Analysis of Various Modes of Stirred Tank Bioreactor Operations

Analysis of various modes of Stirred tank bioreactor operations
 Batch
 Fed-batch
 Continuous
 Continuous with recycle
 Two-stage
VIVEK R BITS Pilani, K K Birla Goa Campus

Batch vs Fed batch vs Continuous cultures in stirred tank reactors
Secondary metabolites Fed batch
 To prolong exponential phase/
stationary phase by feeding
rate limiting substrate
 Pseudo-steady state
/ also called log/exponential phase Continuous
Primary metabolites  To prolong exponential phase/

by feeding rate limiting
substrate
 Steady state

Analysis of continuous operation of stirred tank bioreactor
 Continuous input and output

 Used in brewing, production of baker’s yeast and waste water treatment
 Enzymatic reactions (usually in a packed-bed reactor)
 In well-mixed bioreactor, outflow substrate concentration equal to that of concentration
in reactor
Nutrient limited self balancing culture system, which may be maintained in a steady state
over a wide range of sub-maximum specific growth rates

Steady state conditions in a continuous culture
F
Dilution rate SS substrate Specific growth
S0 F X D= F/V, h-1 concentration, rate h-1
S g/L
D (at F) S µ
D1 (at F1) S1 µ1
Dilution rate
X S D2(at F2) S2 µ2
D = F/V
µ
D2 > D1 > D
S  residual/ steady state µ2 > µ1 > µ
substrate concentration
Volume = V X  steady state biomass concentration

Batch vs Fed batch vs continuous
Continuous
Steady state µ = D
µ2
µ2 𝝁𝒎𝒂𝒙 𝑺
𝝁= µ1
µ1 𝑲𝒔 + 𝑺
S1 S2 time
Strategies of continuous culture
Chemostat
 Constant chemical environment, liquid volume kept constant by setting input and
outlet rates equal
 Dilution rate is constant
 The system adjusts itself to the feed rate so as to maintain steady state condition
Turbidostat
 Constant turbidity: Liquid volume kept constant by setting inflow rate equal to the outlet rate.
 However, inflow rate is adjusted to keep the biomass concentration constant
 Dilution rate adjusts to steady-state value required to achieve the desired biomass concentration
 Complex monitoring and control
Steady-state cell and substrate concentrations as a function of “D” in a chemostat
The curves were obtained using the

following parameter values:
μmax = 0.5 h -1, KS=0.2 kg m-3,
YXS =0.5 kg/kg , and si=20 kg m-3
To avoid washout D < μmax
𝝁𝒎𝒂𝒙 𝑺
𝝁=
𝑲𝒔 + 𝑺
Cells washout
Effect of dilution rates on steady state concentrations
at low and high affinity constants (Monod constant)
At low Ks At high Ks
Steady state biomass concentration

Steady state substrate concentration

Biomass
Substrate
Biomass
Substrate
Dilution rate
Dilution rate
Lower the Ks higher is the affinity of biomass towards the substrate

Effect of increased substrate concentration Si on x and S

Dilution rate

Mass balance: Enzymatic reactions
𝑑𝑀
= 𝑀𝑖 − 𝑀𝑜 + 𝑀𝐺 − 𝑀𝐶
𝑑𝑡
𝑉 𝑑𝑆
= 𝐹𝑆𝑖 − 𝐹𝑆 − 𝑣𝑉 (1)
𝑑𝑡
𝑉 𝑑𝑆 𝑣𝑚 𝑆
= 𝐹𝑆𝑖 − 𝐹𝑆 − 𝑉 (2) ;applying Michaeli’s Menton kine
𝑑𝑡 𝐾𝑚 + 𝑆
𝑉𝑚 𝑆
𝐷 𝑆𝑖 − 𝑆 = (3)
𝐾𝑚 + 𝑆
 If vmax, Km, and Si are known, (3) can be used to calculate “ D” required to achieve a particular
level of substrate conversion.
 The steady-state product concentration can then be evaluated from stoichiometry
For immobilized enzymatic system, equation (3) will have the term ηT
𝑉𝑚 𝑆
𝐷 𝑆𝑖 − 𝑆 = η 𝑇 (4)
𝐾𝑚 + 𝑆
where ηT total effectiveness factor,

