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Fermentation
Technology

Book list:
1
...
Introduction to Fermentation Process
Fermentation: The word fermentation originates from a Latin verb ‘fervere’ which literally means to boil
...
Fermentation in a strict sense is a biological process that occurs in the absence of O 2
...

Fermentation technology: Fermentation technology is a field that involves the utilization of microorganisms and
enzymes for production of compounds that have applications within the energy material, pharmaceutical, chemical
and in the food business
...
Bioprocessing broadly involves a
multitude of enzyme catalyzed reactions carried out by living cells (or cell-free systems) for industrial purposes
...
This demarcation however, is not very rigid
...

Those that produce microbial enzymes
...

Those that produce recombinant products
...


The component parts of a fermentation process: There are 6 basic component parts:
1
...

3
...

5
...


The formulation of media to be used in culturing the process organism during the development of the inoculum
and in the production fermenter
...

The production of an active, pure culture in sufficient quantity to inoculate the production vessel
...

The extraction of the product and its purification
...


Fig: A typical fermentation process
...


Solid state fermentation
Utilizes solid substrates like bran, bagasse and
paper pulp
...


Submerged fermentation
Utilizes free flowing liquid substrates, such as
molasses and broths
...


Substrates are utilized very slowly, need not be
replaced
...


Substrates are utilized quite rapidly, need to be
replaced constantly
...


Best suited for fungi that require less moisture
content
...


Best suited for bacteria that require high moisture
content
...


Culture system involves three phases – solid, liquid
and gaseous
...


Culture system involves two phases – liquid and
gaseous
...

6
...

System may or may not involve agitation
...

6
...

System often needs agitation
...
Fermenter or Bioreactor
The heart of fermentation or bioprocessing technology is the fermenter or bioreactor
...
It is a containment system
designed to give right environment for optimal growth and metabolic activity of the organism
...

Traditional fermenters are open vats made of wood or slate
...
A
high-quality stainless steel that does not corrode or leak toxic metals into the growth medium is used
...

Types of bioreactors: Based on the design of the bioreactors, they can be grouped into the following types:
A
...

C
...

E
...


Continuous stirred tank bioreactors
Bubble column bioreactors
Airlift bioreactors
Fluidized bed bioreactors
Packed bed bioreactors
Photobioreactors

In all type of bioreactors, the ultimate aim is to ensure that all parts of the system are subjected to the same
conditions
...
Continuous stirred tank bioreactors: A continuous stirred tank bioreactor consists of a cylindrical vessel with
motor driven central shaft that supports one or more agitators (impellers)
...
The number of impellers is variable and depends on the size of the bioreactor, i
...
height to
diameter ratio, referred to as aspect ratio
...

However, for animal cell culture applications, the aspect ratio is less than 2
...
The distance between two impellers is approximately 1
...

3

Different types of impellers (Rustom disc, concave bladded marine propeller etc
...

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 along with impellers (agitators) enables better gas distribution system throughout the
vessel
...
This enables the creation of a uniform and homogenous environment throughout the
bioreactor
...
This include:
1
...

2
...

3
...

4
...


B
...
The flow rate of the air or gas
influences the performance factors – O2 transfer, mixing
...
The vessel used for bubble column bioreactors is usually cylindrical
with an aspect ratio of 4⸺6 (i
...
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
...
The other
zone that receives no gas is the downcomer
...
There are two types of airlift bioreactors:
a
...
These bioreactors are simple in design, with
volume and circulation at a fixed rate for fermentation
...


External loop airlift bioreactor: External loop airlift bioreactor possesses an external loop so that the
liquid circulates through separate independent channels
...
In general, the airlift bioreactors are more efficient
than bubble columns, particularly for more denser suspensions or microorganisms
...

Airlift bioreactors are commonly employed for aerobic bioprocessing technology
...
Due to high efficiency, airlift bioreactors are
sometimes preferred e
...
methanol production, waste water treatment, single cell protein production
...


