TITLE : FED-BATCH FERMENTATION

INRODUCTION AND THEORY :

Fed-batch culture is define as a technique in microbial process where one

or more nutrients are supplied to the fermenter during cultivation but no removal
of the culture until the end of the process (Yoshida et al., 1973). The basic
characteristic of fed-batch culture is that the concentration of the nutrient fed
into the culture liquid can be controlled by changing the feed rate. They are 2
basic approaches of fed-batch that can be use. They are fixed volume fed-batch;
variable volume fed-batch and exponential fed-batch.
In the 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 it
is dependent on the requirements, limitations and objectives of the operator.
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 increasing in the specific
growth rate, followed by a gradual decrease as the quasi-steady state is
established.
In the fixed volume fed-batch, the limiting substrate is fed without diluting
the culture. The culture volume can also be maintained practically constant by
feeding the growth limiting substrate in undiluted form, for example, as a very
concentrated liquid or gas (ex. Oxygen). Alternatively, the substrate can beaded
by dialysis or, in a photosynthetic culture, radiation can be growth limiting factor
without affecting the culture volume. A certain type of extended fed-batch the
cyclic fed-batch culture for fixed volume systems refers to a periodic
withdrawal of a portion of the culture and use of the residual culture as the
starting point for a further fed-batch process.
Basically, once the fermentation reaches a certain stage, (for example,
when aerobic conditions cannot be maintained anymore) the culture is removed
and the biomass is diluted to the original volume with sterile water or medium
containing the feed substrate. The dilution decreases the biomass concentration
and result in an increase in the specific growth rate. Subsequently, as feeding
continues, the growth rate will decline gradually as biomass increases

approaches the maximum sustainable in the vessel once more, at which point
the culture may be diluted again.
Fed-batch fermentation is a production technique in between batch and
continuous fermentation (Longobardi, 1994). A proper feed rate, with the right
component constitution is required during the process. Fed-batch offers many
advantages over batch and continuous cultures. From the concept of its
implementation it can be easily concluded that under controllable conditions and
with the required knowledge of the microorganism involved in the fermentation,
the feed of the required components for growth and/or other substrates required
for the production of the product can never be depleted and the nutritional
environment can be maintained approximately constant during the course of the
batch.
The production of by-products that are generally related to the presence of
high concentrations of substrate can also be avoided by limiting its quantity to
the amounts that are required solely for the production of the biochemical. When
high concentrations of substrate are present, the cells get "overloaded", this is,
the oxidative capacity of the cells is exceeded, and due to the Crabtree effect,
products other than the one of interest are produced, reducing the efficacy of the
carbon flux. Moreover, these by-products prove to even "contaminate" the
product of interest, such as ethanol production in baker's yeast production, and
to impair the cell growth reducing the fermentation time and its related
productivity.
Sometimes, controlling the substrate is also important due to catabolic
repression. Since this method usually permits the extension of the operating
time, high cell concentrations can be achieved and thereby, improved
productivity [mass of product/(volume.time)]. This aspect is greatly favored in
the production of growth-associated products. Additionally, this method allows
the replacement of water loss by evaporation and decrease of the viscosity of
the broth such as in the production of dextran and xanthan gum (McNeil and
Harvey, 1990), by addition of a water-based feed.

OBJECTIVES :

1. To train students on how exponential fed-batch fermentation for controlling

specific growth rate of saccharomyces cerevisae at certain value during
fermentation can be operated.
2. To give students exposure on the use of model for process control.

