FED BATCH FERMENTATION OF PHOSPHOBACTERIUM

EXPT NO:

DATE:

AIM:
To determine the feed rate(F), Growth rate(µ) and biomass(x) production rate of
the culture Phospho bacterium

INTRODUCTION:
A fed batch culture is one in which one or more nutrients are fed into
the fermentor during the fermentation period. In this way nutrients can be added
at the same rate as they are used up, so excess of nutrients and any inhibition
resulting from this can be avoided. A classical example is penicillin fermentation
where the different nutritional requirements at different phases can be catered for
feeding in particular nutrients at the time they are required.

Two basic approaches to the fed batch fermentation can be used:

1. Fixed volume fed batch
2. Variable volume fed batch

VARIABLE VOLUME FED BATCH:

As the name implies, a variable volume fed batch is one in which the
volume changes with the fermentation time due to the substrate feed. The way
this volume changes is dependent on the requirements, limitations and objectives
of the operator.

The feed can be provided according to one of the following options:

1. The same medium used in the batch mode is added.
2. A solution of the limiting substrate at the same concentration as that
in the initial medium is added.
Variable volume fed batch can still be classified as:
1. Repeated fed batch process or cyclic fed batch culture.
2. Single fed batch process.

REPEATED FED BATCH PROCESS:

In this method once the fermentation reached a certain stage after
which is not effective anymore, a quantity of culture is removed from the vessel
and replaced by fresh nutrient medium. The decrease in volume results in an
increase in the specific growth rate, followed by a gradual decrease as the quasi
steady state is established.

SINGLE FED BATCH PROCESS:

In this case, supplementary growth medium is added during the fermentation, but
no culture is removed until the end of the batch. This system presents a
disadvantage over the fixed volume fed batch and the repeated fed batch process:
much of the fermentor volume is not utilized until the end of the batch and
consequently, the duration of the batch are limited by the fermentor volume.

ADVANTAGES AND DISADVANTAGES OF THE FED BATCH REACTORS:

ADVANTAGES:

1. Production of high cell densities due to the extension of working of

working time.
2. Controlled conditions in the provision of substrates during the
fermentation, particularly regarding the concentration of specific substrates as for
example the carbon source.
3. Control over the production of byproducts or catabolite repression
effects due to limited provision of substrates solely required for product formation.
4. Allows the replacement of water loss by evaporation.
5. Increase of antibiotic marked plasmid stability by providing the
correspondent antibiotic during the time span of the fermentation.

DISADVANTAGES:
1. It requires previous analysis of the microorganism, its requirements
and the understanding of its physiology with the productivity.
2. In a cyclic fed batch culture, care should be taken in the design of the
process to ensure that toxins do not accumulate to inhibitory levels and that
nutrients other than those incorporated into the feed medium become limiting.
Also if many cycles are run, the accumulation of non-producing or low producing
variants may result.
PUMPS:
There are two types of pumps which are suitable for the aseptic pumping of small
volumes of culture media:
1. Peristaltic pump
2. Diaphragm dosing pump

PERISTALTIC PUMP:
A main body that comprises of both the derive motor and electrics and
the rotating unit of rollers typically constitutes the peristaltic pump. This unit if
rollers occludes the tube which, as it recovers to its original size passes to the next
roller until it is expelled, as the moves around. The flow rate can be varied by
either the speed setting or by changing the diameter of the tube being used.
DIAPHRAGM DOSING PUMP:
The diaphragm-dosing pump consists of a main body and a detectable
heat sterilizable head. The fluid is sucked into the pump head. The suction inlet
tube then closes and the pressure discharge tube opens and forces the fluid out.
The suction and pressure forces in the pump head are generated by the
reciprocating action of both diaphragm plunger and the return spring.

MODELING FED BATCH FERMENTATION:

VARIABLE VOLUME FED BATCH:
In a variable volume fed batch fermentation, an additional element
should be considered, the feed. Consequently, the volume of the medium in the
fermentor varies because there is an inflow and no outflow. Again, the growth of
the fermentation of the microorganism is limited by the concentration of one
substrate.
For the mathematical developments that will be presented, the assumptions are:
1. Specific growth rate is uniquely dependent on the concentration of the
limiting substrate.
2. The concentration of the limiting substrate in feed is constant.
3. The feed is sterile.
4. The yields are constant during the fermentation time.

