation:
○ Nutrient Limits: Synthetic media may lack the variety of nutrients needed for robust
growth of microbes in large quantities.
○ Cost: Some synthetic ingredients can be pricey, making them impractical for large-
scale production.
○ Microbial Needs: Certain microbes need specific nutrients that synthetic media may
not provide adequately.
○ Foaming and Mixing: Synthetic media may not manage foaming and mixing well in
large fermenters, leading to process challenges.
○ Regulations: Some industries require more standardized media for quality control,
which synthetic media may not always meet.
35. Water is a very much important in fermentation industry. Why?
A) water is crucial in the fermentation industry because it:

● Dissolves essential nutrients for microbial growth.

● Serves as a medium for biochemical reactions.
● Regulates temperature fluctuations.
● Acts as a pH buffer, stabilizing acidity.
● Functions as a solvent for product extraction.
● Maintains hydration and viability of microbial cells

36. Why calcium carbonates is added to the medium?

A) calcium carbonate is added to fermentation media because it:

● Helps stabilize pH levels, preventing acidity that can harm microbes.

● Provides essential minerals like calcium and carbonate for microbial growth.
● Promotes the formation of clumps, aiding in the removal of microbes from the
solution.
● Can sometimes serve as a food source for certain microbes, boosting their growth.

37. What is the function of sulphur bacteria?

A) Sure, here are the main points about sulfur bacteria in a simple way:

1. Sulfur Cycling: Sulfur bacteria help recycle sulfur in nature, converting it

between different forms.
2. Energy Production: They get energy by processing sulfur compounds like
hydrogen sulfide.
3.Cleaning the Environment: Some sulfur bacteria help clean up pollutants
containing sulfur.
4.Anaerobic Respiration: They play a role in anaerobic environments, converting
sulfur compounds.
5.Sulfur Deposits: Some sulfur bacteria can create sulfur deposits in certain
environments.
In short, sulfur bacteria help maintain sulfur balance, produce energy, clean the
environment, and contribute to various natural processes.
 38. What is the function of nitrifying bacteria?

A)Nitrifying Bacteria helps in turning the toxic nitrogen compounds (NH3) present in
the soil into useful nitrates by the process of nitrification, so that plants can readily
absorb it. Eg-Nitrobacter, Nitrosomonas

39. If we add phenyl acetic acid in corn steep liquor then which penicillin would
be obtained?
A) Penicillin G
40. What do you mean by Pasteurization?
A) Pasteurization is a heat treatment process used to destroy harmful pathogens (such as
bacteria, viruses, and parasites) in food and beverages while preserving their quality and
extending their shelf life. The process involves heating the product to a specific temperature
for a defined period, followed by rapid cooling to inhibit further microbial growth.
Pasteurization effectively reduces the microbial load in the food or beverage, making it safer
for consumption, especially for products like milk, fruit juices, and other liquid foods

41. What happens if excess of carbon is provided to the medium?

repeat question
42. You have been given with two bacterial cell culture and asked to do sub-
culturing and plot the growth curve. You prepared LB broth and inoculated from
the sample that was provided to you. While plotting the curve you observed that
one of the bacterial sample had a prolonged Lag phase than the other.
Accordance to you what can be the potent reasons for your observation.
repeat question
43. What do you mean by mixed growth associated product formation?
A) Mixed growth associated product formation" refers to the production of multiple products
at different stages of the growth curve, resulting in a mixed profile of product formation over
time.
44. What do you mean by maintenance coefficient of the cell?
45. How primary and secondary metabolite controls the rate of cell growth in a
culture media?
46. How endogenous metabolism regulates the existence of cell during the
stationery phase of cell growth?
47. How can you classify organism depending upon the temperature optima of
cell growth?
A)
● Psychrophiles: Thrive in cold temperatures (0°C to 20°C).
● Mesophiles: Grow best at moderate temperatures (20°C to 45°C), common in
terrestrial environments.
● Thermophiles: Prefer high temperatures (45°C to 80°C or higher), often found in hot
springs and geothermal areas.

48. State any two application of the enzyme amylase.

A) applications are
● Food Industry: Amylase is widely used in the food industry for its ability to break down
starch into simpler sugars such as maltose and glucose. Amylase is also used in
baking to improve dough texture and increase yeast activity by breaking down starch
into fermentable sugars.
● Detergent Industry: Included in laundry detergents to break down starch-based stains
like pasta sauce and chocolate, making them easier to remove during washing
49. Name the crude carbon sources of media
A) Crude carbon sources commonly used in media formulations include:

● Glucose: A simple sugar commonly used as a readily available carbon source for
many microorganisms.
● Sucrose: Another simple sugar often used as a carbon source, especially in media for
yeast and certain bacteria.
● Lactose: Found in milk, lactose is used as a carbon source in media formulations for
lactose-fermenting bacteria such as Escherichia coli.
● Maltose: A sugar derived from malted grains, maltose is utilized as a carbon source in
media for certain fungi and bacteria.
● Starch: A complex carbohydrate commonly used as a carbon source after hydrolysis
by enzymes present in the microbial culture.
● Glycerol: A sugar alcohol used as a carbon source in media formulations for various
microorganisms, including bacteria and yeast

50. How temperature plays a vital role in the growth of bacteria?

A) In general, the higher the temperature, the more easily microorganisms
can grow up to a certain point. Very high and low temperatures both
obstruct the enzyme processes microorganisms depend on to survive.
51. What are symbionts?
 A) Symbionts are organisms that live in close association with each other, often benefiting
from their relationship.Symbiosis refers to the interaction between two different species
living together in close proximity.These interactions can be beneficial, harmful, or neutral.

52. What do you mean by microaerophilic organism?

A)The term 'microaerophile' refers to those microbes, which although requiring
oxygen for growth, are unable to grow at normal atmospheric oxygen tensions;
these organisms are adapted to particular environments that contain low oxygen
concentrations
53. How pH has its impact on the growth of the organisms?
A) The effects of pH on organisms can include changes in cell shape and size,
membrane properties, and the structure and function of intracellular
molecules. pH affects microbial growth, enzyme activity, protein stability,
and structure of biological molecules. The effect of pH on the dynamics of
natural membranes.
54. How can you differentiate between chemostat and turbidostat?

A)
55. What do you mean by synchronous culture?
 A) A synchronous or synchronized culture is a microbiological culture or a cell
culture that contains cells that are all in the same growth stage
56. Name the enzyme that is used for alcoholic fermentation in yeast.
A)Zymase is the enzyme complex that catalyzes alcoholic fermentation in yeast.
Zymase is also known as alcoholase.
57. What do you mean by solid state fermentation?
A) Solid state fermentation (SSF) is defined as the cultivation process in which
microorganisms grow on solid materials without the presence of free liquid.
58. What is idiophase?
A) idiophase (plural idiophases) (biology) The phase in the growth of a culture
during which secondary metabolites are produced.
59. State two applications of plug flow reactor.
A) Plug flow reactors (PFRs) are used in many industrial applications, including
large-scale chemical, pharmaceutical, fertilizer, and petrochemical
production. Two applications of PFRs are:
Biofuels: PFRs are used in the production of biodiesel and other biofuels
with a recycle system.
Bioenergy production: PFRs are preferred for bioenergy production because
of their steady-state operation and lack of agitation or baffling.

