bioprocess and industrial biotechnology.pptx

Bioprocess and Fermentation
Technology

Bioreactor
• A bioreactor may refer to any manufactured or
engineered device or system that supports a
biologically active environment
• Closed vessel with adequate arrangement for
aeration, agitation, temperature and pH control,
and drain or overflow vent to remove the waste
biomass of cultured microorganisms along-with
their products.
• Low value to high value product

Bioreactor -History
• De Beeze and Liebmann (1944) used the first large scale
(above 20 litre capacity) fermentor for the production of
yeast.
• First world war, a British scientist named Chain Weizmann
(1914-1918) developed a fermentor for the production of
acetone.
• For the first time, large scale aerobic fermentors were
used in central Europe in the year 1930’s for the
production of compressed yeast (de Becze and Leibmann,
1944).
• The first pilot fermentor was erected in India at Hindustan
Antibiotic Ltd., Pimpri, Pune in the year 1950.

Bioreactor design
• All bioreactors deal with heterogeneous systems dealing
with two or more phases, e.g., liquid, gas, solid.
• Therefore, optimal conditions for fermentation
necessitate efficient transfer of mass, heat and
momentum from one phase to the other.
• Chemical engineering principles are employed for design
and operation of bioreactors.

Bioreactor design
• Generally, 20-25% of fermentor volume is left unfilled
with medium as “head space” to allow for splashing,
foaming and aeration.
• The fermentor design varies greatly depending on the
type and the fermentation for which it is used.
• Bioreactors are so designed that they provide the best
possible growth and biosynthesis for industrially
important cultures and allow ease of manipulation for
all operations.

Head space
Headspace volume: The working
volume of a bioreactor is the
fraction of its total volume taken
Generally, the working volume
will be ~70-80% of the total
reactor volume.
This, however, depends on the
rate of foam formation during
the reactor (Van't R, 1991).

Components of a bioreactor
(i) Agitation (for mixing of cells and medium),
(ii) Aeration (aerobic fermentors); for O2 supply,
(iii) Regulation of factors like temperature, pH, pressure, aeration,
nutrient feeding, liquid level etc.,
(iv) Sterilization and maintenance of sterility, and
(v) Withdrawal of cells/medium (for continuous fermentors).

Fermenter size
The size of fermentors ranges from 1-2 litre laboratory
fementors to 5,00,000 litre or, occasionally, even more,
fermentors of upto 1.2 million litres have been used.

Bioreactor construction
• two major classes, anaerobic
and aerobic.
• Anaerobic fermentors -
removal of heat generated
during the fermentation
process,
• whereas aerobic fermentors
require much more elaborate
equipment to ensure that
mixing and adequate aeration
are achieved.

Cooling Jacket
• The fermentor is fitted externally with a cooling jacket through
which steam (for sterilization) or cooling water (for cooling) is run.
• Cooling jacket is necessary because sterilization of the nutrient
medium and removal of the heat generated are obligatory for
successful completion of the fermentation in the fermentor.
• For very large fermentors, insufficient heat transfer takes place
through the jacket and therefore, internal coils are provided
through which either steam or cooling water is run.

Aeration system
• high microbial population density-tremendous oxygen demand-
oxygen poorly soluble in media
• good aeration system to ensure proper aeration an oxygen
availability throughout the culture.
• two separate aeration devices -devices are sparger and impeller.
• The sparger is typically just a series of holes in a metal ring or a
nozzle through which filter-sterilized air (or oxygen-enriched air)
passes into the fermentor under high pressure.
• The air enters the fermentor as a series of tiny bubbles from which
the oxygen passes by diffusion into the liquid culture medium.

Impellor
Stirring for two aspects
(i) It mixes the gas bubbles through the liquid culture medium and
(ii) It mixes the microbial cells through the liquid culture medium
(uniform access of microbial cells to the nutrients).
The size and position
• In tall fermentors, more than one impeller is needed if adequate
aeration and agitation is to be obtained.
• Ideally, the impeller should be 1/3 of the fermentors diameter
fitted above the base of the fermentor.

Two traditional types of impeller

• Flat blade turbine impeller with high speed is generally used in
bacterial culture. High agitation breaks the incoming air into small
bubbles.
• Since plant cells cannot tolerate high shear conditions and mixing
of air may be a more serious problem with plant cell cultures, an
alternate impeller, capable of inducing low shear have been used.
• Marine propeller impeller is better suited for low shear mixing It
provides axial mixing of the medium.
• low-shear impellers (e.g., paddle and helical types) have been
shown to more useful for plant cell cultivation.

Baffles
• The baffles are normally incorporated into fermentors of
all sizes to prevent a vortex and to improve aeration
efficiency.
• They are metal strips roughly one-tenth of the fermentors
diameter and attached radially to the walls.
• Baffles are obstructing vertical arranged vanes or
elongated plates inside the vessel needed to stop the
radial swirl inside the fermenter and convert the
rotational flow to axial mixing.
• Without baffles, the tangential velocities coming from any
turbine(s) causes the entire fluid mass to spin creating a
central vortex.
• Baffles, so to speak, increase the friction to the vessel
inner wall surface

Number of baffles in BactoVessel range from 3 to 6 depending on the fermenter
diameter. The baffle width (B1) is typically T/10 - T/12 of the vessel inner diameter (T1).
Baffles are located with a minimum distance of T/72 – T/50 from the wall.

Controlling devices in a bioreactor
• In any microbial fermentation, it is necessary not only to
measure growth and product formation but also to
control the process by altering environmental
parameters as the process proceeds.
• For this purpose, various devices are used in a
fermentor.
• Environmental factors that are frequently controlled
includes temperature, oxygen concentration, pH, cells
mass, levels of key nutrients, and product
concentration.

Air delivery system consists of a compressor, inlet air, sterilization
system, air sparger and exit air sterilization system to avoid
contamination.
Foam control system is an essential element of bioreactor as
excessive foam formation leads to blocked air exit and builds up
pressure in the reactor.
Temperature control system involves temperature probes, heat
transfer system (jacket, coil). Heating is provided by electric heaters
and steam generated in boilers and cooling is provided by cooling
water produced by cooling towers or refrigerants such as ammonia.
pH control system uses neutralizing agents to control pH; these
should be non- corrosive, non-toxic to cells when diluted in the
medium.

• Sampling ports are used to inject nutrients,
water, salts etc. in bioreactors and also for
collecting samples .
• Cleaning and sterilization system is important
to avoid contamination.
• Thermal sterilisation by steam is preferred
option for economical and large-scale
sterilizations of equipment.
• Charging & emptying lines are used for input
of reactants and withdrawal of products in the
bioreactor.

The six types are:
(1)Continuous Stirred Tank Bioreactors
(2) Bubble Column Bioreactors
(3)Airlift Bioreactors
(4) Fluidized Bed Bioreactors
(5) Packed Bed Bioreactors and
(6) Photo-Bioreactors.

Continuous Stirred Tank Bioreactors
Consists of a cylindrical vessel with motor driven central shaft
that supports one or more agitators (impellers).
The shaft is fitted at the bottom of the bioreactor .
The number of impellers is variable and depends on the size of
the bioreactor i.e., height to diameter ratio, referred to as
aspect ratio
The diameter of the impeller is usually 1/3 rd of the vessel
diameter.
The distance between two impellers is approximately 1.2
impeller diameter.
Different types of impellers (Rustom disc, concave bladed,
marine propeller etc.) are in use.

