DEPARTMENT OF BIOPROCESS TECHNOLOGY

FACULTY OF BIOTECHNOLOGY AND BIOMOLECULAR SCIENCES

UNIVERSITI PUTRA MALAYSIA

BTC4305 – 1

BIOPROCESS MODELLING AND OPTIMIZATION

LAB REPORT 1

FERMENTATION DATA ANALYSIS USING MICROSOFT EXCEL

NAME: NUR ANIS AFIFAH MOHD ELIAS

MATRICS NUMBER: 200551

LECTURER: DR. MOHD. SHAMZI MOHAMED

 FERMENTATION DATA ANALYSIS USING MICROSOFT EXCEL

QUESTION 1

Below is a set of data on batch fermentation of Saccharomyces cerevisiae using 2% glucose as a

carbon source. Enter all the data on a sheet (MS-EXCEL) and plot the cell dry weight, glucose and
ethanol as a function of time. For this batch cultivation process, calculate the following kinetic
parameter values:

Time (h) DCW (g/L), X Glucose (g/L), S Ethanol (g/L), P

0 0.22 20.8 0.3
1.5 0.34 15.4 0.2
3.5 0.51 15.0 0.3
6.25 1.07 11.6 1.2
8.20 2.12 5.4 3.45
10.5 3.65 0 6.3
12.0 4.40 0 5.8
22.0 4.50 0 4.2
23.5 4.63 0 3.9

Plot of DCW against Time

5.00
4.50
4.00
3.50
DCW (g/L), X

3.00
2.50
2.00
1.50
1.00
0.50
0.00
0.00 5.00 10.00 15.00 20.00 25.00
Time (h)

Figure 1 Plot of Dry Cell Weight against Time

In growth kinetics, the rate of growth is directly proportional to the concentration of cell. Based
on Figure 6, the growth kinetics of batch cultivation of yeast follows a microbial growth curve
which is divided into lag phase, exponential growth or log phase and stationary phase. The
lag phase can be seen from 0 h to 1.5 h of cultivation, where the cells are adapting to the
environment, causing a low growth rate. From 1.5 h to 10.5 h, log phase was observed,
whereby the yeast cells divide rapidly at a constant rate, causing the cell mass and cell
number increases exponentially. Stationary phase was observed from 10 h onwards, where
growth rate decreases when the nutrients are being used up and by-products accumulate,
thus the microbial growth rate equals to the death rate.

Plot of Glucose Concentration against Time

25.00
Glucose concnetration (g/L), S

20.00

15.00

10.00

5.00

0.00
0.00 5.00 10.00 15.00 20.00 25.00
Time (h)

Figure 2 Plot of Glucose Concentration against Time

Based on Figure 2, the glucose concentration decreased with time, as it acts as a substrate
for the yeast cells, thus were consumed during the batch cultivation process.

Plot of Ethanol Concentration against Time

7.00
Ethanol concentration (g/L), P

6.00

5.00

4.00

3.00

2.00

1.00

0.00
0.00 5.00 10.00 15.00 20.00 25.00
Time (h)

Figure 3 Plot of Ethanol Concentration against Time

Based on Figure 3, the concentration of ethanol, which is the product of yeast fermentation
increases until 10.5h of cultivation. the decrease in ethanol in ethanol concentration beyond
10.5h of fermentation might be due to the consumption of yeast cells on ethanol, as the
substrate, which is glucose depleted, from 10.5h of fermentation.
 1. Specific growth rate (µ)

Based on Figure 1, exponential phase occurs at 1.5h to 10.5h

Xt
ln =μ ( t t−t 0 )
X0
ln X t−ln X 0=μ ( t t−t 0 )
(ln X t −X 0 )
μ=
( t t−t 0 )
μ=¿ ¿
μ=0.264 h−1
Thus, the specific growth rate during the exponential phase of batch fermentation of yeast
0.264 h-1

2. Doubling time (td)

0.693
T d=
μ
0.693
¿
0.264
¿ 2.63 h
Thus, 2.63 h is required for a given quality to double in size at a constant growth rate

