Exercises for lecture biochemical process engineering
I. Enzymatic process engineering .......................................................................................... 2
A. Keyword ...................................................................................................................... 2
B. Exercises ...................................................................................................................... 2
1. Effect of temperature on glucose isomerase ............................................................ 2
2. Incomplete enzyme reaction .................................................................................... 3
3. Kinetic of oxygen uptake ......................................................................................... 4
4. Kinetics of gluconic acid production ....................................................................... 5
5. Enzyme half-life....................................................................................................... 6
6. Enzyme kinetics ....................................................................................................... 8
II. Growth kinetics .................................................................................................................. 9
A. Keywords ..................................................................................................................... 9
B. Exercises .................................................................................................................... 10
1. Yields in acetic acid production ............................................................................. 10
2. Hybridoma doubling time ...................................................................................... 10
3. Thermal death kinetics ........................................................................................... 11
4. *Growth parameter of recombinant [Link] ............................................................. 13
5. *Growth parameters for hairy roots ....................................................................... 14
6. *Ethanol fermentations by yeast and bacteria........................................................ 14
III. Mass transfer ................................................................................................................. 16
A. Keywords ................................................................................................................... 16
B. Exercises .................................................................................................................... 16
1. Cell concentration in an aerobic cell culture .......................................................... 16
2. Estimating kLa using the dynamic method ............................................................. 16
3. *kLa required to maintain critical oxygen concentration ........................................ 17
4. *Single-point kLa determination using the oxygen-balance method ..................... 17
5. *kLa measurement .................................................................................................. 18
IV. Material Balance ........................................................................................................... 19
A. Keywords ................................................................................................................... 19
B. Exercises .................................................................................................................... 19
1. General mass-balance equation.............................................................................. 19
2. Setting up a flow sheet ........................................................................................... 19
3. Continuous filtration .............................................................................................. 19
4. Batch Mixing ......................................................................................................... 23
5. Continuous acetic acid fermentation ...................................................................... 26
6. Xanthan gum production ........................................................................................ 30
7. Stoichiometric coefficients for cell growth ............................................................ 35
8. Production yield and oxygen demand .................................................................... 36
* indicates exercises as homework
I. Enzymatic process engineering
A. Keyword
Michaelis-Mentin-Kinetics, Michaelis-Menten constant, enzyme activity, Lineweaver-Burk
Plot, Eadie-Hofstee Plot, equilibrium constants, activation energy
B. Exercises
0.37
1. Effect of temperature on glucose isomerase
Glucose isomerase is used extensively in the USA for production of high-fructose syrup. The
reaction is:
glucose fructose.
∆H0exn for this reaction is 5.73 kJ gmol-l; ∆S0rxn is 0.0176 kJ gmol-l K-1.
(a) Calculate the equilibrium constants at 50°C and 75°C
(b) A company aims to develop a sweeter mixture of sugars, i.e. one with a higher
concentration of fructose. Considering equilibrium only, would it be more desirable to operate
the reaction at 50°C or 75°C.
2. Incomplete enzyme reaction
An enzyme catalyzes the reaction:
A B
At equilibrium, the reaction mixture contains 63 wt% A.
(a) What is the equilibrium constant?
(b) If the reaction starts with A only, what is the equilibrium yield of B from A?
3. Kinetic of oxygen uptake
Serratia marcescens is cultured in minimal medium in a small stirred fermenter. Oxygen
consumption is measured at a cell concentration of 22.7 g l-1 dry weight.
(a) Microbial oxygen consumption is a zero order reaction. Determine the rate constant for
oxygen uptake.
(b) If the cell concentration is reduced to 12 g l-1, what is the value of the rate constant?
4. Kinetics of gluconic acid production
Aspergillus niger is used to produce gluconic acid. Product synthesis is monitored in a
fermenter; gluconic acid concentration is measured as a function of time for the first 39 h of
culture.
(a) Determine the rate constant.
(b) Estimate the product concentration after 20 h.
5. Enzyme half-life
Amyloglucosidase from Endomycopsis bispora is immobilised in polyacrylamide gel.
Activities of immobilised and soluble enzyme are compared at 80~ Initial rate data measured
at a fixed substrate concentration are listed below.
What is the half-life for each form of enzyme?
6. Enzyme kinetics
Lactase, also known as β-galactosidase, catalyses the hydrolysis of lactose to produce glucose
and galactose from milk and whey. Experiments are carried out to determine the kinetic
parameters for the enzyme. Initial rate data are listed below.