5

D
...
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
...

For an efficient operation of fluidized beds, gas
is sparse to create suitable gas-liquid-solid fluid
bed
...
Recycling of the
liquid is important to maintain continuous
contact between the reaction contents and
biocatalysts
...


E
...
The solids used may be porous
or non-porous gels, and they may be
compressible or rigid in nature
...
The products obtained in the packed
bed bioreactor are released into the fluid and removed
...

The concentration of the nutrients (and therefore the products formed) can be increased by increasing the flow
rate of the nutrient broth
...
However, these bioreactors are preferred for bioprocessing
technology involving product-inhibited reactions
...


F
...
Since artificial illumination is expensive, only the outdoor
photobioreactors are preferred
...
g
...

Photobioreactors are usually operated in a continuous mode at a temperature in the range of 25⸺400 C
...
The organisms grow during day light while the products are
produced during night
...


Stirred tank fermenters: It is a simple and the most widely used
reactor
...
It is
mainly used in brewing industry for yeast fermentation
...

Application: Agitation
...

For volume more than a liter, to drive mechanical motors are
used
...

Problems faced: Major damaging force is bubbling or bubble
bursting
...


Fig: Stirred tank fermenter

Air lift fermenter: It is a simple system without mechanical
components
...

Design and working: The fluid volume of the vessel is divided
into two interconnected zones by means of a baffle or draft tube
...
The
sparged zone is known as riser, the zone that receives no gas is
downcomer
...
8
and 3
...

Performance characteristics of airlift fermenters are linked with
the gas injection rate and the resulting rate of fluid circulation
...

They are highly energy efficient compared to stirred tank
reactors
...

Fig: Airlift fermenter

3
...

Design and working: The top section is expanded to reduce the superficial velocity of the fluidized liquid to a
level below that it is needed to keep the solids in the suspension
...
Therefore, the solids are retained in the reactor whereas
the liquid flows out
...
If the
solid particles are too light, they can be artificially weighted
...
e
...


7

Fig: Fluidized bed bioreactor

4
...

Design and working: It is a static
bed of solid inert particles e
...
glass
beads
...

The depth of the bed is by several
factors including the density and the
compressibility of the solids, the
need to maintain the certain level of
critical nutrient e
...
oxygen through
the entire depth, flow rate that is
needed for a given pressure drop
...
A fluid containing nutrients flow continuously through the
bed to provide the needs of the immobilized catalysts
...
The flow may be upward or downward but downflow under the gravity is normal
...

Problem faced: The environment of a packed bed is non-homogenous as proper mixing is not done
...


5
...
In that case, photobioreactors are preferred
...
Open ponds or
raceways are often used to culture micro-algae
...


8

Photobioreactors are normally operated in continuous mode
...

Problem faced: Due to excess growth shadowing of culture and inhibitory levels of oxygen produced
...


Bubble column fermenter: It has a cylindrical column with aspect
ratio of 4 – 6
...
Mixing is
influenced by gas flow rate and rheological properties of fluid
...
For a
given gas flow rate, the mixing improves with increasing vessel
diameter
...


7
...
They provide high aeration
required pump and recycle air from fermenter and therefore, no
compressor
...
Automatic
temperature control is available
...
They are automatic for
charging alcoholic solution and discharging of completed
fermentation broth
...
Rate of oxygen removal from the
medium by the acetobacter cells is increased and so, there is no
oxidative browning
...
Acetators operate in semibatch mode and
cavitators in continuous mode
...
Whereas, in cavitator, nutrient liquid and air are sucked down a hollow tube
extending from the liquid surface so that agitation and caviation can cause air bubbles
...
The type of aeration-agitation system used in a particular fermenter
depends on the characteristics of the fermentation process under consideration
...
e
...

