Bioprocess Lab Manual Guide

SECTION OF BIOENGINEERING
TECHNOLOGY
UNIKL MALAYSIAN INSTITUTE OF
CHEMICAL AND BIOENGINEERING
TECHNOLOGY
ALOR GAJAH, MELAKA
CBB 20503
PRINCIPLES OF BIOPROCESS
TECHNOLOGY
LABORATORY MANUAL
GUIDELINE:
A. BASIC GOOD LABORATORY PRACTICE
1. Prepare for each laboratory period by reading each exercise and becoming familiar
with the principles and methods involved. By being familiar with the exercise you
decrease the chances of an accident. Also, advance preparation allows you to use
your time efficiently in the laboratory to complete the experiment.
2. No eating, drinking, or smoking is permitted in the laboratory.
3. Laboratory coats or aprons must be worn at all times in the laboratory. This is to
ensure that culture material is not accidentally deposited on your clothes or skin,
and as a safeguard to protect your clothes and yourself from chemical spills and
stains.
4. Only those materials pertinent to your laboratory work, such as laboratory manuals,
laboratory notebooks, and other laboratory materials, should be brought to your
laboratory work space. All other items, such as coats, books, and bags, should be
stored away from your work area.
5. Begin each laboratory session by disinfecting your work area. Saturate the area with
a disinfectant, spread the disinfectant with a paper towel, and allow the area to dry.
Repeat this procedure after you have finished your work to ensure that any material
you have deposited on the work surface is properly disinfected.
6. All culture material and chemicals should be properly labeled with your name, class,
date, and experiment. Labeling is critical to avoid improper use or disposal of
material.
7. Be very careful with Bunsen burners. To avoid injuries, burners should be turned
off when not in use. When reaching for objects, be careful not to place your hands
into the flame.
8. All contaminated material must be disinfected before disposal or reuse. All material
to be autoclaved should be placed in a proper receptacle for collection. Used pipets
should be placed in disinfectant.
9. After the laboratory session, observe good hygiene by washing your hands before
leaving the laboratory.
10. In the event of any accident or injury, report immediately to the laboratory instructor
so that prompt and proper action can be taken.
CBB 20503 PRINCIPLES OF BIOPROCESS TECHNOLOGY Page 2 of 27

EXPERIMENT 1
kLa MEASUREMENT
INTRODUCTION
OBJECTIVES
1. To determine kLa of a fermentation system by non-fermentative static gassing

out techniques.
2. To determine the effect of agitation speed and aeration rate on kLa.
3. To determine the effect of medium viscosity on kLa value.
APPARATUS
Bioreactor including pO2 probe.
Stopwatch
CHEMICALS
NaCl
Antifoam
Distilled water
Calibration of dissolved oxygen electrode

Before calibration the pO2 probe must be polarized. The polarization must be repeated
any time the electrode is disconnected from the amplifier for more than 10 min, but may
require less time then. The calibration of pO2-electrode includes zero and slope
calibration. The “zero” is the electrode’s current, when no oxygen is present in the
culture medium meanwhile the “slope” is usually the pO2 after saturation of the medium
with air at the maximum air supply intended for the process.
The calibration of the pO2 electrode involved several steps:
1. Temperature in the culture vessel is adjusted at the operating temperature.
2. For "zero" calibration, it could be measured the pO2 of the culture medium before
starting the air supply. The medium will be degassed almost completely due to the
heat impact during sterilization and thus should not contain dissolved oxygen.
Alternatively, we can supply an oxygen-free gas (such as nitrogen of 99.98 purity)
to the culture medium to displace the dissolved oxygen until a constant pO2 near 0"
can be read at the measurement and control system.
3. For slope adjustment, the air supply is activated and the stirring speed is adjusted at
the operating value. The medium should be optimally gassed (max. flow rate
intended for the process) and mixed. At a stable display of the measured value we
can calibrate this as 100 pO2".
4. After calibration, the gas supply rate required for the startup of the intended
fermentation process can be adjusted on the rotameter of the control unit. Note that