3. Biomass yield coefficient (YX/S)

YX/S
X m− X 0
¿
S0 −S f
4.63−0.22
¿
20.8−0
cell
¿ 0.212 g substrate
g
Thus, 0.212g of cells will be produced from 1g of substrate, which is glucose

4. Product yield coefficient (YP/S)

YP/S
P m−P0
¿
S 0−S f
6.3−0.3
¿
20.8−0
product
¿ 0.288 g substrate
g
 This, 0.288g of product, which is ethanol, will be produced from 1g of substrate, which is
glucose

5. Productivity of biomass (g/L/h)

Productivity of biomass (applied on exponential phase) = rate of cell growth
= µX
= 0.264 x 3.65
= 0.964 g/L/h
Thus, 0.964 g/L of biomass were produced every hour

6. Productivity of ethanol (g/L/h)

Productivity of ethanol (applied on maximum product formed)
= rate of product formation
P max−P 0
¿
t max−t 0
6.3−0.3
¿
10.5−0
g
¿ 0.571 /h
L
Thus, 0.571 g/L of ethanol were produced every hour

QUESTION 2

Below are the 2 set of data on batch cultivation of microorgansims. Plot the lines curve of the data in
one graph. Compare the growth kinetic performance of each cultivation.

Time (h) X1 (g/L) Time (h) X2 (g/L)

0 1 0 1.065
2 0.56 2 1.994
4 3.59 4 2.8742
6 5.66 6 3.7056
Figure 4 10 3.25 Growth 10 5.222 kinetic
12 7.59 12 5.907
14 5.96 14 6.5432
24 7.89 24 8.9922
28 10.23 28 9.6302
32 10.89 32 10.073
34 9.58 34 10.221
36 10.25 36 10.321
38 10.22 38 10.371
40 10.56 40 10.373

performance against Time

According to Figure 4, it can be observed that data set 1 is an unusual pattern of growth kinetic
performance, while data set 2 gave a usual growth kinetic pattern for the batch cultivation of
microorganisms. The growth of microorgansims might be inhibited by extracellular measures such as
warmth, moisture, pH levels and oxygen level which disrupted the process.

QUESTION 3
Below is a set of data on Lactobacillus plantarum sp growth data in batch cultivation using different
peptone concentration as a nitrogen source. Plot the graph of the bacterium growth in one graph and
discuss the performance of the cultivations.

Table 1: Cell Growth (g/L)

Peptone Concentration (g/L) against Cell growth (g/L)

4
Peptone concnetration (g/L)

0
0 2 4 6 8 10 12 14 16
Cell growth (g/L)

1 5 10 15 20

Figure 5 Peptone concentration against cell growth

Table 2: Glucose Concentration (g/L)

Peptone Concentration (g/L) against Glucose Concentration

(g/L)
12
Peptone concentration (g/L)

10
8
6
4
2
0
0 2 4 6 8 10 12 14 16
Glucose concentration (g/L)

1 5 10 15 20

Figure 6 Peptone concentration against glucose concentration

Table 3: Product Concentration (g/L)

Peptone Concentration (g/L) against Product Concentration

(g/L)
Peptone concentration (g/L) 35
30
25
20
15
10
5
0
0 2 4 6 8 10 12 14 16
Product Concentration (g/L)

1 5 10 15 20

Figure 7 Peptone concentration against product concentration

From the above analysis, it can be observed that the growth of Lactobacillus plantarum and product
concentration increases with the increase of peptone concentration. Consumption of protein by the
cell increases the growth dynamic of its intracellular metabolite thus allowing an increase in the
uptake rate of peptone which accumulates a higher quantity of cell. This occurrence directly affects
the product concentration of the fermentation process because when the number of cells increase the
formation of product decrease. Meanwhile, the concentration of glucose decreases as peptone
increases. As glucose provide energy for the cell metabolism and acts as a substrate, its quantity
decreases with time as more glucose is being consumed throughout the process.