Thus, mechanical agitation is usually required in fungal and actinomycete fermentations
...
In such vessels, aeration is sufficient to produce
high turbulence, but a tall column of liquid does require greater energy input in the production of the compressed air
...
g
...
It should be possible to design a fermenter to achieve these conditions;
this will require knowledge of the most appropriate agitator, air sparger, baffles, the best positions for nutrient feeds,
acid or alkali for pH control and anti-foam addition
...


9

Types:

10

3
...

1
...

3
...

5
...

It must be available in sufficiently large volumes to provide an inoculum of optimum size
...

It must be free of contamination
...


Development of inocula for yeast processes
A
...
The
dangers inherent in this practice are:
• Introduction of contaminants
• The degeneration of the strain
• The degree of flocculence and attenuating abilities of the yeast
During the fermentation, the yeast cells flocculate and float to the surface, the first cells to do this being
the most flocculent and the last cells the least flocculent
...
Therefore, the middle skimmings contain cells which have the desired flocculence
and which have been protected from contamination by the surface layer of the yeast head
...
5 to 3, washing with water,
washing with ammonium persulfate and treatment with antibiotics such as polymyxin, penicillin and
neomycin
...
Sterols are
required for membrane synthesis but they are only produced in the presence of O 2
...
This anomaly is resolved traditionally by aerating the wort before inoculation
...

B
...
Although the production stages of the process may not be operated under strictly aseptic
conditions, a pure culture is used for the initial inoculum, thereby keeping contamination to a minimum in the
early stages of growth
...
The yeast may be pumped from one stage to the next or the seed
cultures may be centrifuged and washed before transfer, which reduces the level of contamination
...

Roll bottle technique: This technique is used for the production of spores of P
...

300 cm3 medium containing 3% agar sterilized in 1 dm3 cylindrical bottles
...


•

•

Sporulation on solid media: Many filamentous organisms will sporulate freely on the surface of cereal grains
from which the spores are harvested
...
The sporulation of a given fungus affected by the amount
of water added to the cereal before sterilization and the relative humidity of the atmosphere, which should be
as high as possible during sporulation (90 – 98% of humidity is required)
...
Sporulation in submerged culture is more
convenient than the use of solid or solidified media because it is easier to operate aseptically and it may be
applied on a large scale
...
For the submerged sporulation of the
griseofulvin-producing fungus P
...
05% - 0
...


13

4
...
If the initial
number of cells is N0 then,
After 1st generation, the cell number will be

= N0 × 21

After 2nd generation, the cell number will be

= N0 × 22

After 3rd generation

= N0 × 23 and so on
...

The term doubling time (td) or mean generation time (MGT) refers to the time taken for doubling the cell number or
biomass
...
If
N is the number of cells at a given time, then the increase in the number of cells (growth rate) with time is given by
the formula:
𝑑𝑁
𝑑𝑡

= μN

(1)

If X is the biomass concentration at a given time, then the increase in the biomass (growth rate)with time is given by:
𝑑𝑋

= μX

𝑑𝑡

(2)

In general, the specific growth rate (μ) is a function of the concentration of limiting substrate (S), the maximum
specific growth rate (μmax) and a substrate specific constant (Ks)
...
g
...

The growth rate (μ) of an organism is not fixed but it is variable depending on the environmental conditions such as
concentration of substrate and temperature
...

In batch culture, the substrate is initially present at a higher concentration i
...
S > Ks, hence the equation (3) is
approximately 1
...
Mode of Fermentation
The growth of microorganisms is a highly complex and coordinated process, ultimately expressed by increase in cell
number or cell mass
...

Doubling time: Doubling time refers to the time period required for doubling the weight of the biomass
...
30⸺1 hour
1⸺2 hours
25⸺48 hours
20⸺70 hours

Generation time: Generation time represents the period of doubling the cell numbers
...
The microorganisms can be grown in batch, fed-batch, semi-continuous and continuous culture systems in a
bioreactor
...

In fed-batch fermentation, substrates are added at short time intervals during fermentation
...