the rotameter is calibrated according to standard conditions (temperature 20°C, with
air at 2 bar abs). If it is important to maintain precise operating air flow-rates for
further calculations, this makes it necessary to recalculate the indicated flowrate with
a correction factor.
The calibration of the pO2-electrode is made in the culture vessel after autoclaving and
under the conditions of fermentation.
Non-fermentative method
In this technique, the oxygen concentration of the solution is lowered by gassing the
liquid out with nitrogen gas, so that the solution is "scrubbed" free of oxygen. Aeration
is then initiated at a constant sir flow rate and the increase in dissolved oxygen tension
(DOT) is monitored using dissolved oxygen electrode. The profile of DOT during
deaeration and aeration is shown in Figure 1. Increase in DOT during aeration can be
expressed by Eq. 1:
dCL
dt
= kLa(CE -CL ) - Qd (1)
Mass balance for the system;
Rate of change in O2 conc. = Rate of O2 in - Rate of O2 out - Rate of usage Qd
Figure 1: Dynamic gassing out for the determination of kLa values. Aeration was
terminated at point A and recommenced at point B.
Since microorganism is not present in the solution, Qd = 0. Eq. 1 becomes
dCL
dt
= kLa (CE -CL ) (2)
Rearrange Eq 2, so

dCL
= kLa.dt
(CE -CL )
Integrate Eq 2, where
1
𝑑𝐶 = kLa dt
(CE -CL )
So,
−[ln(𝐶 − 𝐶 )] + C = k a[t] + C
Then, the final equation become:
ln(C − C ) = −k a. t + ln(C − C )
A plot of ln(C − C ) vs t will produce a straight line where the slope is equal to -kLa.
ln(CE – CL)
EXPERIMENTAL PROCEDURE
1. Set the agitation speed of 500 rpm and 1.0 L/min. Purge the nitrogen gas until reach
0% DO. Determine kLa of stirred tank reactor at different air flow rate (0.5, 1.0, 1.5,
2.0 and 2.5 L/min). For this experiment, set the agitation speed at 500 rpm.
2. Determine the effect of increasing agitation speed (200, 400, 600, 800 and 1000
rpm) on kLa of 2 L stirred tank fermenter. For this experiment, set the air flow rate
at 1 L/min.
3. In experiment 1 and 2, the fermenter is filled with 1.5 L of distilled water.
4. Investigate the effect of salt (NaCI) and antifoam addition to distilled water on kLa.
In this experiment, add 1.5 g of NaCI to 1.5 L distilled water in a fermenter.
Determined the kLa at 500 rpm and air flow rate of 1 L/min. Then, add 5 mL of
antifoam in a salt solution and determine kLa at the same agitation speed and air
flow rate.

Presentation of Results and Discussion
1. Calculate the kLa with unit per hour (h-1) at different agitation speed, aeration rate
and at addition of NaCl and antifoam.
2. Plot a graph to show the effect of air flow rate and agitation speed on kLa. Also
discuss the effect of the addition of salt and antifoam on kLa.
3. Compare the kLa value determined using different rpm and air flow rate.
4. Discuss the possible cause of error in determination of kLa by using this static
gassing out technique.

Results
Non fermentative method

Agitation speed:_______________rpm
Air flow rate: _________________L/min
Volume liquid:________________L
Note: CE = 100% saturation
Time CL CL
∆CL/∆t ln(CE –CL)
(s) (% saturation) (average)
0
20
40
60
80
100
120
130
140
150
160
170
180
190
200
210
220
230
240
250
260
270
280
290
300

EXPERIMENT 2
AEROBIC BATCH CULTURE OF Saccharomyces cerevisiae FOR ETHANOL

PRODUCTION (SHAKE FLASK)
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
favourable 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.
OBJECTIVES
1. To produce ethanol in shake flask through aerobic batch fermentation.
2. To determine the appropriate kinetic parameters to describe the fermentation
process such as yeast growth, product formation and substrate utilization.
EXPERIMENTAL PROCEDURE
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 0 hour.
ANALYSIS
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.
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.

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):
𝑤𝑡. 𝑡𝑢𝑏𝑒 𝑎𝑛𝑑 𝑑𝑟𝑖𝑒𝑑 𝑐𝑒𝑙𝑙𝑠 (𝑔) − 𝑤𝑡. 𝑜𝑓 𝑡𝑢𝑏𝑒 (𝑔)

𝑋(𝑔. 𝐿 = × 10
𝑠𝑎𝑚𝑝𝑙𝑒 𝑣𝑜𝑙𝑢𝑚𝑒 (𝑚𝐿)
The correlation between absorbance and CDW can now being developed. This
standard curve will be used in the latter experiments.
4. Glucose determination
The dinitrosalicylic acid (DNS) method as proposed by Miller et al. (1959) was
employed for total reducing sugar determination. In this method, 1 ml of supernatant
with 1 ml DNS reagent was mixed, and the tubes were placed in a boiling water bath
for exactly 5 min. The tubes were then cooled under running tap water to room
temperature. A 10 ml distilled water was added to a reaction mixture and shaked well.
Finally, the tubes were allowed to stand for at least 20 minutes and the absorbance was
read using spectrophotometer (Shimadzu, Model UV-1601 PC) at 540 nm. The
absorbance (after subtraction of the reagent blank) was then translated into glucose
equivalent using a standard graph obtained by plotting glucose against absorbance.