In stirred tank bioreactors or in short stirred tank reactors (STRs),
the air is added to the culture medium under pressure through a
device called sparger.
The sparger may be a ring with many holes or a tube with a single
orifice.
The sparger along with impellers (agitators) enables better gas
distribution system throughout the vessel.
The bubbles generated by sparger are broken down to smaller
ones by impellers and dispersed throughout the medium.
This enables the creation of a uniform and homogeneous
environment throughout the bioreactor

Advantages
There are many advantages of STRs over other types
• These include the efficient gas transfer to growing
cells, good mixing of the contents
• and flexible operating conditions, besides
• the commercial availability of the bioreactors.

Bubble Column Bioreactors:
• In the bubble column bioreactor, the air or gas is
introduced at the base of the column through perforated
pipes or plates, or metal micro porous spargers
• The flow rate of the air/gas influences the performance
factors —O2 transfer, mixing.
• The bubble column bioreactors may be fitted with
perforated plates to improve performance.
• The vessel used for bubble column bioreactors is usually
cylindrical with an aspect ratio of 4-6 (i.e., height to
diameter ratio).

Airlift Bioreactors:
• In the airlift bioreactors, the medium of the vessel is divided into
two interconnected zones by means of a baffle or draft tube.
• In one of the two zones referred to a riser, the air/gas is
pumped.
• The other zone that receives no gas is the down comer.
• The dispersion flows up the riser zone while the down flow
occurs in the down comer.

Internal-loop airlift bioreactor
• has a single container with a central draft tube that creates
interior liquid circulation channels.
• These bioreactors are simple in design, with volume and
circulation at a fixed rate for fermentation.

External loop airlift bioreactor
• possesses an external loop so that the liquid circulates through
separate independent channels.
• These reactors can be suitably modified to suit the requirements
of different fermentations.
• In general, the airlift bioreactors are more efficient than bubble
columns, particularly for more denser suspensions of
microorganisms.
• This is mainly because in these bioreactors, the mixing of the
contents is better compared to bubble columns.

Airlift bioreactors are commonly employed for aerobic
bioprocessing technology.
• They ensure a controlled liquid flow in a recycle system by
pumping.
• Due to high efficiency, airlift bioreactors are sometimes
preferred e.g., methanol production, waste water treatment,
single-cell protein production.
• In general, the performance of the airlift bioreactors is
dependent on the pumping (injection) of air and the liquid
circulation.

Fluidized Bed Bioreactors:
• Fluidized bed bioreactor is comparable to bubble column bioreactor
except the top position is expanded to reduce the velocity of the
fluid.
• The design of the fluidized bioreactors (expanded top and narrow
reaction column) is such that the solids are retained in the reactor
while the liquid flows out (Fig. 19.3A).
• These bioreactors are suitable for use to carry out reactions
involving fluid suspended biocatalysts such as immobilized enzymes,
immobilized cells, and microbial flocs.

• For an efficient operation of fluidized beds, gas is spared to
create a suitable gas-liquid-solid fluid bed.
• It is also necessary to ensure that the suspended solid
particles are not too light or too dense (too light ones may
float whereas to dense ones may settle at the bottom), and
they are in a good suspended state.
• Recycling of the liquid is important to maintain continuous
contact between the reaction contents and biocatalysts.
• This enable good efficiency of bioprocessing.

Packed Bed Bioreactors:
A bed of solid particles, with biocatalysts on or within the
matrix of solids, packed in a column constitutes a packed bed
bioreactor (Fig. 19.3B).
The solids used may be porous or non-porous gels, and they
may be compressible or rigid in nature.
A nutrient broth flows continuously over the immobilised
biocatalyst.
The products obtained in the packed bed bioreactor are
released into the fluid and removed.
While the flow of the fluid can be upward or downward, down
flow under gravity is preferred.

• The concentration of the nutrients (and therefore the products
formed) can be increased by increasing the flow rate of the
nutrient broth.
• Because of poor mixing, it is rather difficult to control the pH of
packed bed bioreactors by the addition of acid or alkali.
• However, these bioreactors are preferred for bioprocessing
technology involving product-inhibited reactions.
• The packed bed bioreactors do not allow accumulation of the
products to any significant extent.

6. Photo-Bioreactors:
• These are the bioreactors specialised for fermentation that can
be carried out either by exposing to sunlight or artificial
illumination.
• Since artificial illumination is expensive, only the outdoor photo-
bioreactors are preferred.
• Certain important compounds are produced by employing photo-
bioreactors e.g., p-carotene, asthaxanthin.

• The different types of photo-bioreactors are depicted in Fig.
19.4. They are made up of glass or more commonly transparent
plastic.
• The array of tubes or flat panels constitute light receiving
systems (solar receivers). The culture can be circulated through
the solar receivers by methods such as using centrifugal pumps
or airlift pumps.
• It is essential that the cells are in continuous circulation
without forming sediments. Further adequate penetration of
sunlight should be maintained.
• The tubes should also be cooled to prevent rise in
temperature.

• Photo-bioreactors are usually operated in a continuous mode at a
temperature in the range of 25-40°C.
• Microalgae and cyanobacteria are normally used.
• The organisms grow during day light while the products are
produced during night.

Fermentation media
In a fermentation process, the choice of the most optimum
micro-organisms and fermentation media is very important for
high yield of product.
The quality of fermentation media is important as it provides
nutrients and energy for growth of micro-organisms.
This medium provides substrate for product synthesis in a
fermentor.

INTRODUCTION TO THE MEDIA
OF FERMENTATION
•All micro-organisms require water, sources of energy,
carbon, nitrogen, mineral element and vitamin plus
oxygen in their growth medium.
•On a small scale, it is simple to device a medium
containing pure compounds, but the resulting medium
although satisfy the growth, may be unsuitable for use
in a large scale process.

• On a large scale one must use sources of
cheap nutrient to create a medium which
will meet as many as possible of the
following criteria:

1. It will produce a maximum yield of product
at biomass per gram of substrate used.
2. It will produce a maximum concentration of
product or biomass.
3. It will be the minimum yield of undesired
product.
4. It will be cheap and of a consistent quality
and is readily available throughout the year.
5. It will cause minimal problem in other
aspects of production and agitation,
extraction, purification and waste treatment.

Use of molasses, cereal grain, glucose,
sucrose and lactose as carbon sources
and ammonium salts, urea, nitrates, soya
bean meal, slaughter-house waste and
fermentation residues as nitrogen source
have tended to meet the above criteria for
production media.

• The medium selected will affect the design of
the fermenter to be used.
• A laboratory medium may not ideal in a large
fermenter with a low gas-transfer pattern.
• Media with a high viscosity will also need a
higher power input for effective stirring.
• Besides the requirement for growth and
product formation, medium may also
influence pH variation, foam formation,
oxidation-reduction potential and the
morphological form of the organisms.
• It may also be necessary to provide
precursors or metabolic inhibitors.

Fermentation media consists of major and minor
components.
• Major components include Carbon and Nitrogen source.
• Minor components include inorganic salts, vitamins, growth
factors, anti-foaming agents, buffers, dissolved oxygen,
other dissolved gases, growth inhibitors and enzymes.

There are two uses of fermentation media
Growth media
Fermentation media
Growth medium contains low amounts of nutrients.It is useful
in creating raw material for further fermentation processes.
Fermentation media contains high amounts of nutrients. It is
used in creating final products using fermentation.
For example, growth of yeast requires 1% carbon. But during
fermentation of alcohol, yeast requires 12 to 13 % carbon in
the medium.

