1

Quiz
Biology for Engineer (EPF 3103)
Lecturers: Dr.-Ing. Mohd Noriznan Mokhtar/ Dr. Azhari Samsu Baharuddin

Fermentation Technology: Yeast growth curve/ glucose consumption
curve

1. Aim of the experiment

1. To plot the profile of cell concentration and glucose concentration
2. To determine specific growth rate
max
, overall growth yield Y
x/s
and
saturation constant K
s
by employing the integration method (Monod).


2. Equipment and material

In this experiment, the yeast strain saccharvnryces cereoisiae has been cultivated in a
3.2 L bioreactor, and the course of the cell concentration has been monitored by using
different techniques; microscopic count, turbidity measurement and glucose
consumption. The baker's yeast used for the experiment is commercially available at
UNIFERM GmbH & Co. KG, Werner Germany. The dry content of the biomass is
about 27%. About 50% of the dry content of the yeast comprises of protein.


Figure 1: Bakers yeast saccharvnryces cereoisiae

2
The bioreactor is equipped with a pH-controller, p
02
controller, temperature
controller, antifoam controller and agitator. The total volume of the reactor is about 3.1
L, the working volume is about 2 L. The medium can be sterilized in situ. Figure 2
shows the fermenter model KLF 2000 of Bioengineering AG.

1. Heating Finger
2. Temperature sensor
3. Exhaust filter
4. Safety jacket
5. Glass cylinder
6. Safety valve
7. Drive shaft
8. Cooling, finger
9. Cooling water inlet
10. Cooling water outlet
11. Immersion tube
12. Non return valve
13. Inlet air filter
14. Aeration tube
15. Bearing
16. Leak basin
17. Motor
Figure 2: Fermenter
3

3. Measurement of cell number / microscopic count

The number of cells in a sample was measured under a microscope by counting cells
placed in special counting, chambers. The Neubauer-counting chamber has a special
square grid marked on the surface of the glass slide (Figure 3). The distance between
the grid and the deepness were known so that the volume of a square was precisely
known. A sample of cell suspension to be counted is allowed to flow under a cover
slip and to fill the counting chamber. Then the number of cells per unit area of grid
were counted under the microscope. Very dense suspensions were counted if they
were diluted appropriately. The cell concentration was calculated as follows:

cells/ml) of (number ion concentrat
chamber the of deepness area counting
factor dilution cells of number
=

Figure 3: Neubauer-counting chamber







4

4. Measurement of glucose concentration

The change of the cell mass was monitored indirectly by measuring nutrient
consumption. For the determination of glucose concentration a test from Boehringer
Mannheim was used.
Principle:
- ase dehydrogen phosphate 6 Glucose -
Hexokinase
H NADPH phosphate - 6 - gluconate - D NADP phosphate - 6 - glucose - D
ADP phosphate - 6 - glucose - D ATP glucose - D
+ + +
+ +

The NADPH produced has an absorption maximum at 340 nm . The samples were
prepared as follows:
Blank Sample
Solution 1 (Buffer, NADP, ATP, MgSO
4
,
stabilisators)
500L 500L
H
2
O 1000L 950L
Sample (centrifuge, dilute) - 50L
Wait for 3 min, read the absorbance at 340 nm
Solution 2 (Hexokinase, G-6-P-DH) 10L 10L
Wait for 15 min, read the absorbance at 340 nm

Table 1: Determination of glucose concentration

The glucose concentration was calculated as follows:
| |
F A
mol mmol 1000 v d
MG V
c(Glucose)
1 -



=
c

V = total volume [mL]
MG = molecular weight of glucose [g/moll
c = absorption coefficient of NADPH [L

mmol
-
' cm
-1
]
d = light path length [cm]
v = sample volume [mL]
A = Absorption
F = dilution factor
5
With the data given the glucose concentration can be calculated as: c[g/L] =0.864 A F


5. Procedure

Culture media:

Material Weight (g)
Glucose 100
Yeast extract 8.5
NH
4
Cl 1.32
MgSO
4
0.11
CaCl
2
0.06

Table 2: Typical growth medium for yeast (for 1L)

The amount of glucose and medium needed were weighted for 2 liters. The
glucose and medium were added to the 2 L-flask. Glucose and medium were
dissolved in 2 L distillated water

Inoculum:
20 g of the baker yeast was dissolved in 100 ml distillated water

Operating Manual:
1. Valve of cooling water and the valve of air supply were opened.
2. The agitation was set, temperature controller to on``.
3. The contents of the bioreactor was emptied with pressure (the air outlet was
closed and the sampling pipe was opened)
4. The medium was transferred to the culture vessel
5. The pH-probe was installed to the reactor
6. The medium was adjusted to pH 4.5 with 1 M HCl
7. The inoculum was added to the culture vessel
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8. All inlets and outlets of the vessel were closed
9. The speed controller was set to 500 rpm
10. The temperature was adjusted to 37 C and the aeration rate was adjusted to
0.5 vvm
11. The air outlet was opened
12. In order to obtain the yeast growth curve, a sample was taken every hour (the
air outlet was closed and the sample line was opened slowly)
13. The optical density was determined at 660 nm (cell concentration) and 340
nm (NADPH absorbance)

Cleaning:
1. After the cultivation, the contents of the culture vessel was emptied and
rinsed with the distilled water three times.
2. All the switches were placed in the OFF position, except the pH-controller.
3. All glassware were rinsed with distilled water and let dry.

6. Observation
Data from the experiment were tabulated into the Table 3.
No
Time
(min) pH
Cell nu
(1:100)
Adsorbance NADPH for
glucose determination
(1:100)
1 0 4.53 4 1.16
2 60 4.26 9 0.75
3 120 3.98 18 0.3
4 180 3.31 20 0.12
5 240 2.98 19 0.05
6 300 2.94 18 0.01
T= 37
o
C

Table 3: The observation of the experiment

7. Calculation and analysis of experiment

7
Measurement of cell number/microscopic count:

cells/mL) of (number ion concentrat
chamber the of deepness area counting
factor dilution cells of number
=

Sample calculation for time = 0,
Counting area = 0.0025 mm
2
Deepness (chamber) = 0.1 mm
Cell number (cells/mL) =
3
10 1 . 0 0025 . 0
100 4

Measurement of glucose concentration:

| |
F A 0.864 F A
mol mmol 1000 v d
MG V
c(Glucose)
1 -
=


=
c

Sample calculation,
When t = 0, A = 1.16
g/L 100.2 100 1.16 0.864 c(Glucose) = =


Kinetic for batch cultivation:

The biomass can be described,
( )
( )
X
X X
Y
S K
X X
Y
S
dt
dX
o
S X
o S
S X
o

+

=
/
0
/
max
1
1

Where, X = biomass concentration, S =
substrate concentration (g/L), Y
X/S
= yield,

max
= maximum growth rate (h
-1
), K
S
=
saturation constant (g/L)

The integration gives:
o S X o o
S
o S X o o
S
S
S
Y X S
K
X
X
Y X S
K
t ln
/
ln 1
/
/ /
max

|
|
.
|

\
|
+

|
|
.
|

\
|
+
+
=
then
8
o
o
o
X
X
S
S
A A
X
X
t
ln
ln
1
ln
max max
+
+
=


the constant
max
and K
S
can be calculated from the axis intersection (A+1)/
max
and the
slope A/
max

S X o o
S
Y X S
K
A
/
/ +
=
Y
X/S
calculation base on the final stage operation.
S S
X X
Y
o
Y X

=
0
/


Then,
o
X
X
t
ln
versus
o
o
X
X
S
S
ln
ln
must be plotted