5. Ethanol determination (Spectrophotometer technique)
In order to use this method, the alcohol present in the sample reacts with an orange-
yellow chemical called potassium dichromate (K2Cr2O7). When the alcohol reacts with
the potassium dichromate, a blue-green compound called chromium(III)sulfate is
produced. The reaction is as follows:
2K2Cr2O7 + 8H2SO4 + 3CH3CH2OH → 2Cr2(SO4)3 + 2K2SO4 + 3CH3COOH + 11 H2O
orange-yellow alcohol blue-green
The spectrophotometer is set to absorb the blue-green (560 nm) colour of light that is
produced in the reaction.
Part I Calibration curve
1. Label four 150 mL or 250 mL beakers 1 to 4.
2. Using a 10 mL graduated cylinder, measure 5.0 mL of 0.25 M potassium

dichromate (K2Cr2O7) solution into each beaker.
3. Add 1 drop of 0.1 M silver nitrate (AgNO3) to each beaker. Swirl the beaker
immediately after addition.
4. Using a buret or automatic dispenser, add 5.0mL of 6 M sulfuric acid (H2SO4) to

each beaker. Swirl the beaker immediately after addition. Be careful, sulfuric acid
can cause severe burns to your skin.
5. Add distilled water and alcohol to the beakers as indicated below:
Beaker # Alcohol Concentration Alcohol Water
1 (BLANK) 0% none 20 drops

2 2.5% 5 drops of 10% 15 drops
alcohol solution
3 5.0% 10 drops of 10% 10 drops
alcohol solution
4 10.0% 20 drops of 10% none
alcohol solution
BE SURE TO SAVE YOUR BLANK SOLUTION FOR PART II!!!!!!!
6. Allow the chemical reaction to proceed for 5 minutes. Swirl the beakers several
times during the five minutes.
7. Using the graduated cylinder, dilute the contents of each beaker by adding 39.0
mL of distilled water to each beaker. Stir each beaker with a clean stirring rod.
8. On the spectronic 20, set the wavelength number to 560 nm.

9. With the cover of the sample holder closed and no cuvette in place, turn the left
knob (the zero control) until you read 0% transmittance.
10. Using your BLANK solution (beaker #1), fill the cuvette two-thirds full. Place
the cuvette in the sample holder and set your instrument, using the right knob
(light control) to 100% transmittance.
11. Remove and save your BLANK to adjust the transmittance before EVERY
sample reading.
12. In another cuvette, fill it two-thirds full with the contents from beaker #2 and read
the absorbance. After the reading, pour the contents of the cuvette into a waste
container.
13. Repeat step 12 for beakers #3 and #4.
14. Obtain graph paper from your laboratory instructor and create a calibration curve.
Take the absorbance values and plot the corresponding alcohol concentrations.
Make sure you draw your line of best fit going through the 0/0 point of the x and
y axes.
Part II Sample detection
1. Write the number of the unknown sample on your data sheet and label your
beaker with the unknown number.
2. Repeat steps 2 through 4 from Part I.
3. To your unknown beaker add 20 drops of the unknown sample and no distilled
water.
4. Repeat steps 6 through 12 as in Part I.
5. Use the absorbance value you obtain for your sample and using your calibration
plot from Part I, determine the alcohol concentration in your unknown sample.

EXPERIMENT 3
AEROBIC BATCH CULTURE OF Saccharomyces cerevisiae FOR ETHANOL

PRODUCTION (2L BIOREACTOR)
INTRODUCTION
alcohol. The yeast cell contains enzyme catalysts that provide an
energetically favourable 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 2 L bioreactor through aerobic batch fermentation.
2. Determine the appropriate kinetic parameters to describe the fermentation
3. To compare the kinetic parameters between shake flask and 2l bioreactors
fermentation.
EXPERIMENTAL PROCEDURES
A. Inoculum preparation
1. Prepare the 150mL volume of YPG medium comprises of:

YPG Medium
Component Mass (g/L)
Glucose 20
Peptone 20
Yeast extract 10
Commercial antifoam 0.5mL
* Note: The glucose should be prepared and sterilized separately.
2. Transfer the medium in 500mL Erlenmeyer flask (bottom tubing). 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 15
mL of yeast cell suspension.
5. Incubate the culture at 30°C for ? hours (get the result from Experiment 1) and
190 rpm rotational speed.
B. Fermentation in 2 L bioreactor
1. Prepare the 1350mL volume of YPG medium comprises of the above

ingredients (section 2.1).
2. Adjust the pH to 4.5 by the addition of 2N NaOH or 2N HCl. Transfer the
medium to 2L bioreactor. The appropriate probes, pH and DO should be placed
in the bioreactor. Take note that pH probe should be calibrated prior to
sterilization (refer section 2.2.1).
3. Make sure that all clips are tighten and filters are covered with aluminium foil.
This is to prevent from backflow as well as preventing the filters from getting
wet and blocked. It is very important that the exhaust outlet remains open and
unblocked. This is to prevent pressure build up that may lead to rupture of the
glass bioreactor.
4. The bioreactor is autoclave for at 121°C for a period of 20 minutes.
5. After autoclaving, when the vessel is cool, connect it to the control module.
Switch on the water supply to the condenser unit, the stirrer motor to the
required rpm (500-700 rpm), and the air inlet filter to the air supply, and adjust
the flow rate to between 0.1 - 1.0vvm.
6. Switch on the temperature control unit to provide a temperature of 30°C. Wait
until the temperature has reached 30°C and calibrate dO2 probe (refer section
2.2.2).
B.1 Calibrate pH probe (using pH 4.0 and pH 7.0 buffer)

Calibration of pH electrode is carried out before the bioreactor is autodaved. This is
because calibration of the pH electrode is done externally. To calibrate the pH electrode,
the electrode must be connected top the control imit. The pH electrode is then taken out
from the top plate and mount on a retort stand in a buffer with pH 7.0. Under the
calibration mode on the control panel/ the pH of the buffer is read until it remains
constant. For Biostat B, the cursor on the LCD display will jump automatically to the
next buffer (pH 4.0). The electrode is then immersed in buffer pH 4.0 until it remain
constant.
Notes: Care has to be taken to ensure that the pH electrode is not left submerged in
water/buffer for a long period of time.
B.2 Calibrate dissolved oxygen probe.

There are two basic types of dissolved O2 probe, galvanic and polarographic. Most, if
not all of the probes used are of the Ingold polarographic type. The principles for both
types, however, are the same.
•Before sterilization of the bioreactor check that the Teflon membrane on the end
of the dO2 probe is not damaged or kinked, or is leaking electrolyte.
•Top up the probe with electrolyte if necessary, or change the membrane.
•Autoclave the bioreactor with the dO2 probe in place.
•After cooling/ set the temperature control module to 30°C, aeration rate to 0.1 –
1.0vvm, and agitation rate to ~700 rpm.
•When the bioreactor has reached 30°C and dO2 probe display has stabilized with
the dO2 adjustment control set the display to 100, that is fully saturated with
O2.
•Switch off the air and sparge reactor with O2-free N2, or simply switch the probe to
zero setting. After the sígnal has stabilized at a low value, use the zero
adjustment control to set the display to zero. The output of the probe is now
calibrated over the range 0 to 100 dissolved oxygen. The zero adjustment varies
from one bioreactor to another, so consult the manual for the model used before
beginning.

•Set the dO2 set-point control to 0 or 100, depending on the experiment, and set the
minimum and maximum limits for the agitation speed and/or aeration rate
depending on whether the dO2 control is through agitation or aeration
respectively. Consult the bioreactor manuals.
C. Inoculum transfer
1. Inoculate the bioreactor with inoculum which has been prepared in section 2.1.
Wait 5 minutes to ensure complete mixing and take a 20mL sample of the
culture as the t0 sample.
2. 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. Continue to sample the culture at 3-hour interval for 24 hours.
ANALYSIS
Refer to analysis method in Experiment 2
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 (YX/s)
c. The product yield coefficient (YP/s)
d. The productivity of biomass (g/L.h)
e. The productivity of ethanol (g/L.h)
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

EXPERIMENT 4
ANAEROBIC BATCH CULTURE OF Saccharomyces cerevisiae FOR

ETHANOL PRODUCTION (2L BIOREACTOR)
INTRODUCTION
alcohol. The yeast cell contains enzyme catalysts that provide an
energetically favourable pathway for the reaction.
Fermentation refers to catabolic processes where organic molecules, such as sugars or
amino acids, are broken down to produce energy without the use of a membrane-bound
electron transport chain. Depending upon the organism, fermentation can occur in the
presence (aerobic) and/or in the absence (anaerobic) of oxygen. Fermentation pathways
produce by-products such as carbon dioxide, ethanol (alcohol), or organic acids (lactic
acid or acetic acid, for example). Yeast cells can ferment sugar and in the process
produce carbon dioxide and alcohol.
Yeast can respire in anaerobically and aerobically. However, yeast gets more energy
from aerobic respiration, but in the absence of oxygen it can continue to respire
anaerobically, though it does not get as much energy from the substrate. Yeast produces
ethanol when it respires anaerobically and ultimately the ethanol will kill the yeast (find
out why is yeast continue to produce ethanol even the last is an inhibitor).
C6H1206 → 2 CH3CH2OH + 2 CO2 + 2 ATP (anaerobic)