S  bulk substrate concentration, and
vmax and Km  the intrinsic kinetic parameters.
ηT can be calculated for constant S using the theory for heterogeneous reactions

Cell culture: Biomass balance
𝑑𝑀
𝑑𝑡
𝑉𝑑𝑥
= 𝐹𝑥𝑖 − 𝐹𝑥 + 𝜇. 𝑥. 𝑉 − 𝐾𝑑 . 𝑥. 𝑉 (5)
𝑑𝑡
At steady state, dx/dt = 0 𝜇. 𝑥. 𝑉 = 𝐹𝑥 ;since no biomass is fed into the system
𝝁=𝑫 (6)

Substrate balance
𝑑𝑀
𝑑𝑡
𝑉𝑑𝑆 𝜇. 𝑥. 𝑉 𝑞𝑃 . 𝑥. 𝑉
= 𝐹𝑆𝑖 − 𝐹𝑆 − − 𝑚𝑆 . 𝑥. 𝑉 − (7)
𝑑𝑡 𝑌𝑋 𝑌𝑃/𝑆
𝑆
At steady state, dS/dt =0,
𝜇. 𝑥. 𝑉 𝑞𝑃 . 𝑥. 𝑉
𝐹 𝑆𝑖 − 𝑆 = + 𝑚𝑆 . 𝑥. 𝑉 + (8)
𝑌𝑋 𝑌𝑃/𝑆
𝑆

𝜇 𝑞𝑃
𝐷 𝑆𝑖 − 𝑆 = 𝑥 ( + + 𝑚𝑆 ) (9)
𝑌𝑋 𝑌𝑃
𝑆 𝑆
𝐷(𝑆𝑖 − 𝑆)
𝑥= 𝜇 𝑞 (10)
( + 𝑃 + 𝑚𝑆 )
𝑌𝑋 𝑌𝑃
𝑆 𝑆
Ignoring maintenance and product formation/ directly linked to energy metabolism
𝒙 = 𝒀𝑿 (𝑺𝒊 − 𝑺) (11)
𝑺
Where steady state substrate concentration S is 𝐾𝑆 𝐷

𝑆= (12)
obtained from Monod expression 𝜇𝑚 − 𝐷

Product balance
𝑑𝑀
𝑑𝑡
𝑉𝑑𝑝
= 𝐹𝑝𝑖 − 𝐹𝑝 + 𝑞𝑃 . 𝑥. 𝑉 (13)
𝑑𝑡
At steady state dp/dt = 0,
𝑞𝑃 𝑥
𝑝 = 𝑝𝑖 + (14)
𝐷

Steady state conditions in a continuous culture
F
Dilution rate SS substrate Specific growth
S0 F X D= F/V, h-1 concentration, rate h-1
S g/L
D (at F) S µ
D1 (at F1) S1 µ1
Dilution rate
X S D2(at F2) S2 µ2
D = F/V
µ
D2 > D1 > D
S  residual/ steady state µ2 > µ1 > µ
substrate concentration
Volume = V X  steady state biomass concentration

Batch vs Fed batch vs continuous
Continuous
Steady state µ = D
µ2
µ2 𝝁𝒎𝒂𝒙 𝑺
𝝁= µ1
µ1 𝑲𝒔 + 𝑺
S1 S2 time
Effect of dilution rates on steady state concentrations
at low and high affinity constants (Monod constant)
At low Ks At high Ks


Biomass
Substrate
Biomass
Substrate
Dilution rate
Dilution rate
Lower the Ks higher is the affinity of biomass towards the substrate

Effect of increased substrate concentration Si on x and S

Dilution rate

Steady-state cell and substrate concentrations as a function of “D” in a chemostat
To avoid washout, D < μmax

