PPT 3: CELL KINETICS d Cn

rn = =μ C n
Growth Kinetics dt
Where:
 Important for the design and operation of
fermentation system r n – growth rate of cells on a number basis
 Deals with the rate of cell growth C n – cell number density, number of cells per unit
 How it is affected by various conditions volume
μ – specific growth rate [=] hr-1
Various Models for Cell Kinetics
Cell Components
Population 3.2 Specific Growth Rate
Unstructured Structured
Cells are represented by a Multiple cell 1 d C n d ln C n
single component, which components, uniformly μ= =
Distributed is uniformly distributed distributed throughout the
C n dt dt
throughout the culture. culture and interact with If μ is constant with time,
each other. C n
dCn t
Segregated
Cells are represented by a Cells are composed of
single component, but multiple components and ∫ C =∫ μ dt
C n t
they form a heterogenous from a heterogenous n0 0

mixture. mixture. C n=C n 0 exp [ μ ( t−t 0 ) ]

Growth Cycle for Batch Cultivation 3.3 Doubling Time
 Lag phase – a period of time when the change of cell
ln 2
number is zero. t d=
 Accelerated growth phase – the cell number start to μ
increase and the division rate increases to reach a
maximum. 3.4 Factors affecting Specific Growth rate
 Exponential growth phase – the cell number increases  Substrate Concentration
exponentially as the cells start to divide. o Monod equation – one of the most widely
 Decelerated growth phase – after the growth rare employed expression for the effect of
reach a maximum, it is followed by the deceleration substrate concentration on μ
of both growth rate and the division rate. μmax C s
 Stationary phase – the cell population will reach a μ=
K s+C s
maximum value and will not increase further.
Where:
 Death phase – after nutrient depletion, cells will start
to die and the number of viable cells will decrease. C s – limiting substrate concentration in the medium
K s – system coefficient
1. Lag Phase  Product Concentration

( )( )
 The initial stationary or latent phase. Cs Kp
 The cells may grow in size even though the cell μ=μ max
number does not increase. K s +C s K p +C p

( )( )
 Factors affecting lag phase: Cs Cp n
o Type and age of the microorganisms μ=μ max 1−
o Size of the inoculum K s +C s Cp m

o Culture conditions Where:

C pm – maximum product concentration above which
2. Accelerated Growth Phase cells cannot grow
 Growth begins and division rate increases gradually
and reaches a maximum value in the exponential 4. Stationary Phase
period.  The cell growth rate has leveled off and become
constant.
3. Exponential Growth Phase
 The number of cells multiplying equals the number
 The progressive doubling of cell number results in a of cells dying.
continually increasing rate of growth in the microbial
 Cells in this phase have different chemical
population.
composition from those cells in the exponential
 Mimics a first0order autocatalytic chemical reaction phase.
of balanced growth.
5. Death Phase
3.1 Growth rate
 Occurs due to the depletion of cellular reverse energy
 The rate of the cell population increase at any or the accumulation of toxic products.
particular time.
 Death is also an exponential function.
 ( )
K Y
s X/S x s X/S s C K Y C
( t−t 0 ) μmax = C +C Y +1 ln C + C +C Y ln C 0

x s
0 X/S x x
0 s X/S s 0 0 0

2. Ideal Continuous Stirred-tank Fermenter

 Chemostat – flow rate is set at a particular value;
easier to operate.
 Turbidostat – turbidity is set at a constant level;
recommended for continuous fermentation at high
dilution rates.

2.1 Material Balance

d Cx
F C x −F C x +V r x =V
i
dt

Stirred-tank Fermenter At steady state,

V C x −C x
τ m= = i

F rx

If the input stream is sterile (C x i =0 ) and the cells are

growing exponentially (r x =μ C x ),
1 1
τ m= =
μ D
If growth rate can be expressed by Monod equation,
1 μmax C s
D=μ= =
τ K s+C s
1. Batch or Plug-flow Fermenter Given the residence time,
Change of the cell concentration Ks
d Cx C s=
=r x =μ C x τ m μmax −1
dt
Integrating
Cx Cx t
Note: valid only when τ m μmax >1.
d Cx d Cx
∫ =∫
r x C μ Cx t
=∫ dt =t−t 0 If the growth yield is constant,
Cx 0

( Ks
)
x0 0

C x =Y X /S C si −
Note: the performance equation only applies when r x τ m μ max−1
is larger than zero. Thus, t 0 is the time at the

( Ks
)
beginning of the accelerated growth phase.
C p=C p +Y X /S C si−
1.1 Autocatalytic Reactions
i
τ m μ max −1
S+ X → X + X

The rate for an autocatalytic reaction is slow at the

start.
Cx
( K s +C s ) d C X t

∫ μ max C s C x
=∫ dt
Cx 0 t0
1.2 Growth Yield
∆ Cx C x −C x0
Y X / S= =
−∆ Cs −( C s−Cs 0 )