There are two types of fermentation media used in industries.
• Synthetic media
• Crude media

Ingredients of Crude Media
1) Inorganic nutrients
• Crude media contains inorganic salts containing cations
and anion along with a carbon source.
• Sometimes, fermentation micro-organisms have a specific
requirement of ions like magnesium ions, phosphates or
sulphates.
• These requirements are fulfilled by addition of these ions
to balance the crude media.

Carbon source
Simple to complex carbohydrates can be added to media as a
source of carbon. We can add different sugars like mannitol,
sorbitol, organic acids, fatty acids, proteins, peptides we can
choose any of these as a source of carbon.
Simple carbohydrates – simple sugars are semi purified
polysaccharides and sugar alcohol are added. Sources of
simple carbohydrates are Black strap molasses, Corn
molasses, Beet molasses, sulphite waste liquor, Hydrol (corn
sugar molasses), Cannery waste.
Complex carbohydrates – Source of complex carbohydrates
are Starch, Corn, Rice, Rye, Milo, wheat potatoes etc. Source
of starch cellulose are corn cobs, straws, wood waste, saw
meal etc.

Nitrogen source
• Salts of urea, ammonia, and nitrate can be used as a
nitrogen source.
• When fermentation organisms are non-proteolytic in
nature, pure form of urea, ammonia and nitrate are used
as a source of nitrogen.
• When fermentation organisms are proteolytic in nature,
animal and plant raw material is used; like distillery dried
solubles, Casein, Cereal grains, peptones, yeast extract,
hydrolysate, and soybean meal etc.

Growth factors
• Crude media constituents provides enough amount
of growth factors so no extra addition of growth
factor is required.
• If there is a lack of any kind if vitamins or nutrients,
growth factors can be added to media. Examples
are yeast extract, and beef extract.

Precursors
• Precursors are generally present in the media as crude
constituents.
• Precursors are added in the fermentation media at time
of fermentation as it get incorporated in the molecules of
product without bringing any kind of change to the final
product.
• This helps in improving yield and quality of product.
Sometimes, precursors are added in pure form
depending upon the need of product.
• For example, Cobalt chloride is added less than 10 ppm
in fermentation of vitamin B12.

Buffers
• Buffers are used to control drastic changes of pH. Sometimes,
media components may act as buffers.
• For example, protein, peptides, amino-acids act as good
buffers at neutral pH.
• Sometimes inorganic buffers like K2HPO4, KH2PO4, and
CaCO3 etc, can be added as required.
• Generally, during the fermentation process, pH changes to
acidic or alkaline pH.
• The cheapest and easily available buffer is CaCO3.

Definition
– Scale up studies refers to the act of using
results obtained from laboratory studies for
designing a prototype and a pilot plant
process;
– construction a pilot plant and using pilot plant
data for designing and constructing a full
scale plant or modifying an existing plant.
Scale up

• A pilot plant allows investigation of a product and
process on an intermediate scale before large amounts
of money are committed to full-scale production.
• It is usually not possible to predict the effects of a many-
fold increase in scale.
• Scale up studies are studies carried out at the laboratory
or even pilot plant scale fermentors to yield data that
could be used to extrapolate and build the large scale
industrial fermentors with sufficient confidence it will
function properly with all its behaviours anticipated.
Why conduct Scale up Studies?

• More important during scale up exercises is that we are trying
to build industrial size fermentor capable or close of producing
the fermentation products as efficient as those produced in
small scale fermentors.
• It must be appreciated as the size of fermentation increases
during scale up various parameters measured might not show
a predictable linear co-relationships.
• Certain parameters changes, while some remained constant.
Some parameters need to be modified and adjusted during
scale up studies.
• The objective is to try to get the same fermentation efficiency
as obtained in small scale fermentors at the most economical
values.
Importance of scale up studies

There are few crucial studies which will only be
answered by carrying it out on the pilot plant such
as:
• Determining the various operational parameters
for optimized oxygen supply to the fermentation
process.
• Selection of optimum operative modes of the
fermentor
• Determining the changes in rheological properties
and its effect on the fermentation process.
• Modeling and formulation of process controls
• Sensors and controls

• Define product economics based on projected market size
and competitive selling and provide guidance for allowable
manufacturing costs
• Conduct laboratory studies and scale-up planning at the same
time
• Define key rate-controlling steps in the proposed process
• Conduct preliminary larger-than-laboratory studies with
equipment to be used in rate-controlling step to aid in plant
design
• Design and construct a pilot plant including provisions for
process and environmental controls, cleaning and sanitizing
systems, packaging and waste handling systems, and
meeting regulatory agency requirements
• Evaluate pilot plant results (product and process) including
process economics to make any corrections and a decision
on whether or not to proceed with a full scale plant
development
Steps in Scale-Up

• Most scale up studies are usually carried at different
phases involving different scales of fermentors.
• Preliminary work are carried out at the level of petri
dishes and small scale laboratory fermentors to establish
whether the process is technically viable, meaning it is
possible to produce such fermentation process and the
products on the small scale.
• Additional parameters not provided by petri dishes
studies and for more confidence are obtained by carrying
further studies using submerged liquid fermentation
using various sizes laboratory scale fermentors and even
a pilot plant fermentor.
INITIAL SCALE UP STUDIES

• There are a few rules of the thumb followed when
doing scale up studies such as:
• Similarity in the geometry and configuration of
fermentors used in scaling up
• A minimum of three or four stages of increment in the
scaling up of the volume of fermentation studies.
• Each jump in scale should be by a magnitude or power
increase and not an increase of a few litres capacity.
• Slight increase in the working volume would not yield
significant data for scale up operation
Rules followed while doing scale-up

• Evaluating the results of laboratory studies and making
product and process corrections and improvements
• Producing small quantities of product for sensory,
chemical, microbiological evaluations, limited market
testing or furnishing samples to potential customers,
shelf-live and storage stability studies
• Determining possible salable by-products or waste
stream requiring treatment before discharge
• Providing data that can be used in making a decision on
whether or not to proceed to a full-scale production
process; and in the case of a positive decision, designing
and constructing a full-size plant or modifying an existing
plant
A pilot plant can be used for

1. Inoculum development
2. Sterilization establishing the correct sterilization cycle
at larger loads
3. Environmental parameters such as nutrient
availability, pH, temperature, dissolved oxygen,
dissolved carbon dioxide,
4. Shear conditions, foam production
Studies carried out during scale up

ANTIBIOTICS
Penicillin
• Penicillin (PCN or pen) is a group of antibiotics which
include penicillin G (intravenous use), penicillin V (use
by mouth), procaine penicillin, and benzathine penicillin
(intramuscular use).
• Penicillin antibiotics were among the first medications to
be effective against many bacterial infections caused by
staphylococci and streptococci.
• They are still widely used today, though many types of
bacteria have developed resistance following extensive
use.
• About 10% of people report that they are allergic to
penicillin; however, up to 90% of this group may not
actually be allergic.
• Serious allergies only occur in about 0.03%.All penicillins
are beta-lactam antibiotics.

• Penicillin was discovered in 1928 by Scottish scientist Alexander
Fleming.
• People began using it to treat infections in 1942.
• There are several enhanced penicillin families which are effective
against additional bacteria;
• these include the antistaphylococcal penicillins, aminopenicillins
and the antipseudomonal penicillins.