Washout occurs at critical dilution rate Dcrit
From Eq 11
𝐾𝑆 𝐷
𝑥= 𝑌𝑋/𝑆 (𝑆𝑖 − )
𝜇𝑚 −𝐷
At D = Dcrit, x = 0
𝜇𝑚 𝑆𝑖
𝐷𝐶𝑟𝑖𝑡 = (15)
𝐾𝑆 + 𝑆𝑖
For most cell cultures KS << si;

therefore Dcrit ≈ μmax Cells washout
Optimal dilution rate, Dopt
 Near washout, the system is very sensitive to small changes in D, causing relatively greater
changes in “x” and “S”
So Dopt needs to be evaluated to operate the chemistat

at safe dilution rates
Volumetric biomass productivity Qx is given by
𝐹. 𝑥
𝑄𝑥 = = D. 𝑥 (16)
𝑉
𝐾𝑆 𝐷
Substituting 𝑥 = 𝑌𝑋/𝑆 (𝑆𝑖 − )
𝜇𝑚 −𝐷

𝐾𝑆 𝐷
𝑄𝑥 = D. 𝑌𝑋/𝑆 (𝑆𝑖 − ) (17)
𝜇𝑚 − 𝐷
𝒅𝑸𝒙
𝐴𝑡 𝐷 = 𝐷𝑜𝑝𝑡, =𝟎 (18)
𝒅𝑫
Differentiating equation w.r.t. D and equating to 0
𝑲𝑺
𝑫𝒐𝒑𝒕 = 𝝁𝒎 (𝟏 − )
𝑲𝑺 + 𝑺𝒊
Chemostat operated at Dopt gives maximum biomass productivity

Evaluation of kinetic and yield parameters in chemostat culture
In a chemostat under steady state

D=µ
𝜇𝑚 𝑆
𝐷= 1/D
𝐾𝑆 + 𝑆
Slope= Ks/µm
Rearranging the above equation
1/µm
𝟏 𝑲𝑺 𝟏 𝟏
Lineweaver-Burk plot = +
𝑫 𝝁𝒎 𝑺 𝝁𝒎 1/S

Chemostat is also used as a tool to calculate the true yields and maintenance coefficients
𝑟𝑋 𝜇
𝑌′𝑋/𝑆 = = 𝜇
𝑟𝑆 ( + 𝑚𝑆 )
𝑌𝑋/𝑆
1/Y’X/S
1 1 𝑚𝑆 Slope= ms
= +
𝑌′𝑋/𝑆 𝑌𝑋/𝑆 𝜇
Under steady state condition µ = D
1/YX/S
𝟏 𝟏 𝒎𝑺
= + 1/D
𝒀′𝑿/𝑺 𝒀𝑿/𝑺 𝑫

Choice of cultivation method - Batch vs Continuous

Factors to consider
A. Productivity
B. Genetic instability
C. Operability and reliability
D. Market economics

A) Productivity
Batch productivity
Total batch cycle time
𝑡𝑇 = 𝑡𝑏 + 𝑡𝑑𝑛 (1)
1 𝑥𝑚
𝑡𝑇 = ln + 𝑡𝑑𝑛 (2)
𝜇𝑚 𝑥0
Batch biomass productivity is given by
𝑑𝑥 𝑥𝑚 − 𝑥0 𝑌𝑋/𝑆 𝑆0
𝑟𝑥,𝑏 = = = (3)
𝑑𝑡 𝑡𝑇 1 𝑥
( ln 𝑚 + 𝑡𝑑𝑛 )
𝜇𝑚 𝑥0

Continuous productivity
𝑟𝑥,𝑐 = 𝐷𝑜𝑝𝑡 𝑥𝑜𝑝𝑡 (4)
𝐾𝑠
𝐷𝑜𝑝𝑡 = 𝜇𝑚 1− (5)
𝑆0 + 𝐾𝑆
𝐾𝑆 𝐷𝑜𝑝𝑡
𝑥𝑜𝑝𝑡 = 𝑌𝑋 𝑆0 − 𝑆 = 𝑌𝑋 (𝑆0 − ) (6)
𝑆 𝑆 𝜇𝑚 − 𝐷𝑜𝑝𝑡
Substituting for Dopt and rearranging
𝑥𝑜𝑝𝑡 = 𝑌𝑋/𝑆 {𝑆0 + 𝐾𝑆 − 𝐾𝑆 𝑆0 + 𝐾𝑆 } (7)