RESULTS :
Table 1 : Overall results
Time(h

OD

V(ml)

V(L)

VX(g)

ln(VX

S(g/L)

DOT%

)
0
2

600
0.119
0.15

(g/L)
0.068
0.100

700
700

0.7
0.7

0.048
0.070

)
-3.045
-2.654

19.9
19.65

100
98.8

4
6
8

0.28
0.8
10.5

5
0.237
0.783
10.97

700
700
700

0.7
0.7
0.7

0.166
0.548
7.680

-1.796
-0.601
2.039

18.2
16.5
5.05

97.5
92.1
78.5

12.6

2
13.17

700

0.7

9.225

2.222

1.67

87.1

12

13.8

8
14.43

700

0.7

10.107

2.313

1.012

52.6

13

14.9

9
15.59

700

0.7

10.916

2.390

0.881

43

14

15

4
15.69

728

0.728

11.429

2.436

0.268

45

15

15.5

9
16.22

766

0.766

12.428

2.520

0.197

60.2

16

16.5

4
17.27

804

0.804

13.889

2.631

0.153

70.1

17

21

5
22.00

846

0.846

18.613

2.924

0.141

74.3

31

1
32.50

875

0.875

28.442

3.348

0.004

78.2

35

5
36.70

941

0.941

34.542

3.542

0.08

80.3

10

18
19

20

45

8
47.21

21

50

2
52.46

1055

1.055

55.350

4.014

0.019

84.5

22
24

65
85.6

4
68.22
89.85

1299
1500

1.299
1.5

88.618
134.78

4.484
4.904

0.012
0.009

84.3
84.3

1002

1.002

47.306

3.857

0.054

83.1

y = 0.952x + 0.0543

DISCUSSIONS :
1. Plot graph (refer to graph 1)
For the cell concentration X(g/L) versus time (hour) graph, it gives
sigmoid-like graph. From hour 0 to 6, there were so little in increasing cell
concentration. This period is called as a lag phase which is the adaptation period.
During this stage, the cells in the bioreactor were trying to adapt the surrounding
conditions like, pH, temperature, nutrients provided and more. The adaptation
process slowed the growth process. It takes a time for it to establish itself and
begin adding to the population. After that, the population began to expand
rapidly in what is known as logarithmic growth. It was started from the 6 th hour
until the end of the fermentation process. The cell concentration was increasing
rapidly until the 16th hour but it became much slower after the 16th hour due to
the optimum growth rate of the cell.
For the glucose concentration,S(g/L) versus time (hour), the decreasing of
the value was occurred. The concentration of the glucose decreased as the
glucose was cosumed by the cell in the tank. At 0 to 6 th hour, the amount of

glucose consumed is small. It is because at this period, the tank has small
number of cells population in it. At 6 th to 16 hour, amount of glucose fall
extremely as cell concentration increase. But the question is, during 6 th to 16th
hour, the cell concentration not really in optimum condition(refer to graph 1).
The population of the cell increase actively started from the 16 th hour. If we look
at graph 3, there is small changes in glucose consumption. Cells not actively
consume the glucose when the cell concentration extremely increase. Maybe
when the population of the cells become bigger, the needs of glucose no longer
required even it is slightly impossible.
On the other hand, graph for Cell concentration times the culture volume
(VX) versus time (hour) give almost the same graph shape as cell concentration
X (g/L) versus time (hour) graph. Changes in cell concentration related to the
culture volume. If the cell concentration increases, the culture volume will also
increase. It is because, the size of cell population will fill up the bioreactor tank,
thus increase the volume of the culture. From 0 hour to the 6 th hour still no big
changes in cell concentration and culture volume. From 6 th hour to the 16 hour
there is only some improvement in concentration and the volume. And the
highest improvement was started from 6th hour until 24th. This means the growth
of the cells are optimum.