1.3 Changes in cell concentration with respect to time

 PPT 4: FERMENTERS 1 CX
t c = ln +t (6) m

Cell Culture μ CX l 0

 Batch Culture Where:

o No addition or removal t l – lag + harvesting + preparation; varies with size of
o Simple and widely used equipment and nature of the fermentation; normally 3-10
 Fed-batch Culture hours.
o Addition but no removal
 Continuous Culture
o Addition and removal 1.5 Productivity for Cell Mass
C X −C X Y C Sf
1. Cell Growth in Batch Culture Productivity= m 0
=
tc tc

Substituting equation 6 to the equation above,

Y X / S C Sf
Productivity= (7)
1 CX
ln m
+t
μ CX l 0

2. Continuous Operation (Chemostat)

1.1 Exponential Growth Phase

d Cx
=μ C x (1)
dt
Where C x =C x0 at t=0
Cx
ln =μt (2)
Cx0

C x =C x e μt (3) Where:
0
F(L/h) – flow rate
V (L) – working volume
1.2 Doubling Time
μt X (g/L) – cell concentration
C x =C x e 0
S (g/L) – substrate concentration
μτ
2 C x =C x e d

0 0
2.1 Mass Balances
ln 2=μ τ d Acc=¿−Out +Gen−Con
ln 2 0.693
τ d= = (4)
μ μ  Cell
d ( XV )
1.3 Exponential Period =0− XF+ μXV −0(1)
dt
From equation 2,  Substrate
CX d ( SV ) 1
=S F F−SF +0− μXV (2)
ln m
=μ t e dt Y
CX 0

1 CX At steady state,
t e = ln (5) m

0=−XF+μXV (3)
μ CX
1
0

Where: 0=( S F−S ) F− μXV (4 )

C X – maximum cell concentration Y
m
Equation 3 becomes,
t e – exponential period
F
μ= =D(5)
1.4 Batch Cycle Time (t c ) V
Equation 4 becomes,
t c =t e +t l Y F
X=
μ
( S F −S ) (6)
V
 Substituting equation 5 to equation 4, Productivit y chemo Y μm S
X =Y ( S F −S ) ( 7) = F

Productivit y batch Y SF
1 Xm
ln +t
2.2 Concentrations at Steady-state μb X0 l
Monod equation,

μ ( ¿ D )=
μm S
Ks+ S
(8)
Productivit y chemo
Productivit y batch
=μm
1
(ln
Xm
μb X 0 l
+t (16)
)
DKS In most commercial fermentations,
S= (9)
μm −D Xm Xm
Substituting equation 9 to equation 7, ≈ 10−20 , ln ≈ 2.3−3.0
X0 X0
(
X =Y S F −
D KS
μm −D )
(10)
Continuous systems have a significant productivity
advantage for primary products.
2.3 Productivity of Chemostat
X ( g/ L ) ∙ F ( L/hr ) Example:
Productivity= =DX
V ( L) Xm −1
Substituting equation 10 to the equation above, =20 , μm=μ b=1.0 h , t i =5 h
X0

( )
2
D KS Productivit y chemo
DX =Y S F D− (11) =8
μm −D Productivit y batch

2.6 Reasons why most Commercial Bioprocesses are batch

 Equation 16 applied only to growth-associated
products.
o Secondary product is only made at very low
dilutions, far below those values optimal for
biomass production.
 Genetic Instability
2.4 Maximizing Productivity  Sterility, operability, reliability
d ( DX )  Market economics
=0 o Batch systems provide much greater
dD
flexibility.
D opt =μ m 1−( √ KS
K S+ SF )
(12) 2.7 Examples of Continuous Operation
 Production of single-cell protein (SCP)
Substituting equation 12 to equation 10,
 Waste Treatment
X opt =Y ( S F + K S−√ K S ( S F + K S ) ) (13)  Some other large-volume growth-associated products

( √
Dopt X opt =Y μ m 1−
KS
KS+ SF
( )
S F + K S− √ K S ( S F + K S ) ) (14) 3. Fed-batch Operation

Usually, S F ≫ K S
Dopt X opt =Y μ m ( 1−0 ) ( S F +0−0 )
Dopt X opt =Y μ m S F (15)
Where:
2.5 Comparison of Chemostat and Batch (or Fed-batch)
F(L/h) – flow rate
Cultures
V (L) – working volume
X (g/L) – cell concentration
S (g/L) – substrate concentration
3.1 Variable-Volume Semi-batch operation  Total
 A semi-batch operation in which the feed containing dV
sources of carbon, nitrogen and other are fed either =F (3)
dt
intermittently or continuously during the operation.
With,
dS
3.2 Products using Fed-batch Operation =0
 Antibiotics  Organic acids dt
 Baker’s yeast  r-DNA products Then equation 2 becomes,
 Enzymes  solvents μX V
F= (4)
 Microbial cells  Vitamins Y S F −S
 Natural lipids  Yeasts  Cell (equation 1)
 Nucleotides d ( XV )
=μXV
3.3 Specific Growth Rate dt
μt
1 d ( XV ) XV =X 0 V 0 e (5)
μ=
XV dt
Substituting equation 5 to equation 4,
 Batch
1 dX μ X 0 V 0 e μt
μ= F= (6)
X dt Y ( S F−S )
 Fed-batch
1 dX 1 dV 4. Multistage Chemostat Systems
μ= + The growth and product formation steps need to be
X dt V dt
separated if optimal conditions for each step are different.
3.4 Growth Yield  Particularly for secondary metabolite production.
∆ X −dX  The culture of genetically engineered cells.
Y= =
∆S dS

