Bioprocess Engineering

Stoichiometry
• Pauline Doran : Bioprocess Engineering Principles
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 of kanamycin in the
slurry is 0.05% by weight. Liquid filtrate is collected at a
rate of 112 kg h-1; the concentration of kanamycin 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 of kanamycin in the filter-cake liquid is the
same as in the filtrate, how much kanamycin is absorbed
per kg filter aid?
(a) The liquid content of the filter cake is 4.4%.

Dividing the mass of water in the filter cake by the total mass of this
stream, the percentage liquid is: 0.79 kg/18 kg x 100 = 4.39%.

• (b) The amount of kanamycin absorbed by the filter aid is 9.6 x 10-4 kg
kg-1
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?
1 gm glucose + 0.23 gm O2+ 0.01 gm NH3

0.75 gm gum + 0.09 gm cells + 0.27 gm CO2 +

0.13 gm H2O

mass in + mass generated = mass out + mass

consumed
• Air – A kg
• The mass of O2 in the inlet air is 0.233A; the mass of N2 is 0.767A.
• F - total mass of feed medium added;
• P - total mass of product
• F+A= 1250 + P
• Gum generated = (0.035P) kg

• From reaction stoichiometry, synthesis of (0.035P) kg gum requires:

• (0.0467P) kg glucose
• (0.0107P) kg O2
• (0.00047P)kg NH3

• and produces:
• (0.0042P) kg cells
• (0.0126P) kg CO2
• (0.00607P) kg H2O.
• (0.233A) kg O2 in + 0 kg O2 generated = O2 out + (0.0107P) kg O2 consumed.
• O2 out = (0.233A - 0.0107P) kg.

• N2 out = (0.767A) kg.

• 0 kg CO2 in + (0.0126P) kg CO2 generated = CO2 out + 0 kg CO2 consumed.

• CO2 out = (0.0126P) kg

• The total mass of gas out is 1250 kg. Therefore, adding the amounts of O2, N2 and CO2
• A= 1250- 0.0019P
• glucose in + 0 kg glucose generated = 0 kg glucose out + (0.0467P) kg
glucose consumed
• Glucose in = (0.0467 P) kg

• NH3 in + 0 kg NH3 generated = 0 kg NH3 out + (0.00047P) kg NH3 consumed

• NH3 in = (0.00047P) kg

• F= glucose in + NH3 in + water in

• F= (20000 + 0.04717P) kg
• P = 20 948.3 kg.
• A = 1210.2 kg
• F= 20 988.1 kg
• O2 in = 282.0 kg
• N2 in = 928.2 kg.
• O2 out = 57.8 kg
• N2 out = 928.2 kg
• CO2 out = 263.9 kg
• Glucose in = 978.3 kg
• NH3 in = 9.8 kg.
• Cell balance
• 0 kg cells in + (0.0042P) kg cells generated = cells out + 0 kg cells consumed
• Cells out = (0.0042P) kg Cells out
• = 88.0 kg.

• H2O balance
• 20 000 kg H20 in + (0.00607P) kg H2O generated = H2O out + 0 kg H2O consumed
• H2O out = 20000 + (0.00607P) kg.
• H2O = 20127.2 kg.
Mass of oxygen required to react completely with 978.3 kg glucose is 225.0 kg

The mass provided is 282.0 kg;

% excess air = 25.3%.

(a) 980 kg glucose and 9.8 kg NH3 are required.

(b) 25% excess air is provided.
Acetobacter aceti bacteria convert ethanol to acetic
acid under aerobic conditions. A continuous
fermentation process for vinegar production is
proposed using 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?
O2 content = (0.21) (200) (32) = 1344 g = 1.344 kg

N2 content = (0 79) (200) (28) = 4424 g = 4.424

kg.

Therefore, the total mass of air in = 5.768 kg.

Total mass of the product stream must be 2/0.12 =

16.67 kg kg.
Sterilization
• Destruction of microorganism by heat

• -dN/dt = kN
• N = number of viable microorganism present
• t = time of sterilization treatment
• k = specific death rate

• ln(Nt/No) = -kt

• If Nt/N0 is plotted against time, viable microorganism decreases exponentially.

