CBB 20104 PRINCIPLES OF BIOPROCESS ENGINEERING TECHNOLOGY

EXPERIMENT 1
AEROBIC BATCH CULTURE OF SACCHAROMYCES CEREVISIAE FOR
ETHANOL PRODUCTION (SHAKE FLASK)

LECTURER: NORHANI JUSOH

TECHNICIAN: SYAHNIZAM MD PADOLLAH
MUHAMMAD YUSOF ABDUL HADI
SHUKRI ZAKARIA
NORHASLIANA CHE HASSAN
VENUE: LAB 13 A & LAB 21

MICET Confidential Page 1 of 5

CBB 20104 PRINCIPLES OF BIOPROCESS ENGINEERING TECHNOLOGY

1.0 Introduction:

Fermentation processes are used extensively in the biotechnology, pharmaceutical,

food and beverage industries. Typically, fermentations utilize microorganisms
(bacteria, yeast) to produce a desired product from a substrate. Butyl alcohol, acetone,
citric acid, hydrogen, glycol, fuel alcohol, and beer are examples of the hundreds of
biochemicals produced by fermentation. In many cases, fermentation is the more cost
effective means to manufacture products.

In this experiment, Saccharomyces cerevisiae yeast is used to convert glucose into

ethyl alcohol. The yeast cell contains enzyme catalysts that provide an energetically
favorable pathway for the reaction.

There are many environmental conditions that affect yeast cell growth and the kinetics
of chemical reactions within living cells. These include the availability of major and
minor nutrients, the temperature, pH, and dissolved oxygen concentration, and the
possible presence of competing organisms.

Hence, this experiment is aimed to

1. produce ethanol in shake flask through aerobic batch fermentation.
2. determine the appropriate kinetic parameters to describe the fermentation
process such as yeast growth, product formation and substrate utilization.

2.0 Procedures:

1. Prepare the appropriate volume of YPG medium comprises of:

YPG Medium
Component Mass (g/L)
Glucose 20
Peptone 20
Yeast extract 10
Commercial antifoam 0.5mL

2. Distribute the medium in two Erlenmeyer flask (each 500mL Erlenmeyer flask
containing 200mL of YPG medium). Adjust the pH to 4.5 by the addition of
2N NaOH or 2N HCl.
3. Sterilize the medium.
4. After autoclaving, when the medium is cool, inoculate the medium with 20mL
of yeast cell suspension.
5. Incubate the culture at 30°C for 24 hours and 190 rpm rotational speed.
6. Take 10mL sample for every 3 hours interval starting from o hour.

MICET Confidential Page 2 of 5

CBB 20104 PRINCIPLES OF BIOPROCESS ENGINEERING TECHNOLOGY

3.0 Analysis

3.1 Sampling

Take 10mL sample of the culture after complete mixing for time = 0. Immediately
determine the absorbance of the culture (600nm), retain 2 x 1.5mL for cell dry weight
(CDW) determination. The supernatant from the CDW determination can be used to
estimate glucose and ethanol.

3.2 Absorbance

The growth of microbial cells can be determined by measuring the absorbance and
relating this value to a calibration curve of absorbance against cell dry weight.
Generally 600nm is used for yeasts, whereas 400-450nm may be used for bacteria
(Fig. 1). One of the major sources of error in such measurements is the nonlinearity of
the absorbance measurements at high cell densities. If the absorbance is above 0.3,
carefully and accurately dilute the culture until a suitable value has been obtained.
Measure the absorbance against a medium blank.

Figure 1: The relationship between absorbance of yeast cultures against cell dry
weights.

MICET Confidential Page 3 of 5

CBB 20104 PRINCIPLES OF BIOPROCESS ENGINEERING TECHNOLOGY

3.3 Dry weight determination

This measurement forms the basis for the determination of most of the important
growth parameters such as productivities and yields, since the concentration of
parameters can be directly related to a constant state of reference, that is the cell
composition. Thus, microbial cells are like all living systems, composed of 80-80%
water. The actual water content within the cells can vary considerably, depending on
their physiological state, whilst the intracellular water content will depend on the
method used to separate the cells from the medium, as well as the nature of the
medium itself and the organism.

Dry weight methods aim to completely remove both intra- and intercellular water
completely, so that the non viable cell material remaining is composed of
carbohydrates, fats, proteins and nucleic acids.

One method is as follows:

1. Carefully and precisely weigh two dry clean microcentrifuge tubes (1.5mL
volume size).
2. Carefully mix the 5mL culture sample and accurately pipette 1.5mL into each
tube.
3. Centrifuge the tubes at 3000rpm for 10 minutes.
4. Carefully decant the supernatant and resuspend cell pellet in approximately
1.5mL saline (0.9% NaCl) and re-centrifuge.
5. Decant supernatant and place tubes in 90°C oven for 20 hours.
6. Remove tubes from oven and immediately place in a dessicator containing a
drying agent until cool.
7. Reweigh tubes.
8. Calculate CDW (X):

. ( − . (
( . = × 10!
(

The correlation between absorbance and CDW can now being developed. This
standard curve will be used in the latter experiments.

3.4 Ethanol determination

The concentration of ethanol can be measured by means of gas chromatography or

ethanol kit. Follow the instruction manual provided.

MICET Confidential Page 4 of 5

CBB 20104 PRINCIPLES OF BIOPROCESS ENGINEERING TECHNOLOGY

3.5 Glucose determination

The residual glucose concentration in the culture medium can be determined by a

number of analytical methods including glucose analyzer, HPLC, chemical assay
(DNS method), TLC and enzyme assays. The choice of assay depends upon the
availability of equipment and the presence of compounds in the medium which may
interfere with the assay system.

4.0 Results
Enter all the data on MS EXCEL and plot absorbance, CDW, glucose and ethanol as a
function of time. With these data the following can be determined:
a) The specific growth rate (µ)
b) The biomass yield coefficient ("#% )
$
c) The product yield coefficient ("&% )
$
d) The productivity of biomass (g.L-1.h-1)
e) The productivity of ethanol (g.L-1.h-1)

5.0 References:
1. A.H. Scragg (1991). Bioreactors in Biotechnology, a practical approach. Ellis
Horwood.
2. Shuler, M.L. and Kargi, F. (2009). Bioprocess Engineering: Basic Concepts
(2nd Ed). New Delhi: PHI Learning Private Limited

MICET Confidential Page 5 of 5