Substituting (5) and (7) in (4)
𝐾𝑆
𝐷𝑜𝑝𝑡 𝑥𝑜𝑝𝑡 = 𝑌𝑋/𝑆 𝜇𝑚 1− [𝑆0 + 𝐾𝑆 − 𝐾𝑆 𝑆0 + 𝐾𝑆 ] (8)
𝑆0 + 𝐾𝑆
Under normal circumstances S0 >> KS,
𝑟𝑥,𝐶 = 𝐷𝑜𝑝𝑡 𝑥𝑜𝑝𝑡 = 𝜇𝑚 𝑌𝑋/𝑆 𝑆0 (9)

Ratio of continuous to batch productivities
𝑟𝑥,𝑐 𝑥𝑚
= 𝑙𝑛 + 𝜇𝑚 𝑡𝑑𝑛 (10)
𝑟𝑥,𝑏 𝑥0
Most commercial fermentations will have xm/x0 ≈10-20.
For example, an E.coli with xm/x0 =20, tdn = 5 h and µm = 1 h-1
we get
rx,c/rx,b = 8

B) Genetic instability
Chemostat
 Chemostat imposes strong selection pressure for the most rapidly growing cell
 Back mutation of productive stains to less productive strains possible
 Less productive mutant dominates in the chemostat, decreasing productivity
Batch
 Batch culture- number of generations (< 25) available for the revertant cell to outgrow the
most productive strain is limited

C) Operability and reliability
Batch
Batch to batch variability in case of batch process
Chemostat
Continuous process may suffer from breakage in pumps, failure in controllers, and so on.
Maintenance of sterility for long time is very difficult

D) Market economics
Dedicated bioreactor required for continuous production, whereas, the single system can be
used for scheduling the production of more than one products per year

Problem #15
A 5 m3 fermenter is operated continuously with feed substrate concentration 20 kg m-3. The

microorganism cultivated in the reactor has the following characteristics µmax = 0.45 h -1, Ks
=0.8 kg m-3, Yxs =0.55 kg kg-1.
(a) What feed flow rate is required to achieve 90% substrate conversion?
(b) How does the biomass productivity at 90% substrate conversion compare with the
maximum possible?

Problem #16: Batch and continuous biomass production
Pseudomonas methylotrophus is used to produce single-cell protein from methanol in a 1000

m 3 pressure-cycle airlift fermenter. The biomass yield from substrate is 0.41 g g- 1,
KS is 0.7 mg l- 1, and the maximum specific growth rate is 0.44 h- 1. The medium contains
4% (w/v) methanol. A substrate conversion of 98% is desirable. The reactor may be operated
either in batch or continuous mode. If operated in batch, an inoculum of 0.01% (w/v) is used
and the downtime between batches is 20 h. If operated continuously, a downtime of 25 d is
expected per year. Neglecting maintenance requirements, compare the annual biomass
production from batch and continuous reactors.

 Batch
 Fed-batch
 Continuous
 Two-stage chemostat
 Single stage chemostat with recycle

Two staged-chemostat
Application 1: Secondary metabolite production
 Beneficial for products (secondary metabolite) requiring distinct optimal processing conditions
for growth and production
Lactose medium
Glucose medium
T = 30°C Growth Production T = 33°C

pH = 7.0 pH = 6.5
Agitation = 250 rpm Agitation = 200 rpm
Reactor 1 Reactor 2
Application 2: Recombinant protein production
 During recombinant protein production, in the un-induced state, plasmid-containing cells

and plasmid-free cells grow at the same rate.
 However, during induction, plasmid-free cells have growth advantage over plasmid-
containing cells
 Thus, single-staged chemostat not suitable for rec-protein production because of problems
resulting from genetic stability
 In a two-stage chemostat, antibiotic is added (no inducer added) in the first stage to maintain
dominant population of plasmid-containing cells.