C6H1206 + 6O2 → 6CO2 + 6H2O + 16-18 APT (aerobic)

1. Produce ethanol in 2L bioreactor through anaerobic batch fermentation.
2. Determine the appropriate kinetic parameters to describe the fermentation
3. To compare the kinetic parameters between aerobic and anaerobic batch
fermentation of glucose.
EXPERIMENTAL PROCEDURES:
Refer to laboratory manual for EXPERIMENT 2: Aerobic batch culture of

Saccharomyces cerevisiae using 2% glucose as a carbon source (2L bioreactor) on
the inoculum preparation and medium preparation.
The difference is on the setting of bioreactor:
Reduce the agitation speed to 300 rpm, shut off air supply or set dO2 control to 0%,
inoculate, and run as before.

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:
b. The specific growth rate (µ)
c. The biomass yield coefficient (YX/s)
d. The product yield coefficient (YP/s)
e. The productivity of biomass (g/L.h)
f. The productivity of ethanol (g/L.h)
REFERENCES

Horwood.
2. Shuler, M.L. and Kargi, F. (2009). Bioprocess Engineering: Basic Concepts
(2nd Ed). New Delhi: PHI Learning Private Limited

EXPERIMENT 5
FED-BATCH CULTURE OF MICROBIAL CELL
INTRODUCTION
In fed-batch culture, nutrients are continuously or semi-continuously fed, while effluent

is removed discontinuously (Figure 1). Such a system is called a repeated fed-batch
culture.
Figure 1 Schematic of a fed-batch culture
Fed-batch culture is usually used to overcome substrate inhibition or catabolite

repression by intermittent feeding of the substrate. If the substrate is inhibitory,
intermittent addition of the substrate improves the productivity of the fermentation by
maintaining the substrate concentration low. A fed-batch culture is established initially
in batch-mode and is then fed according to one of the following strategies:
i. The same medium used to establish the batch culture is added, resulting in an
increase in volume (variable volume system).
ii. A solution of the limiting substrate at the same concentration as that in the initial
medium is added, resulting in an increase in volume (variable volume system).
iii. A concentrated solution of the limiting substrate is added at a rate less than in (i)
and (ii), resulting in an increase in volume (intermediate system).
iv. A very concentrated solution of the limiting substrate is added at a rate less than in
(i), (ii) and (iii), resulting in an insignificant increase in volume (fixed volume
system).
OBJECTIVES
1. Produce ethanol in fed-batch culture

2. Effect of feed rate on ethanol production

Medium preparation
1. Prepare 5000 mL of medium with the following compositions:
Medium Amount
Refined Sugar 100 g
Yeast Extract 10 g
Peptone 10 g
KH2PO4 4g
Na2HPO4.7H2O 4g
Distill water 1L
pH 5.5
2. Separate the medium into 4000 mL in carboy, 900 mL in 2 L bioreactor and

100 mL in 250 mL shake flask.
3. Autoclave the medium for 15 min at 121C.
Inoculum preparation
1. Inoculate the 100 mL medium with yeast from slant agar and incubate in an
incubator shaker for 12-14 h to reach the exponential growth at 150 rpm and
30C.
2. The 100 mL inoculum will be used to inoculate the 900 mL medium in a 2 L
bioreactor.
Fed-batch culture procedure

1. Let the 1000 mL culture to grow for 12 hours.
2. At 12 hours of incubation starts the feed pump.
3. At completion of each pumping time, start the outlet pump at 100% pump
setting to pump out the medium until it reach the initial volume which is 1000
mL.
4. Once the remaining medium reach 1000 mL, stop the outlet pump.
5. Then set the feed pump as stated in table at step no 2.
6. Take 10 mL sample at every 3 hours starting from 0 hour of incubation.
7. Analyse the sample as per method stated in experiment 2.