QUESTION 4
Assume that the kinetic parameter values for batch fermentation of Saccharomyces cerevisae are as
follow: max = 0.4, Ks=0.1, Yx/s=0.21 X0=0.22, Xm=4.63, S0=20.8, Yp/s = 0.29, =0.05, =0.02
Simulate the growth of the microorganism using Logistic growth model using spreadsheet (MS-
EXCEL) computer program
 Growth of Microoganisms against Time
5
4.5
4
3.5
3
2.5
X(t)

2
1.5
1
0.5
0
0 5 10 15 20 25 30
Time (H)

Figure 8 Growth of microorganisms against time

QUESTION 5
Exponential Fed-batch Fermentation Data for batch fermentation of lysine by Saccharomyces
cerevisae are given in Table below. Find the relationship between specific growth rate and specific
lysine production rate for this fermentation process.
Specific growth rate,  = 1/X(dX/dt)
Specific lysine production rate,  = 1/X(dP/dt)

Batch Fermentation Run 1

Specific growth rate, µ against Specific lysine production rate,


1.40
Specific lysine production rate, 

1.20
1.00
0.80
0.60
0.40
0.20
0.00
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50
Specific growth rate, µ

Figure 9 Specific growth rate against specific lysine production rate for run 1

Batch Fermentation Run 2

Specific growth rate, µ vs Specific lysine growth, 

1.40

1.20
Specific lysine growth, 

1.00

0.80

0.60

0.40

0.20

0.00
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50
Specific growth rate, µ

Figure 10 Specific growth rate against specific lysine growth for run 2

QUESTION 6
Information on the fermentation kinetics is important in the selection of mode of fermenter operation
for the improvement of the process. Give reasons why continuous culture is not suitable or
appropriate for the non-growth associated fermentation process.
Based on Gilbert P. (1985), a continuous culture is an approach that provides a spectrum of exciting
opportunities for studying bacteria under conditions closer to natural growth than continuous culture.
In continuous culture, microorganisms are placed in an environment where the rate of supply to and
from the system is fixed. Therefore, microorganisms experience a constant and stable supply of
restricted substrates and nutrients. A non-growth associated product is not suitable for continuous
fermentation as it requires a high concentration of cells in the growth phase, and then switch the
metabolism of the cell to arrest cell growth by feeding product precursors, carbon, and oxygen at a
rate sufficient to meet the maintenance and product synthesis requirement. A non-growth associated
product is more suitable to be done in fed-batch fermentation technique.
QUESTION 7
From the kinetic analysis of batch ethanol fermentation by Zymomonas mobilis using glucose as a
carbon source at 30oC and pH 4, the following kinetic parameter values are obtained.

Maximum specific growth rate, m = 0.3 h-1

Cell yield coefficient based on glucose consumed, Yx/s = 0.5 g cell/ g glucose
Ethanol yield coefficient based on glucose consumed, Yp/s = 0.6 g ethanol/ g glucose
Monod saturation constant, Ks = 5 mg/L
Coefficient for growth associated product formation,  = 4.8 g ethanol/g cell
Coefficient for non-growth associated product formation,  = 0 g ethanol/g cell.h-1

Predict the optimal dilution rate for the highest productivity of ethanol production in continuous culture
at the same temperature and pH as for batch fermentation if the concentration of glucose in the feed
of 40 g/L is used. Use continuous fermenter model to calculate steady-state value of S, X and P at
different dilution rate

Dilution rate against Productivity

30.00

25.00
Productivity (µ/h)

20.00

15.00

10.00

5.00

0.00
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35
Dilution rate (1/h)

Figure 11 Dilution rate against productivity

From the above figure, we can conclude that the cell productivity increases as there is an increase of
the dilution rate until it reaches its max value. Otherwise, a high enough dilution rate will cause the
cells to be flushed out of the reactor and cannot be sustained. The optimal dilution rate for the above
analysis is 0.29 h-1, where the productivity is 27.74 µ/h.
At steady state,
Where, µm = 0.3, D = 0
KsD
S=
U m −D
0.005 x 0
S=
0.3−0
S=0
X =Y x ( S i−S )
s

X =0.5 ( 40−0 )
g
X =20
L
β
( ( ))
P= α +
D
X

0
( ( ))
P= 4.8+
0
20

P=96 g / L