In case of continuous fermentation, as the fermentation proceeds, fresh growth medium is added continuously
...
This
enables the cells to grow optimally and continuously
...
The sterile nutrient culture medium in the bioreactor is inoculated
with microorganisms
...
It may be necessary to add acid or alkali to maintain pH and anti-foam agents to minimize foam
...

2
...

4
...

6
...


Lag phase: The initial brief period of culturing after inoculation is referred to as lag phase
...
There is no increase in
the cell number although the cellular weight may slightly increase
...
For instance, lag phase may not occur if the culture inoculated is at exponential
phase (i
...
log phase), and growth may start immediately
...


Acceleration phase: This is a brief transient period during which the cells start growing slowly
...


3
...
The cells
undergo several doublings and the cell mass increases
...
Growth rate of microbes
in log phase is independent of substrate (nutrient supply) concentration as long as excess substrate is present,
and there are no growth inhibitors in the medium
...
This is explained on the basis of extra energy needed
to split long chain substrates
...
This happens since one of the substrates is preferentially
metabolized first which represses the breakdown of second substrate
...
Now a second log phase occurs
...


Deceleration phase: As the growth rate of microorganisms during log phase decreases, they enter the
deceleration phase
...


5
...
The microbial growth may either slow down
or completely stop
...
This phase, however,
frequently associated with dramatic changes in the metabolism of the cells which may produce compounds
(secondary metabolites) of biotechnological importance e
...
production of antibiotics
...


Death phase: This phase is associated with cessation of metabolic activity and depletion of energy reserves
...
In the commercial and industrial fermentations, the growth
of the microorganisms is halted at the end of the log phase or just before the death phase begins, and the cells
are harvested
...
Periodical substrate addition prolongs log and stationaey phases which results in an increased
biomass
...
g
...

As it is difficult to directly measure substrate concentration in fed-batch fermentation, other indicators that correlate
with substrate consumption are used
...
In general, fed-batch fermentation requires more careful
monitoring than batch fermentation, and is therefore, not a preferred method by industrial biotechnologists
...
Depending on the microorganism
and the nature of recombinant protein, the fed-batch fermentation can increase the product yield from 25⸺1000%
compared to batch fermentation
...

Limitations: The major limitation of fed-batch culture is that the microorganisms in the stationary phase produce
proteolytic enzymes or proteases
...
By carefully
monitoring the fermentation, the log phase can be prolonged and the onset of stationary phase can be delayed
...

Fed-batch cultures for higher organisms: Fed-batch cultures are successfully employed for mammalian and insect
cells
...

Semi-continuous culture or semi-continuous fermentation
Some of the products of the fermentation are growth-linked and such products are formed from at the end of the log
phase e
...
ethanol production
...
This process of culture medium

16

change can be repeated at appropriate intervals
...
The product output is much higher compared to batch culture systems
...

There are however, certain disadvantages of semi-continuous fermentation
...

Continuous culture or continuous fermentation
Continuous fermentation is an open system
...
Both addition and removal are done at the same rate so that the
working volume remains constant
...

Types: Commonly two types
...


2
...
Chemostat bioreactors: The concentration of any one of the substrates (carbohydrate, nitrogen
source, salts, O2) is adjusted to control the cell growth and maintain a steady state
...
Turbidostat bioreactors: In this case, turbidity measurement is used to monitor the biomass
concentration
...

Plug flow bioreactors: In plug flow bioreactors, the culture solution flows through a tubular reaction vessel
without back mixing
...
Microorganisms along with nutrient medium are continuously
added at the entrance of the bioreactor
...

2
...

4
...

The yield of the product is more consistent since the physiological state of the cells is uniform
...

Continuous fermentation can be run in a cost-effective manner
...

2
...


Continuous fermentation may run continuously for a period of 500 to 1,000 hours
...