 In the second stage, inducer is added to induce the plasmid to express the protein
 Cells defective in product synthesis will not overtake the culture, as fresh genetically unaltered
cells added from stage 1 to stage 2 continuously
Inducer
Antibiotic
Glucose medium
Growth Protein
expression

First stage
Substrate balance
𝐾𝑆 𝐷1
𝑆1 = (1)
𝜇𝑚 − 𝐷1
Biomass balance
𝑥1 = 𝑌𝑋/𝑆 (𝑆0 − 𝑆1 ) (2)

Second stage
Biomass balance
𝑑𝑥2
𝐹𝑥1 − 𝐹𝑥2 + 𝜇2 𝑥2 𝑉2 = 𝑉2 (3)
𝑑𝑡
𝑑𝑥2
At steady state =0
𝑑𝑡
𝑥1
𝜇2 = 𝐷2 1 − (4)
𝑥2

Substrate balance
𝜇2 𝑥2 𝑉2 𝑑𝑠2
𝐹𝑆1 − 𝐹𝑆2 − = 𝑉2 (5)
𝑌𝑋/𝑆 𝑑𝑡
𝑑𝑆2
At steady state =0
𝑑𝑡
𝜇2 𝑥2
𝑆2 = 𝑆1 − (6)
𝐷2 𝑌𝑋/𝑆
where
𝐹 𝜇𝑚 𝑆2
𝐷2 = 𝑎𝑛𝑑 𝜇2 =
𝑉2 𝐾𝑆 + 𝑆2
Equations (4) and (6) can be solved simultaneously for x2 and S2

 Batch
 Fed-batch
 Continuous
 Two-stage chemostat
 Single stage chemostat with recycle

Chemostat with recycle

Chemostat with recycle
 In chemostat, at D > µ washout occurs, reducing the productivity
 Microbial growth  autocatalytic  rate of conversion increases with cell concentration
 Cell recycle increases the productivity and stability of the

chemostat (e.g waste water treatment plants by reducing the
effects of process perturbation
 Cells in the effluent stream centrifuged/filtered/settled in a

conical tank for recycling

F, x0, S0 where
(1+α) F, x1, S α  recycle ratio based on volumetric flow rates,
C concentration factor or ratio of cell concentration
αF, S
in the cell recycle stream to the cell concentration in
the reactor effluent,
F  nutrient flow rate, V is culture volume,
V F, x2
x0 and x1 are cell concentrations in feed and recycle
Cell streams,
separator
x2 is cell concentration in effluent from the cell
Recycle stream with cell concentrate
Cx1 separator.

Biomass balance
0
F, x0, S0 0 𝑑𝑥
𝐹𝑥0 + 𝛼. 𝐹. 𝐶. 𝑥1 − 1 + 𝛼 𝐹. 𝑥1 + 𝑉. 𝜇. 𝑥1 = 𝑉
(1+α) F, x1, S 𝑑𝑡
𝑑𝑥 (1)
αF At steady state = 0;
𝑑𝑡
𝑥0 = 0, sterile feed
Since D = F/V
V F, x2
𝜇 = 1 + 𝛼 − 𝛼𝐶 𝐷 = 1 + 𝛼 1 − 𝐶 𝐷
(2)
Cell
separator
Since C > 1 and α (1-C) < 0, then µ < D
Recycle stream with cell concentrate Thus with cell recycle, chemostat can be operated
Cx1 at dilution rates higher than the specific growth rates

