7BIOL001W – Fermentation Technology Assessments

There are three assessments in this module:

Pilot plant report: The pilot plant week/report introduces students to practical aspects of
bioprocess development/technology transfer, including kLa determination to determine the
volumetric mass transfer in bioreactors important in assessing the aeration efficiency of
bioreactors, in-situ sterilisation processes to assess the efficiency of sterilisation regimes
and enzyme production in bioreactors to show how bioreactors are set up, monitored and
controlled. Data gathered is interpreted and written in form of a report. The exercise should
equip students with skills needed to work in research and development biotech roles in
industry or academia. This assessment will contribute to LOs 1, 2 and 6.

Data handling/interpretation exercise: During process development or manufacturing of

biotech products or diagnostics, a lot of data is generated and students should be able to
handle and interpret this data. This assessment will introduce students to handling data
generated during bioprocess development, including data generated from multi-factor
optimisation of bio-processes and growth kinetics studies. The applicable LO is 6.

Term paper: This assessment will enable students to discuss a given application within bio-
manufacturing. By taking a focused look at a given topic, students will gain an in-depth
understanding of important topics in bio-manufacturing such as: scale up, scale down,
process control of bioreactors and strain engineering / cell line development contributing to
LOs 3, 4 and 5.

Assessment criteria: What criteria will be used to assess my work on this module?
Pilot plant report: Students will carry out practicals on batch sterilisation, oxygen transfer in
stirred tank bioreactors and batch fermentation for production of an enzyme
betagalactosidase. They will then write a report by answering set questions associated with
each practical and will be assessed based on the answers to those questions.

Data handling/interpretation exercise: This will be assessed based on how well the data is
transformed into graphs and how well the data is discussed.

Term paper: Students will be assessed based on their ability to demonstrate knowledge,
comprehension and critical understanding of the set topics. An excellent term paper should
show a masterful exposition of the topic showing command of the relevant concepts and
facts, normally including considerable well-chosen supplementary material and providing
very good independent, critical, analytical and/or synthetic treatment of the information.
Generic Grade Descriptors at Level 7

Details of assessments

1) Pilot plant report. This is based on the experiment on enzyme

production as indicated below.

BATCH FERMENTATION AND b-GALACTOSIDASE REGULATION IN

E. coli B

Introduction

Microbial growth:

Microorganisms grow in a wide spectrum of chemical and physical environments.

Many biochemical processes involve batch growth of microorganisms. Microbial
growth is usually described by the time needed to double mass or cell number. In a
simple batch culture, different phases of growth can be monitored and classified as:
lag, exponential, stationary and death:

The Diauxic Growth Curve of E. coli grown in limiting concentrations of a

mixture of glucose and lactose.

The number of living cells in a culture changes with time. In the exponential
(logarithmic) phase, growth is described as:

dx/dt = mx Þ dx/x = mdt where:

x = cell concentration (g/l)

t = time (h)
m = specific growth rate (h-1) Integration of the above
equation results in:
 ò dx/x = ò mdt

If m is constant, then : ln (x2/x1) = m Dt

To find out the time for doubling of cell mass:

t = td where td = doubling time (h)

x2 = 2x1 ln2 = mtd but ln2 = 0.693
So:
td = 0.693/m

Regulation of enzymes:

Bacteria have the ability to synthesise enzymes to utilize different nutrients.

Enzymes may be constitutive or inducible. The microorganisms independent of their
environment synthesize constitutive enzymes. Biosynthesis of inducible enzymes
however, depends on environmental influence. In order to economise on energy
utilisation, bacteria synthesise certain enzymes only in response to the specific
substrate the enzyme metabolises. An example of the inducible enzymes is the
synthesis of b-galactosidase. This enzyme catalyses the hydrolysis of lactose:

Lactose b-galactosidase glucose + galactose

Enzyme induction and repression is regulated at the level of transcription of the

corresponding genes. The lac operon (figure shown below) includes all the genes
involved in lactose uptake and breakdown. The cluster of the three lac structural
genes, lacZYA encodes protein products, which enable the cells to take up and
metabolise β-galactosides such as lactose. The lacZ gene encodes β-galactosidase,
which breaks a -galactoside into its component sugars; the lacY gene encodes β-
galactoside permease, which transports β-galactosides into the cell; the lacA gene
encodes a -galactoside transacetylase, an enzyme that transfers an acetyl group
from acetyl-CoA to β-galactosides. The regulator protein synthesised by the lacI
gene, called the lac repressor, controls transcription of these structural genes. The
lac repressor functions by binding to the operator at the start of the lacZYA cluster,
which lies between the promoter and the structural genes. When the repressor
binds to the operator, it prevents RNA polymerase from initiating transcription at the
promoter. Thus, in the absence of lactose the lac repressor switches off the
structural genes. In the presence of lactose, lactose binds to the lac repressor,
causing a conformational change in the latter, rendering it unable to bind the
operator, thus switches on the lac structural genes resulting in the production of
active enzymes. Hence, lactose acts as an inducer for the synthesis of these
enzymes when it is the only source of carbon. Lactose is then transported into the
cell and hydrolysed to glucose and galactose.
 lac repressor binding
operator
The lac
operon
P Plac Olac

lacI lacZ lacY lacA

CAP binding site

Figure 2: The lac operon and the lacI region in the E. coli genome
P,Plac correspond to the promoter for the lacI gene and the lacZYA genes
respectively; Olac corresponds to the operator region responsible for
repressor binding.

However, in the presence of both glucose and lactose, glucose is used preferentially
as a carbon source. This is achieved by preventing expression of the lac structural
genes. This effect is called catabolite repression. Catabolite repression is possible
because of the ability of glucose to reduce the level of cyclic AMP (cAMP) in the cell.
The cAMP binds to the catabolite activator protein (CAP), which in turn binds to a
specific region upstream of the lac promoter to allow transcription (shown in the
figure above). Hence in the absence of cAMP (in the presence of glucose), the CAP
protein is unable to activate the lac promoter resulting in catabolite repression.

In this experiment the wild type strain Escherichia coli B (E. coli B, NCIB 10243) is
used.
In a medium containing glucose and lactose, E. coli first consumes glucose. During
this period, lactose does not induce b-galactosidase. When the glucose has been
utilized, there is a short delay in growth called the diauxie lag. During the lag period,
lactose-utilizing system is induced, then growth resumes again. This time, lactose is
used as carbon source.
Procedure

A stirred tank fermenter with a volume of 2 litres is sterilized containing a defined

medium for growth of E. coli B. The composition of the medium is as follows:

g/l
Ingredients

glucose 0.5
lactose 0.5
ammonium sulphate 5.0
sodium chloride 3.0
disodium hydrogen orthophosphate (hydrate) 13.6
potassium dihydrogen orthophosphate 1.6
magnesium sulphate 0.1

pH is adjusted at 7.0

Glucose and lactose are sterilized separately and added to the fermenter after
sterilization. This is done to avoid caramelization.

Add 50 ml of an overnight grown inoculum of E. coli B to the fermenter. This should

be done aseptically. Take 5 ml samples every 15-30 minutes (as appropriate) under
sterile conditions.

Growth of E. coli B:

Measure the growth of E. coli by monitoring the absorbance of the cell suspension at
670 nm using a spectrophotometer. The measurement should be carried out
immediately after the sample is taken. The samples are read against a water blank.
The absorbance readings are directly proportional to the cell concentration up to an
absorbance of 0.4. Samples giving a value above 0.4 should be diluted with distilled
water. An absorbance reading of 1.0 is equivalent to 0.5 g/l of E. coli dry weight.

b-Galactosidase assay:

b-Galactosidase is an interacellular enzyme. Sonication can be used for cell

disruption. A colorimetric assay is carried out to detect the levels of b-Galactosidase
at different stages of the growth of E. coli B.
Sonicate under supervision (see the procedure for sonication), 5 mls of the
sample taken from the fermenter for 10 sec. under maximum power setting. The
sample should be kept in ice during sonication. Weigh out 0.083 g of o-
nitrophenylgalactoside and dissolve it in 100 ml of the buffer labelled A. This buffer
solution contains: 0.02M phosphate, pH 7; 1.0 mM MgCl 2 ; 10.0 mM
mercaptoethanol. After addition of o- nitrophenylgalactoside to the buffer, label the
container as B.
Procedure for Sonication:

Important Notes
1. The unit contains high voltage.
2. Absolutely avoid all bodily contact with the probe tip end while the unit is on. Skin
and nerve damage can result to the area contacted.
3. Use gauze masks to avoid aerosols.
4. The audible sound level is annoying but falls well within the safety limits. You may
feel more comfortable to use an earmuff. Make sure the door to the chamber is
closed before you start the sonicator.
5. During operation the probe tip must be immersed into the culture.
6. Make sure not to drop or hit the titanium tip of the probe.

