2007 Bio 301Final test
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30 Total Marks (4 +16+10)
Section 1: (4 marks)
Answer all of the questions on Algal Biotechenology below:
1.1 (0.2 marks): In mixotrophic growth, what is the energy source(s) for algal
growth?
1.2 (0.2 marks): At pH 6.1 what are the main species of inorganic C present in the
medium?
1.3 (0.6 marks): List the 3 most common forms of N added to algal culture media?
1.4 (0.2 marks): Why is Germanium sometimes added to algae cultures?
1.5 (0.4 marks): Give two examples each of macronutrients; micronutrients, trace
elements and vitamins which are a common component of algae media.
1.6 (0.4 marks): Define the doubling time in terms of the specific growth rate, μ.
What is the unit for μ ?
1.7
(2 marks): The growth curve below is for a culture of Dunaliella salina grown
in a 20 L, stirred carboy-type photobioreactor in the laboratory. The growth
conditions were:
Temperature:
Light:
Initial pH:
o
C
185 μmol photons.m-2.sec-1 (supplied by cool-white
fluorescent lights) on a 12h light: 12h dark cycle.
pH 9.1
2007 Bio301 final test 1/13
Composition of the medium
(all concentrations are in g.L-1)
NaCl
MgCl2.2H2O
MgSO4.7H2O
CaCl2.2H2O
KCl
NH4Cl
K2HPO4
H3BO3
MoO3
CuSO4.5H2O
MnCl2.4H2O
ZnCl2
125.0
1.5
0.5
0.2
0.2
0.1
0.01
0.061
0.015
0.006
0.004
0.004
Write brief answers to each of the following questions (all are of equal value):
(i) How would you change the culture conditions to increase the growth rate;
(ii) How would you change the culture conditions to achieve a higher final cell yield;
(iii) What could be done to maximise the content of β-carotene per cell;
2007 Bio301 final test 2/13
Section 2: 16 marks
Answer 4 out of the following 5 questions. Each question carries 4 marks
2.1 . Nitrogen removal (10 min)
Nitrogen removal from wastewater is important to avoid pollution of freshwater and
marine environments.
1. Explain the consequences of pollution caused by emission of dissolved
nitrogen compounds into the environment
2. Nitrification and Denitrification are processes that play a role in nitrogen
removal from wastewater. Explain these reactions
a. Show the reaction equations and point out the electron donor and
electron acceptor
b. Refer to the 3 physiological groups of bacteria necessary to enable
nitrogen removal.
c. State the critical process conditions needed for the reactions to occur.
Refer to the role of dissolved oxygen
3. Explain why it is difficult to enable both processes to occur in the same
treatment process
4. Explain 2 possible ways to enable nitrification as well as denitrification to
occur in the same process.
5. Explain the principle of Simultaneous Nitrification and Dentrification via
nitrite (NO2-)
a. Point out the possible role of the storage polymer PHB (poly-hydroxybutyrate)
b. Explain how an online process controller based on dissolved oxygen
monitoring can be used to maximise nitrogen removal via SND.
c. Explain the likely consequences if the dissolved oxygen set-point is
kept too high or to low.
2.2 Anaerobic Digestion
Anaerobic digestion is a process than can convert organic material into a renewable
fuel (CH4). However it is a process that can go wrong resulting in digester failure
due to overloading.
1. Explain the principle of electron flow during anaerobic digestion during
successful operation and during failure.
2. Explain in detail the reasons for digester failure by referring to the energetics
(Gibbs Free Energy Change) of the key reactions.
3. Describe a possible on-line control strategy that could help controlling the
anaerobic digestion process to avoid failure due to overeloading.
a. What will be measured
b. What will be controlled
c. What type of algrorithm (use words or flow diagram) could be used
2.3. 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.
2007 Bio301 final test 3/13
2.4 High Cell Density Culture of E. coli using Fed Batch reactors
For the production of recombinant proteins E. coli is grown at extremely high
concentrations. Both, feed and oxygen supply are critical for successfully
product formation. E. coli can form actetate as an undesired byproduct when glucose
is present in excess, even though dissolved oxygen is not limiting.
1
2
Explain the physiological reasons why this can happen.
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.
2.5 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 0.3 mg/L. The reactor was run at room temperature so the
oxygen saturation concentration was 8 mg/L.
1. What is the current conversion rate of the substrate glycerol (CH2OH- CHOHCH2OH) in this reactor assuming the complete oxidation of the substrate to
CO2?
2. If the kLa of the reactor is not changed but the dilution rate increased
resulting in a lowered dissolved oxygen concentration of 1 mg/L, what would
be the new substrate oxidation rate?
3. In the above calculations the microbial yield coefficient and assimilation of
glycerol has been neglected. Consider a significant yield coefficient of 0.4 g of
biomass formed per g of substrate taken up (assume that the biomass sum
formula is identical to that of the substrate). What would be the expected
glycerol uptake rate now?
2007 Bio301 final test 4/13
Section 3: Short answer questions 10 marks
Answer 5 out of the following 7 questions. Each question carries 2 marks
3.1. 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 ?
3.2 (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.
3.3 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.
3.4 (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) ?
3.5 How much methane will be produced from the anaerobic digestion of hydroxybutyrate (CH3-CHOH-CH2-COOH)? What is the expected ratio of CH4/CO2 from
this conversion?
3.6 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.
3.7 Process monitoring and control (10 min)
A proportional- integral- differential controller (PID) can be used in bioprocesses to
control 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.
Sulfur cycle; Explain reactions t
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) ?
Section4: Chemostat Laboratory session
2007 Bio301 final test 5/13
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.
2007 Bio301 final test 6/13
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 temperature not only influences
the oxygen solubility but also the diffusivity (and hence the kLa value) of the process.
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 be 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 in a chemostat (biomass plotted as
a function of dilution rate)
2007 Bio301 final test 7/13
1.6. Process monitoring and control (10 min)
A proportional- integral- differential controller (PID) can be used in bioprocesses to
control 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.
Process control can be critical to some bioprocesses in order to maintain
consistent performance. Explain the type sensors (measuring the process control
variable) and the actuators (the action caused to keep the process control
variable
2007 Bio301 final test 8/13
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”
Explain 3 different examples of significant reactions (to the environment or
industrially) in which microbes use inorganic electron donors.
Show the reaction equations
State the critical process conditions
Biogechemical cycles
Microbially catalysed reactions can cause the oxidation as well as reduction of
2007 Bio301 final test 9/13
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)
2007 Bio301 final test 10/13
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 .
Help provided:
Reaction :
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.
2007 Bio301 final test 11/13
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