Evaluate vmax and KM from the michaelis menten plot and with Lineweaver-Burk plot
Michaelis Menten Plot
Vmax = 0.00195 mol l-1 min-1, Km = 0.003 mol l-1
Lineweaver-Burk plot
Vmax = 0.00226, Km = 0.0039
II. Growth kinetics
A. Keywords
Yield of product formation, biomass yield, doubling time, growth rate,
B. Exercises
1. Yields in acetic acid production
The equation for aerobic production of acetic acid from ethanol is:
C2H5OH + O2 ---> CH3CO2H + H2O
Acetobacter aceti bacteria are added to vigorously-aerated medium containing 10 g l-1
ethanol. After some time, the ethanol concentration is 2 g l-1 and 7.5 g l-1 acetic acid is
produced. How does the overall yield of acetic acid from ethanol compare with the theoretical
yield?
2. Hybridoma doubling time
A mouse-mouse hybridoma cell line is used to produce monoclonal antibody. Growth in batch
culture is monitored with the following results.
(a) Determine the specific growth rate during the growth phase.
(b) What is the culture doubling time?
3. Thermal death kinetics
The number of viable spores of a new strain of Bacillus subtilis is measured as a function of
time at various temperatures.
(a) Determine the activation energy for thermal death of B. subtilis spores.
(b) What is the specific death constant at 100°C.
(c) Estimate the time required to kill 99% of spores in a sample at 100°C
4. *Growth parameter of recombinant [Link]
Escherichia coli is being used for production of recombinant porcine growth hormone. The
bacteria are grown aerobically in batch culture with glucose as growth-limiting substrate.
Cell and substrate concentrations are measured as a function of culture time; the results are
listed below.
(a) Plot µ as a function of time.
(b) What is the value of µ max ?
(C) What is the observed biomass yield from substrate? Is Y´X/S constant?
5. *Growth parameters for hairy roots
Hairy roots are produced by genetic transformation of plants using Agrobacterium rhizogenes.
The following biomass and sugar concentrations were obtained during batch culture of
Atropa belladonna hairy roots in a bubble-column fermenter.
(a) Plot ju as a function of culture time. When is the growth
rate maximum?
(b) Plot the specific rate of sugar uptake as a function of time.
(c) What is the observed biomass yield from substrate? Is Yxs
constant?
6. *Ethanol fermentations by yeast and bacteria
Ethanol is produced by anaerobic fermentation of glucose by Saccharomyces cerevisiae. For
the particular strain of S. cerevisiaeemployed, the maintenance coefficient is 0.18 kg kg-1 h-1,
Yx/s is 0.11 kg kg-1, YPX is 3.9 kg kg-1 and µ max is 0.4 h-1. It is decided to investigate the
possibility of using Zymomonas mobilis bacteria instead of yeast for making ethanol. Z.
mobilis is known to produce ethanol under anaerobic conditions using a different metabolic
pathway to that employed by yeast. Typical values of Yxs are lower than for yeast at about
0.06 kg kg-1; on the other hand, the maintenance coefficient is higher at 2.2 kg kg -1 h-1. Ypx
for Z. mobilis is 7.7 kg kg-l; µ max is 0.3 h- 1.
(a) From stoichiometry, what is the maximum theoretical yield of ethanol from glucose?
(b) Y'PS is maximum and equal to the theoretical yield when there is zero growth and all
substrate entering the cell isused for maintenance activities. If ethanol is the sole extracellular
product of energy-yielding metabolism, calculate mp for each organism.
(c) S. cerevisiae and Z. mobilis are cultured in batch fermenters. Predict the observed product
yield from substrate for the two cultures.
(d) What is the efficiency of ethanol production by the two organisms? Efficiency is defined
as the observed product yield from substrate divided by the maximum or theoretical
product yield.
(e) How does the specific rate of ethanol production by Z. mobilis compare with that by S.
cerevisiae?
(f) Using Eq , compare the proportions of growthassociated and non-growth-
associated ethanol production by Z. mobilis and S. cerevisiae. For which organism is
nongrowth associated production more substantial?
(g) In order to achieve the same volumetric ethanol productivityfrom the two cultures, what
yeast concentration is required compared with the concentration of bacteria?
(h) At zero growth, the efficiency of ethanol production is the same in both cultures. Under
these conditions, if the same concentration of yeast and bacteria are employed, what
size fermenter is required for the yeast compared with the bacteria in order to achieve the
same total productivity?
(i) Predict the observed biomass yield from substrate for the two organisms. For which
organism is biomass disposal less of a problem?
(j) Make a recommendation about which organism is better suited for industrial ethanol
production, and give your reasons.