The recombinant cells with plasmid constructs cannot function continuously and therefore the product yield
decreases
...
Nutrient variations will
alter the growth and physiology of the cells, and consequently the product yield
...
However, it is expected that continuous fermentation will also
become popular in due course
...

2
...

4
...

6
...

8
...

10
...

12
...

14
...

16
...

18
...

20
...


Batch fermentation
It is a closed system
...

The process is stopped once the product is
formed
...

Less control over the growth of the microbes
and the production of desired products
...

Turnover rate (conversion of the substrate to
desired product) is less
...

Microbes in the fermenter show lag, log and
stationary phase
...

The fermenter is washed and cleaned before the
next step fermentation
...

Huge application in the industrial production
...
g
...

More common method for the large-scale
production of cell biomass and products
...

Less initial investment is required
...

Labor demand is less
...

Easy and quick control methods
...

2
...

4
...

6
...

8
...

10
...

12
...

14
...

16
...

18
...

20
...


Continuous fermentation
It is an open system
...

The process is not stopped for the collection of
the products, but it is continuously taken out
from the fermenter
...

More control over the growth of the microbes
and the production of desired products
...

Turnover rate is high
...

Microbes in the fermenter show exponential
growth rate in the fermenter
...

The fermenter is not washed and cleaned before
the next step fermentation
...

Limited application in the industrial production
...
g
...

Less common method for the large-scale
production of cell biomass and products
...

More initial investment is required
...

Labor demand is more
...

Hard and time-consuming control methods
...
Sterilization of Fermenters and Liquid
Media
A fermentation product is produced by the culture of a certain organism, or organisms, in a nutrient medium
...

2
...

4
...


6
...

If the fermentation is a continuous one then the contamination may outgrow the production organism and
displace it from the fermentation
...
g
...

The contaminant may produce compounds which make subsequent extraction of the final product difficult
...
g
...

Contamination of a bacterial fermentation with phase could result in the lysis of the culture
...

2
...

4
...

Sterilizing the fermenter vessel
...

Maintaining aseptic conditions during the fermentation
...
However, for practical reasons, stem is used almost universally for the sterilization of fermentation
media
...

The destruction of microorganisms by steam (moist heat) may be described as a first order chemical reaction and thus,
may be represented by the following equation:
(1)
Where N is the number of viable organisms present
t is the time of sterilization treatment
k is the reaction rate constant of the reaction, or the specific death rate
It is important at this stage to appreciate that we are considering the total number of organisms present in the volume
of medium to be sterilized, not the concentration – the minimum number of organisms to contaminate a batch is one,
regardless of the volume of the batch
...
Thus, k is
a true constant only under constant temperature conditions
...

On taking natural logarithms, equation (5) becomes:
(6)
By combining equation (3) and (5), the following expression may be derived for the heat sterilization of a pure culture
at a constant temperature:
(7)
Deindoerfer and Humphrey (1959) used the term ln (N0/Nt) as a design criterion for sterilization, which has been
variously called the Del factor, Nabla factor and sterilization criterion represented by the term ∇
...
Therefore,
(8)
Advantages of continuous sterilization over batch sterilization:
1
...

3
...

5
...


Superior maintenance of medium quality
...

Easier automatic control
...

The reduction of sterilization cycle time
...


Advantages of batch sterilization over continuous sterilization:
1
...

3
...


Lower capital equipment costs
...

Easier manual control
...


The early continuous sterilizers were constructed as plate heat exchangers and these were unsuitable on two accounts:
1
...


Failure of the gaskets between the plates resulted in the mixing of sterile and unsterile streams
...


20

However, modern continuous sterilizers use double spiral heat exchangers in which two streams are separated by a
continuous steel division
...
However, a major limitation
to the adoption of continuous sterilization was the precision of control necessary for its success
...
However, batch sterilization is still used in
many fermentation plants
...
The highest temperature which appears to be feasible for batch sterilization is 121 0
C so the procedure should be designed such that exposure of the medium to this temperature is kept to a minimum
...
Deindoerfer and Humphrey (1959) presented a method of asses the contribution made by the
heating and cooling periods
...