2. Calculate the yield coefficient (Yx/s) and productivity (P) obtained

from the cultivation and compare with the results obtained on the
batch fermentation (Practical 5)
Yx/s

(Xm X0/ Si St)

(89.859 0.068/ 19.9 0.009)

4.514 g/L/h

Productivity of biomass = max biomass concentration

Time
= 89.859 / 24
= 3.744 g/L/h

For biomass yield coefficient, the value for batch fermentation is

0.104 g/L while the value for fed-batch fermentation is 4.524 g/L. The
value for biomass yield coefficient is higher than the batch fermentation.
Biomass yield coefficient is obtained by:
(Maximum cell concentration initial cell concentration)/ (initial substrate
concentration final substrate concentration).
The higher value for fed-batch fermentation is due to the bigger amount of
the cell obtain and the least the amount of the substrate due to the
increase in feeding while for the batch fermentation, the amount of
substrate consume is high and cell concentration is low compare to the
fed-batch due to the no other feeding during the fermentation process.
The productivity from the batch fermentation is 0.357 g/L/h and the
productivity for fed-batch fermentation is 3.744 g/L/h. Productivity is
measure by maximum cell concentration/time. In the fed-batch, the
amount of the cell concentration obtain is higher that is 89.85 g/L and the
cell is obtain in shorter time that is 24h hour compare to the batch
fermentation. In batch fermentation, the amount of the cell obtains is 10.7
in 30 hour. So, the productivity of the fed-batch fermentation is higher
than the batch fermentation.
3. Plot (VX) versus time (t). From the equation, controlled real
value

which

was

controlled

during

the

cultivation

can

be

calculated as a slope of the graph. Compare the real value of

with the calculated *.

Graph 2 : Graph ln (VX) versus Time (h)

From the graph, we can determine the specific growth rate () of the
inoculums that we use. From the equation dx/dt=x, we have to integrate the
equation and we get a new equation that is ln(x/x0)=x where the is the
specific growth rate. This formula is applied for batch fermentation.
The slope from the graph determines the specific growth rate or . The
slope from the graph is 0.1837. Comparing the specific growth rate of the batch
fermentation and the fed-batch fermentation, the specific growth rate of the fedbatch fermentation is slightly higher than the batch fermentation. To determine
the specific growth rate for the fed-batch, the formula that being used is in
ln(VtXt)/(V0X0) = t and the can be obtain by using the slope. In the fedbatch fermentation, the value for the specific growth rate because we take into
consideration of the value of volume in the calculation. Besides that the growth
rate of the inoculums is being control to desired exponential growth by
controlling the feeding rate.
In fed-batch fermentation, the amount of substrate is continuously being
added into the fermenter, this may result in increase cell concentration because
there is always food for them to grow. Compare to the batch fermentation, the
medium is not being added into the bioreactor, thus cell concentration will
decrease when there is depletion of the substrate. In fed-batch also the cell

specific growth rate also higher because we control the growth rate at the log
phase which mean we prolong the log phase to get the maximum cell
proliferation cause at the log phase there is maximum cell division.
The graph shows the whole fermentation where by the batch fermentation
is taking into consideration but starting from 18 hours, the fed-batch
fermentation is taking into consideration. Its starts from 18 hour of the
fermentation and the graph are in the exponential growth. In the first 17 hour the
fermentation is carryout in batch fermentation because in order to do the fedbatch fermentation, we have to start inoculate the yeast with batchfermentation. After that, the fermentation process is taken by the fed-batch
fermentation where the medium start to be inserting and the growth rate of the
inoculums is controlled.

REFERENCES :
1. Agrawal P., Koshy G. and M. Ramseier: An algorithm for Operating a FedBatch Fermentor at Optimum Specific Growth Rate. Biotechnology and
Bioengineering, 33, 115 -125, (1989).
2. Atkinson B., and F. Mavituna: Biochemical Engineering and Biotechnology
Handbook, Stockton Press, (1991)
3. Jong J., Hsiun D. and W. Wu: Fed-Batch Culture of Bacillus thuringiensis for
Thuringiensin Production in a Tower Type Bioreactor. Biotechnology and
Bioengineering, 48, 207 - 213, (1995)
4. Longobardi G. P.: Fed-Batch versus Batch Fermentation. Bioprocess
Engineering, 10, 185 - 194, (1994)

5. McNeil B. and L. M. Harvey: Fermentation, a Practical Approach. IRL Press,

Tokyo, (1990)

Diagram of fed-batch culture