3.5 Mass Balance

Acc=¿−Out +Gen−Con
 Cell
d ( XV )
=μXV (1)
dt
 Substrate
d ( SV ) 1
=S F F− μXV (2)
dt Y 4.1 Mass Balances for the First Stage
 Total Acc=¿−Out +Gen−Con
dV  Cell
=F (3)
dt d ( X1V 1)
3.6 Determination of Feed Rate – using mass balance =0−X 1 F+ μ 1 X 1 V 1−0 (1)
dt
equations
 Substrate
 Cell
d ( S1V 1) 1
d ( XV ) =S0 F−S 1 F +0− μ X V (2 )
=μXV dt Y 1 1 1
dt
dX
dt (
= μ−
F
V )
X (1) 4.2 Concentrations at Steady state
D1 K S
S1 = (3)
 Substrate μm −D 1
d ( SV ) 1
dt
=S F F− μXV
Y
dS ( S F −S ) F μX
(
X 1 =Y S0−
D1 K S
μm −D1
(4 )
)
= − (2) Where,
dt V Y D1=F /V 1
 { αC −( 1+α ) D+ μ } X 1 =0
4.3 Mass Balances for the Second Stage μ= (1+ α −αC ) D (3)
 Cell
d ( X 2 V 2) C> 1→ α ( 1−C ) <0 →1+ α ( 1−C ) <1
= X 1 F− X 2 F+ μ X 2 V 2 −0(5)
dt μ< D(4)
d X2
= ( X 1 − X 2 ) D 2 + μ2 X 2
dt A chemostat can be operated at dilution rates higher than
the (maximum) specific growth rate when the cell cycle is
At steady state, used.

μ2=D2 1−
( X1
X2 )
(6)  Substrate

Where, d ( SV ) μ X1V
X 1 / X 2<1 =S0 F+ SαF−S ( 1+α ) F− (5)
dt Y
μ2 < D2
At steady state,
 Substrate μ X1
d ( S2 V 2 ) S0 D+SαD−S ( 1+α ) D=
1 Y
=S1 F−S 2 F +0− μ X V (7 ) μ X1
dt Y 2 2 2
d S2 ( S0 −S ) D= (6)
1 Y
=( S 1−S 2 ) D2− μ2 X 2
dt Y
At steady state, Substituting equation 3 to equation 6,
μ2 X 2 Y ( S 0−S )
S2=S 1− (8) X1= (7)
D 2Y 1+α −αC
Compare with the X in chemostat without cell recycle,
5. Chemostat with Recycle X =Y ( S 0−S )
 Higher cell concentration
 Longer residence time The steady-state cell concentration in a chemostat is
o High specific productivity increase by a factor of ( 1+α −αC )=k by cell recycle. (Not
 Higher stability exactly!)
o Example: wastewater treatment –
minimizing the effects of process 5.2 Concentrations at Steady state
perturbation ( 1+α −αC ) D=μ
By Monod equation,
μm S
μ=
K S +S
Then,
kD K S
S= (8)
μm −kD

Substituting equation 8 to equation 7,

5.1 Mass Balance

 Cell
Acc=¿−Out +Gen−Con
X1=
Y
k (
S0 −
kD K S
μ m−kD
(9) )
5.3 Cell Mass Balance around the Cell Separator
d ( X1V ) ( 1+α ) F X 1=F X 2+ αFC X 1
=αFC X 1−( 1+α ) F X 1+ μ X 1 V (1)
dt X 2 =( 1+ α −αC ) X 1
X 2 =k X 1 (10)
At steady state,
{ αFC −( 1+ α ) F+ μV } X 1 =0(2)
5.4 Productivity
X2F
productivity= =D X 2
V
Substituting equation 10 to the equation above,
D X 2=kD X 1

Then substitute equation 9 to it,

(
D X 2=Y S 0 D−
k D2 K S
μ m−kD )
( )
2
D KS
D X 2=Y S 0 D− (11)
1
μ −D
k m

Compare equation 11 with the productivity without cell

recycle.

(
DX =Y S 0 D−
D2 K S
μm −D )
5.5 Chemostat with and without Recycle

5.6 Cell Recycle System

Cell concentrations and productivities are higher with cell
recycle, resulting in higher rates of substrate consumption.
Recycle system is used extensively in waste treatment and
is increasing use in ethanol production.