EXPERIMENT 6
CONTINUOUS CULTURE OF Saccharomyces cerevisiae FOR ETHANOL

PRODUCTION UNDER GLUCOSE (2%) LIMITATION (2L BIOREACTOR)
INTRODUCTION
Most bioreactors can be used for continuous culture if provided with some form of
control of culture volume, and often a simple weir system is used. The schematic
diagram of chemostat is shown in Figure 1. Chemostat is operated by supplying an
essential growth-limiting nutrient at a constant rate with the result that the culture
density and growth rate adjust themselves to the supply. Generally, small bioreactors
of 200ml to 2 l (working volume) are used for continuous cultures, as this reduces the
amount and frequency that the medium supply has to be recharged.
Figure 1 Schematic representation of a chemostat
A basic requirement of a continuous culture is a method for controlling a constant

bioreactor volume. One way of doing it is by using a level tube with the outlet pump
rate at approximately 2 – 3 times of the inlet pump (Figure 2).

Figure 2 Maintenance of a constant chemostat volume using a level tube with the outlet
pump rate at approximately 2 – 3 times that of the inlet pump.
Thus the material balances of a single-stage of chemostat is written as:

Cell accumulation = cell in – cell out + growth – minus death
dx Fxo Fx
= - +μx - αx
dt V V
Where
F = flow rate of medium supply (L/h))

V = Constant working volume in bioreactor (L)
xo = cell concentration in medium supply (g/L)
x = cell concentration in bioreactor (g/L)
 = specific growth rate (h-1)
 = specific death rate (h-1)
The medium supply is usually sterile (assuming no cell recycle) and  ≫ , so the
equation is simplified to:
dx Fx
=- +μx
dt V
The term F/V is termed the dilution rate that is the number of culture volume passing
through the reactor per hour. The equation can be rewritten as
dx
=-Dx + μx = x (μ-D)
dt
Where D is the dilution rate. During steady state, dx/dt = 0 consequently  = D. So, by
varying the medium supply rate, the growth rate can be varied and this hold until D >
m (the maximum specific growth rate). Under these conditions the nutrient is no longer
limiting and the expression ( – D) becomes negative. The result is a fall in cell
concentration and wash out of the culture.
The mechanism underlying the controlling effect of the dilution rate is essentially the
relationship expressed by Monod equation.

μm S
μ=
(K s +S)
At steady state dx/dt = 0 and dS/dt = 0, so x and S (limiting substrate concentration in
chemostat, g/L) become constant and are represented by x and S and  = D. So the
equation become
μm 𝑆̅
D=
(K s +𝑆̅)
Solving for S
Ks D
S=
(μm -D)
And the concentration of cells at in the chemostat at steady state is described by

equation:
x = Yx/S (SR -S)
Where SR is the initial substrate concentration (g/L).

1. Produce ethanol in 2 L bioreactor through anaerobic continuous fermentation.
2. Determine the appropriate kinetic parameters to describe the fermentation process
such as 𝜇 , 𝐾 , 𝑌 ⁄ at various dilution rate, the maximum biomass yield and
maintenance coefficient from plot of 1/Y versus 1/D.
1. Prepare seed medium and dispense 600 mL to adjust pH 5.5 with 1N HCl and 1N
NaOH. Dispense 35 mL in a 100 mL Erlenmeyer flask and autoclave for 15 min at
121C.
Medium Amount
Glucose 50 g
Yeast Extract 5g
Peptone 5g
KH2PO4 2g
Na2HPO4.7H2O 2g
Distill water 1L
pH 5.5
2. Transfer a loopful of yeast culture from agar slant into the medium and incubate
for 24 h at 30OC.This culture will be used as inoculum
3. Prepare another seed medium of volume 250 mL in 500 mL Transfer the
inoculum at the 5% v/v to the flask and incubate for 12-14 h to reach the
exponential growth at shaking 150 rpm.

4. Prepare production medium for 1350 ml.
Medium Amount
Refined Sugar 100 g
Yeast Extract 10 g
Peptone 10 g
KH2PO4 4g
Na2HPO4.7H2O 4g
Distill water 1L
pH 5.5
5. The culture is then inoculated about 150ml in sterile fermenter containing 1350
ml medium.
6. Autoclave the fermenter altogether with its instruments and buffer.
7. A 2 L tank bioreactor is used for this fermentation. For aeration, ethanol
fermentation does not need oxygen so the aeration is shut off. The fermenter is
equipped with temperature, dissolved oxygen and pH controllers. During all
fermentation, agitation speed is fixed at 400 rpm and the temperature within the
fermenter was controlled at 320C and pH at 5.5.
8. Calibrate the pump rate before starting any continuous fermentation.
9. Prepare same solution (production medium) in a 20L carboy; the media is
prepared with 10L distilled water and autoclave.
10. Once the absorbance begin to rise, switch on the medium supply pump according
to media flow rate in table below.
Media flow rate Retention time,
(F), ml/hr , hrs
83 16
125 12
167 9
250 6
500 3
D= F/V; V= 1500ml
𝜏=1 𝐷
11. Take sample every 3 hrs.
12. Analysis to be done on samples:
1. Optical density
2. Cell dry weight
3. Glucose concentration
4. Ethanol concentration
5. Record pH