• Relation between temperature and reaction rate constant is given by
Arrhenius equation
• d(ln k)/dt = E/RT2

• E is activation energy
• T temperature
• On integration
• k = Ae-E/RT

• A is Arrhenius constant
• ln k = ln A – E/RT

• Plot of ln k vs 1/T will give straight line and enable us to calculate A and E.

• From this, the following expression has been derived for heat sterilization
at constant temperature
• ln N0/Nt = A t e-E/RT

• ln N0/Nt is called design criteria for sterilization and is called del factor
• For design, Bacillus stearothermophilus is taken as standard due to
high heat resistance

• Loss of nutrient quality due to sterilization

• 1. interaction between nutrient component of media – maillard type
browning reaction (reaction between reducing sugar with amino
group of amino acids and proteins). So sugar is sterilized separately .
• 2. degradation of heat labile components – filter sterilization
Advantages of continuous sterilization over batch
sterilization

• (i) Superior maintenance of medium quality.

• (ii) Ease of scale-up.
• (iii) Easier automatic control.
• (iv) The reduction of sterilization cycle time.
• (v) Reduction of fermenter corrosion.
Advantages of batch sterilization over continuous
sterilization

• (i) Lower capital equipment costs.

• (ii) Lower risk of contamination — continuous processes require the
aseptic transfer of the sterile broth to the sterile vessel.
• (iii) Easier manual control.
• (iv) Easier to use with media containing a high proportion of solid
matter
 advantages of a separate medium sterilization vessel

• (i) One cooker may be used to serve several fermenters and the
medium may be sterilized as the fermenters are being cleaned and
prepared for the next fermentation, thus saving time between
fermentations.
• (ii) The medium may be sterilized in a cooker in a more concentrated
form than would be used in the fermentation and then diluted in the
fermenter with sterile water prior to inoculation. This would allow the
construction of smaller cookers.
• (iii) In some fermentations, the medium is at its most viscous during
sterilization and the power requirement for agitation is not met by
aeration only. So powerful motor needed.
• (iv) The fermenter would be spared the corrosion which may occur
with medium at high temperature.
disadvantages of a separate
medium sterilization vessel
• (i) The cost of constructing a batch medium sterilizer is much the
same as that for the fermenter.
• (ii) If a cooker serves a large number of fermenters complex pipework
would be necessary to transport the sterile medium, with the
inherent dangers of contamination.
• (iii) Mechanical failure in a cooker supplying medium to several
fermenters would render all the fermenters temporarily redundant.
Filter sterilization
• Suspended solids may be separated from a fluid
• during filtration by the following mechanisms:
• (i) Inertial impaction.
• (ii) Diffusion.
• (iii) Electrostatic attraction.
• (iv) Interception.
• (i) Inertial impaction

• Suspended particles in a fluid stream have momentum. The fluid in

which the particles are suspended will flow through the filter by the
route of least resistance. However, the particles, because of their
momentum, tend to travel in straight lines and may therefore become
impacted upon the fibres where they may then
remain.
• (ii) Diffusion
• Extremely small particles suspended in a fluid are subject to Brownian
motion which is random movement due to collisions with fluid
molecules. Thus, such small particles tend to deviate from the fluid
flow pattern and may become impacted upon the filter fibres.

• (iii) Electrostatic attraction

• Charged particles may be attracted by opposite
• charges on the surface of the filtration medium.
• (iv) Interception

• The fibres comprising a filter are interwoven to

• define openings of various sizes. Particles which are larger than the
filter pores are removed by direct interception. However, a significant
number of particles which are smaller than the filter pores are also
retained by interception. This may occur by several mechanisms
• — more than one particle may arrive at a pore simultaneously or an
irregularly shaped particle may bridge a pore etc.
• An ideal filtration system for the sterilization of animal cell culture media must fulfil the
following criteria:

• (i) The filtered medium must be free of fungal,

• bacterial and mycoplasma contamination.

• (ii) There should be minimal adsorption of protein

• to the filter surface.

• (iii) The filtered medium should be free of viruses.

•
• (iv) The filtered medium should be free of endotoxins
• One example is a case of four filters arranged in sequence.