● Have β-Lactam functional group, thus belong to the β-
Lactam antibiotic group.
● They all have a basic ring-like structure (a β-Lactam)
derived from two amino acids (valine and cysteine) via a
tripeptide intermediate. The third amino acid of this
tripeptide is replaced by an acyl group (R).
● The nature of this acyl group produces specific
properties on different types of penicillin.

• The by-product, l-lysine, inhibits the production of
homocitrate, so the presence of exogenous lysine
should be avoided in penicillin production.
• The Penicillium cells are grown using a technique
called fed-batch culture, in which the cells are
constantly subject to stress, which is required for
induction of penicillin production.
• The available carbon sources are also important:
glucose inhibits penicillin production, whereas
lactose does not.
• The pH and the levels of nitrogen, lysine,
phosphate, and oxygen of the batches must also
be carefully controlled.
• Semisynthetic penicillins are prepared starting from
the penicillin nucleus 6-APA (aminopenicillanic acid)

Mediumforpenicillin
1. The Penicillium chrysogenum usually contain its carbon
source, which is found in corn steep liquor and glucose.
2. A medium of corn steep liquor and glucose are added to the
fermenter. Medium also consists of salts such as MgSO4,
K3PO4 and sodium nitrates. They provide the essential ions
required for the fungus metabolic activity.

★ Heat sterilization
Medium is sterilized at high heat and high pressure, usually through
a holding tube or sterilized together with the fermenter.
The pressurized steam is used and the medium is heated to 121°C at
30 psi or twice the atm. pressure
Sterilisation machine

Fermentation
• It is done in a fed-batch mode as glucose must not be added in
high amounts at the beginning of growth The fermentation
conditions for the Penicillium mold, usually requires
temperatures at 20-24°C while pH conditions are kept at 6.5
• The pressure in the bioreactor is much higher than the
atmospheric pressure (1.02atm).
• This is to prevent contamination from occurring as it prevents
external contaminants from entering.
• . It is necessary to mix the culture evenly throughout the culture
medium. Fungal cells are able to handle rotation speed of
around 200 rpm.

★ Seed Culture
9. The seed culture is developed first in the lab by the addition
of Penicillium chrysogenum spores into a liquid medium. When
it has grown to the acceptable amount, it is inoculated into
the fermenter.
The medium is constantly aerated and agitated. Carbon and
nitrogen are added sparingly along the precursor.
Typical parameters such as pH, temperature, stirrer speed and
dissolved oxygen concentration, are observed.

★ Seed Culture
11.After about 40 hours, penicillin begins to be secreted by
the fungus.
12.After about 7 days, growth is completed, the pH rises to
8.0 or above and penicillin production ceases.
The Penicillium fungus

★ Additionofsolvent
16.Organic solvents such as amyl acetate /
butyl acetate are added to dissolve the
penicillin present in the filtrate.
17.At this point, penicillin is present in the
solution and any other solids will be
considered as waste (can be used as
fertilizers and animal feed).

★ Centrifugal Extraction
Centrifugation is done to separate the
solid waste from the liquid component
which contains the penicillin.
Usually a disk centrifuge is used at this point.
The supernatant will then be
transferred further in the downstream
process to continue with extraction.
Disk centrifuge - One of the most
common type of centrifuge for large
scale production

21.A series of extraction processes are carried upon the
dissolved penicillin, to obtain a better purity of the penicillin
product.
22.The acetate solution is first mixed with a phosphate buffer,
followed by a chloroform solution, and mixed again with a
phosphate buffer and finally in an ether solution.
23.Penicillin is present in high concentration in the ether
solution and it will be mixed with a solution of sodium
bicarbonate to obtain the penicillin-sodium salt, which allow
penicillin to be stored in a stable powder form at rtp.
Extraction

Extraction
24. The penicillin-sodium salt is obtained from the liquid material
by basket centrifugation, in which solids are easily removed.
Batch extraction unit Basket Centrifuge: Extremely useful in the
removal of solids in this case Penicillin salt

★ Fluidbeddrying
25. Drying is necessary to remove any
remaining moisture present in the powdered
penicillin salt.
In fluid bed drying, hot gas is pumped from the
base of the chamber containing the powdered
salt inside a vacuum chamber.
Moisture is removed this way, and this
result in a much drier form of penicillin.
Powdered penicillinbeing
blown by hot air
Fluid bed drying
tube

• Penicillin is stored in containers and kept in a dried environment.
The White Penicillin-Sodium salt
• The resulting penicillin (called Penicillin G) can be chemically and
enzymatically modified to make a variety of penicillins with slightly
different properties.
• These can be semi-synthetic penicillins, such as; Penicillin V,
Penicillin O, ampicillin and amoxycillin.
Storage

Streptomycin
• Streptomycin is an antibiotic used to treat a number of bacterial
infections.
• This includes tuberculosis, Mycobacterium avium complex,
endocarditis, brucellosis, Burkholderia infection, plague,
tularemia, and rat bite fever.
• For active tuberculosis it is often given together with isoniazid,
rifampicin, and pyrazinamide.
• It is given by injection into a vein or muscle.

MEDIUM
The culture medium for streptomycin consists of –
1. Carbon source : starch, dextrin, glucose,
2. Nitrogen source :
• natural agricultural by-products, soybean meal, corn steep
liquor, cotton seed flour, casein hydrolyte, or yeast & its extract.
• Inorganic N salts like ammonium sulphate & ammonium
nitrates are also used.

Animal oils, vegetable oils and mineral oils are also
used.
Inoculum -S. griseus spores maintained in soil stocks
or lyophilized in carrier are inoculated into sporulation
medium, which builds up mycelial inoculum.
THE HOCKENHUL MEDIUM
Glucose, extracted soya meal, distillers dried soluble □
sodium chloride, pH as 2.5% 4%, 0.5%, 0.25%, 7.3 -
7.5 respectively.

FERMENTATION PROCESS
Spores of S. griseus are inoculated into a medium to establish a
culture with high mycelial biomass for introduction into inoculum
tank, using inoculum to initiate the fermentation process.
• Yield in production vessel responds to high aeration &
agitation conditions.
Other conditions involve-Temperature range 25-30°C pH
range 7-8 Time 5-7 days
The fermentation process for production of
-Streptomycin involves 3 phases.

PHASE 1
• Initial fermentation phase and there is little production of
streptomycin.
• Rapid growth with production of mycelial biomass.
• Proteolytic enzymatic activity of S.griseus releases
NH3 from soya meal, raising the pH to 7.5
Characterized by release of ammonia.
• Carbon nutrients of soya meal are utilized for growth.
• Glucose is slowly utilized with slight production of
Streptomycin.

PHASE 2
• Little production of mycelia.
• Glucose added to the medium & the NH3,released from soya meal
are consumed.
• pH remains fairly constant ranging □ between 7.6 to 8.
PHASE 3-Final phase of fermentation.
• Depletion of carbohydrates from medium.
• Streptomycin production ceases & bacterial cells begin to lyse.
• Ammonia from lysed cells increase the pH.

RECOVERY & PURIFICATION
• Mycelium is separated from broth by filteration &
streptomycin is recovered. Recovery process - broth is
acidified, filtered & neutralized.
• Then its subjected to column containing cation exchange
resin to adsorb Streptomycin from the broth & column is
washed with water & streptomycin eluted with HCl before
concentration in vacuum almost to dryness.