60. Write two advantages of plug flow reactor.

A) Two advantages of plug flow reactor are:
● Efficient utilization of reactor volume
● Optimum reaction yield and purity
● Low start-up and shutdown losses
● Easy maintenance
● High conversion rate
● Mechanically simple
● Unvarying product quality
● Good for studying rapid reactions
● Low operating (labor) cost
● Continuous operation
● Good heat transfer

61. Write two limitations of fluidized bed reactor.

A) Two limitations of fluidizes bed realtor are:
i) Complexity: Fluidized bed reactors can be complex to operate.
 ii) Difficult separation of the fine catalyst particles from the exhaust gas
iii) Erosion: The high linear velocities of the fluidized material can cause
erosion of the reactor components.
62. State two advantages of bubble column reactor.
A) Two advantages of bubble column reactor are:
i) Low maintenance and operating costs.
ii) Excellent heat and mass transfer.
63. What are the advantages of recombinant cell culture?
A) The advantages of recombinant cell culture are:
i) Scalability
You can produce large volumes of starting material in a short, predictable
period of time. This keeps protein purifications on-schedule and finished
products in stock
ii) Fast production
You can produce recombinant cell lines in nonviral expression vectors
64. Why do you think moist heat sterilization is better than dry heat
sterilization?
A) Dry heat sterilization uses exceptionally high temperatures, 170ºC at
minimum, to inactivate microorganisms. Steam sterilization is often
preferred over dry heat sterilization as steam sterilization can kill microbes
at lower temperatures, thus minimizing material degradation following
sterilization.
65. How can you differentiate between steady and unsteady culture?
A)
66. What is the difference between net specific growth and gross specific
growth?
A) Net specific growth rate is the relative change in bacterial biomass, while
gross specific growth rate is the rate of cell mass loss due to cell death or
endogenous metabolism.
67. Write the structure of penicillin.
A) It is made up of an enclosed dipeptide formed by the condensation of L-
cysteine and D-valine. This results in the formations of β-lactam and
thiazolidinic rings. The key structural feature of the penicillins is the four-
 membered β-lactam ring; this structural moiety is essential for penicillin's
antibacterial activity.

68. Name two organisms those are involved in the production of antibiotics.
A) Two groups of microorganisms that can produce antibiotics are bacteria and
fungi. Some examples of organisms that produce antibiotics include:
i) Streptomyces: The largest genus of organisms that produce antibiotics,
streptomyces produces over two-thirds of the clinically useful antibiotics of natural
origin. Streptomyces produces antibacterial, antifungal, and antiparasitic drugs, as well
as other bioactive compounds.
ii) Penicillium: A group of fungi that can produce antibiotics. Penicillin V is an
antibiotic in the penicillin group that helps fight bacteria in the body system.
69. Why do you think fungi require acidic pH for their growth?
A)
70. Differentiate between defined and undefined media.
A) Defined media is a type of culture media that contains specific chemicals at
known concentrations. The exact chemical composition of defined media is
known because it's made from pure ingredients dissolved in double distilled
water.
Undefined media is a type of culture media that has some parts that are
not entirely defined. This is due to the presence of extracts from microbes
or animals, which have an unknown chemical composition. Undefined
media is also known as complex media or basal media.
71. Differentiate between batch and continuous culture.
A)
72. How can you transform a parabolic curve of Monod’s Kinetic to straight

line ?A)
73. Write the equation for KLa.
A) The volumetric oxygen mass transfer coefficient – kLa, is the parameter that
controls the rate of how oxygen transitions from the gas phase into the liquid
phase. kLa shows numerically how efficiently oxygen, which is introduced
through a sparger in the vessel, is dissipated and distributed in the medium by
the mixer.
KLa = kL * A
Where:
KLa is the overall mass transfer coefficient, in s-1.
kL is the liquid-side mass transfer coefficient, in m/s.
A is the specific surface area of the gas-liquid interface, in m2/m3.

74. What are the factors affecting the value of KLa?

A) The volumetric mass transfer coefficient (KLa) is a measure of aeration
capacity in fermentation processes. KLa is affected by several factors,
including:
Bubble size: In most fermentation broths, if the bubbles have diameters
less than 2 to 3 mm, surface tension effects dominate the behavior of the
bubble surface.
 Temperature: KLa increases as temperature is increased.
Gas holdup: Larger bubbles represent large air volume but have a shorter
residence time due to a faster ascent rate.
Mass transfer: KLa depends on factors like aeration rate, agitation rate, and
impeller design.
Oxygen demand of cells: Optimal oxygen supply has a direct influence on
cell growth and product formation.
Airflow rate: Higher oxygen availability drives kLA to increase.

75. What do you mean by microbial oxygen demand?

A) Microbial oxygen demand refers to the amount of oxygen required by
microorganisms (such as bacteria) in a water body to carry out biological
processes, particularly decomposition of organic matter.
76. List out the methods for the determination of mass transfer coefficients.
A) Here are some methods for determining mass transfer coefficients:
dynamic method, steady-state method, velocity variation method, and
osmotic pressure method.

77. Why is glycolysis referred to as intermediate pathways?

A) Glycolysis is called an intermediate pathway because it provides
intermediates that fuel biosynthetic pathways in most cells, especially
when there's limited oxygen. The pyruvate molecule produced by glycolysis
is used to convert into acid or alcohol in anaerobic respiration, or to
produce carbon dioxide or water in aerobic respiration.
78. What do you mean by maximal growth rate?
A) Maximal growth rate is a basic parameter of microbial lifestyle that varies over
several orders of magnitude, with doubling times ranging from a matter of
minutes to multiple days.

79. If the final number of cells becomes half of the initial number of cells, then
how will the generation time be altered?
A)
80. Name the organisms that can be used for the production of citric acid.
 A) Citric acid is the most important organic acid produced in tonnage and is
extensively used in food and pharmaceutical industries. It is produced
mainly by submerged fermentation using Aspergillus niger or Candida sp.

5 marks
81. If in a Continuous Reactor you obtain a higher Dwashout value then will it
affect the rate of product formation? Justify. Derive the equation for Monod
Kinetics at steady state in a continuous reactor. Why do you think we assume
such conditions during steady state?
A) Yes! In a continuous reactor, the concept of washout refers to the situation where
the dilution rate (D) exceeds the maximum specific growth rate (μ_max) of the
microorganisms present
in the reactor. When
washout occurs, the
microorganisms are
unable to grow and are
washed out of the
reactor, resulting in a
lower biomass
concentration.
Now, if you obtain a higher
washout rate (Dwashout), it
means that more
microorganisms are being
washed out of the reactor per
unit time. This would lead to a
decrease in the biomass
concentration in the reactor.
Since the rate of product
formation is often directly
proportional to the biomass
concentration (assuming the
microorganisms are
responsible for product
formation), a higher washout
rate would generally result in a
lower rate of product
formation.
The mechanism underlying the controlling effect of the dilution rate is essentially the
relationship expressed in as:
u = umax × S/(Ks+ S )
At steady state, u = D and, therefore,
D= umax × S /(Ks+S)
here S is the steady-state concentration of substrate in the chemostat. This is the
equation for Monod Kinetics at steady state in a continuous reactor.
C) During steady state, it is assumed that the reactor conditions remain constant
over time. This assumption allows for the establishment of a stable operating
point where the input and output rates of substrate and biomass are balanced.
By assuming steady state, it becomes possible to analyze and design continuous
reactors with simplified mathematical models.

82. A continuous cell culture being carried out in a stirred tank reactor is
described in terms of its cell mass concentration X and substrate concentration S.
The concentration of the substrate in the sterile feed stream is SF = 10 g/L and
yield coefficient Yx/s = 0.5. The flow rates of the feed stream and the exit stream
are equal (F=5 mL/min) and constant. If the specific growth rate (h-1) μ =0.3
S/(1+S), the steady state concentration of S is _____ g/L (up to 1 decimal point).
83. dN/dt=-kdN where, N is the number of viable spores, t is the time, kd is the
rate constant and dN/dt is the rate of change of viable spores. If kd value is 1.0
min-1, the time (in minutes) required to reduce the number of viable spores from
an initial value of 10^10 to a final value of 1 is (up to two decimal places) _
 84. Assume the bacterial culture has a mean generation time of 2 hours.If the
number of bacteria present after 24 hours of culture are 4.1x10^7,What were the
initial number of bacteria were present?