Substrate balance
F, x0, S0 𝜇. 𝑉. 𝑥1 𝑑𝑆
𝐹𝑆0 + 𝛼𝐹. 𝑆 − − 1 + 𝛼 𝐹. 𝑆 = 𝑉 (3)
(1+α) F, x1, S 𝑌𝑋 𝑑𝑡
𝑆
𝑑𝑆
αF At steady state =0
𝑑𝑡
𝐷
𝑥1 = 𝑌𝑋 (𝑆0 − 𝑆) (4)
𝜇 𝑆
V F, x2 Substituting for µ from eq (2)
𝑌𝑋/𝑆 (𝑆0 − 𝑆)
Cell 𝑥1 = (5)
(1 + 𝛼 − 𝛼𝐶)
separator
Recycle stream with cell concentrate Thus steady state biomass concentration
Cx1 is increased by the factor 1/(1+ α-αC)
From eq (2) and from monod expression in terms of substrate concentration, the steady state
substrate concentration is given by
Effluent cell concentrations and productivities
𝐾𝑆 𝐷 (1 + 𝛼 − 𝛼𝐶) in a chemostat with and without cell recycle
𝑆= (6)
𝜇𝑚 − 𝐷(1 + 𝛼 − 𝛼𝐶)
With recycle
 Cell concentration and productivities are higher Without recycle

with cell recycle  higher rates of substrate consumption
 Application in wastewater treatment and ethanol production
R biomass output rate (g/L/h)

Problem #18: Two stage- chemostat
In a two-stage chemostat system, the volumes of the first and second reactors are V1 = 500 l and
V2 = 300 l, respectively. The first reactor is used for biomass production and the second is
for a secondary metabolite formation. The feed flow rate to the first reactor is F = 100 l/h, and the
glucose concentration in the feed is S = 5.0 g/l. Use the following constants for the cells.
µm = 0.3 h-1, KS = 0.1 g/l, YX/S = 0.4 g dw cells/g substrate
a. Determine cell and glucose concentrations in the effluent of the first stage.
b. Assume that growth is negligible in the second stage and the specific rate of product formation
is qP = 0.02 g P/g cell h, and YP/S = 0.6 g P/g S. Determine the product and substrate
concentrations in the effluent of the second reactor.

Problem #16: Chemostat with recycle
In a chemostat with cell recycle, the feed flow rate and

culture volumes are F = 100 ml/h and V = 1000 ml,
respectively. The system is operated under glucose
limitation,
and the yield coefficient, YX/S, is 0.5 gdw cells/g substrate.
Glucose concentration in the feed is S0 = 10 g glucose/l. The
kinetic constants of the organisms are µm = 0.2 h-1,
Ks = 1 g glucose/l. The value of C is 1.5, and the recycle
ratio is α = 0.7. The system is at steady state.
a. Find the substrate concentration in the recycle stream (S).
b. Find the specific growth rate (µ)of the organisms.
c. Find the cell (biomass) concentration in the recycle stream.
d. Find the cell concentration in the centrifuge effluent x2).
Problem #17: Single stage- chemostat vs Chemostat with recycle
Consider a 1000 L CSTR in which biomass is being produced with glucose as the substrate.
The microbial system follows a Monod relationship with µm = 0.4 h-1, KS = 1.5 g/l (an unusu-
ally high value), and the yield factor YX/S = 0.5 g biomass/g substrate consumed. If normal
operation is with a sterile feed containing 10 g/l glucose at a rate of 100 l/h:
a. What is the specific biomass production rate (g/l-h) at steady state?
b. If recycle is used with a recycle stream of 10 l/h and a recycle biomass concentration five
times as large as that in the reactor exit, what would be the new specific biomass
production rate?
c. Explain any difference between the values found in parts a and b.
Lecture No. Learning Objectives Topics to be covered
 Overview of the course
Introduction to Biochemical  What is a Biochemical Engineer?
1–3
Engineering  Cellular metabolism
 Biological basics: Cells
 Energy source, carbon sources, nitrogen sources and
other minor and trace nutrients
Formulation of growth medium,
4-6  Medium optimization
inoculum
 Criteria for development of inocula
 Development of inoculum for bacterial processes
 Elemental balances
Stoichiometry of microbial growth  Electron balances
7-11
and product formation  Degree of reduction, Theoretical oxygen demand
 Theoretical prediction of Yield coefficients
Kinetics of substrate utilization,  Ideal reactors (batch, CSTR)