FILTERATION
• The streptomycin is dissolved in acetone, methanol &
filtered.
• Acetone is used in filtrate to precipitate the antibiotic.
& dried in vacuum.
• Purification is done by dissolving in methanol to form pure
Streptomycin chloride complex.
• Further by, adsorbing it onto activated charcoal & eluting
with acid alcohol.

• Citric acid is a usually occurring acid found primarily in
Several Varieties of fruits and vegetables with citrus
fruits such as lemons and limes containing the highest
amounts of citric acid.
• This Organic acid has many uses, including as a food
additive /Preservative, ingredient in Cosmetic products
and as a powerful cleaving agent.

HISTORY:
• The alchemist Jabir Ibn Hayyan (Geber).
• Citric acid was first isolated in 1784 by the Swedish chemist
carl Wilhelm Scheele, who crystallize it from lemon juice.
• Industrial scale citric acid production began in 1890 based on
the Italian citrus fruit industry.
• In 1893, C. Wehmer discovered penicillin mold could produce
citric acid from sugar.
• However, microbial production of citric acid did not become
industrially important until world war I disrupted Italian
citrus exports.

Industrial production of citric acid:
• 99% of world production microbial processes surface
or submerged culture.
• 70% of total production of 1.5 million tons per year is
used in food and beverage industry as on acidifier or
antioxidant to preserve or enhance the flavors and
aromas of fruit juices, ice cream and marmalades.
• 20% used pharmaceutical industry as anti oxidant to
preserve vitamins, effervescent, pH corrector, blood
preservative, or in the form of iron citrate.

Micro-organisms used for citric acid production:
Large number of micro-organisms including bacteria,
fungi and yeasts have been employed to produce citric acid.
The main advantages of using this micro-organisms are:
a) Its easy of handling
b) Its ability to ferment a variety of cheap raw
materials
c) High yields

Micro organisms:
Fungi:
Aspergillus niger
A. aculeatus
A. awamori
A. carbonarius
A. wentii
A. foetidus
Penicillium janthinelum
Bacteria:
Bacillus licheniformis
Arthrobacter paraffinens
Corynebacterium sp.
Yeasts:
Saccahromicopsis lipolytica
Candida tropicalis
C. oleophila
C. guilliermondii
C. parapsilosis
C. citroformans
Hansenula anamosa

Citric acid production:
Fermentation is the most economical and widely
Used for synthesis citric acid production.
The industrial citric acid production can be
carried in three different ways:
• surface fermentation
• submerged fermentation
• solid state fermentation

Surface Fermentation:
Surface fermentation using Aspergillus niger may be
done on rice bran as is the case in Japan, or in liquid
solution in flat aluminium or stainless steel pans.
Special strains of Aspergillus niger which can produce
citric acid despite the high content of trace metals in
rice bran are used.

SUBMERGED FERMENTATION:
• In this case , the strains are inoculated of about 15cm
depth in fermentation tank.
• The culture is enhanced by giving aeration using air
bubbles.
• And its allowed to grow for about 5 to 14 days at 27 to
33 degree Celsius.
• The citric acid produced in the fermentation tank and it
is purified.

Solid state fermentation:
It is simplest method for citric acid production.
Solid state fermentation is also known as koji
process, was first developed in Japan.
Citric acid production reached a
maximum(88g/kg dry matter)when fermentation
as carried out with cassava having initial moisture
of 62% at 26 degree Celsius for 120 hours.

Separation:
• The biomass is separated by filtration.
• The liquid is transferred to recovery process
• Separation of citric acid from the liquid
• precipitation.
• Calcium hydroxide is added to obtain calcium citrate.

• Acetic acid (CH3COOH) is also called as vinegar.
• Vinegar fermentation is one of the oldest fermentations
known to man.
• It is formed naturally due to spoilage of wine.
• Therefore, literally vinegar means “sour wine.”
• Technically vinegar is fermented food product consisting
of about 4 g of acetic acid per 100 ml.
• Vinegar was produced only for local consumption until
the middle ages.
Introduction to Acetic Acid:

• From 1949, submerged process was employed in the
fermentative production of vinegar.
• Presently both these processes that is surface fermentation and
submerged fermentation are employed worldwide.
• The surface fermentation is used still today because of the
better flavour of the product.

The production of vinegar actually involves two fermentation
processes-
• the first utilizing yeast to produce alcohol from sugar and the
• second utilizing acetic acid bacteria to oxidize ethyl alcohol acetic
acid through acetaldehyde (Fig. 4.7).
Biosynthesis of Acetic Acid:

• The microbial oxidation of ethanol to acetic acid is an aerobic
fermentation that has high oxygen requirement.
• Acetobacter bacteria are employed for industrial production of
vinegar. Acetobacter bacteria can be divided into two groups
• Gluconobacter and Acetobacter.
• Gluconobacter oxidizes ethanol to acetic acid, while Acetobacter
oxidizes ethanol first to acetic acid and then to CO2 and H2O.
• Species of the Acetobacter used commercially are Acetobacter
aceti and A. pasteurianum.
• Similarly, Gluconobacter oxydans and its subspecies are employed
in the commercial production of vinegar.
• Mixed cultures, sometimes appear during production even
though pure culture is used especially in surface process.

• Two oxidation steps occur during the conversion of ethanol to
acetic acid.
• In the first step ethanol is oxidized to acetaldehyde in the
presence of NAD or NADP and
• in the second step acetaldehyde is changed to acetic acid and
render the catalytic action of the enzyme alcohol
dehydrogenase (Fig. 4.7).
• In this oxidation one liter of 12% acetic acid is produced from
one liter of 12% alcohol
• that is one mole of acetic acid is formed from one mole of
alcohol.
Two step oxidation

• Commercially acetic acid is produced by two methods, surface
fermentation process and submerged fermentation process.
• If materials with low alcohol content are used such as wine,
whey, malt or cider there is no need of addition of any
component to constitute a complete nutrient solution. However,
if potato or grain spirits or technical alcohol is used, nutrients
must be added to obtain optimal growth and acetic acid
production.
• Nutrient concentration that is used in submerged fermentation is
generally five times greater than surface fermentation.

• made up of wood and has a total volume up to 60 m3 and its inner
surface is lined with birch wood shavings.
• The starting material, that is, ethanol is passed into the generator
from top which trickles through the birch wood shavings
containing bacteria into bottom basin
• where the partially converted solution is cooled and pumped back
again to the top of the generator and passed again through it.
• Thus, the process is repeated again and again until 88-90% of
alcohol is changed to acetic acid.
Surface Fermentation Process: (Trickling generator)

• The starting material should contain both acetic acid and ethanol
for optimal growth of Acetobacter.
• However, ethanol supply is critical because if it is less than 0.2%
(v/v) in solution the death rate of bacteria increases, but its
concentration should not increase above 5%.
• Presently higher yielding strains are employed in vinegar
fermentation which are able to yield 13-14% of acetic acid.

• The production rate of ethanol with submerged fermentation
process is ten times higher per cubic meter than the surface
fermentation process.
• Other advantages include lower capital investment for production,
20% plant area required for installation, conversion to other mashes
in a short time and low manual cost because of fully automatic
control.
• Material with low alcohol concentration such as fruits, wines and
special mashes were first used in the initial stages of submerged
fermentation process, which generally do not require aeration.
• But presently high yielding materials are employed which are
capable of yielding 13% acetic acid.
• However, the process with such high yielding material requires high
aeration upto 50 m3 oxygen.
Submerged Fermentation Process:

• Fermenters constructed with stainless steel are employed and they
are stirred from bottom.
• Aeration is provided with a suction rotor, with the incoming air
coming down through a pipe from the top of the vessel .
• Heat exchanger is provided to control the temperature along with
foam eliminators.
• Domestic vinegar is produced through semi continuous fully
automatic process under continuous stirring and aeration.
• The starting material 100 ml of 5% ethanol are used in the process
to get 7-10 g acetic acid/100 ml.
• The fermentation process is carried up to 35 hours at 40°C
temperature.