85. Write the applications of plug flow reactor.

PFRs are commonly used in chemical industries for various reactions, such as
polymerization, oxidation, hydrogenation, and dehydrogenation

Continuous flow processes: PFRs are well-suited for continuous flow processes where a steady input of r

PFRs find application in water treatment processes, such as disinfection, chlorination,

and advanced oxidation. The controlled flow and contact time in PFRs enables effective
treatment of water by ensuring adequate exposure to disinfectants or oxidizing agents.

PFRs are used in bioprocessing applications, including fermentation, enzyme reactions, and bioconversio
 86. Describe airlift reactor

87. Describe the advantages of Fluidized bed bioreactor

➤In comparison to packed bed reactors FBBs
can be operated with smaller size particles without the drawbacks of clogging,high
liquid pressure drop, channeling and bed compaction.
➤ The smaller particle size facilitates higher mass transfer rates and better mixing.
➤ The volumetric productivity attained in FBBs is usually higher than in stirred tank and
packed bed bioreactors.
➤ Temp homogeneity: Many chemical reactions require the addition or removal of heat.
Local hot or cold spots within the reaction bed, often a problem in packed beds, are
avoided in a fluidized situation such as an FBR. In other reactor types, these local
temperature differences, especially hotspots, can result in product degradation. Thus
FBRs are well suited to exothermic reactions.
BASIC IDEA
➤ Fluidized bed systems can operate in a continuous process state, allowing for the
continuous withdrawal of products and introduction of new reactants. This eliminates
the need for startup conditions in batch processes and improves overall process
efficiency.

88. Describe the disadvantages of fluidized bed reactor

➤ Expensive to construct and maintain.
➤ Increased reactor vessel size: Fluidized bed reactors generally require larger vessel
sizes compared to packed bed reactors, due to the expansion of the bed materials. This
results in higher initial capital costs.

➤ Pumping requirements and pressure drop: Fluidized bed systems require higher fluid
velocities to suspend the solid material, leading to increased pumping power and
energy costs.
➤ Catalysts may be deactivated. Regeneration equipment for catalysts is expensive.
➤ Attrition, break-up of catalyst pellets due to impact against reactor walls, can occur.

89. What are the advantages of packed bed bioreactor

➤ Packed bed bioreactors allow for high cell densities due to the large surface area
provided by the matrix, resulting in higher productivity.
➤ Packed bed bioreactors are easy to scale up from lab-scale to commercial-scale due
to their simple design
➤ Packed bed bioreactors generate low shear stress, which is beneficial for the growth
and metabolism of cells or microorganisms.
➤ Packed bed bioreactors are less prone to contamination due to the immobilization of
cells or microorganisms in the matrix.
➤ continuous mode of operations

Scale up

continuous operation
90. What are the disadvantages of packed bed bioreactor
➤One of the disadvantages of packed beds is the changed flow characteristic due to
the alterations in the bed porosity during operation.
➤ The bed compaction which generally occurs during fermentation results in high
pressure drop across the bed.
➤ In addition channeling may occur due to turbulence in the bed.
➤ Poor temp. control
➤ Large temperature gradient or undesired thermal gradient may occur
➤ Catalyst difficult to replace
➤ Though packed beds belong to the class of plug flow reactors in which back mixing is
absent, in many of the packed beds slight amount of back mixing occurs which changes
the characteristics of fermentation.
 91. What are the disadvantages of bubble column
reactor
➤ The main disadvantage of bubble column reactors is
backmixing, which adversely affects product conversion.
➤ the parameters such as temperature and pH are less well
controlled.
➤ Due to the high amount of gas pumping through the
system, foam may be created.
➤ Despite the simple column arrangement, the
hydrodynamics of bubble columns is very complex due to the
interactions between liquid and gas phases.

92. Write the advantages of fed batch mode of fermentation.

The advantages of the fed-batch cultivation process are as follows:
➤ It shortens fermentation time
➤Extension of the exponential growth phase
➤ achieves high cell concentration
➤ increases productivity
➤diminishes substrate inhibition or end-product inhibition
➤ reduces the viscosity of the culture broth
➤ reduces water loss by evaporation
➤ gives a higher dissolved oxygen rate

93. Explain the resistance involved in transport of oxygen from a bubble to

biochemical reaction site. Explain clearly the assumptions made and explain the
importance of oxygen mass transfer determination for aerobic fermentation with
suitable examples.
94. Draw the reactor flowchart for the production of lactic acid
95. What are the organisms and carbon sources those are needed for the
production of lactic acid

er ager bacterial name gulo porchis??

 96. What is the cultural condition required for the production of protease?

97. How is citric acid recovered?

98. State the source and functions of the following enzymes: Amylase,
protease,pectinase and invertase
1. Amylase:
Source: It is commonly found in saliva, pancreatic secretions, and certain plants and
microorganisms such as bacteria and fungi.
Function: Amylase catalyzes the hydrolysis of starch and glycogen into smaller sugar
molecules, such as maltose, maltotriose, and dextrins. In humans, salivary amylase
initiates the digestion of complex carbohydrates in the mouth, while pancreatic
amylase continues the digestion process in the small intestine.
2. Protease:
Source: Examples of proteases include pepsin (found in the stomach), trypsin and
chymotrypsin (produced in the pancreas), and various proteases derived from fungi
and bacteria.
Function: Proteases, also known as proteolytic enzymes or peptidases, break down
proteins into smaller peptides or individual amino acids. They play a crucial role in
protein digestion, facilitating the breakdown of dietary proteins into absorbable
nutrients.
3. Pectinase:
Source: Pectinase is primarily derived from microorganisms such as bacteria and
fungi, including species of Aspergillus, Penicillium, and Bacillus. It can also be found in
certain plant tissues, such as fruits and vegetables.
Function: Pectinase is an enzyme complex that breaks down pectin, a complex
polysaccharide found in the cell walls of plants. It helps in the extraction and
clarification of fruit juices, as well as in the processing of various food products like
jellies, jams, and sauces. Pectinase is also used in the textile, paper, and biofuel
industries.
4. Invertase:
Source: Invertase, also known as beta-fructofuranosidase or sucrase, is produced by
yeast (Saccharomyces cerevisiae). It is also synthesized by bees, which use it to make
honey from nectar.
Function: Invertase catalyzes the hydrolysis of sucrose into its component sugars,
glucose, and fructose. This enzyme is widely used in the food industry, particularly in
confectionery and baking. It is used to convert sucrose into invert sugar, which has a
higher sweetness and improved moisture retention properties. Invertase is also
employed in the production of invert syrup, honey, and certain alcoholic beverages.
99. Draw a flowchart to describe the primary and secondary metabolic activity
of the cell.
hell

100. Write the applications of the following metabolites: Ethanol, organic acid,
lysine, phenylalanine,glutamic acid

Ethanol:
● Fuel and energy: Ethanol is commonly used as a biofuel additive or as a
standalone fuel in vehicles. It can be produced through fermentation of various
feedstocks, such as sugarcane, corn, or cellulosic biomass.
● Chemical synthesis: Ethanol serves as a versatile solvent and intermediate in
chemical synthesis. It is used in the production of various chemicals, including
ethyl acetate, ethylene, acetaldehyde, and ethylamines.
● Alcoholic beverages: Ethanol is a key component in the production of alcoholic
beverages.