To carry out sonication:

1. Assemble the probe following your demonstrator's instructions.

2. Make sure that 1 inch of the probe tip is immersed into the cultures where
possible.
3. Use maximum power setting in all cases.
4. Wash the probe with distilled water after use.

To carry out the b-galactosidase assay:

Warm up 2.3 ml of solution B to 37° C in a test tube. Add 0.2 ml of the supernatant
from the sonicated sample and mix well. Leave the test tube in 37° C bath to
incubate for exactly 5 min. Then add 2.5 ml of solution C. This solution contains 0.2
M Na2Co3 and is used to stop the reaction. If a precipitate is observed, centrifuge
samples for 5 min. at 10,000 r.p.m. Read the absorbance of clear samples at 420 nm
in a spectro-photometer. Use the following blank: 2.3 ml solution B+ 0.2 ml water +
2.5 ml solution C. If the absorbance exceeds 0.7, repeat the assay on appropriately
diluted samples. An absorbance reading of 1.0 is equivalent to 0.285 mmol o-
nitrophenol /ml in the solution being measured.

The following enzymatic reaction relates to the above assay:

o- nitrophenylgalactoside b-galactosidase® o- nitrophenol + galactose

Protein assay: Bradford

Carry out total protein assay (Bradford method) on the selected samples (samples
taken for b-galactosidase assay).
Bradford assay is a protein determination method that involves the binding of the
Coomassie Brilliant Blue G-250 dye to proteins (Bradford, 1976). The dye exists in
three different forms: Cationic (red), neutral (green) and anionic (blue) (Compton and
Jones, 1985). Under acidic conditions the dye is mostly in the protonated red
cationic form (Amax 470nm). However, when the dye binds to protein it is converted
to an unprotonated stable blue form (Amaz 595nm) (Fazekes de St. Groth et al.
1963, Sedmack and Grossberg 1977).
In Bradford assay it is the blue form (protein-dye) that is detected at 595nm using a
spectrophotometer.
The standard protocol can be performed in three different volumes: 5 ml and 1 ml
cuvette assay, and a 250 μlmicroplate assay. In this experiment we will be using the
1ml cuvette assay.

Materials:
1. Bovine serum albumine (BSA): 1mg/ml solution
2. Coomassie blue 1x dye solution
3. Protein with Unknown concentration (sample) [20µl sample is needed for the
assay]

Protocol

1. BSA will be used to create a standard range of protein concentrations. The

linear range of this assay for BSA is in the range 125 – 1,000 μg/ml (mg/l).

2. Remove the 1x dye reagent from 4°C storage before use.

3. Pipette each standard and sample solution into separate clean 1.5ml
eppendorf tubes. Add the 1xdye reagent to each tube and vortex.

Content of the cuvettes Volume of Volume of 1x dye

standard/sample reagent
1 ml 20µl 1ml

Note! Blank samples (0 μg/ml) should be made using distilled water and dye reagent.

4. Incubate at room temperature for at least 5 min.

5. Set the spectrophotometer to 595 nm. Zero the instrument with the blank.
Measure the absorbance of the standards and the samples.

6. Create a standard curve by plotting the 595 nm absorbance values (y-axis)

versus their concentration in μg/ml (x-axis). Determine the sample
concentration using the standard curve. If the samples were diluted, adjust the
final concentration of the samples by multiplying by the dilution factor used.

Reporting

Answer the following questions. Number your answers according to the number of
the question.