• The first filter is a positively charged polypropylene pre-filter of 5

micron size for the removal of coarse contaminants

• Second filter is also positively charged polypropylene of 0.5

micron for bulk microbial removal

• Third filter is a single layered, nylon/polyester positively charged

filter with a 0.1 micron size for further microbial and endotoxin
removal

• Fourth filter is similar to the third but is double layered and

removes mycoplasmas and rest of the contaminants

• For virus removal, 0.04 micron nylon/polyester membrane added

Air sterilization
• 0.2 micron PTFE filter is used.

• dN/dx = -KN
• ln(N/N0) = -Kx
• N is the concentration of particles in the air at
• a depth x in the filter and
• K is a constant.
• The efficiency of the filter is given by the ratio of
• the number of particles removed to the original number present

• E = (N0-N)/N0
• = 1 – e-Kx

• For 90% removal, X90, N0 = 10 and N = 1

• Then
• X90 = 2.303/K
Heat transfer
• Hot, freshly-sterilised nutrient medium is cooled in a double-pipe heat
exchanger before being used in a fermentation. Medium leaving the steriliser
at 121°C enters the exchanger at a flow rate of 10 m3 h-1; the desired outlet
temperature is 30°C. Heat from
the medium is used to raise the temperature of 25
m3 h-1 water initially at 15°C. The system operates at steady state. Assume
that nutrient medium has the properties of water.
(a) What rate of heat transfer is required?
(b) Calculate the final temperature of the cooling water as it leaves the heat
exchanger.
Heat capacity of water is 4.19x 103 J kg-1 °C-1
• Q = Mc Cpc (Tco-Tci) = Mh Cph (Thi-Tho)

• M is mass flow rate

• Q is rate of heat removal from system

• Mh = 2.78 kg s-1
• Mc = 6.94 kg s-1
• Q = 1060 KW

• The exit water temperature is 52°C

• A 150 m3 bioreactor is operated at 35°C to produce fungal biomass
from glucose. The rate of oxygen uptake by the culture is 1.5 kg m-3 h-
1
; the agitator dissipates heat at a rate of I kW m-3. 60 m3 h-1 cooling
water available from a nearby river at 10°C is passed through an
internal coil in the fermentation tank. If the system operates at steady
state, what is the exit temperature of the cooling water?
• Given, 460 kJ heat is released for each gmol oxygen consumed
• At steady state, the temperature of the hot fluid, e.g. fermentation
broth, does not change.
• Q = Mc Cpc (Tco-Tci)
• Now,
• Q = - ∆Hrexn -Mv∆hv + Ws
• In most fermentation systems the only source of shaft work is the
stirrer; therefore Ws is the power P dissipated by the impeller.
• Mv is the mass flow rate of evaporated liquid
leaving the system, ∆hv is the latent heat of evaporation.
• ∆Hrexn = -460 x1000 x 1.5 x 150
• 32 x 3600
• = -898 KJ s-1 = -898 KW (negative sign is due to exothermic
reaction)
• The rate of heat dissipation by the agitator is:
• (1 kW m-3) 150 m3 = 150 kW.
• Q = ? kW.
• Mc = ? kg s-1
• Q = Mc Cpc (Tco-Tci)
• Q = 1048 kW.
• Mc = 16.7 kg s-1
• The water outlet temperature is 25°C
• A single-pass shell-and-tube heat exchanger is used to heat a dilute
salt solution used in large-scale protein chromatography. 25.5 m3 h-l
solution passes through 42 parallel tubes inside the heat exchanger;
the internal diameter of the tubes is 1.5 cm and the tube length is 4
m. The viscosity of the bulk salt solution is 10-3 kg m-1 s-1, the density is
1010 kg m-3, the average heat capacity is 4 kJ kg-1 °C-l and the thermal
conductivity is 0.64 W m-l °C-l. Calculate the heat-transfer coefficient.
• Total cross sectional area 42 πR2

• Re = Dvρ/µ

• Pr = Prandtl number = Cpµ/k

• Nusselt number = 0.023(Re)0.8(Pr)0.4

(Flow in tubes without phase change)

• Nu = hD/k
• Re 1.44x104
• Pr 6.25
• Nu 101.6

• The heat-transfer coefficient is 4.3 kW m-2 °C-l .