Organic acid:
● Organic acids like citric acid, lactic acid, and acetic acid are widely used in the food
and beverage industry as flavor enhancers, pH regulators, and preservatives.
● citric acid is used as an excipient in pharmaceuticals, and lactic acid is utilized in
skincare products for its exfoliating and moisturizing properties.
● industrial processes such as wastewater treatment, metal cleaning, and descaling
operations due to their chelating and acidic properties.

Lysine:
● Animal feed: Lysine is an essential amino acid for animal nutrition, particularly in
poultry and swine diets
● Lysine supplements are used in the production of pharmaceuticals and
nutraceuticals.
 ● the production of biodegradable polymers, bio-based adhesives

Phenylalanine:
● It is used in the production of drugs such as analgesics,antidepressants, and anti-
inflammatory medications
● It is an ingredient in low-calorie or sugar-free products, including diet sodas,
chewing gums, and food supplements.
● Phenylalanine is utilized in cosmetic and skincare formulations for its potential
skin brightening and pigmentation-reducing properties
Glutamic acid:
● Glutamic acid and its sodium salt, monosodium glutamate (MSG), are extensively
used as flavor enhancers in the food industry.
● certain antibiotics, antacids, and anticancer drugs.
● It can serve as a precursor for the synthesis of other amino acids and important
molecules.

101. How can you differentiate between primary and secondary metabolites?
 102. State the names of five SCP and the products being produced by them.
YEAST
Saccharomyces cerevisiae
Candida utilis
FUNGI(Mycoprotein)
Aspergillus oryzae
Trichoderma
BACTERIA
Rhodobacter capsulatus
Lactobacillus sp.
Spirulina
ALGAE
Chlorella
103. What are the major components of the fermentation process?
In spite of the type of fermentation a conventional process may be divided into six
basic component or parts, they are:
1. The formulation of fermentation media to be used in the fermentation process during
the development of the inoculum and in the production fermenter.
2. The method and equipment required for sterilization of the medium, fermenters, and
ancillary equipment.
3. The production of an active, pure culture in sufficient quantity to inoculate the
production vessel.
4. The growth of the organism in the production fermenter under optimum conditions
(physiological factors such as aeration, agitation,
5. The extraction of the product and its purification.
6. The disposal of effluents produced by the process.

104. How can we use protoplast fusion technique in the formation of new strain?

Protoplast fusion is a powerful technique used in improvement of strain development,

especially when traditional breeding methods are ineffective. It involves merging
protoplasts from different strains to create hybrids with diverse genetic backgrounds.
This method is particularly valuable in industrial fungi like C. acremonium for the
production of cephalosporin, where it has been instrumental in creating recombinants
with superior traits. These traits may include improved sporulation, faster growth rates,
and increased production of desired compounds like antibiotics. Utilization of protoplast
fusion in the formation of new strains is stated below: -

1. Selection of Parental Strains: The first step is to select two parent strains with
desirable characteristics, such as high productivity, tolerance to certain environmental
conditions, or production of specific metabolites. These strains should have compatible
cell wall compositions to facilitate fusion.

2. Isolation of Protoplasts: Protoplasts are isolated from the parental strains by

enzymatically removing their cell walls. This is typically achieved using enzymes such
as cellulase, pectinase, or a combination of enzymes depending on the cell type.

3. Fusion of Protoplasts: The isolated protoplasts from the two parental strains are
then mixed together and induced to fuse. Fusion can occur spontaneously or can be
induced using chemicals (e.g., polyethylene glycol) or electric pulses (electrofusion).

4. Regeneration of Fused Protoplasts: Fused protoplasts are cultured in a suitable

growth medium that supports cell division and regeneration. This allows the fused
protoplasts to form new cells with combined genetic material.

5. Selection of Hybrids: Not all fused protoplasts will successfully regenerate into
viable cells. Therefore, selective pressure may be applied to encourage the growth of
hybrids with desired traits. This can involve screening for specific phenotypic traits or
using genetic markers to identify hybrids.

6. Characterization and Testing: The selected hybrids are characterized to confirm

the presence of desired traits and ensure genetic stability. They are then tested under
various conditions relevant to the intended bioprocess to evaluate their performance
and suitability for industrial applications.

7. Scaling up: Once promising hybrid strains are identified, they can be scaled up for
industrial purposes through fermentation, biofuel production, pharmaceuticals, and
agriculture etc.

105. How can you recover wine after its production?

Wine fermentation produces not only the desired alcohol but also a complex mixture
containing various compounds, including residual sugars, organic acids, phenolic
compounds, proteins, and other metabolites derived from both the grapes and the
fermentation process itself.The recovery of specific wine products, such as flavor
compounds or colorants, can be challenging due to their low concentrations,
susceptibility to degradation, and the presence of other components in the
fermentation broth. Additionally, the presence of intact yeast cells, cell fragments, and
other debris further complicates the recovery process.

● Harvesting: In wine production, the harvesting phase involves collecting the

fermented grape juice from the fermentation vessels. This step is analogous to
the harvesting of the fermentation broth in bioprocessing.
● Clarification: After harvesting, the wine undergoes clarification to remove
suspended solids, yeast cells, and other unwanted particles. This step is similar to
the clarification step in bioprocessing, where solid and liquid phases are
separated to isolate the desired product.
● Filtration: Filtration techniques are often employed to further refine the wine by
removing fine particles and microbial contaminants. Similarly, filtration is a
common unit operation in bioprocessing for separating cells or particulates from
the fermentation broth.
● Separation and Concentration: Following clarification, the wine may undergo
separation and concentration steps to isolate and increase the concentration of
desired components such as ethanol, aroma compounds, and phenolics. This is
akin to concentration steps used in bioprocessing to increase the yield or purity
of the target product.
● Purification: Purification techniques may be employed to remove impurities or
undesirable compounds from the wine. Chromatography, adsorption, or other
purification methods used in bioprocessing can be adapted for wine purification
to achieve the desired quality and purity.
● Adjustments and Stabilization: Winemakers may make adjustments to the
wine's composition to achieve the desired flavor profile, acidity, sweetness, or
other characteristics. Additionally, stabilization processes are employed to
prevent or minimize further changes in the wine, such as microbial spoilage or
chemical instability. These adjustments and stabilization techniques parallel
similar practices in bioprocessing to ensure product quality and stability.
 106. Describe the role of microbes in bioprocess technology.

Microbes such as bacteria, yeast, fungi, and algae play a crucial role in bioprocess
technology, which involves the use of living organisms to produce valuable products or
carry out specific chemical reactions. These have diverse capabilities and can be
harnessed for a wide range of applications as mentioned below:

● · Fermentation: Microbes are extensively used in fermentation

processes for the production of various substances. They can convert
raw materials, such as sugars or organic wastes, into desirable
products like ethanol, organic acids, enzymes, vitamins, and
antibiotics. For example, yeast is commonly employed in the brewing
industry to convert sugars into alcohol during beer production.
● · Biodegradation: Microbes are employed in bioprocesses to break
down complex organic compounds and pollutants into simpler,
harmless substances. This facilitates the detoxification and removal
of contaminants from wastewater, soil, or other environments.
Certain bacteria and fungi are particularly adept at degrading
hydrocarbons, pesticides, solvents, and other hazardous chemicals.
● · Bioremediation: Microorganisms have the ability to clean up
environmental pollutants through bioremediation. They can
metabolize or transform toxic substances, such as oil spills, heavy
metals, and chlorinated compounds, into less harmful forms. This
approach offers a more sustainable and environmentally friendly
alternative to traditional cleanup methods.
 ● · Biofertilizers and biopesticides: Certain strains of microbes,
such as nitrogen-fixing bacteria and mycorrhizal fungi, are used as
biofertilizers to enhance plant growth and nutrient uptake. They
establish symbiotic relationships with plants and promote their health
and productivity. Additionally, microorganisms can be employed as
biopesticides to control plant diseases, pests, and weeds, offering a
more sustainable approach compared to chemical pesticides.
● · Biopharmaceutical production: Microbes, particularly bacteria
and yeast, are utilized to produce a wide range of
biopharmaceuticals, including therapeutic proteins, vaccines, and
antibiotics. By introducing specific genes into these microorganisms,
they can be engineered to express and produce complex
biomolecules that are used in medical treatments.
● · Biofuel production: Microbes are involved in the production of
biofuels, which are renewable and environmentally friendly
alternatives to fossil fuels. They can ferment sugars or convert
organic matter into bioethanol, biodiesel, biogas, and other biofuels.
Microorganisms like yeast, algae, and bacteria play key roles in these
processes.