2
...


A profile of the increase and decrease in the temperature of the fermentation medium during the heating and
cooling periods of the sterilization cycle
...

The thermal death characteristics of the design organism
...


Knowing the original number of organisms present in the fermenter and the risk of contamination considered
acceptable, the required Del factor may be calculated
...
It is worth reinforcing at this stage that we are considering the total
number of organisms present in the medium and not the concentration
...
2
...
Thus,
knowing the Del factors contributed by heating and cooling, the holding time may be calculated to give the required
overall Del factor
...
Deindoefer and Hymphrey
(1959) produced integrated forms of the equation for a variety of temperature-time profiles, including linear,
exponential and hyperbolic
...
Richards (1966) considered the relative merits of in situ medium sterilization and the use of a
special vessel
...

2
...


4
...

The medium may be sterilized in a cooker in a more concentrated form than would be used in the fermentation
and then diluted in the fermenter with sterile water prior to inoculation
...

In some fermentations, the medium is at its most viscous during sterilization and the power requirement for
agitation is not alleviated by aeration as it would be during the fermentation proper
...
Obviously, the sterilization kettle would have to be equipped with a powerful motor, but this would
provide sterile medium for several fermenters
...


Disadvantages of a separate medium sterilization:
1
...

3
...

If a cooker serves a large number of fermenters complex pipework would be necessary to transport the sterile
medium, with the inherent dangers of contamination
...
The provision of contingency equipment may be prohibitively costly
...
Thus, sterilization in dedicated vessels is the method of choice for
batch sterilization
...
Thus, two of the major
objections to continuous systems (capital cost and aseptic transfer) may be considered as no longer relevant
...
The continuous system includes a time period during which
the medium is heated to the sterilization temperature, a holding time at the desired temperature and a cooling period
to restore the medium to the fermentation temperature
...
The length of the
holding period is dictated by the length of the coil and the flow rate of the medium
...
The
major advantage of the continuous process is that a much higher temperature may be utilized, thus reducing the holding
time and reducing the degree of nutrient degradation
...

Types of continuous sterilizer: Two types:
•
•

The indirect heat exchanger and
The direct heat exchanger (steam injector)

They are discussed below:
•

The indirect heat exchanger: The most suitable indirect heat exchangers are of the double-spiral type which
consists of two sheets of high-grade stainless steel which have been curved around a central axis to form a
double spiral
...

Process:
To achieve sterilization temperatures, steam is passed through one spiral and medium through the
other in countercurrent streams
...


22

↓
Incoming unsterile medium is used as the cooling agent in the first cooler so that the incoming medium
is partially heated before it reaches the sterilizer and thus, heat is conserved
...


2
...

This makes cross contamination between the two streams unlikely
...
The exchanger tends to be self-cleaning which reduces the
risk of sedimentation, fouling and burning-on
...
The plates are separated by gaskets and failure of these gaskets can cause
cross-contamination between the two streams
...
However, the plate exchanger is more adaptable than the spiral system in that extra plates may
be added to increase its capacity
...

Advantages:
1
...

3
...

5
...

It may be used for media containing suspended solids
...

Easy cleaning and maintenance
...


Disadvantages:
1
...


Foaming may occur during heating
...


Sterilization of the fermenter: If the medium is sterilized in a separate batch cooker, or is sterilized separately before
the sterile medium is added to it
...
Steam pressure held at 15 psi in the vessel for approximately 20 minutes
...

Sterilization of the feeds: A variety of additives may be administered to a fermentation during the process and it is
essential that these materials are sterile
...
If the additive is fed in large quantities then continuous sterilization may be
desirable
...

Whatever the sterilization system employed it is essential that all ancillary equipment and feed pipework associated
with the additions are sterilizable
...
Thus, waste biomass of such
organisms must be sterilized before disposal
...

