Biochemical Engineering

8CH01

Dr. Sudhir Ranganath

Contact Hours/Week: 3 (Lecture) Credits: 3.0

CIE Marks: 50
Total Lecture Hours: 39 SEE Marks: 50
UNIT IV
KINETICS OF CELL GROWTH & PRODUCT FORMATION

Major Cellular metabolic pathways

Growth kinetics in batch culture (Monod)

Substrate-limited growth kinetics

Kinetic models with growth inhibitors

Transient cell growth kinetics

Cell growth in continuous culture

Models for Transient Behavior
In reality, environmental & culture conditions vary with time
Changes in cellular composition & biosynthetic capabilities
Changes are over an observable period of time & not instantaneous

Chemically structured models

Models capture important kinetic interactions b/w cellular subcomponents
Need at least three components appear necessary
20 to 40 components (sophisticated models)
Chemically Structured Models
Understanding of the physical system for making appropriate assumptions
Two important guidelines for writing such models
All reactions expressed in terms of intrinsic concentrations
Dilution of intrinsic concentration by growth be considered

Intrinsic concentration: Amount of compound per unit cell mass or cell

volume
Extrinsic concentration: Amount of compound per unit reactor volume

In a non-flow reactor, we use

! " # $%
= () * x ,-. …(1)
!&
rate of change in total biomass rate of formation of i per unit
amount of i in the reactor in reactor. biomass based on intrinsic
concentrations

where, VR is the total reactor volume

X is the extrinsic biomass concentrations
Ci is the extrinsic concentration of component i
Chemically Structured Models
Eq (1) can be written in terms of intrinsic concentrations using mass fractions
(Ci/X):
! #$ ⁄% ( !#$ !%
= − *+
!& % !& !&

, *+ ⁄- 1 ,*+ ,-⁄,.
= − *+ ⁄-
,. - ,. -

( !% ! #$ ⁄% ( !#$
since, 0 = we get = − 0 *+ ⁄-
% !& !& % !&

assuming VR is a constant, and dividing Eq (1) by VRX and

( !#$ 1 2 3 ⁄4 89:5 23
substituting for
% !&
, we get
15
= 673 − 4

Rate of formation of
component i (intrinsic Dilution by
Rate of change
concentration) growth
of component i
Cell Growth in Continuous Culture
Batch growth
Culture conditions change with time
Termination of growth, P formation & S utilization after time “ t “

Characteristics of continuous culture

Well stirred culture with continuous supply of fresh nutrient medium
Continuous withdrawal of products & cells
Prolonged growth & product formation
Reaches steady state: X, [P] and [S] remain constant
Constant environment for growth & product formation
Provides uniform-quality product

Devices for continuous culture

Chemostat
Turbidostat
Plug flow reactor (PFR)
Devices for Continuous Culture of Cells
Chemostat Turbidostat
One limiting nutrient X is maintained constant by controlling
Steady state OD of the culture & feed flow rate
Constant chemical Constant volume is maintained
environment Useful for selection of subpopulations
able to withstand a desired stress

Plug Flow Reactor (PFR)

No axial mixing, hence active cells in fluid elements can’t inoculate other
fluid elements
So, liquid recycle is required for continuous inoculation of nutrient media
X, [P] and [S] vary with axial position
Ideal Chemostat
It is a perfectly mixed continuous-flow, stirred-tank reactor (CFSTR).
Most chemostats require some control elements, such as pH and DO
control units, to be useful.
Fresh sterile medium is fed to the completely mixed and aerated reactor,
and cell suspension is removed at the same rate.
Liquid volume in the reactor is kept constant.
A critical feature of a chemostat is that a steady state arises spontaneously
where the growth rate is determined by the feed flow rate to per unit of
reactor volume.
A material balance on the cell
concentration around the chemostat
yields:
-"
!"# − !" + &' () " − &' *+ " = &'
-.
….. (1)

Where,
! is the flow rate of nutrient solution
in L/h
Ideal Chemostat
!" is the culture volume in L, # is the cell concentration in g/L, $% is growth rate
and &' is endogenous (or death) rate constants, respectively in h-1.
'(
The previous equation is rearranged as: = +#, + $% − &' − + # … (2)
')

Where, + is the dilution rate given by: + = //!" and + is the reciprocal of
hydraulic residence time (!//).

Since the feed medium is sterile, #, = 0, and if the endogenous metabolism or

death rate is negligible compared to the growth rate (&' ≪ $% ) and if the
'(
system is at steady state ( = 0), then we get the following:
')

$% = + (if &' = 0) ….. (3)

In a chemostat, cells are removed at a rate equal to their growth rate, and the
growth rate of cells is equal to the dilution rate.

This property allows the manipulation of growth rate as an independent

parameter, hence chemostat is a powerful experimental tool.
Ideal Chemostat
Since growth rate is limited by at least one substrate in a chemostat, a simple
description of chemostat performance can be made by substituting the Monod
equation for growth rate in equation (3) becomes:

%& '
!" = $ = () *'
…… (4)

Here, + is the steady-state limiting substrate concentration in g/L.

If $ is set at a value greater than !, , the culture cannot reproduce quickly

enough to maintain itself and is washed out (ie., no substrate is used, + = +-
and no biomass is formed, . = 0. A plot of 1⁄!" versus 1⁄+ can be used to
estimate values for !, and 23 .