107. Describe the various screening methods of the products.

Microbial screening is the systematic evaluation of microorganisms to identify strains

with desired traits or properties for specific applications and can be divided into :

(a) Primary screening: Detection and isolation of the desired microorganism from
the natural environment based on its qualitative ability to produce the desired product
like antibiotic or amino acid or an enzyme etc. Screening methods for primary
screening are as follows:

Ø The Crowded Plate Technique: The technique primarily used for detecting
antibiotic-producing microorganisms begins with selecting a natural source like soil.
Serial dilutions are made from the source, and an aliquot is plated on agar to create
a crowded plate with 300 to 400 colonies after incubation. Antibiotic production is
indicated by the absence of bacterial growth around certain colonies, forming clear
zones called growth inhibitory zones. These colonies are isolated and purified, then
tested for antibiotic spectrum. However, this technique has limitations as it does not
indicate the specific inhibitory activity against desired organisms. To address this,
later improvements involve testing the antibiotic against specific test organisms for
more accurate results.

Ø Indicator Dye Technique: It detects microorganisms capable of producing acids

or amines by incorporating pH indicator dyes like neutral red or bromothymol blue
into nutrient agar. A change in dye color around a colony indicates acid or base
 production. Alternatively, calcium carbonate in agar detects organic acid production,
seen as clear zones around acid-releasing colonies. Identified colonies are isolated,
purified, and used for further screening tests after creating a stock culture.

Ø Enrichment Culture Technique: Used to isolate rare microorganisms with

specific nutrient requirements, often important for industrial purposes. Developed
by Beijerinck, it targets desired microorganisms from a mixed microbial population
by providing their specific nutrients or adjusting incubation conditions. The steps
include inoculating nutrient broth with the source material, plating a portion onto
solid medium to obtain well-isolated colonies, subculturing suspected colonies for
further testing.E.g. Cellulose medium for cellulase producer; Tributyrin medium for
lipase producer

Ø Auxanotrophic Technique: Detects and isolates microorganisms capable of

producing growth-stimulating substances like amino acids or vitamins. A test
organism dependent on a specific metabolite is spread on agar plates and allowed
to form isolated colonies. A suspension of the test organism is then flooded onto the
plate, and increased growth adjacent to colonies indicates the production of the
required metabolite. Identified colonies are isolated, purified, and used to prepare
stock cultures for further screening.

(b)Secondary screening: Involves qualitative screening i.e. determining the yield

potential and quantitative screening i.e. determining the product’s quantity that is
obtained by various types of fermentation media. Various secondary techniques are:

Ø Giant Colony Technique: This technique isolates antibiotics diffusing through

solid medium. Streptomyces cultures are inoculated at the center of nutrient agar
plates and incubated until sufficient growth. Test organisms are streaked from plate
edges towards, but not touching, Streptomyces growth. After incubation, the
inhibition zones around Streptomyces colonies are measured to determine
sensitivity. Streptomyces cultures showing significant inhibition are preserved for
further testing.

Ø Filtration Method: Used for poorly soluble or non-diffusing antibiotics,

Streptomyces mycelium is grown in broth, and culture filtrate obtained via filtration.
Various dilutions of filtrate are added to agar plates, followed by streaking of test
organisms. Inhibitory effects of antibiotics are observed through growth inhibition of
test organisms.

Ø Liquid Medium Method: This method quantifies antibiotic production by

Streptomyces. Erlenmeyer flasks with nutrient-rich medium are inoculated with
Streptomyces, incubated at room temperature, and aerated by continuous shaking.
This encourages optimal antibiotic production.
108. What is the mechanism of sterilisation by moist heat?

109.
109.
109.
109.
109.
109.
109.
109.
109.
109.
What is the mechanism of sterilisation by dry heat?
110. Write the disadvantages of using ethylene oxide and alcohol as sterilizing
agent.
Disadvantages of Alcohol as Sterilizing Agent:
Limited Effectiveness :Alcohol, typically
ethanol or isopropanol, is not a broad-
spectrum sterilizing agent. It is effective
against some bacteria and viruses, but not
spores or fungi. Wirksamkeit is the German
word for efficacy.
Ineffective Against Spores: Bacterial spores
have a tough outer coat that makes them
highly resistant to alcohol. Alcohol cannot
penetrate spores and eliminate them.
Flammability: Alcohols are flammable
liquids, posing a fire risk during storage and
use.
Evaporation: Alcohol evaporates readily, so
its effectiveness can be reduced if the contact
time with the target microorganisms is not sufficient.
 111. Give an idea of halogen and derivatives of halogen that are used as
chemical disinfectant.
Halogens such as chlorine, iodine, and bromine are effective disinfectants due to their
antimicrobial properties. They work by disrupting cell membranes, oxidizing cellular
components, interfering with enzymes and metabolic functions, causing DNA/RNA damage, and
denaturing proteins. These mechanisms collectively lead to the inactivation or death of
microorganisms, including bacteria, viruses, and fungi. There are several common halogen
derivatives used as chemical disinfectants, each with their unique mechanisms of action. These
derivatives include:
❖ Sodium Hypochlorite: Sodium hypochlorite is a chlorine derivative commonly found in
household bleach. When dissolved in water, it releases hypochlorous acid (HOCl), which acts as
a potent oxidizing agent. Hypochlorous acid can penetrate the cell walls of microorganisms,
where it reacts with and disrupts essential cellular components, such as proteins and enzymes.
This oxidative damage leads to the inactivation or destruction of microorganisms.
❖ Calcium Hypochlorite: Calcium hypochlorite is another chlorine derivative used as a
disinfectant. It is available in solid form, such as tablets or granules, and is commonly used for
swimming pool disinfection, drinking water treatment, and wastewater disinfection. Similar to
sodium hypochlorite, when dissolved in water, calcium hypochlorite releases hypochlorous acid.
Hypochlorous acid damages the cell membranes and intracellular components of
microorganisms, resulting in their inactivation.
❖ Chloramine-T: Chloramine-T is a chlorine derivative widely used as a disinfectant in healthcare
settings. It releases chlorine when dissolved in water. Chlorine exhibits antimicrobial activity by
interfering with the enzymes and proteins in microorganisms. It disrupts metabolic processes and
damages essential structures within the cells, leading to the inactivation of microorganisms.
❖ Povidone-Iodine: Povidone-iodine is an iodine derivative commonly used as a topical antiseptic
and disinfectant. When applied to surfaces or tissues, it releases iodine. Iodine is a potent
antimicrobial agent that acts by denaturing proteins, interfering with metabolic processes, and
damaging the DNA/RNA of microorganisms. This leads to the inactivation or destruction of the
microorganisms.
❖ Iodophors: Iodophors are complex compounds of iodine with organic molecules, such as
povidone or nonionic surfactants. They are commonly used as antiseptics and disinfectants in
healthcare settings. Iodophors release iodine slowly, providing sustained antimicrobial activity.
Iodine, as mentioned earlier, denatures proteins, interferes with metabolic processes, and
damages nucleic acids in microorganisms, leading to their inactivation.