Batch sterilization involves the sparging of steam into holding tanks, whereas continuous processes would
employ the type of heat exchangers
...

↓
The sterilization process have to be validated and are designed using the Del factor approach
...

stearothermophilus
...
Thus, the sterilization regime used for destruction of the process organism will be
different from that used in sterilizing the medium
...

2
...

4
...


2
...

4
...
The fluid in which the particles
are suspended will flow the filter by the route of least resistance
...
Inertial impaction is more significant in the filtration of gases than in the filtration of liquids
...
Thus, such small particles tend to deviate from the fluid flow
pattern and may become impacted upon the filter fibers
...

Electrostatic attraction: Charged particles may be attracted by opposite charges on the surface of the filtration
medium
...
Particles which
are larger than the filter pores are removed by direct interception
...
This may occur by several mechanisms
– more than one particle may arrive at a pore simultaneously, an irregularly shaped particle may bridge a pore,
once a particle has been trapped by a mechanism other than interception, the pore may be partially occluded
enabling the entrapment of smaller particles
...

Filters have been classified into two types: those in which the pores in the filter are smaller than the particles
which are to be removed and those in which the pores are larger than the particles which are to be removed
...
Filters of the latter type
are frequently referred to as depth filters and are composed of felts, woven yarns, asbestos pads and loosely
packed fiberglass
...
Terms which bear more relationship to the construction of filters are non-fixed pore
filters (corresponding with depth filters) and fixed pore filters (corresponding with absolute filters)
...
The packing material contains innumerable tortuous routes through the
filter but removal is a statistical phenomenon and thus, sterility of the product is predicted in terms of the
probability of failure (similar to the situation for steam sterilization)
...
Also, because the fibers are not cemented into position an
increase in the pressure to which the filter is subjected may result in movement of the material, producing
larger channels through the filter
...


24

Fixed pore filters are constructed so that the filtration medium will not be distorted during operation so that
the flow patterns through the filter will not change due to disruption of the material
...
e
...
Thus, interception is the major mechanism by which particles are removed
...
Fixed pore filters are superior for most purposes in that they have
absolute ratings, are less susceptible to changes in pressure and are less likely to release trapped particles
...
However, modern absolute filter
cartridges which have been developed by many filtration companies contain pleated membranes with very
large surface areas, thereby minimizing the pressure drop across the filter
...
Thus, the materials
must be stable at high temperatures and the steam must be free of particulate matter because the filter modules
are particularly vulnerable to damage high temperatures
...


Filter sterilization of fermentation media: Media for animal cell culture cannot be sterilized by steam because they
contain heat-labile proteins
...
An ideal filtration system for the sterilization of animal cell culture media must fulfill the following
criteria:
1
...

3
...


The filtered medium must be free of fungal, bacterial and mycoplasma contamination
...

The filtered medium should be free of viruses
...


Filter sterilization of air: Aerobic fermentations require the continuous addition of considerable quantities of sterile
air
...

Fixed pore filters (which have an absolute rating) are very widely used in the fermentation industry and several
manufacturing procedure filtration systems for air sterilization
...
The most common
construction material used for the pleated membranes for air sterilization is PTEE, which is hydrophobic and is
therefore resistant to wetting
...
As was seen for the filter sterilization of liquids it is
essential that a prefilter is incorporated up-stream of the absolute filter
...
The use of a coalescing prefilter also
ensures the removal of water from the air; entrained water is coalesced in the filter (air flow being from the inside of
the filter to the outside) and is discharged via an automatic drain
...
With the advent of the use of
recombinant organisms and a greater awareness of the safety and emission levels of allergic compounds the
contaminant of exhaust air is more common (and in the case of recombinant organisms, compulsory)
...
Also, foam may overflow from
the fermenter into the air exhaust line
...
This pretreatment may be a hydrophobic prefilter or a mechanical separator to remove water, aerosol
particles and foam
...
2-μm hydrophobic filter
...