Considering the overall mass balance

(Input) = (output) + (accumulation)
F = 0 + dV/dt
F = dV/dt (1)
Where,
V – the time volume of the fermentor
T – time
F – the feed rate (volume/time)
Considering now the balance to the biomass
(Accumulation) = (in) + (produced) – (lost by cell death)
d(Vx)/dt = F. Xo + rx.V – rd.V (2)
where,
x – biomass concentration (mass of biomass/volume)
Xo - biomass concentration in the feed (mass of biomass in
feed/volume)
rx- biomass production rate (mass of biomass/volume. time)
rd – biomass death rate (mass of biomass /volume. time)
As the feed is considered to be sterile, Xo amounts to zero.
Therefore eqn (2) becomes
d (Vx)/dt = rx.V – rd.V
V.dx/dt + xdV/dt = rx.V – rd.V
 V.dx/dt + xF = rx.V – rd.V (using eqn
1)
But,
rx.V = µ.x.Vand rd.V = kd.x.V
where,
µ =specific growth rate (time-1)
k d = specific death rate (time-1)
Therefore
V.dx/dt + xF = µ.x.V- kd.x.V
Rearranging the equation
dx/dt = x (µ.V- kd.V - F)/ V
Specific growth rate is smaller so it can be neglected.
Consider now the balance to limit substrate
(accumulation) = (in) + (consumed)
d(V.s)/dt = F.so – rs.V
Where,
s is the substrate concentration in the fermentor (mass of substrate / volume)
so is the substrate concentration in the feed (mass of substrate in feed/ volume)
rs is the consumption rate of substrate (mass of substrate/volume. time)
Vds/dt + s.dV/dt = F.so – rs. V
It should be noted that the consumption rate of substrate includes the specific
consumption used for biomass production, product formation and maintenance of
the cell. Including now the concept of yield
rs. V = µ. x. V/Yx/s
Where,
Yx/s is the ratio between the mass of cells produced per mass of substrate
consumed.
Substituting rs. V = µ. x. V/Yx/s
Vds/dt + s. F = F.so – µ. x. V/Yx/s
Rearranging the equation,
ds/dt = F.(so – s)/ V - µ.x./Yx/s
The table below summarizes the equations that have just been developed:
Mass balance for the main components for a fed batch reaction:

Component Mass balance equation

Overall F = dV/dt
Biomass dx/dt = x (µ.V- kd.V - F)/ V
Substrate ds/dt = F.(so – s)/ V - µ.x./Yx/s
Product dp/dt = Qp.x – P.F/V

V is the volume of the fermentor

T is the time
F is the feed rate (Volume/time)
X is the biomass concentration (mass of biomass / volume)
µ is the specific growth rate. (time–1)
kd is the specific death rate (time-1)
s is the substrate concentration in the fermentor (mass of substrate / volume)
so is the substrate concentration in the feed (mass of substrate /volume)
Yx/s is the yield factor (mass of biomass / mass of substrate)
Γ is product concentration (mass of product / volume)
Qp is the specific production rate of product (mass of product /mass of biomass.
time)

MATERIALS AND METHODS:

BIOREACTOR (Ex-situ type)
The studies that will be reported here were conducted on 5lit (working volume
2.5lit) reactor. This reactor is equipped with flat four-blade Rushton turbine.
Height 265mm, diameter of tank 146mm, diameter of impeller 58mm, inter
impeller distance 93mm, distance between last impeller and tank 212mm. The
tank has two baffles with a width and height of 16 and 25mm.
The organism used was Phosphobacterium.