RESULTS
1. Plot a graph of
- Cell dry weight and residual glucose versus dilution rate
- Ethanol versus dilution rate
- Biomass and ethanol productivity versus dilution rate
2. Determine the following
- The maximum specific growth rate
- The Monod constant
- The biomass yield at each dilution
- The maximum biomass yield and maintenance coefficient from a plot of 1/Y
versus 1/D
- The dilution rate giving maximum productivity of biomass and alcohol (Dm)
REFERENCES

Horwood.
2. Shuler, M.L. and Kargi, F. (2009). Bioprocess Engineering: Basic Concepts
(2nd Ed). New Delhi: PHI Learning Private Limited

EXPERIMENT 7
PREPARATION OF CARROT SEEDLING CULTURES AND CALLUS

CULTURES
INTRODUCTION
Plant tissue culture has been used in a variety of contexts including: organogenesis
(Dodds and Roberts, 1985); somatic embryogenesis (Dodds and Roberts, 1985);
protoplast isolation and culture (Boitino, 1981); effects of hormone balance on growth
and morphogenesis of explants (Mineo, 1990); an exercise to analyze growth (Cooper,
et al., 1993); and to demonstrate the genetic variation caused by plant tissue culture
(Morgan and Marcotrigiano, 1987/1988). In the experiment described here, we used
plant tissue culture as a means to study cell differentiation and the permanence of
developmental fate in plant tissue. Moreover, we collected both qualitative and
quantitative results.
The first part of the experiment is to prepare healthy tissue that can be used to establish
callus cultures. This material must be sterile and so seedling preparation is done using
aseptic technique.
The instructional objectives of this experiment were to:

 introduce students to the technique of plant tissue culture;
 permit students to become skilful in aseptic technique;
 learn about cell differentiation and dedifferentiation (the loss of specialized
characteristics) in plant tissue;
 carry out a long-term, team project; and collect and analyze a large data set.
MATERIALS
15% silver nitrate solution

Carrot seeds
Sterile filter paper
Petri dish containing sterile standard medium
1. To sterilize the carrot seeds, place them in a small beaker and cover them with a 15
percent silver nitrate solution. Since carrot seeds are small, 10 cm3 of solution is
adequate to sterilize 20 – 30 seeds.
2. Swirl the seed suspension for 15 sec and then quickly pour the mixture through a
funnel lined with a cone of filter paper.
3. Allow the filter to drain, open it and let the seeds air dry for 2-4 hrs.
4. After the seeds have dried adequately, sprinkle 20-30 on to a Petri dish containing
standard culture medium (Table 1).
5. Seal the edge of the Petri dish with parafilm and incubate in the light. The silver
nitrate often darkens the medium right around the seeds. Germination is not
affected; it will commence 4-7 days after the seeds are sown.

Preparation of callus cultures: general instructions
After the seeds have germinated, students can proceed to the next stage of the
experiment, the establishment of the callus culture. As with the preparation of seedlings,
this procedure will succeed only if sterile technique is used.
1. To initiate a callus culture, use sterile forceps to medium, and place it in an empty
sterile Petri dish.
2. With a sterile scalpel, separate leaf, stem, and root tissue. Cut the desired tissue
into sections 1-5 mm long.
3. Transfer the cut tissue sections, with sterile forceps, to callus initiation medium
(table 1). (Students are either provided with Petri dishes containing callus initiation
medium or with melted medium with which they pour their own plates.)
4. Put 4-5 tissue sections on each of 2-3 plates.
5. Record the length and width of all tissue pieces.
6. Seal the edge of the Petri dishes with parafilm and incubate in the dark at 25-28°C.

Data collection and analysis
1. Measure the length and width of each tissue in millimetres. Use a ruler to make
these measurements but do not open the dishes.
2. Repeat these measurements weekly for at least 10 weeks. Keep a careful record.
REFERENCE:
Donna M. Bozzonea (1997). Using tissue culture to investigate plant cell differentiation
and Dedifferentiation. Journal of Biological Education, 31 (4).