• The vinegar produced in a submerged fermentation process is
turbid due to presence of bacteria.
• It is clarified by filtration.
• Plate filters and filter aids are generally used.
• After filtration K4[Fe(CN)6] is used to decolorize the final
product, if required.
(c) Recovery:
• The ethanol concentration is continuously measured and when
its concentration goes down to 0.05%-0.3%, 50%-60% of the
solution is removed and replaced with a new mash with 0.2 g
acetic acid/100 ml and 10-15% ethanol.
• The yield of acetic acid is about 98% in fully continuous process.

Microbial Fermentation and Production of Amylases
Presences of major two classes of starch-degrading enzymes i.e.
alpha amylase
& glucoamylase (gamma amylase) -microorganisms.
Alpha amylase is also referred as endo - 1, 4-α-D-glucan
glucanohydrolase, which randomly splits the 1,4-α-D-glycosidic
linkages between the adjacent glucose units in linear amylose chain.
Unlike alpha -amylase, most glucoamylases are also able to
hydrolyze the 1,6-alpha - linkages at the branching points of
amylopectin, although at a slower rate than 1, 4- linkages.
Various bacteria and fungi produce amylases, although the
enzymes produced from these two sources are not identical.

Fungal and bacterial amylases are used commercially
in various fields like,
1 preparation of sizing agents and removal of starch
sizing from woven cloth in textile industries
2 preparation of starch sizing pastes for use in paper
coating in paper industries
3 in liquefaction of heavy starch pastes formed during
manufacturing of chocolates &
corn syrups in processed-food industries
4 in brewing industries and removal of food spots in
the dry- cleaning industries.

Amylase Fermentation
Fungal amylase production utilizes solid state fermentation or
submerged aerated- agitated fermentation.
Solid-state fermentation
Solid-state fermentation (SSF) is well-known for the production of
enzymes by filamentous fungi.
As these molds have specific morphology and physiology, they can
penetrate and colonize various solid substrates.
Various agro-industrial wastes are utilized as substrate in SSF that
provides both physical support and source of nutrients in the
production of numerous fermented products and enzymes.

SSF offers advantages such as,
1 great volumetric productivity,
2 superior product recovery and product characteristics,
3 small capital investment,
4 use of agricultural industrial wastes reduce pollution problems
and a lesser amount of
effluent generation,
5 Level of catabolite repression gets reduced.
Agricultural wastes which are used in enzyme production
includes starch, wheat bran, rice bran, maize bran, sugar cane
bagasse, wheat straw, rice straw, rice husk, banana waste, tea
waste, cassava waste, , cassava flour, corn flour, wheat flour,
steamed rice, etc.

Submerged Fermentation
Industrial enzymes can be produced using submerged fermentation in
which microorganisms are cultivated in liquid nutrient broth.
This process involves growth of selected microorganisms in closed
containers having a rich fermentation broth of nutrients and a high
concentration of oxygen.
As microorganisms are growing, they release the desired enzymes into
solution.
Nowadays large-scale fermentation technologies are developed which
allows significant production of microbial enzymes.
Large fermenters (vessels) with capacity of up to 1,000 cubic meters
volumes are used for large scale fermentation.
Common raw materials like maize, sugars and soya are used in the
fermentation media.
microorganisms secrete industrial enzymes into the fermentation
medium -degrade the carbon and nitrogen sources.

There are two common processes:
batch-fed and continuous fermentation.
To allow growth of the biomass in the batch-fed process, sterilized
nutrients are added to the fermenter.
But in the continuous process, sterilized liquid nutrients are added
into the fermenter at the same proportion as the fermentation broth
leaving the system which lead to steady- state production of enzymes.
To optimize the fermentation process various parameters like pH,
temperature, oxygen depletion and carbon dioxide creation are
measured and controlled.
First step in harvesting of enzymes from the fermentation broth is
removal of insoluble products like microbial cells which is generally
carried out by centrifugation.

Most of industrial enzymes are extracellular, which remain in the
fermentation medium after removal of the biomass.
The biomass is treated with lime to deactivate the microorganisms
and stabilize it during storage & then can be used as a fertilizer.
The enzymes remaining in the broth are then concentrated by
various methods like
evaporation,
membrane filtration
or crystallization.
Submerged Fermentation offers advantages like ease of
sterilization as well as process control is simple.
The enzyme produced can be constitutive or inducible which
depends on the strain and the culture conditions.

Fungal Amylases
Fungal amylases can produced by any of these two fermentation
processes.
Generally strains of Aspergillus oryzae are utilized for stationary
culture with wheat bran.
For this process, the wheat bran spread in relatively thin layers in
trays, is moistened with water or dilute acid, sterilized and
inoculated with spores of a fungus.
amylase is extracted by water from the wheat bran culture,
precipitated from the aqueous solution by the addition of alcohol,
and dried at 55°C or less.

The Submerged Fermentation for amylase production have recently
become economically feasible - strains of both Aspergillus niger &
Aspergillus oryzae have been studied extensively for their possible use
in this process.
Fungal amylases are produced by submerged culture method
employing the following (g/L)
Corn Starch 24 ,Corn steep liquor 36, KCL 0.2,Na2HPO4 47
CaCL2, MgCL2.6H2O 0.2
There is a problem of aeration & agitation because of a very high
viscosity of the medium due to the presence of mycelial body.
Amylases biosynthesis is inhibited when the medium contains
glucose.

Bacterial Amylases
Various bacteria elaborate amylases, but only amylases from
Bacillus subtilis and Bacillus diastaticus have been produced on a
commercial basis.
Bacterial amylases are employed under those conditions in which
fungal amylases, or amylases from other sources, hydrolyze starch less
well.
For instance, bacterial amylase has an optimum temperature for
activity at near about 55°C, and the enzyme is relatively heat
resistant.
As a result, bacterial enzyme finds particular application in
situations in which starch hydrolysis must be conducted at higher
temperatures.
Strains of Bacillus subtilis are specially selected for amylase with
high starch liquefying and dextrinizing activity.
Subsequently, this amylase produces comparatively less
fermentable sugars when acting on starch.

High yields of bacterial amylase are obtained when Bacillus
subtilis is grown in stationary culture.
• Fermentation medium contains a high level of crude protein
because high carbohydrate level in the medium stimulates
protease production and depresses amylase production.
• high aerated submerged culture by using a special medium having
high content of starch.
• The pH of the medium is near neutrality and the fermentation
proceeds for approximately 6 days at an incubation temperature
of 25 to 30°C.
Bacillus subtilis in this stationary liquid culture produces a heavy
surface-pellicle growth, which apparently is associated with high
amylase yield, and fresh sterile air is circulated over the pellicle to
improve aeration.