25

7
...
To achieve this goal it is important to understand what is happening to a fermentation process and how to
control it to obtain optimal operating conditions
...
The provision of such conditions requires
careful monitoring (data acquisition and analysis) of the fermentation so that any deviation from the specified optimum
might be corrected by a control system
...
Thus, the pH may be measured and recorded but not maintained at a specified pH or
the dissolved O2 concentration may be determined but no attempt will be made to prevent O 2 depletion
...

There are three main classes of sensor:
1
...

3
...
g
...

Sensors which operate samples which are continuously withdrawn from the fermenter e
...
exhaust gas
analyzers
...
g
...


It is also possible to characterize a sensor in relation to its application for process control:
1
...


3
...

On-line sensor: Although the
sensor is an integral part of the
fermentation equipment, the
measured value cannot be used
directly for control
...

Off-line sensor: The sensor is not
part
of
the
fermentation
equipment
...
An operator is
needed
for
the
actual
measurement
(e
...

medium
analysis or dry weight sample)
and for entering the measured
values into the control system for
process control
...


26

Methods of measuring process variables:
Temperature: The temperature in a vessel or pipe is one of the most important parameters to monitor and control in
any process
...
Accurate mercury-in-glass
thermometers are used to check and calibrate the other forms of temperature sensors, while cheaper thermometers are
still used with laboratory fermenters
...
In larger fermenters, it would be necessary to insert it into a
thermometer pocket in the vessel, which introduces a time lag in restricting the vessel temperature
...

Electrical resistance thermometer: It is well known that the electrical resistance of metals changes with
temperature variation
...

Thermistors: Thermistors are semiconductors made from specific mixtures of pure oxides of iron, nickel and
other metals
...
The
change in resistance is a friction of absolute temperature
...
Thermistors are
relatively cheap and have proved to be stable, give reproducible readings and can be sited remotely from the
read-out point
...


Flow measurement and control: Flow measurement and control of both gases and liquids is important in process
management
...

The most commonly used example is a rotameter
...

By rotameters
By electrical flow transducer

Pressure measurement: Pressure is one of the crucial measurements that must be made when operating many
processes
...
Industrial
and laboratory equipment is designed to withstand a specified working pressure plus a factor of safety
...
The measurement
of pressure is also important in media sterilization
...
One of the standard pressure measuring
sensors is the Bourbon tube pressure gauge
...
The valve should be set to release the pressure as soon as it increases markedly
above a specified working pressure
...

Agitator shaft power: A variety of sensors can be used to measure the power consumption of a fermenter
...
This measuring
technique becomes less accurate as there is a decrease in in scale to pilot scale and finally to laboratory fermenters, the
main contributing factor being friction in the agitator shaft bearing
...
Since the dynamometer has to be placed on the shaft outside the fermenter, the measurement will once
again include the friction in the bearings
...

Foam sensing and control: The formation of foam is a difficulty in many types of microbial fermentation which can
create serious problems if not controlled
...
A probe is inserted through the top plate of the fermenter
...

When the foam rises and touches the probe tip, a current is passed through the circuit of the probe, with the foam acting
as an electrolyte and the vessel acting as an earth
...

Microbial biomass:
•
•
•
•

By dry weight samples (made quicker with microwave ovens)
Cell density (spectrophotometers)
Cell numbers (coulter Counters)
By the use of gateway sensors

Measurement of dissolved O2: At equilibrium, the probe signal of an electrode will be determined by:

P(O2) = C(O2) × PT
Where
P(O2) = partial pressure of dissolved oxygen sensed by the probe
C(O2) = volume or mole fraction of oxygen in the gas phase
PT = total pressure
pH measurement and control: In batch culture, the pH of an actively growing culture will not remain constant for very
long
...
Rapid changes in pH can often be reduced by the careful design of media, particularly in
the choice of carbon and nitrogen sources, and also in the incorporation of buffers or by batch feeding
...


28



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