2006 Bio 301Final test
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30 Total Marks
Section 1: 12 marks
Answer 6 out of the following 8 questions. Each question carries 2
marks
1.1. Oxygen transfer (10 min)
The oxygen transfer from air to solution is affected by a number of factors, amongst
them are temperature and the partial pressure of oxygen in the atmosphere (21 kPa for
air). Explain in your own words how an increase in temperature and a doubling in the
O2 partial pressure e.g. from 21 kPa to 42 kPa would effect the OTR plotted against
either the dissolved oxygen concentration (cL) or the driving force (saturation deficit,
cS-cL). Sketch the three graphs as one figure (normal, increase of temperature,
doubling of oxygen partial pressure). Assume that not only the oxygen solubility is
affected but that a temperature increase increases the diffusivity (and hence the kLa
value) of the process.
2006 Bio301 final test 1/16
1.2. Growth competition (10 min)
(a) List the four growth constants of microbes that allow the prediction of their
growth. (b) Give suitable units of the growth constants. (c) Explain for each growth
constant, whether a higher value or a lower value would prove a competitive
advantage against a competing organisms and why. Sketch the effect of an increase in
each of the growth constants on the biomass concentration (plotted as a function of
dilution rate of a chemostat)
2006 Bio301 final test 2/16
1.3. Fermentation pathways (10 min)
Outline two of the following glucose fermentation pathways: heterolactic acid
fermentation, propionic acid fermentation, butyric acid fermentation, butanol/acetone
fermentation, homoacetogenesis. Explain how they differ in terms of which electron
acceptor is used for reducing equivalents (NADH) from glycolysis and point out the
differences in ATP gain.
2006 Bio301 final test 3/16
1.4. Organic acid production (10 min)
Explain the significance of anaplerotic sequences (replenishing the TCA cycle) in
some biotechnological processes. Give examples (bioprocess and substrate used).
Name the key enzymes involved. Show the stoichiometric equation for one of the
reactions assuming glucose as the substrate.
2006 Bio301 final test 4/16
1.5. Nitrogen removal (10 min)
Simultaneous nitrification and denitrification is a novel way or nitrogen removal from
wastewater. Explain what the difficulties are to have both processes running in
parallel and how SND can be accomplished. Point out the three different groups of
bacteria involved. And explain how an online oxygen controller can be used to
maximise nitrogen removal. Explain the likely consequences if the dissolved oxygen
setpoint is kept too high or to low.
2006 Bio301 final test 5/16
1.6. Process monitoring and control (10 min)
A proportional- integral- differential controller (PID) can be used to control important
process parameters such as dissolved oxygen concentration or pH to a desired
setpoint. Explain how the three different elements (P, I, D) work and give at least one
advantage and one disadvantage of each element.
2006 Bio301 final test 6/16
1.7. Biogeochemistry (10 min)
Give 3 examples of microbial processes that use anaerobic bacterial respiration. Point
out
 the electron donor and electron acceptor of the reactions.
 the environmental significance of the process
 what specifically makes the process a “respiration”
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1.8. Methanogenesis, Anaerobic Digestion
The anaerobic digestion process involves methane producing bacteria that can only
use acetate and hydrogen gas as the substrate for methane production. The hydrogen
uptake by these bacteria plays a particularly important role as it helps other groups of
bacteria in their own metabolism. A syntrophic relationship develops between
obligate hydrogen producing acetogens (OHPA) and methane producing bacteria
develops. Explain the principle of this relationship and its dependence on
thermodynamics by referring to the plot below. Start by labeling the two lines in the
figure below.
0
-1
-2
Log of H2
concentratio
n
(atm)
-3
-4
-5
-6
-7
+50
0
-50
Gibbs Free Energy Change
(G)
2006 Bio301 final test 8/16
Section 2: Short answer questions 10 marks
Answer 8 out of the following 10 questions. Each question carries
1.25 marks
2.1. Chemostat (5 min)
Plot the typical trends of specific growth rate, substrate concentration and productivity
of a chemostat as a function of (a) dilution rate (b) time. Indicate in your plot where
the critical dilution rate and the maximum productivity are.
2.2. Product formation, stoichiometry of bio-reactions (5 min)
Explain the differences in primary and secondary metabolites. Which ones are suited
to chemostats and batch cultures and why ?
2006 Bio301 final test 9/16
2.3. (5 min) Under which conditions are reducing equivalents (e.g. reduced electron
carriers such as NADH) an energetic advantage or disadvantage for bacterial cells?
Give examples.