At harvest, the culture is filtered or centrifuged, the recovered
aqueous portion is concentrated by evaporation to yield an amylase
concentrate, and salt and an antiseptic are added.
As an alternate recovery procedure, the amylase can be precipitated
from aqueous solution by the addition of cold acetone, ethanol,
isopropanol, or ammonium sulfate.
Bacillus subtilis amylases can also be produced in highly aerated
submerged culture by using a special medium having high content of
starch.
Component Amount (g/litre)
Ground soya bean meal- 18.5
Amber BYF (Brewer’s Yeast Fraction) 15
Distillers dried soluble 7.6
Enzymatic casein hydrozylate 6.5
Lactose 47.5
MgCL2.6H2O 0.4
Antifoam 0.5

Microbial Fermentation and Production of Proteases
Proteolytic enzymes are produced by various bacteria, such as
species of Bacillus, Pseudomonas, Clostridium, Proteus, and Serratia,
and by fungi such as Aspergillus niger, Aspergillus oryzae, Aspergillus
flavus, and Penicillium roquefortii.
However, the enzymes associated with these microorganisms are
actually mixtures of proteinases and peptidases, with the
proteinases usually being excreted to the fermentation medium
during growth, while the peptidases often are liberated only on
autolysis of the cells, since they are endoenzymes.
Complex mixtures of true proteinases & peptidases are usually
called proteases.
At present, the proteinases are of more commercial interest than
are the peptidases.
Among the commercial applications of proteolytic enzymes is the
bating of hides in the leather industry.

• Proteolytic enzymes also are employed in the textile industry to
remove proteinaceous sizing, and in the silk industry to liberate the
silk fibers from the naturally occurring proteinaceous material in
which they are imbedded.
• In addition, these enzymes are employed in the tenderizing of meat,
and they also are the active ingredient in spot-remover preparations
for removing food spots in the dry-cleaning industry.
• The fungal proteases present a wider pH activity range than do
animal or bacterial
• proteases, and to a certain extent this results in a wider range of
uses for the fungal proteases.
• There are two types of proteases: (a) alkaline serine proteases and
(b) acid proteases.
• Alkaline serine proteases are mainly produced by Bacillus
licheniformis by submerged culture method.

Acid proteases are mostly produced by fungi by either semisolid
culture or submerged culture method, depending upon the fungal
species employed.
Fungal Protease- Surface fermentation
Various fungi produce protease enzyme in good yield, and the
commercial production of fungal protease has utilized Aspergillus flavus,
Aspergillus wentii, Aspergillus oryzae, Mucor delemar, Mucor miehei and
Amylomyces rouxii.
The fungus is usually grown on wheat bran, although other media are
sometimes employed, under fermentation conditions similar to those
for amylase production.
At sporulation, the various fungal proteolytic enzymes are present in
the medium, and the proteases are recovered by procedures similar to
those for mold amylases.
The optimum temperature of the fermentation is 30°C & requires 3
days for completion.

Submerged fermentation procedures for fungal protease
production have been studied, and the indications are that such
fermentations may become commercially feasible.
Mucor miehei is used to produce acid proteases by submerged
culture method using following media:
Component/ Amount (g/litre)
Starch -40
Soya bean meal-30
Ground barley -100
Calcium Carbonate-5
The optimum temperature of the fermentation is 30°C, but
requires 7 days for completion.

Bacterial Protease
Bacterial protease production again utilizes strains of Bacillus
subtilis, and the fermentation conditions are similar to those for
amylase production by this organism.
However, the Bacillus subtilis strains are specially selected for high
protease activity and not for amylase activity.
As stated previously, a high carbohydrate content medium is utilized
to stimulate protease activity and depress amylase production,
although the final product does contain some amylase activity.

• The fermentation is incubated 3 to 5 days at 37°C in pans
containing a shallow layer of fermentation medium,
• and the harvest procedure is similar to that for bacterial
amylase, except that concentration of the broth is carried out
at reduced pressure and at temperatures of less than 40°C in
order to protect the enzyme from denaturation.
• Alkaline serine proteases are obtained from Bacillus
licheniformis by submerged culture method using following
media:
Component Amount (g/litre)
Starch hydrolysate -50
Soya bean meal-20
Casein-20
Na2HPO4-3.3

Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) catalyze the
hydrolysis and the synthesis of esters formed from glycerol and long-
chain fatty acids.
Lipases occur widely in nature, but only microbial lipases are
commercially significant. Microbial lipases are high in demand due to
their specificity of reaction, stereo specificity and less energy
consumption than conventional methods.
Many microorganisms such as bacteria, yeast and fungi are known to
secrete lipases. Lipase-producing microorganisms have been found
in diverse habitats such as industrial wastes, vegetable oil processing
factories, dairies, soil contaminated with oil, etc.
Lipases

• Bacterial lipases are mostly extracellular and are greatly influenced
by nutritional and physico- chemical factors, such as temperature,
pH, nitrogen and carbon sources, inorganic salts, agitation and
dissolved oxygen concentration.
• The sources of lipase enzyme are generally found in nature such as
plants, animals, yeast, fungi and bacteria, for example, Candida
rugosa, Thermomyces lanuginosus, Fusarium oxysporum f. sp.Lini,
Candida antarctica, Rhizopus oryzae, Lactobacillus spp., Bacillus
stearothermophilus L1, and Burkholderia sp. C20 .
• Bacterial lipases are important enzymes applications in various
industries, because of friendly for environment, non-toxic and no
harmful Residues.
• widely uses in dairy industry and pharmaceutical industry, detergent
and Surfactant, taste or flavor industry , agricultural industry,
chemical, cosmetic and perfume .

Production and media development for lipase:
• Microbial lipases are produced mostly by submerged culture , but
solid state fermentation methods can be used also.
• The Solid State Fermentation (SSF) due to the possibility of using
residues and by-products of agro-industries as nutrient sources and
support for microorganism development.
• use of by-products as substrates for lipase production, adds high
value and low-cost substrate may reduce the final cost of the
enzyme
• production of lipase through submerged fermentation needs, large
space, complex media and also needs complex machinery,
equipment and control systems.
• Moreover, submerged fermentation for production of lipase at large
scale demands high energy demand, higher capital and recurring
expenditure

• In several cases, immobilization of microbial cells producing
lipases increase the extent of reaction and facilitate the
downstream processing.
• This is because it avoids washout of the cells at dilution rates, it
helps to increase cell concentration in the reactor and easy
separation of cells from the product containing solution
• Generally, lipase production is influenced by the type and
concentration of carbon and nitrogen sources, the culture pH,
the growth temperature and dissolved oxygen concentration .
• Lipidic carbon sources seem to be generally essential for
obtaining a high lipase yield;
• however, a few authors have produced good yields in the
absence of fats and oils

Purification of lipases:
• Currently many lipases have been widely purified and characterized
without losing their activity and stability profiles depending to pH,
temperature and effects of metal ions and chelating agents.
• The methods used for purification of lipases are nonspecific
techniques, some of which are extraction, precipitation,
hydrophobic interaction, chromatography, gel filtration,
crystallization and ion exchange chromatography.
• Affinity chromatography is significant to reduce most purification
steps needed .
• If the objective is production of lipase for industrial use, the
purification technique should be inexpensive, rapid, high-yielding
and liable to large-scale operations.
• The degrees of quality of the products are entirely to depend on the
purpose and the economic point of view.
• For instance, the lipase for synthetic reactions in pharmaceutical
industry needs further purification

History of Glutamic Acid:
• The history of the first amino acid production dates back to 1908
when Dr. K. Ikeda, a chemist in Japan, isolated glutamic acid from
kelp, a marine alga, after acid hydrolysis and fractionation.
• He also discovered that glutamic acid, after neutralization with
caustic soda, developed an entirely new, delicious taste.
• This was the birth of the use of monosodium glutamate (MSG) as a
flavour-enhancing compound.
• The breakthrough in the production of MSG was the isolation of a
specific soil-inhabiting gram-positive bacterium, Corynebacterium
glutamicum, by Dr. S. Ukada and Dr. S. Kinoshita in 1957.
• The successful commercialization of monosodium glutamate (MSG)
with this bacterium provided a big boost for amino acid production
and later with other bacteria like E. coli as well.