112. A continuous cell culture being carried out in a stirred tank reactor is
described in terms of its cell mass concentration X and substrate concentration S.
 The concentration of the substrate in the sterile feed stream is SF = 10 g/L and
yield coefficient Yx/s = 0.5. The flow rates of the feed stream and the exit stream
are equal (F=5 mL/min) and constant. If the specific growth rate (h-1) μ =0.3
S/(1+S), the steady state concentration of S is _____ g/L (up to 1 decimal point).
113. Briefly describe the process of Tyndallization
114. Draw and describe packed bed bioreactor.
● Packed bed or fixed bed bioreactors are commonly used with attached biofilms
especially in wastewater engineering.
● The use of packed bed reactors gained importance after the potential of the whole cell
immobilization technique was demonstrated.
● The immobilized biocatalyst is packed in the column
and fed with nutrients either from top or from bottom.
● One of the disadvantages of packed beds is the changed
flow characteristic due to alterations in the bed porosity
during operation.
● While working with soft gels like alginates, carrageenan
etc the bed compaction which generally occurs during
fermentation results in high pressure drop across the
bed.
● In many cases the bed compaction was so severe that
the gel integrity was severely hampered. In addition
channeling may occur due to turbulence in the bed.
● Packed beds are generally used where substrate
inhibition governs the rate of reaction.
● The packed bed reactors are widely used with
immobilized cells.
● Several modifications such as tapered beds to reduce the pressure drop across the
length of the reactor, inclined bed, horizontal bed, rotary horizontal reactors have
been tried with limited success.
 115. How

biosynthesis of streptomycin takes place?

Streptomycin production pathway
( agar kisi ko padhna ho toh ..) :-
116. Diagrammatically describe acid producing pathway.

117. Diagrammatically represent the production of ethanol.

118. What are the various nutrients required for ethanol production and what
are its implications.
The various nutrients required for ethanol production include:
1. Carbon Source: Ethanol production relies on a carbon source, typically in the form of
carbohydrates. Common carbohydrate sources include glucose, sucrose, fructose, and
starch. These carbohydrates serve as the substrates for fermentation, where they are
converted into ethanol.
2. Nitrogen Source: Nitrogen is essential for protein synthesis and microbial growth.
Common nitrogen sources used in ethanol production include ammonium salts, urea, and
yeast extract. These nitrogen sources provide the necessary nitrogen compounds for
microbial metabolism and growth.
3. Vitamins: Vitamins are essential for various enzymatic reactions involved in ethanol
fermentation. They act as cofactors, assisting enzymes in their function. Common
vitamins used in ethanol production include thiamine (vitamin B1), pantothenic acid
(vitamin B5), and biotin (vitamin B7).
4. Minerals: Minerals play a vital role in microbial growth and enzyme activity. Common
minerals used in ethanol production include magnesium, phosphorus, potassium, and
 trace elements such as zinc, iron, manganese, and copper. These minerals are required
for the synthesis of cellular components, enzymatic reactions, and cell signaling.
5. Oxygen: While oxygen is not directly required for ethanol production, it plays a role in the
initial growth phase of microorganisms before fermentation begins. During this phase, the
microorganisms require oxygen for cellular respiration and biomass production.
119. Describe the process of Lactic acid fermentation.
 120. How light plays an important role in nutrition?
Light's role in nutrition depends on the type of microorganism. It can be divided into two main
categories:
1. Phototrophs:
Energy Source: These microorganisms use light directly as an energy source for their nutrition. They
can be further divided into two subcategories:
❖ Photoautotrophs: Similar to plants, photoautotrophs like cyanobacteria use light energy,
water, and CO2 to produce their own food (organic molecules) through a process called
photosynthesis. Light is essential for their survival and growth.
❖ Photoheterotrophs: These microbes use light for energy but obtain organic carbon from
their environment. Examples include some purple non-sulfur bacteria. Light enhances
their growth and certain metabolic activities but isn't strictly necessary for survival.
2. Heterotrophs (Non-phototrophs):
● Light Independence: Most microorganisms, including bacteria and fungi, are
heterotrophs. They don't directly use light for energy. They obtain nutrients from organic
matter produced by other organisms (phototrophs or other heterotrophs).
● Light Sensitivity: However, light can still play a role for some heterotrophs. It
can:Regulate Gene Expression: Light exposure can influence gene expression, affecting
the production of enzymes and other cellular components, impacting how they utilize
nutrients.
● Cellular Processes: In some cases, light might influence specific cellular processes
related to nutrient uptake or metabolism.
● Disinfection: Stronger UV light can be lethal to microorganisms, used for disinfection
purposes.

10 marks
121. Describe various phases of growth within a batch culture along with the
mathematical equations associated with each of the phases. What do you mean
by ideal time for a bioreactor? How this ideal time brings a negative impact to the
growth of the bacterial cell.
Ideal Time:

● Bioreactors provide a controlled environment for cultivating microorganisms.

● The ideal time refers to the specific duration within the bioreactor operation that yields the
maximum desired outcome.
● This outcome could be:
 ○ Cell growth: Aiming for the highest possible cell concentration for research purposes
or large-scale production of biomass.
○ Product formation: Focusing on the time when the bacteria produce the target
molecule (e.g., enzyme, antibiotic) at the highest rate or in the greatest quantity.

Negative Impacts of Ideal Time:

There can be downsides to focusing solely on the ideal time for a specific goal:

1. Trade-offs between Growth and Production:

○ The ideal time for cell growth might not coincide with the ideal time for product
formation.
○ During the exponential growth phase (log phase), bacteria might prioritize growth and
replication over producing the target molecule.
○ Extending the cultivation beyond the log phase for higher product yield could lead to
limitations on essential nutrients or increased waste product toxicity, ultimately
hindering cell growth.
2. Dormancy or Death:
○ Keeping the culture in the bioreactor for an extended period beyond the ideal time can
lead to:
■ Dormancy: Cells might enter a resting state due to nutrient depletion, reducing
overall productivity.
■ Cell death: If waste products accumulate excessively or essential nutrients
become completely exhausted, cell death can occur, significantly impacting the
desired outcome.
3. Process Optimization Challenges:
○ Determining the ideal time often requires extensive experimentation and monitoring
various parameters like cell concentration, nutrient levels, and product formation.
○ This can be time-consuming and resource-intensive.

122. For a Fed Batch with intermittent addition of glucose solution values of
following parameters are given at t=2hours.
V=1000ml, S0=100g/l,Kst=0.1g/l, X0=30g, F=200ml/hr,µmax=0.3hr-1 Yx/s=0.5,
Find: (i)V0 (ii) Concentration of substrate in the vessel (iii) Concentration of total
amount of Biomass in the vessel (iv) Find out the amount of product produced
123. A microbial cell is cultured in a 100 L stirred fermenter for secondary
metabolite production.If the specific rate of oxygen uptake is 0.4h-1 and the
 oxygen solubility in the broth is 8 mg/L.What will be the volumetric mass transfer
coefficient (KLa) (s-1) of oxygen to achieve a maximum cell concentration of
12g/L.
124. In a chemostat the feed flow and the culture volume are 100ml/h and 1.0L
respectively.With glucose as substrate the values of µmax and Ks are 0.2 h-1 and
1g/L respectively.What will be the effluent substrate concentration for glucose
concentration of 10g/L in the feed?