2.4 (5 min) Competition. An organism with a maximum specific growth rate µmax of
0. 2 h-1 is cultivated under substrate limiting conditions (substrate concentration of
0.8 mM) resulting in a much slower than maximum specific growth rate of 0.05 h-1 .
What is its substrate half saturation constant (kS) ?
2006 Bio301 final test 10/16
2.5. (2 min) In a continuous culture bioprocess how can you control the biomass
concentration (X) and the specific growth rate (µ) respectively ?
2.6. (2 min) During the aerobic degradation of a wastewater containing an undefined
mixture or easily degradable matrial it was established that 0.12 mol of oxygen were
used per L of wastewater. If this waste water was anaerobically treated, how much
methane gas could one possibly expect (LCH4/Lwastewater) ?
2006 Bio301 final test 11/16
2.7. (5 min) How much methane will be produced from the anaerobic digestion of
hydroxy-butyrate (CH3-CHOH-CH2-COOH)? What is the expected ratio of
CH4/CO2 from this conversion?
2.8. Write comments on the significance of poly hydroxy alkanoates (PHA) such as
poly-hydroxy-buryate (PHB) in microbial bioprocesses. Point out the precursors for
synthesis of PHB.
2006 Bio301 final test 12/16
2.9. What is the advantage of biomass feedback in bioprocesses. Give an example.
2.10. Anaerobic respirations lead to reduced endproducts which can serve as the
electron donor for aerobic chemolithotrophic bacteria. Give and example of the above
process and point out its environmental or industrial significance.
2006 Bio301 final test 13/16
Section 3: Calculations 8 marks
Answer 1 out of the following 4 questions:
3.1 Bioenergetics (20 min)
The anaerobic conversion of butyrate (CH3-CH2-CH2-COO-) into two moles of
acetate (CH3-COO-) and hydrogen gas is an important reaction but is energetically
close to the equilibrium. Calculate the driving force ΔG of this reaction when the
concentrations of reactants are:
butyrate (1 mM),
acetate (100 mM),
hydrogen partial pressure (100 ppm = 0.0001 bar)
The values of ΔG of formation (ΔGfo)of the reactants are as follows: butyrate
-352.63 kJ/mol, acetate: 369.41 kJ/mol, H2: 0 kJ/mol, H+: 0 kJ/mol, H20:
237.178 kJ/mol .
Formula sheet needed.
To obtain the the ΔGo (for standard conditions) Subtract ΔGfo of substrates from ΔGfo
of products.
To obtain the actual ΔG values insert the product and substrate concentrations by
using the Nernst equation: ΔG= ΔGo + 5.69 kJ * log (P/S)
3.2 Growth constants calculation: (20 min) A continuous aerobic process
(chemostat) was used to convert a medium containing 90 g/L of methanol CH3CH2OH) into biomass for Vitamin B12 production (35 mg B12/ g of dry biomass).
The biomass concentration and the residual substrate concentration in the reactor (27
L working volume) were monitored as a function of the dilution rate D and are
displayed below:
D
h-1
0.015
0.038
0.075
0.150
0.375
0.500
0.625
0.682
0.732
Acetate
g/L
0.01
0.02
0.05
0.11
0.42
0.84
2.10
4.21
16.89
Biomass
g/l
19.51
19.68
19.73
19.75
19.70
19.61
19.33
18.87
16.08
Determine the 4 growth constants (maximum specific growth rate (µmax), the
substrate half saturation constant kS, the maximum growth yield coefficient (Ymax)
and the maintenance coefficient (mS) of the organism used. Use the graph paper
supplied if needed. Do not forget the units when stating your results.
3.3 Chemostat performance(20 min) An aerobic chemostat is operated at an oxygen
steady state concentration of 2.5 mg/L. After interruption of the airflow the DO
decreased every second by 0.2 mg/L. The reactor was run at room temperature so the
oxygen saturation concentration was 8 mg/L. What is the current productivity of
2006 Bio301 final test 14/16
acetic acid (CH3-COOH) from this reactor considering that ethanol (CH3-CH2OH) is
the substrate? If the kLa of the reactor is not changed, what is the maximum possible
acetate production rate that this reactor would allow, if the bacteria were much more
active (e.g. by increasing the substrate concentration in the feed inflow).
3.4 “Starvostat” control of high cell density fermentation of recombinant E.coli
strains. E. coli can form actetate as an undesired byproduct when glucose is present
in excess, even though dissolved oxygen is not limiting. Explain why this can happen.
More importantly explain how in a sophisticated process control loop (that is based on
DO readings and the kLa value) the feed supply to a fed batch high cell density
culture can be supplied such that it is always limiting and hence avoiding the build-up
of undesired acetate.
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