Commercial Production of Glutamic Acid:
• Glutamic acid commercial production by microbial fermentation
provides 90% of world’s total demand, and remaining 10% is met
through chemical methods.
• For the actual fermentation the microbial strains are grown in
fermentors as large as 500 m3.
• The raw materials used include carbohydrate (glucose, molasses,
sucrose, etc.), peptone, inorganic salts and biotin.
• Biotin concentration in the fermentation medium has a significant
influence on the yield of glutamic acid.
• Fermentation completes within 2-4 days and, at the end of the
fermentation, the broth contains glutamic acid in the form of its
ammonium salt.

• In a typical downstream process, the bacterial cells are separated
and the broth is passed through a basic anion exchange resin.
• Glutamic acid anions get bound to the resin and ammonia is
released.
• This ammonia can be recovered via distillation and reused in the
fermentation.
• Elution is performed with NaOH to directly form monosodium
glutamate (MSG) in the solution and to regenerate the basic
anion exchanger.
• From the elute, MSG may be crystallized directly followed by
further conditioning steps like decolourization and serving to yield
a food-grade quality of MSG.

• α-ketoglutaric acid serves as the precursor of glutamic acid and the
conversion of the α- ketoglutaric acid to glutamic acid occurs in
presence of enzyme glutamic acid dehydrogenase.
• It has been found that if penicillin is added in he medium, the
glutamic acid production can be increased manifold.

Uses of Glutamic Acid:
As stated earlier, glutamic acid is widely used in the production of
monosodium glutamate (MSG) which is commonly known as the
‘seasoning salt’.
The world production of glutamic acid is to the tune of 800,000
tonnes/year.
Monosodium glutamate is condiment and flavour-enhancing agent, it
finds its greatest use as a common ingredient in convenient food-
stuffs.

Production of L-Lysine:
Total world production of L-lysine is around 35,000 metric tons per
year.
Industrially it is produced by two different fermentation methods.
They are:
(a) Indirect fermentation
(b) Direct fermentation.

(a) Indirect Fermentation:
• It is also called as dual fermentation as two different
microorganisms are employed in this fermentation process.
• Auxotrophic mutant of Escherichia coli is used in the first half of
the fermentation
• and wild type or prototrophic E. coli or Aerobacter aerogenes is
employed in the second half of the fermentation.

• Diaminopimelic acid produced in the first half of fermentation by
auxotroph of E. coli,
• is converted into L-lysine by A. Aerogenes in the second half of
the fermentation (Fig. 5.2).
• A. Aerogenes should also be deficient of lysine decarboxylase
• so that further decarboxylation of lysine to cadaverine is
prevented and accumulation of lysine is facilitated.

Direct Fermentation:
• L-lysine can also be fermentatively produced from any of the
substrates directly and the process is called as direct
fermentation.
• Direct fermentation processes are presently employed
throughout the world for the production of L-lysine.
• Direct production of l-lysine from carbohydrate was developed
first with a homoserine or threonine plus methionine auxotroph
of Corynebacterium glutamicum.
• Production of lysine by this bacterium is regulated by the
mechanism as depicted in Fig. 5.4.
• The prototroph of this bacterium produces L-glutamic acid in
large quantities. The same type of process was reported with a
homoserine auxotroph of Brevibacterium flavum.

• The homoserine auxotroph was later recognized as threonine
sensitive mutant because growth was inhibited by the excess of
threonine and the inhibition was released by the addition of
methionine.
• This phenomenon is due to feedback inhibition of residual
homoserine dehydrogenase by threonine.

• Homoserine auxotroph of other bacteria were also found to
produce L-lysine but the yields were lower than that from
homoserine auxotroph of Coryneform bacteria.
• Threonine and leucine auxotrophs produce fairly large amounts
of L-lysine but they are inferior to the homoserine auxotroph.
• Other auxotrophs of Corynebacterium glutamicum and other
bacteria were also inferior to the homoserine auxotroph of C.
glutamicum.
• Therefore, this bacterium is extensively used for the L-lysine
production on commercial basis by fermentation process.

• Double auxotrophs, which require atleast one of the amino acids,
threonine or isoleucine or methionine in addition the
homoserine, for growth have been found highly stabilized,
showing little tendency to revert the homoserine independence.
• It is possible not only to prevent reversion of the culture to a wild
type, but also to produce lysine in higher yields since many of the
microorganisms are double mutants in the homoserine pathway.

Fermentation Process of L-Lysine:
This process consists of four stages.
They are:
(i) Preparation of inoculum,
(ii) Preparation of medium,
(iii) Fermentation process,
(iv) Harvest and recovery,

(i) Preparation of Inoculum:
Suitable and high yielding mutant strain of C. glutamicum usually (strain 901)
is used from the stock culture for the production of inoculum. Seed cultures
are raised twice, in which two different media are used.
The medium for first seed culture contains:
The medium is prepared in tap water.
The medium for second seed culture contains:
It is also prepared in tap water. This prepared inoculum is employed for
fermentation.

(ii) Preparation of Medium:
• The medium with the following composition is used as
fermentation medium.
• Reducing sugar (expressed as inverted cane molasses), 20%,
Soyabean meal hydrolysate (as weight of meal before hydrolysis
with 6NH2SO4 1.8% and neutralization with ammonia water) are
dissolved in tap water and sterilized.

(iii) Fermentation Process:
• The fermentation is carried out at 28°C and is allowed up to 60
hours.
• The amount of growth factors, homoserine or threonine and
methionine should be appropriate for the production of L-lysine
and suboptimal quantity to support the optimal growth.
• The biotin concentration in the medium should be greater than 30
mg per liter.
• If biotin is supplied in limited quantities there will be
accumulation of L- glutamic acid instead of L-lysine. Cane
molasses generally supplies enough biotin.
• There will be 30-40% yield of L-lysine as monohydrochloride in
relation to the initial sugar concentration. Foam production in the
aerated culture can be controlled by adding suitable antifoam
agent.

(iv) Harvest and Recovery:
• The same process of recovery of L-lysine that is employed in indirect
fermentation process is also used in this process.
• Mutant strains of Bacillus licheniformis are also employed for the
production of L-lysine.
• The mutant strains were obtained by the introduction of both
analogue-resistance and auxotrophy.
• The medium containing 10% cane molasses is used. A temperature
of 40°C is suitable for L- lysine production.
• The sporulation activity which reduces yield, can be suppressed by
the addition of certain antibiotics like tetracycline and
chloramphenicol.
• These mutants yield approximately 30 mg of L-lysine per ml of
carbon source used.

Uses of L-Lysine:
L-lysine is useful in many fields:
1. L-lysine is an essential amino acid required for the human
nutrition.
2. It is used as supplementary for cereal proteins.
3. Protein quality of certain foods like wheat (based foods) is
improved by addition of L-lysine which results in the improved
growth and tissue synthesis.
4. It is used as a nutraceutical.

bioprocess and industrial biotechnology.pptx

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