1. Write a brief introduction explaining the importance of betagalactosidase enzyme

and the scientific basis of the experiment.
2. Specify the period of diauxie lag, giving the starting and finishing time. Show the
relevant points on the graph
3. Plot loge of cell concentration (g/l) vs time for the duration of the fermentation.
4. Specify cell concentration in g dry cell weight/l: (a) - when glucose was
completely utilized (b) - when lactose was utilised (end of fermentation).
5. Calculate td for the two exponential phases.
6. Calculate specific growth rate (max) for the two exponential phases.
7. Calculate total protein concentration for a chosen point in fermentation.
8. Calculate β-Galactosidase activity for a chosen point in fermentation
9. Calculate glucose concentration for a chosen point in fermentation.
10. Draw cell concentration, total proteins concentration, glucose concentration and
-Galactosidase activity (mol/l.sec.) against time on the same plot for the duration
of the fermentation. Discuss.
11. Calculate x/s, enz/s, enz/x, prot/s, prot/x and enz/prot for the two exponential growth
phases. Tabulate the yields and discuss.
12. Calculate qp and Qp for production of -Galactosidase.
13. Discuss any other specific point relevant to your practical.

Important:

Remember to take into account the dilution factors!

Data from the experiment

Table 1. OD, total protein, enzyme activity and glucose raw data (absorbance
values)
Sampl Time Optical Optical Absorbanc Protein Glucose
e (minute density density 2 e after 5 assay assay
numb s) 1(670 nm) (670 nm) min for the (absorbanc (absorbance)
er enzyme e) . Samples
assay were diluted
10 times
1 0 0.065 0.064 0.05 0.124 1.5282
2 30 0.065 0.065 0.004 0.021 1.455
3 60 0.069 0.07 0.012 0.006 1.2362
4 90 0.091 0.093 0.006 0.042 0.7982
5 120 0.158 0.153 0.008 0.071 0.5062
6 150 0.222 0.22 0.019 0.186 0.2142
7 180 0.22 0.225 0.256 0.249 0
8 210 0.367 0.36 0.289 0.486 0
9 240 0.446 0.445 0.302 0.845 0
10 270 0.478 0.475 0.408 0.802 0
11 300 0.626 0.65 0.421 0.911 0
12 330 0.662 0.662 0.455 1.272 0
13 360 0.716 0.712 0.566 1.252 0

Table 2. Total protein calibration curve measurements using Bovine serum albumin
BSA Standard Curve   (BSA) as a representative protein
Tube no. OD 1 OD2
1(1g/L) 0.56 0.57
2(500 mg/L) 0.313 0.35
3(250 mg/L) 0.159 0.162
4(125 mg/L) 0.084 0.09
5(62.5 mg/L) 0.053 0.055
6(31.25 mg/L) 0.025 0.03
Table 3. Calibration curve for determination of glucose
Standard OD (1) OD (2) Glucose
Concentration
(mg/ml)
1 0.352 0.339 0.02
2 0.669 0.665 0.04
3 0.966 0.951 0.06
4 1.231 1.212 0.08

References

1) Bradford MM, A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding, Anal
Biochem, 72, 248–254 (1976).
2) https://www.bioproduction-sekisui.com/about-us/
3) Peter Stanbury, Allan Whitaker, Stephen J. Hall. Principles of fermentation
technology. eBook ISBN: 9780444634085; Paperback ISBN: 9780080999531
Data handling/interpretation questions
For the data handling assessment , please answer the following questions:
1) The CAF corporation is developing a new antibiotic Antimadoxin. Antimadoxin is produced by
Bacillus antimadoxae but yields are poor. The gene Am44 encodes for the production of
Antimadoxin and is located on the plasmid AMdxp14. The CAF corporation has introduced the
gene Am44 into Bacillus hendonae and this has given improved yields. Bacillus hendoae has one
large plasmid with 38 kbp The feasibility of using either batch or continuous culture for the
production of Antimadoxin has been explored. Fermentation processes were designed at pilot
plant stage which could then be easily scaled up to mass production stage. To ensure the strain
was actively growing, Bacillus hendonae was cultured in 50 mL of Dextrose Yeast Broth medium
containing the organism, was then inoculated into 500 mL of a fermentation medium. This was
incubated at 32oC in either batch or continuous culture. Conditions in the batch fermenter were
maintained at pH 7.2 – 7.3, oxygen was continuously injected and the medium was stirred at 165
rpm. Production was ceased at 96 h. In the continuous culture vessel, the dilution rate was 1
volume change per day. The pH, oxygen levels and stirrer speed were maintained at the same
level as the batch culture vessel. Samples were taken regularly to measure levels of antibiotic
production and results are shown in Tables 1 and 2:

Table 1. Production of antimadoxin in batch culture

Time (hours) 0 8 16 24 32 40 48 56 64 72 80 88 96
Antimadoxin 0 0 0 2.3 10.6 18.7 29.4 33.2 36.8 37.4 36.9 35.7 36.1
(µg/ml)

Table 2. Production of antimadoxin in continuous culture

Time (days) 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Antimadoxi 0 2. 10. 12. 11. 12. 10. 13. 12. 11. 11. 11. 12. 12. 11. 6. 2. 2.
n 5 6 0 0 6 8 5 5 5 9 7 7 3 9 2 3 1
(µg/ml)

If you were the CAF production manager, suggest whether you would choose batch or continuous
culture for the production of antimadoxin. Justify your arguments.
2) After graduation you accept a position in a biotech company, where your first assignment
is to scale up an aerobic microbial culture from the well-characterized 50 L pilot scale to
the 1,500 L process scale.  You discuss the problem with another recently hired
bioreactor specialist, who received his degree from Mars University.
Him: "That's easy.  Just maintain the same Reynolds number."
You: "You mean the impeller Reynolds number, Rei?"
Him: "Uh… yeah."
You decide to perform an analysis of this strategy.  Calculate the following ratios, where
scale II is 1,500 L and scale I is 50 L.  
a. Stirring speed, NII/NI.
b. Power imparted per volume fluid, (P/V)II/(P/V)I.
c. State two reasons why scaling up in this manner is a really bad idea.

3. Biosurfactants have advantages over synthetic ones because of their high specificity and
biodegradability. They have applications in many industries, ranging from food to
petrochemicals. However, biosurfactants are expensive to produce. Therefore, any means to
improve production at lower cost is welcomed. You have just been recruited as a bioprocess
development scientist at a new startup company developing biosurfactants with one of your
responsibilities being to design, operate and interpret bioreactor experiments with yeasts and
bacteria. You decide to determine whether type of nitrogen source has an impact on the yield of
biosurfactant produced. Yield is determined using two responses: the maximum variation of
surface tension (ST), in mN/m and the emulsification index (EI), in percentage. The higher these
measures, the better the yield of biosurfactant. The nitrogen sources you are investigating are:
peptone (factor 1), yeast extract (factor 2), ammonium sulfate (factor 3) and urea (factor 4)
using the following levels:
The run combinations (24 full factorial design) and results for the nitrogen source experiment
are given in the table below.

Factor 1 Factor 2 Factor 3 Factor 4 Response 1 Response 2

Std Run x1 x2 x3 x4 ST EI
17 1 0 0 0 0 13 26
4 2 1 1 -1 -1 4 1
12 3 1 1 -1 1 4.6 6.2
7 4 -1 1 1 -1 19.5 40.6
11 5 -1 1 -1 1 9.3 13
15 6 -1 1 1 1 21.1 60.4
10 7 1 -1 -1 1 6 30.6
19 8 0 0 0 0 11 25.4
13 9 -1 -1 1 1 15.2 40.3
1 10 -1 -1 -1 -1 6.5 37.3
8 11 1 1 1 -1 13 50
18 12 0 0 0 0 11.4 24
9 13 -1 -1 -1 1 7.5 35.7
2 14 1 -1 -1 -1 14.5 26.1
3 15 -1 1 -1 -1 5.1 27
6 16 1 -1 1 -1 22 52.2
5 17 -1 -1 1 -1 16.6 45.3
16 18 1 1 1 1 9.4 43.2
14 19 1 -1 1 1 16.3 50.1

Using Design Expert, analyse the data to determine which factors/interactions are statistically
significant at the 5% level and write down the regression equations for both ST and EI in terms of the
significant effects identified.

Term paper

Review the use of single use bioreactors in biomanufacturing.