Using equation (4), we can relate effluent substrate concentration to dilution

rate for $ < !, :

() 5
+= …… (5)
%& 65
Ideal Chemostat
A material balance on the limiting substrate yields the following:

0
!"# − !" − %& '( ) 1⁄,-// − %& 12 1⁄,2// − 3/ )%& = %& (6"⁄67) …… (6)

Here, "# and " are feed and effluent substrate concentrations in g/L, 12 is the
specific rate of extracellular product formation in g P/g X-h.

0
,-// and ,2// are maximum yield coefficient and yield coefficient (g X/g S and g
P/g S), and 3/ is the maintenance coefficient based on substrate (g S/g X-h).

9(:; <:) BC @G
Eq (6) can be written as: = @A = + + HA ……. (7)
=> ? DE
?/A DG/A

Eq (7) is the linear equation of substrate consumption.

Ideal Chemostat
Case 1: Extracellular product formation and maintenance are negligible:
System is at steady state (dS/dt = 0) and !" and #$ are negligible, then
equation (7) becomes:

*+ ,
% &' − & = 1 ……. (8)
-./0
Since 23 = % at steady state, if 45 = 0, then we have the following:

9
7 = 8,/$ (&' − &) ……. (9)

Using equation (5), the steady-state cell concentration can be expressed as

follows:

9 <0 =
7= 8,/$ &' − …….. (10)
*> ?=

In equations (8) and (9), the yield coefficient 8,/$ is assumed to be constant
9
and equal to 8,/$ , which is an approximation, since endogenous metabolism
has been neglected.
Ideal Chemostat
Case 2: The presence of endogenous metabolism and death rate: Usually,
!"/$ varies with the limiting nutrient and growth rate.

When endogenous metabolism or death rate are not negligible, Eq (3) takes
the following form:

% = '( − *+ = ',-. ……. (11) or '( = % + *+ ……. (12)

By substituting equation (12) into the steady-state substrate balance, assuming

no extracellular product formation (Eq (8)), we find the following:

2345 "
% 01 − 0 − = 0 ……. (13)
68:⁄9

<
!"/$ is constant and independent of growth rate.

Equation (13) can be rearranged as follows:

=> ?= 2345
% − = 0 …….. (14)
" 68:⁄9
Ideal Chemostat
" * ,-
! − − = 0 …….. (15)
#%'(
⁄& #%+⁄& #%+⁄&

" ,- " " " 2&

+ + = …….. (16) or = + * …….. (17)
#%+⁄& #%⁄& .* #%'(
⁄& #%'(
⁄& #%+⁄&

Where, 34 = 56 ⁄789⁄4 ……. (18) and 34 is the maintenance coefficient based

on substrate S.

78:;
⁄4 is the apparent yield.

When 78/4 is written, it should be interpreted as 78:;

⁄4 .

While 789⁄4 is a constant, 78:;

⁄4 varies with growth conditions if 56 > 0.
Ideal Chemostat
Values of !#%⁄$ and &$ can be obtained from chemostat experiments by plotting
1⁄!#() %
⁄$ against 1/D. The slope is &$ and the intercept is 1⁄!#⁄$ .
Ideal Chemostat
&' (
Here, we have: !"#$ = (*+ ,()
− /0 , we can show the following:

*+ (2,34 ) 2
1= …… (18) and 6 = 79;⁄: 1< − 1 . ………. (19)
&' 52534 2,34

Case 3: Extracellular product formation:

In addition to the effects of maintenance, it is often appropriate to consider the
conversion of extracellular substrate into extracellular product.

We assume instantaneous conversion of substrate to extracellular product,

with the cell acting as a catalyst. The balance on product formation is:

>? = @A 6 ………. (20)

Where, @A is the specific rate of product formation, which is a constant (B) for
non-growth associated product formation and is a function of !C for growth-
associated product formation.
Ideal Chemostat
Therefore, in the presence of product formation and with negligible
maintenance, Eq (7) becomes:

& &
! "# − " = ! + -. / + 1 / ……….. (21)
+
'(/* '0/* 2

The cell mass balance is unchanged from the case with endogenous
metabolism (Equation 18) and gives:

34 (! + -. )
"=
78 − ! − -.

; <
Eq (21) can be solved for / as: / = 9:/4 ("# − ") …… (22)
B+
(/*
<=>? =@A B
0/*

Productivity of a chemostat for product is calculated using !C

Productivity of a chemostat for cell mass is calculated using !/

Ideal Chemostat
The dilution rate that maximizes productivity is found by differentiating !" or
!# with respect to ! and setting the derivative equal to zero.

The optimal value of ! which is !$%& will depend on whether endogenous

metabolism and/or product formation are considered.

When '( = 0 and *% = 0, Eqs (8) and (10) apply.

/0
Then !$%& for cell mass production becomes: !$%& = +, 1 − …. (23)
/0 123

Since 45 ≫ 78 , !$%& will approach ! = +, which is the washout point.

Stable chemostat operation with the above condition is very difficult unless the
flow rate and liquid volume can be maintained exactly constant.

Hence, a value of ! < !$%& may be a good compromise between stability and
cell mass production.
Thank you