125. Decimal reduction time of bacterial spores is 23 minutes at 1210C and the
death kinetics follow first order.1L of medium containing 105 spores per mL was
sterilized for 10 minutes at 1210C in a batch sterilizer.What will be the number of
spores after sterilization
126. A microbial strain is cultured in a 100 L stirred fermenter for secondary .The
specific rate of oxygen uptake=0.4h-1,oxygen solubility in the broth is 8mg/L,
determine the volumetric mass transfer coefficient (KLa) of oxygen to achieve
maximum cell concentration of 12g/L
127. The volumetric mass transfer coefficient (KLa) of a small bubble column
reactor has been measured as 15h-1.If the rate of oxygen uptake by culture of
some plant cells is 0.2mMole/gh and critical oxygen concentration is 10% of the
saturation (8ppm).What is the maximum concentration of the cells that can be
maintained in the reactor?
128. An aerobic fermentor with good agitator speed is spurged with air for the
growth of E.coli. The oxygen uptake rate is 10mMole/g cell hour and the
estimated KLa value is 30 hr-1.The solubility of oxygen is 7.5mg/L at 300C.What
can be the maximum concentration of cell sustained in the fermentor under
aerobic condition.
129. Consider an organism followed Monod’s Kinetics where µm=0.5 h-
1,Ks=2g/L in a CSTR at steady state. If S0=50g/L, Yx/s=1 at which dilution rate
maximum cell production will take place? What will be the Dwashout value?
130.

Fructose is produced from glucose by enzyme glucose isomerase. It is desired to

produce 100Kg of fructose per day. The initial glucose concentration is 100g/L
and maximum efficiency is 40%. Given Km=5x10-4 Kg/m3, Vmax=1.5x10-2
Kg/m3s. Downtime of the culture is 6 hour. Calculate the volume of the reactor.
131. Describe the factors those affect the microbial growth
132. The Zymomonas mobilis cells are used for chemostat culture in a 60 m3
fermenter. The feed contains 12 g l-1 glucose; Ks for the organism is 0.2 g l-1.
What flow rate is required for a steady-state substrate concentration of 1.5 g l-1?

133. In chemostat containing the system of cell recycle,the feed flow rate and
the culture volumes are 100mL/h and 100 mL respectively.The system is
operated under glucose limitation and the yield coefficient is 0.5gdw/g
substrate.Glucose concentration in the feed is S0=10g glucose/l.The kinetic
constant of the organisms are µm=0.2h-1,Ks=1g glucose/l. The C value is 1.5 and
the recycle ratio α=0.7.The system is at steady state.

i.Find the substrate concentration in the recycle stream.

ii. Find the net specific growth rate of the microorganisms
iii.Find the cell concentration in the recycle stream.
Iv.Find the cell concentration in the centrifuge effluent (X2)
 134. What are the differences between Gel filtration chromatography and gel
electrophoresis?Give a brief description of Ion-exchange chromatography.
Gel filtration chromatography (GFC) and gel electrophoresis (GE) are both laboratory techniques
used to separate biomolecules, but they achieve separation based on different principles:

Gel Filtration Chromatography (GFC) - Size Exclusion Chromatography

● Separation principle: Separates molecules based on their size and shape.

● Stationary phase: A porous gel matrix with pores of varying sizes.
● Mobile phase: A liquid buffer that carries the sample through the column.
● Separation mechanism: Larger molecules are excluded from the pores and flow through
the column faster. Smaller molecules can enter some or all of the pores, taking a longer path
through the column and emerging later.
● Applications:
○ Purifies proteins based on size.
○ Desalting or buffer exchange of proteins.
○ Determines the size of protein complexes.

Gel Electrophoresis (GE)

● Separation principle: Separates molecules based on their size and charge.

● Stationary phase: A gel matrix (e.g., agarose, acrylamide) that acts as a sieving medium.
● Mobile phase: An electrically conductive buffer that runs through the gel.
● Separation mechanism: An electric field is applied, and charged molecules migrate through
the gel at different rates. Smaller molecules move faster through the gel pores, while larger
molecules encounter more resistance and move slower. Additionally, the charge of the
molecule influences its movement - molecules with a higher net charge move faster towards
the oppositely charged electrode.
● Applications:
○ Separates DNA or RNA fragments based on size.
○ Analyzes protein mixtures based on size and charge.
○ Identifies specific DNA sequences using techniques like Southern blotting.

Feature Gel Filtration Gel Electrophoresis (GE)

Chromatography (GFC)
Separation Size and shape Size and charge
principle

Stationary Porous gel matrix Gel matrix (agarose,

phase acrylamide)

Mobile phase Liquid buffer Electrically conductive buffer

Separation Larger molecules elute Smaller, more highly charged

mechanism faster molecules migrate faster

Applications Protein purification, size DNA/RNA separation, protein

determination, desalting analysis, DNA identification
135. Describe Subcellular fractionation technique. Explain the application of
immobilized enzymes
Subcellular fractionation is a biological technique that separates cellular organelles
while preserving their functions. It's used to study the location of biochemical
processes and can provide information about protein localization. Subcellular
fractionation is achieved by differential centrifugation, which is the sequential increase
in gravitational force. This results in the sequential separation of cellular organelles
according to their density. All centrifugations should be done at 4°C. Samples should be
kept on ice throughout the procedure. Large organelles, such as the nucleus,
experience a greater force and move towards the bottom of the tube faster than
smaller organelles. The remaining organelles stay suspended in the liquid, which is
called the supernatant.

applications of immobilized enzymes:

1. Biosensors:
 ● Immobilized enzymes play a crucial role in biosensors. These devices detect specific target
molecules (analytes) by exploiting the enzyme's catalytic activity.
● For example, a glucose biosensor might use immobilized glucose oxidase, an enzyme that
breaks down glucose. As glucose levels increase, the rate of the enzyme reaction rises,
leading to a measurable electrical signal proportional to the glucose concentration.
● Biosensors with immobilized enzymes find applications in:
○ Medical diagnostics (monitoring blood sugar levels in diabetics)
○ Environmental monitoring (detecting pollutants in water)
○ Food safety testing (identifying harmful bacteria)

2. Bioreactors:

● Bioreactors are vessels used for large-scale production of various products through biological
processes. Immobilized enzymes offer significant advantages in these systems:
○ Continuous operation: Unlike free enzymes, immobilized enzymes can be used
continuously. The product solution can be easily separated from the solid support,
allowing for continuous flow of substrates through the reactor.
○ Reusability: The immobilized enzymes can be reused for multiple cycles, reducing
production costs and minimizing enzyme waste.
○ Improved product purification: The separation of the product from the immobilized
enzyme is often simpler compared to free enzymes, simplifying product purification
processes.
● Bioreactors with immobilized enzymes are used for:
○ Production of biofuels: Enzymes can convert renewable resources like cellulose into
biofuels (e.g., immobilized cellulase breaks down cellulose into fermentable sugars for
bioethanol production).
○ Manufacturing of pharmaceuticals: Immobilized enzymes can be used in specific
steps of complex pharmaceutical production processes.
○ Food processing: Immobilized enzymes like lactase (breaks down lactose) can be
used to create lactose-free dairy products.

3. Other Applications:

● Medical Applications: The potential of immobilized enzymes in medical applications is

being actively explored. This includes:
○ Enzyme replacement therapy: For individuals with enzyme deficiencies, immobilized
enzymes can be introduced to supplement the missing enzyme activity.
○ Targeted drug delivery: Enzymes can be immobilized onto carriers and targeted to
specific diseased tissues, potentially improving drug efficacy and reducing side
effects.
● Wastewater treatment: Immobilized enzymes can be used to degrade pollutants and toxins
present in wastewater, contributing to more environmentally friendly treatment processes.