III. Mass transfer
A. Keywords
Rate of oxygen uptake, kLa, oxygen transfer rate
B. Exercises
1. Cell concentration in an aerobic cell culture
A strain of Azotobacter vinelandii is cultured in a 15 m3 stirred fermenter for alginate
production. Under current operating conditions kLa is 0.17 s-1. Oxygen solubility in the broth
is approximately 8 x 10-3 kg m-3.
(a) The specific rate of oxygen uptake is 12.5 mmol g-1 h-1. What is the maximum possible
cell concentration?
(b) The bacteria suffer growth inhibition after copper sulphate is accidently added to the
fermentation broth. This causes a reduction in oxygen uptake rate to 3 mmol g-1 h-1. What
maximum cell concentration can now be supported by the fermenter?
2. Estimating kLa using the dynamic method
A 20-1 stirred fermenter containing a Bacillus thuringiensis culture at 30°C is used for
production of microbial insecticide, kLa is determined using the dynamic method. Air flow is
shut offfor a few minutes and the dissolved-oxygen level drops; the air supply is then re-
connected. When steady state is established, the dissolved-oxygen tension is 78% air
saturation. The following results are obtained.
(a) Estimate kLa.
(b) An error is made determining the steady-stateoxygen level which, instead of 78%, is taken
as 70%. What is the percentage error in kLa resulting from this 10% error in CAL?
3. *kLa required to maintain critical oxygen concentration
A genetically-engineered strain of yeast is cultured in a bioreactor at 30°C for production of
heterologous protein. The oxygen requirement is 80 mmol 1-1 h-1; the critical oxygen
concentration is 0.004 mM. The solubility of oxygen in the fermentation broth is estimated to
be 10% lower than in water due to solute effects.
(a) What is the minimum mass-transfer coefficient necessary to sustain this culture if the
reactor is sparged with air at approximately 1 atm pressure?
(b) What mass-transfer coefficient is required if pure oxygen is used instead of air?
4. *Single-point kLa determination using the oxygen-balance
method
A 200-1itre stirred fermenter contains a batch culture of Bacillus subtilis bacteria at 28°C. Air
at 20°C is pumped into the vessel at a rate of 1 vvm; (vvm stands for volume of gas per
volume of liquid per minute). The average pressure in the fermenter is 1 atm. The volumetric
flow rate of off-gas from the fermenter is measured as 189 1 min-1. The exit gas stream is
analysed for oxygen and is found to contain 20.1% 0 2. The dissolved-oxygen concentration
in the broth is measured using an oxygen electrode as 52% air saturation. The solubility of
oxygen in the fermentation broth at 28~ and 1 atm air pressure is 7.8 x 10-3 kg m-3.
(a) Calculate the oxygen transfer rate.
(b) Determine the value of kLa for the system.
(c) The oxygen analyser used to measure the exit gas composition has been incorrectly
calibrated. If the oxygen content has been overestimated by
5. *kLa measurement
Escherichia coli bacteria are cultured at 35°C in the followingmedium:
The stirred fermenter used for this culture has an operating volume of 20 m3 and a liquid
height of 3.5 m. Air at 25°C is sparged into the bottom ofthe vessel at a rate of 25 m3 min-1.
Oxygen tension in the fermenter is measured using polarographic electrodes located at the top
and bottom of the vessel. At the top the reading is 50% air saturation; the reading at the
bottom is 65%. The gas flow rate leaving the fermenter is measured with a rotary gas meter
and is found to be 407 1 s-1. The oxygen content of the off-gas is 20.15%.
(a) Calculate the pressure at the sparger. (Static pressure Ps due to the height of liquid is given
by the equation: Ps=ρ g h -p g h, where ρ is liquid density, g is gravitational acceleration, and
h is liquid height.)
(b) Estimate the solubility of oxygen in the fermentation broth at 35°C and 1 atm air pressure
using the correlation of Eq.
(c) Estimate the solubility of oxygen at the bottom of the tank.
(d) Calculate the logarithmic-mean concentration driving
force, (C*AL - CAL)L.
(e) What is the oxygen transfer rate?
(f) Determine the value of kLa.
(g) What is the maximum cell concentration that can be supported in this fermenter if the if
the oxygen demand of the organism is 7.4 mmol g- 1 h- 1 ?
IV. Material Balance
A. Keywords
Mass balance equation, flow sheet
B. Exercises
1. General mass-balance equation
A continuous process is set up for treatment of wastewater. Each day, 105 kg cellulose and 103
kg bacteria enter in the feed stream, while 104 kg cellulose and 1.5 x 104 kg bacteria leave in
the effluent. The rate of cellulose digestion by the bacteria is 7 x 104 kg d-1. The rate of
bacterial growth is 2 x 104 kg d-1; the rate of cell death by lysis is 5 x 102 kg d-1. Write
balances for cellulose and bacteria in the system.