CULTURE MEDIA
COMPOSITION g/lt
Beef extract 3
Peptone 5
NaCl 5
Distilled water 1000ml
INOCULUM PREPARATION:
A single loop of Phosphobacterium culture was scrapped using a sterile
inoculation loop and then aseptically transferred into a 15ml nutrient medium. This
starter culture was grown overnight. This was mixed with 300ml of media (having
same composition as the reactor media i.e. nutrient medium) and then inoculated
into 2.5lt reactor media to start fed batch fermentation.

PARAMETERS:
Time of sterilization 9:00am
Temperature 121ºC
Pressure 1bar
Hold time 20min
Inoculum 300ml
Time of feed 2:00pm

FED BATCH FERMENTATION:

The experimentation was conducted in a 5L fermentor equipped with
temperature, pH, airflow, DO, pressure and agitation controllers. The experiment
was conducted at 30 ºC. DO was measured by a DO probe. The culture media of
the above composition was used.
When the substrate is completely consumed by the biomass, ds/dt = 0
Hence the substrate balance equation reduces to
FoS/V =µ.x.Yx/s
F = µ. x. V/S0 Yx/s
RESULT:

Hence the feed rate (F), growth rate (µ)and biomass concentration(x) are
determined for the culture of Phospho bacterium.

INFERENCE:
Through the fed batch method following graphs were determined for

OD Vs X :
From this standard graph corresponding biomass concentration are determined
with respect to OD values.

Feed rate Vs Time:

We obtain a graph of increasing order to the corresponding values of feed and
time, which concludes the increase in the growth rate of the organism.
Growth rate Vs Time:
From this graph we obtain a linear increase in growth
corresponding to the time till the substrate is completely utilized, then the growth
rate decreases.

OD Vs time:
There exists a linear increase in OD with respect to time.

ln OD Vs time:
There exists a linear decrease in OD with respect to time.

BATCH HEAT STERILIZATION AND THERMAL DEATH KINETICS

EXPT.NO :

DATE :

AIM:
To determine the holding time of reactor in batch kinetics.
THEORY:
A fermentation product is produced by a culture of certain organisms
in a nutrient medium. If a foreign microorganism invades the fermentation, the
following may be the consequences:
• There would be competition between the foreign microorganism and
the production microorganism for the same nutrient medium, thus leading to loss
of productivity.
• If the fermentation is of continuous mode, the contaminant may
outgrow the production microorganisms, and displace it from fermentation, thus
leading to the loss of money, time and energy.
• Final product may be contaminated.
• Extraction of final product may be very difficult
• Degradation of the desired product may take place due to metabolism
of the contaminant. This is especially true in the case of the production of
antibiotics. If the contaminant happens to be a bacterial strain, which is resistant
to the action of the antibiotic, and this antibiotic also happens to be a part of the
normal metabolic pathway of the contaminant, then it leads to a problem. Eg.
Degradation of β - lactam antibiotics by β - lactamase producing bacteria.
• Contamination of bacterial fermentation with bacteriophages could
result in lysis of the bacterial cells. But this does not occur very often.
Avoidance of contamination may be achieved by:
• Using a pure inoculum to start out with.
• Sterilizing the medium, fermentor vessel and all the materials used in
the process.
• Maintaining aseptic conditions during the fermentation.

Liquid medium is most commonly sterilized in batch mode in the same fermentor
where it will be eventually used. The liquid is heated to sterilization temperature
by introducing steam into the coils or the jacket of the vessel. Other ways of doing
this could be heating the vessel electrically or by bubbling steam into the medium.
If direct steam injection is used, allowance must be made for dilution of the
medium by condensate, which typically adds 10-20% to the liquid volume quality
of the steam must also be high enough to avoid contamination of the medium by
metal ions or organics.
Depending upon the rate of heat transfer from the steam or electrical
element, raising the temperature of the medium in large fermentors can take a
significant period of time. Once the holding or sterilization temperature is reached,
the temperature is held constant for the period of time thold. Cooling water in the
coils or jacket of the fermentor is then used to maintain the temperature.
For operation of batch sterilization systems, we must be able to
estimate the holding time required to achieve the desired level of cell destruction.
As well as destroying contaminants, heat sterilization may also destroy the
nutrients in the medium. To minimize this loss, holding times at the sterilization
temperature must be kept as short as possible.