12-17 product formation and biomass  Growth kinetics (balanced, transient)
production in cell cultures  Kinetic models
 Gas-liquid mass transfer in cellular systems
 Determination of oxygen transfer rates
Transport phenomena in  Determination of volumetric oxygen transfer
18-24
bioprocess systems coefficient
 Power consumption for sparged and agitated vessels
 Heat transfer correlations
 Ideal bioreactors
Design and analysis of  Sterilization reactors
25-30
bioreactors  Multiphase bioreactors: Packed-bed, bubble
column, fluidized bed, trickle bed reactors
 Scale-up criteria
Optimization and Scale up of
31-33  Challenges in scale-up
stirred tank bioreactors
 Scale-down models
 Simple enzyme kinetics with single substrate
 Determining rate parameters for Michaelis-Menton
The kinetics of enzyme
34-40 type kinetics
catalyzed reactions
 Immobilized enzyme kinetics for zero and first order
systems
VIVEK R
Bioreactors classification
Based on
1. The presence or absence of oxygen 4. Microbial agent used
and requirement of stirring - living cells
- stirred/non stirred - enzymes
- aerated/un-aerated 5. Process requirements
2. The mode of operation - Aerobic
- batch -Anaerobic
- fed-batch/ continuous -Solid state
3. The method of growing microbes - Immobilized
-suspended
-immobilized

Packed bed and fluidized bed bioreactors

Fluid recirculation rates
 High liquid recirculation rates  for immobilized animal-cell fluidized bed reactors
 approaches CFSTR
 Low recirculation rates  for waste water treatment requiring longer hydraulic retention time
(slow biochemical process)  approaches plug flow model

To analyze such a system, consider a material balance on the rate- limiting substrate over a
differential element
−𝐹𝑑𝑆 = 𝑁𝑆 . 𝑎. 𝐴 . 𝑑𝑧 (1)
Where
S  bulk substrate concentration (mg S cm-3) and is a function of height
F  nutrient flow rate (cm3 h-1)
Ns  flux of substrate into the biofilm (mg S cm2 h-1)
a  biofilm/support particle surface area per unit reactor volume (cm2 cm-3)
dz  differential height of an element of the column (cm)
A cross sectional area of the bed (cm2)

In the presence of diffusion limitation, the rate of substrate consumption or flux is
expressed in terms of the effectiveness factor
𝑟𝑚 𝑆
𝑁𝑆 = η 𝐿 (2)
𝐾𝑆 + 𝑆
Where
rm  maximum rate of substrate consumption (mg S cm3 h-1)
L  biofilm thickness or characteristic length of the support particle (L = Vp/Ap)
Substituting (2) in (1) and integrating

𝑆𝑓 𝐾𝑆 +𝑆 𝐻
−𝐹 𝑆0 𝑟𝑚 𝑆
𝑑𝑆 = η. 𝑎. 𝐴 0
𝑑𝑧 (3)
𝑆0 η. 𝑟𝑚 . 𝐿. 𝑎. 𝐴. 𝐻
𝐾𝑆 𝑙𝑛 + 𝑆0 − 𝑆𝑓 = (4)
𝑆𝑓 𝐹
H  total height of the packed bed

For low substrate concentrations in the feed, the rate of substrate consumption is first order and eq.
(4) has the following form:
𝑆0 η. 𝑟𝑚 . 𝐿. 𝑎. 𝐴. 𝐻
𝐾𝑆 𝑙𝑛 = (5)
𝑆𝑓 𝐹

Problem #21: Ethanol by immobilized S. cerevisiae cells in packed bed
Glucose is converted to ethanol by immobilized S. cerevisiae cells entrapped in Ca-alginate
beads in a packed column. The specific rate of ethanol production is qP = 0.2 g ethanol/ g cell/ h,
and the average dry-weight cell concentration in the bed is x = 25 g/l bed. Assume
that growth is negligible (i.e., almost all glucose is converted to ethanol) and the bead size is
sufficiently small that η = 1. The feed flow rate is F = 400 l/h, and glucose concentration in
the feed is S0 = 100 g glucose/l. The diameter of the column is 1 m, and the product yield
coefficient is YP/S = 0.49 g ethanol/g glucose.
a. Write a material balance on the glucose concentration over a differential height of the column
and integrate it to determine S = S(z) at steady state.
b. Determine the column height for 98% glucose conversion at the exit of the column.
c. Determine the ethanol concentration in the effluent. VIVEK R BITS Pilani, K K Birla Goa Campus