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SECTION OF BIOENGINEERING

A. BASIC GOOD LABORATORY PRACTICE

CBB 20503 PRINCIPLES OF BIOPROCESS TECHNOLOGY Page 2 of 27

1. To determine kLa of a fermentation system by non-fermentative static gassing

Calibration of dissolved oxygen electrode

The calibration of the pO2 electrode involved several steps:

1. Temperature in the culture vessel is adjusted at the operating temperature.

CBB 20503 PRINCIPLES OF BIOPROCESS TECHNOLOGY Page 3 of 27

Mass balance for the system;

Rate of change in O2 conc. = Rate of O2 in - Rate of O2 out - Rate of usage Qd

Since microorganism is not present in the solution, Qd = 0. Eq. 1 becomes

CBB 20503 PRINCIPLES OF BIOPROCESS TECHNOLOGY Page 4 of 27

Then, the final equation become:

CBB 20503 PRINCIPLES OF BIOPROCESS TECHNOLOGY Page 5 of 27

CBB 20503 PRINCIPLES OF BIOPROCESS TECHNOLOGY Page 6 of 27

Non fermentative method

CBB 20503 PRINCIPLES OF BIOPROCESS TECHNOLOGY Page 7 of 27

AEROBIC BATCH CULTURE OF Saccharomyces cerevisiae FOR ETHANOL

Fermentation processes are used extensively in the biotechnology, pharmaceutical, food

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

CBB 20503 PRINCIPLES OF BIOPROCESS TECHNOLOGY Page 8 of 27

CBB 20503 PRINCIPLES OF BIOPROCESS TECHNOLOGY Page 9 of 27

One method is as follows:

𝑤𝑡. 𝑡𝑢𝑏𝑒 𝑎𝑛𝑑 𝑑𝑟𝑖𝑒𝑑 𝑐𝑒𝑙𝑙𝑠 (𝑔) − 𝑤𝑡. 𝑜𝑓 𝑡𝑢𝑏𝑒 (𝑔)

CBB 20503 PRINCIPLES OF BIOPROCESS TECHNOLOGY Page 10 of 27

2K2Cr2O7 + 8H2SO4 + 3CH3CH2OH → 2Cr2(SO4)3 + 2K2SO4 + 3CH3COOH + 11 H2O

orange-yellow alcohol blue-green

Part I Calibration curve

1. Label four 150 mL or 250 mL beakers 1 to 4.

2. Using a 10 mL graduated cylinder, measure 5.0 mL of 0.25 M potassium

4. Using a buret or automatic dispenser, add 5.0mL of 6 M sulfuric acid (H2SO4) to

5. Add distilled water and alcohol to the beakers as indicated below:

Beaker # Alcohol Concentration Alcohol Water

1 (BLANK) 0% none 20 drops

BE SURE TO SAVE YOUR BLANK SOLUTION FOR PART II!!!!!!!

8. On the spectronic 20, set the wavelength number to 560 nm.

13. Repeat step 12 for beakers #3 and #4.

Part II Sample detection

2. Repeat steps 2 through 4 from Part I.

4. Repeat steps 6 through 12 as in Part I.

CBB 20503 PRINCIPLES OF BIOPROCESS TECHNOLOGY Page 12 of 27

AEROBIC BATCH CULTURE OF Saccharomyces cerevisiae FOR ETHANOL

Hence, this experiment is aimed to

1. Prepare the 150mL volume of YPG medium comprises of:

* Note: The glucose should be prepared and sterilized separately.

1. Prepare the 1350mL volume of YPG medium comprises of the above

B.1 Calibrate pH probe (using pH 4.0 and pH 7.0 buffer)

B.2 Calibrate dissolved oxygen probe.

CBB 20503 PRINCIPLES OF BIOPROCESS TECHNOLOGY Page 14 of 27

Refer to analysis method in Experiment 2

1. A.H. Scragg (1991). Bioreactors in Biotechnology, a practical approach. Ellis

CBB 20503 PRINCIPLES OF BIOPROCESS TECHNOLOGY Page 15 of 27

ANAEROBIC BATCH CULTURE OF Saccharomyces cerevisiae FOR

C6H1206 → 2 CH3CH2OH + 2 CO2 + 2 ATP (anaerobic)

Hence, this experiment is aimed to

Refer to laboratory manual for EXPERIMENT 2: Aerobic batch culture of

CBB 20503 PRINCIPLES OF BIOPROCESS TECHNOLOGY Page 16 of 27

1. A.H. Scragg (1991). Bioreactors in Biotechnology, a practical approach. Ellis

CBB 20503 PRINCIPLES OF BIOPROCESS TECHNOLOGY Page 17 of 27

FED-BATCH CULTURE OF MICROBIAL CELL

In fed-batch culture, nutrients are continuously or semi-continuously fed, while effluent

Figure 1 Schematic of a fed-batch culture

Fed-batch culture is usually used to overcome substrate inhibition or catabolite