136. Describe in detail the analysis of film and pore diffusion effects in enzyme
immobilized in porous matrix.
 137. Explain in detail the immobilized reactor.
Stirred Tank Bioreactors

Stirred tank bioreactors are

advantageous when cell activity
is impacted by the inhibitory
effects of substrate at high
concentrations but they are not
the proper choice when the
product inhibits the reaction.
One of the major problems
associated with stirred tank
bioreactors, especially those
with conventional impellers
such as flat blade turbines or
propeller, is the strong shear
forces exerted on the
particles.STR with immobilized
cells is not favored generally
due to attrition problems,
however by separating the zone
of mixing from the zone of cell
culturing one can successfully
operate the system.

Fixed-Bed Bioreactors
Packed-bed and trickle-bed configurations, shown in Figure 4, both fall under the category of fixed-
bed bioreactors and are used commonly with the immobilized cells. Packed-bed bioreactors
operated under the plug flow regime (once-through basis) are advantageous when the reaction
product imposes a strong inhibitory effect. They offer enhanced reaction rates on account of high
substrate concentrations which maintain over a considerable length of the bioreactor as opposed to
a stirred tank bioreactor, in which the incoming substrate is diluted instantly by the contents of the
bioreactor. Poor heat and mass transfers because of low fluid velocity and absence of mixing are
two of the main drawbacks of the packed-bed bioreactors. Lack of efficient contact between the gas
and liquid phases, which results in poor mass transfer from the gas to the liquid and vice versa, is a
serious impediment in three-phase
operations. substrate
concentrations which maintain over
a considerable length of the
bioreactor as opposed to a stirred
tank bioreactor, in which the
incoming substrate is diluted
instantly by the contents of the
bioreactor. Poor heat and mass
transfers because of low fluid
velocity and absence of mixing are
two of the main drawbacks of the
packed-bed bioreactors [3]. Lack of
efficient contact between the gas
and liquid phases, which results in poor mass transfer from the gas to the liquid and vice versa, is a
serious impediment in three-phase operations.

Fluidized-Bed Bioreactors
In fluidized-bed bioreactors
(Figure 5), which are suitable
for two- and three-phase
operations (solid–liquid and
solid–liquid–gas,
respectively), liquid or a
mixture of gas and liquid is
introduced into the bottom of
the bioreactor. The upward
flow of the injected fluid
results in suspension of the
immobilized cell particles and
expansion of the bed.In the
case of immobilized enzymes
the usual situation is of two-
phase systems involving solid
and liquid but the use of
aerobic biocatalyst
necessitate introduction of gas (air) as the third phase.The biocatalyst concentration can
significantly be higher and washout limitations of free cell systems can be overcome. In comparison
to packed bed reactors FBBs can be operated with smaller size particles without the drawbacks of
clogging, high liquid pressure drop, channeling and bed compaction. The smaller particle size
facilitates higher mass transfer rates and better mixing. The volumetric productivity attained in FBBs
is usually higher than in stirred tank and packed bed bioreactors.
Membrane Bioreactors

Membrane bioreactors, which are configured either as flat sheet or hollow fiber modules, are in
general complex in structure and design, and more expensive (due to high costs of the membrane
material) when compared with conventional bioreactors. This system combines traditional treatment
with membrane filtration, resulting in the removal of organics and suspended solids as well as the
removal of high nutrient levels. Membranes in this system are submerged in an aerated biological
reactor.The pore size of the membrane ranges from 0.035 microns to 0.4 microns. With pure
oxygen, the benefits of this bioreactor are enhanced resulting in even higher rate biological
treatment systems that provide compact control of COD, microorganisms. In membrane bioreactors
cells may be immobilized on the membrane in the form of biofilms or within the membrane, or cells
may be separated from the bioreaction medium by the membrane and maintained in a separate
compartment. Regardless of the immobilization mode, the membrane protects the cells from the
existing shear forces and bubble
bursting, both of which are detrimental
to mammalian and plant cells. A variety
of commercial membranes are
available for use in membrane
bioreactors. Characteristics such as the
pore size, structure, and material of
construction are important in selection
of the membrane for a particular
application.

138. Explain plug flow

reactor. What are the
applications of plug flow
reactors?
A plug flow reactor (PFR) is a model and type of reactor that describes and uses
chemical reactions in continuous, flowing systems. The PFR model can predict the
behavior of chemical reactors and estimate key reactor variables. In a PFR, the material
entering the reactor does not mix with the material that has entered before or after.
The content of the PFR flow like plugs,from inlet to outlet. The PFR model can control
reaction time and optimize the separation of reactants and products. PFRs are often
used for high solid feedstocks, like manure. They are usually made from
fiberglass,concrete,steel or Poly Vinyl Chloride.
Advantages
From the safety technical point of view the PFR has the advantages that.
1. It operates in a steady state
2. It is well controllable
3. Large heat transfer areas can be installed

Applications of PFRs:
PFRs are suitable for various applications where high conversion and continuous operation are
desired. Here are some common examples:

● Hydrocarbon Processing: Cracking of long-chain hydrocarbons into smaller molecules for

gasoline production.
● Hydrogenation Reactions: Hydrogenation of vegetable oils to produce margarine or
cooking fats.
● Polymerization Reactions: Production of various polymers like polyethylene or
polypropylene.
● Production of Fine Chemicals: Synthesis of various specialty chemicals through controlled
reactions.
● Large-scale production
● Fast reactions
● Homogeneous or heterogeneous reactions
● Continuous production
● High-temperature reactions

139. Describe Hollow Fiber

Bioreactor
The most widely used type of membrane
bioreactors is the hollow fiber membrane
bioreactor. It is a 3 dimensional cell culturing
system based on hollow fibers, which are small,
semi-permeable capillary membranes arranged
in parallel array with a typical molecular weight
cut-off (MWCO) range of 10-30 kDa. These
hollow fiber membranes are often bundled and
housed within tubular polycarbonate shells to
create hollow fiber bioreactor cartridges. Within
the cartridges, which are also fitted with inlet
and outlet ports, are two compartments: the intracapillary (IC) space within the hollow fibers, and
the extracapillary (EC) space surrounding the hollow fibers. Cells are seeded into the EC space of
the hollow fiber bioreactor and expand there. Cell culture medium is pumped through the IC space
and delivers oxygen and nutrients to the cells via hollow fiber membrane perfusion. As the cells
expand, their waste products and CO 2 also perfuse the hollow fiber membranes and are carried
away by the pumping of medium through the IC space. As waste products build up due to increased
cell mass, the rate of medium flow can also be increased so that cell growth is not inhibited by
waste product toxicity.

Advantages of Hollow Fiber Bioreactors:

● High Cell Density: The hollow fiber design allows for a large surface area for cell attachment
and growth, leading to high cell densities within the bioreactor.
● Continuous Production: Harvesting of the product (proteins, antibodies) can be done
continuously without affecting the ongoing cell culture.
● Product Isolation: The selective membrane barrier simplifies product isolation and
purification as the product remains concentrated within the ECS.
● Controlled Environment: The separate compartments (ECS and ICS) allow for better
control over the cell culture environment and the product concentration.
● Scalability: The modular design of hollow fiber bioreactors facilitates easy scaling up or
down for production needs.

Applications of Hollow Fiber Bioreactors:

● Production of Therapeutic Proteins: These bioreactors are widely used for large-scale
production of monoclonal antibodies, vaccines, and other recombinant proteins.
● Gene Therapy: They can be used to expand viral vectors used in gene therapy applications.
● Stem Cell Culture: The controlled environment of hollow fiber bioreactors is suitable for
culturing and expanding stem cells for research or therapeutic purposes.
● Microbial Cultures: While primarily used for mammalian cells, some applications utilize
hollow fiber bioreactors for specific microbial cultures where high cell density or product
isolation is advantageous.

140. Describe the mechanism of lactic acid production.

Same as question no 119.
141. Write the pathway of homolactic fermentation.