Analysis of Various Modes of Stirred Tank Bioreactor Operations

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Analysis of various modes of Stirred tank bioreactor operations

VIVEK R BITS Pilani, K K Birla Goa Campus

/ also called log/exponential phase Continuous

Primary metabolites  To prolong exponential phase/

VIVEK R BITS Pilani, K K Birla Goa Campus

 Continuous input and output

VIVEK R BITS Pilani, K K Birla Goa Campus

VIVEK R BITS Pilani, K K Birla Goa Campus

The curves were obtained using the

To avoid washout D < μmax

Steady state biomass concentration

Steady state substrate concentration

Steady state substrate concentration

Lower the Ks higher is the affinity of biomass towards the substrate

VIVEK R BITS Pilani, K K Birla Goa Campus

Steady state substrate concentration

VIVEK R BITS Pilani, K K Birla Goa Campus

where ηT total effectiveness factor,

VIVEK R BITS Pilani, K K Birla Goa Campus

At steady state, dx/dt = 0 𝜇. 𝑥. 𝑉 = 𝐹𝑥 ;since no biomass is fed into the system

VIVEK R BITS Pilani, K K Birla Goa Campus

At steady state, dS/dt =0,

VIVEK R BITS Pilani, K K Birla Goa Campus

Where steady state substrate concentration S is 𝐾𝑆 𝐷

VIVEK R BITS Pilani, K K Birla Goa Campus

At steady state dp/dt = 0,

VIVEK R BITS Pilani, K K Birla Goa Campus

VIVEK R BITS Pilani, K K Birla Goa Campus

Steady state biomass concentration

Steady state substrate concentration

Steady state substrate concentration

Lower the Ks higher is the affinity of biomass towards the substrate

VIVEK R BITS Pilani, K K Birla Goa Campus

Steady state substrate concentration

VIVEK R BITS Pilani, K K Birla Goa Campus

To avoid washout, D < μmax

For most cell cultures KS << si;

So Dopt needs to be evaluated to operate the chemistat

Volumetric biomass productivity Qx is given by

VIVEK R BITS Pilani, K K Birla Goa Campus

Differentiating equation w.r.t. D and equating to 0

Chemostat operated at Dopt gives maximum biomass productivity

VIVEK R BITS Pilani, K K Birla Goa Campus

In a chemostat under steady state

VIVEK R BITS Pilani, K K Birla Goa Campus

VIVEK R BITS Pilani, K K Birla Goa Campus

VIVEK R BITS Pilani, K K Birla Goa Campus

VIVEK R BITS Pilani, K K Birla Goa Campus

Batch biomass productivity is given by

VIVEK R BITS Pilani, K K Birla Goa Campus

𝑟𝑥,𝑐 = 𝐷𝑜𝑝𝑡 𝑥𝑜𝑝𝑡 (4)

VIVEK R BITS Pilani, K K Birla Goa Campus

Under normal circumstances S0 >> KS,

𝑟𝑥,𝐶 = 𝐷𝑜𝑝𝑡 𝑥𝑜𝑝𝑡 = 𝜇𝑚 𝑌𝑋/𝑆 𝑆0 (9)

VIVEK R BITS Pilani, K K Birla Goa Campus

Most commercial fermentations will have xm/x0 ≈10-20.

For example, an E.coli with xm/x0 =20, tdn = 5 h and µm = 1 h-1

VIVEK R BITS Pilani, K K Birla Goa Campus

VIVEK R BITS Pilani, K K Birla Goa Campus

VIVEK R BITS Pilani, K K Birla Goa Campus

VIVEK R BITS Pilani, K K Birla Goa Campus

A 5 m3 fermenter is operated continuously with feed substrate concentration 20 kg m-3. The

VIVEK R BITS Pilani, K K Birla Goa Campus