CELL DEATH KINETICS

In a lethal environment, cells in a population do not all die at once.
Deactivation of the culture occurs over a finite period of time depending on the
initial number of viable cells and the severity of conditions imposed. Loss of cell
viability can be described mathematically as follows.
Cell death is assumed to be a first order process

rd = -dN/dt = kd N (1)
Where rd is the cell death rate, N is the number of variable cells, and kd is the
specific death rate.
Instead of N, we can also express the rate of cell death in terms of biomass (X) of
variable cells.
If kd is constant we can integrate eqn, (1) as follows to derive an expression for N
as a function of time.
N t
∫ (1/N) dN = kd ∫t dt (2)
No o
To get
ln Nt = ln No-kdt (3)
(-k t)
Nt = No e d (4)
Thus, if first order kinetics applies, a plot of ln Nt versus t gives a
straight line with slope kd. However, first order kinetics does not always hold,
particularly for bacterial spores immediately after exposure to heat. The value of
kd is not only species dependent, but also dependent on the physiology of the cell
itself. For eg. Bacillus stearothermophilus is the most heat resistant bacteria
known.
Like other kinetic constants, the specific death constant also depends on
temperature, T. this effect can be described using the Arrhenius relationship:

Kd = Ae(-Ea/RT) (5)

Where A is the Arrhenius constant or frequency factor, Ea is the activation energy

for thermal cell death and R is the universal gas constant.

DESIGN CONSIDERATIONS
DEL FACTOR
Combining eqns 3 & 5, we get:
ln (No / Nt) = Ae (-Ea/RT)* t (8)
This term, ln (No/Nt), has been described as Humphrey and
Deindoerfer as the Del factor, ∇ , or the Nebla factor or the sterilization criterion.
 Thus, the Del factor is a measure of the fractional reduction in viable
organism count by a certain heat and time regime.
Rearranging (6), we get:
ln t = E/RT + ln (∇ /A) (7)
Thus, a plot of ln t versus T world yield a straight line of slope E/R and
y intercept ln (∇ /A).
Thus, the same degree of sterilization may be obtained by heating the substance
at a high temperature for a short time, or a low temperature for a long time.

NUTRIENT DESTRUCTION:
Two types of reactions contribute to loss of nutrient quality during sterilization.

INTERACTIONS BETWEEN SOME NUTRIENT COMPONENTS OF THE MEDIUM

A common reaction during sterilization is the Maillard type browning reaction. This
results in discoloration of the medium as well as loss of nutrient quality. These
reactions are normally caused by the reaction of carbonyl groups, usually from
reducing sugars with the amino acids of proteins. Sterilizing the sugar separately
and mixing it with the rest of the sterilized medium after cooling resolve this
problem.

DEGRADED HEAT- LABILE COMPONENTS:

Certain amino – acids and proteins may be degraded during a heat sterilization
regime. Thermal destruction of nutrient components conforms with first order
kinetics and may be degraded by a from similar to the Arrhenius equation:
(Xt/Xo) = e (-kt) (8)
Values of activation energy Ea for thermal destruction of vitamins and amino acids
are 84 to 92 kJ/gmol, for proteins it is about 165 kJ/gmol. As these values are a
bit lower than that of microorganisms, raising the temperature has a greater effect
on cell death than nutrient destruction. This means that sterilization at higher
temperatures (and shorter times) has the advantage of killing cells with limited
destruction of medium components.

THE PROBABILITY OF CONTAMINATION, N:

Let No denote the number of contaminants present in the raw
medium . During the heating period, the holding period and at the end of the
cooling period, the final number is reduced to N. Ideally N should be zero. But this
is practically impossible to achieve, as absolute sterility would take infinitely long
to reach. Normally the target level of contamination is expressed as a probability
of contamination, and this value is fixed at 0.001 i.e. one batch in every 1000 is
expected to be non-sterile. To apply this kinetics, we need to know some of the
thermal death characteristics of the bacteria present in the medium. The
assumption that Bacillus stearothermophilus one of the most heat resistant
bacteria known, is the only microbial contaminant present, is a safe one(we cannot
practically calculate the average thermal death kinetic values of every known
taxon that might constitute a contamination).Thus a considerable safety factor can
be built into the design by adopting B.stearothermophilus as the design organism.
The death characteristics of this organism are:
Ea = 283000 J/mol (9)
37
A = 9.5 * 10 / min
We must keep in mind that these values will vary considerably
depending on the type of medium used. This is particularly relevant when
considering the sterilization of oils and fats (common fermentation substrates)
where relative humidity may be relatively low (spores of B. stearothermophilus are
ten times more resistant when dry, then when wet).

HOLDING TIME DESIGN:

If No and N known. We can determine the holding time required to reduce the
number of cells by considering the kinetics of cell death.

∇ total = ∇ heating + ∇ holding +∇ cooling (10)

∇ total = ln (No/N) from eqn (6)

We know that for the organism B.stearothermophilus, a good estimate of the

number cells per unit volume of reactor would be = 106
spores / mL
The reactor volume we had was = 750mL
Thus, initial number of organisms we start out with = No = 106 * 750 = 7.5
* 108

∇ heating and ∇ cooling are collected as follows:

We know that ∇ heating = k * t from eqns (5) & (6)
k=Ae (-Ea/RT) eqn (5)

For the organism B. stearothermophilus Ea and A are known. We also have a set of
readings of T (temperature) vs. t (time) for heating and for cooling. Thus for each
value of T, we can get the corresponding value of k (from eqn.5)
If we now plotted k against t, we would obtain a graph whose AUC (or
area under the curve) is = k* t, which in turn, is equal to ∇ . Depending on which
set of readings we took of T vs. t (whether the heating or the cooling) we would
get ∇ heating or ∇ cooling.
Thus, from eqn 10. we can calculate ∇ holding. We know that the holding temperature
is going to be 121°C. from this, we can calculate the corresponding value of k and
t hence, the holding period for the sterilization.

NON – HOMOGENOUS MEDIA:

 These design procedures apply to batch sterilization of medium when
the temperature is uniform throught the vessel. However, if the liquid contains
contaminant particles in form of flocs or pellets, temperature gradients may
develop. Because heat transfer within solid particles is slower than in the liquid,
temperature at the center of the solid will be lower than that in the liquid for some
portion of the sterilizing time. As a result, cell death inside the particles is not as
effective as in the liquid. Longer holding times are required to treat solid- phase
substrates and media containing particles.

PROCEDURE:

• The experimental batch data is obtained using a 2L batch fermentor. It

is filled to the 750mL mark with water.
• The reactor is heated using a heating finger, and temperature was
noted down regularly. A point to remember at this stage the safety valve has to be
removed during the heating stage to prevent reactor bursting.
• Once the temperature has reached 121°C, the heating element is
disconnected, and cooling is begun. Once again, temperature is noted down
regularly. We must remember to plug safety valve back in again at this cooling
stage to prevent a vacuum from developing inside the reactor, which would induce
it to suck in (non-sterile) air from outside.
• This is the first part of the experiment. In the second part, 10ml
culture was exposed to a temperature of 60 °C for 5 min, 10 min and 15 min. At
the end of each time interval, 1 mL of culture was withdrawn and plated in LB
plates at dilutions 1:102, 1:104, and 1:106. The same procedure was repeated for
80ºC.
• The plates were incubated overnight and the colonies were counted
the next day.

RESULT:

The holding time and thermal death rate constant (kd) of batch heat sterilization
are determined

INFERENCE:
Thus tholding can be considerably reduced by considering heating and
cooling parts of cycles in the sterilization process.
The main aim of designing a process is to achieve the probability of obtaining
sterility with minimum loss of nutritive quality. Hence, exposure of medium is kept
to be minimum. An important observation is that the del factor does not include
the absolute number of contaminant concentration and concentration of survival.
Moreover , the holding time is kept as minimum as possible since
heating sterilization may also destroy the medium nutrients. Thus by including
heating and cooling cycles ,the holding times are kept minimum which minimizes
the loss