Solutions in lecture
2. Setting up a flow sheet
Humid air enriched with oxygen is prepared for a gluconic acid fermentation. The air is
prepared in a special humidifying chamber. 1.5 I h- 1 liquid water enters the chamber at the
same time as dry air and 15 gmol min-1 dry oxygen gas. All the water is evaporated. The
outflowing gas is found to contain 1% (w/w) water. Draw and label the flow sheet for this
process.
Solution in lecture
3. Continuous filtration
A fermentation slurry containing Streptomyces kanamyceticus cells is filtered using a
continuous rotary vacuum filter. 120 kg h- 1 slurry is fed to the filter; 1 kg slurry contains 60 g
cell solids. To improve filtration rates, particles of diatomaceous-earth filter aid are added at a
rate of 10 kg h- 1. The concentration ofkanamycin in the slurry is 0.05% by weight. Liquid
filtrate is collected at a rate of 112 kg h-1; the concentration ofkanamycin in the filtrate is
0.045% (w/w). Filter cake containing cells and filter aid is continuously removed from the
filter cloth.
(a) What percentage liquid is the filter cake?
(b) If the concentration ofkanamycin in the filter-cake liquid is the same as in the filtrate, how
much kanamycin is absorbed per kg filter aid?
• Draw the flowsheet showing all data with units.
• Define the system boundary by drawing on the flowsheet
• Analyse (i) State any assumptions. (ii) Collect and state any extra data needed.
• Calculate: (i) Set up a calculation table showing all components of all streams passing
across system boundaries. State the units used for the table. Enter all known quantities.
(ii) Calculate unknown quantities, apply the mass-balance equation.
• Finalise (i) Answer the specific questions asked in theproblem. (ii) State the answers
clearly and unambiguously, checking significant figures.
Define the system boundary by drawing on the flowsheet.
4. Batch Mixing
Corn-steep liquor contains 2.5 % invert sugars and 50% water; the rest can be considered
solids. Beet molasses containing 50% sucrose, 1% invert sugars, 18% water and the
remainder solids, is mixed with corn-steep liquor in a mixing tank. Water is added to produce
a diluted sugar mixture containing 2% (w/w) invert sugars. 125 kg corn-steep liquor and 45
kg molasses are fed into the tank.
(a) How much water is required?
(b) What is the concentration of sucrose in the final mixture?
5. Continuous acetic acid fermentation
Acetobacter aceti bacteria convert ethanol to acetic acid under aerobic conditions. A
continuous fermentation process for vinegar production is proposed using non-viable A. aceti
cells immobilised on the surface of gelatin beads. The production target is 2 kg h- 1 acetic
acid; however the maximum acetic acid concentration tolerated by the cells is 12%. Air is
pumped into the fermenter at a rate of 200 gmol h- 1.
(a) What minimum amount of ethanol is required?
(b) What minimum amount of water must be used to dilute the ethanol to avoid acid
inhibition?
(c) What is the composition of the fermenter off-gas?
6. Xanthan gum production
Xanthan gum is produced using Xanthomonas campestris in batch culture. Laboratory
experiments have shown that for each gram of glucose utilised by the bacteria, 0.23 g oxygen
and 0.01 g ammonia are consumed, while 0.75 g gum, 0.09 g cells, 0.27 g gaseous CO2 and
0.13 g H20 are formed. Other components of the system such as phosphate can be neglected.
Medium containing glucose and ammonia dissolved in 20 000 litres water is pumped into a
stirred fermenter and inoculated with X. campestris. Air is sparged into the fermenter; the
total amount of off-gas recovered during the entire batch culture is 1250 kg. Because of the
high viscosity and difficulty in handling xanthan-gum solutions, the final gum concentration
should not be allowed to exceed 3.5 wt%.
(a) How much glucose and ammonia are required?
(b) What percentage excess air is provided?
7. Stoichiometric coefficients for cell growth
Production of single-cell protein from hexadecane is described by the following reaction
equation:
where CH1.66 O0.27 N0.20 represents the biomass. If RQ= 0.43, determine the stoichiometric
coefficients.
8. Production yield and oxygen demand
The chemical reaction equation for respiration of glucose is:
Candida utilis cells convert glucose to CO2 and H20 during growth. The cell composition is
CH1.84 00.55 N0.2 plus 5% ash. Yield of biomass from substrate is 0.5 g g-1. Ammonia is used as
nitrogen source.
(a) What is the oxygen demand with growth compared to that without?
(b) C. utilisis also able to grow with ethanol as substrate, producing cells of the same
composition as above. On a mass basis, how does the maximum possible biomass yield from
ethanol compare with the maximum possible yield from glucose?