Professional Documents
Culture Documents
Chapter 6: How Cells Grow: David Shonnard Department of Chemical Engineering Michigan Technological University
Chapter 6: How Cells Grow: David Shonnard Department of Chemical Engineering Michigan Technological University
David Shonnard
Department of Chemical Engineering
Michigan Technological University
David R. Shonnard
Presentation Outline:
Introduction
David R. Shonnard
Introduction
Cell growth is the primary response of viable cells to
substrates and nutrients.
Substrates/nutrients + cells products + more cells
1 dX
X dt
X = cell mass concentration (g / L)
t = time (h)
specific growth rate (h -1),
David R. Shonnard
David R. Shonnard
David R. Shonnard
David R. Shonnard
light
luciferase
Inoculum [Xo]
[So]
Batch
Reactor
X = cell
Concentration
(cells/mL)
David R. Shonnard
Lag Phase
David R. Shonnard
t
X = Xoe max or ln
= max t
Xo
doubling time of cells (t d ),
td =
ln 2
max
or
max =
ln
X
Xo
o
o
o
Slope = max
t
X
= 2 ln(2) = max t d
Xo
ln 2
td
Deceleration Phase
David R. Shonnard
10
Stationary Phase
no net growth of cell numbers or cell mass (no cell division)
cell growth rate = cell death rate
secondary metabolites (products) produced
endogenous metabolism of energy stores can result in
maintaining cell viability
removal of inhibitory compounds will result in further growth
if additional substrate is provided
David R. Shonnard
11
Death Phase
1. Cell lysis (spillage) may occur
2. Rate of cell decline is first-order
dX
= - kd t, X = X S at t = 0
dt
X
X = XS e -kd t or ln
= - kd t
Xo
3. Growth can be re-established by transferring to fresh media
David R. Shonnard
12
( max - kd )
max = A e -E a / RT and
kd = A e
-E d / RT
David R. Shonnard
13
pH Effects
acceptable pH is 1 to 2 pH units
pH range varies by organism
bacteria (most) pH = 3 to 8
yeast
pH = 3 to 6
plants
pH = 5 to 6
animals
pH = 6.5 to 7.5
microorganism have the ability to control pH inside the cell,
but this requires maintainance energy
pH can change due to
utilization of substrates; NH4+ releases H+, NO3- consumes H+
production of organic acids, amino acids, CO2, bases
David R. Shonnard
14
pH Effects (cont.)
David R. Shonnard
15
Dissolved O2 Effects
O2 may be a limiting substrate for aerobic fermentation,
since O2 is sparingly soluble in water
critical O2 concentration
5 to 10% of saturation ( 7 mg O2/L) for bacteria/yeast
growth exhibits saturation kinetics with respect to O2
concentration (see next page)
David R. Shonnard
16
Saturation kinetics
Facultative
aerobic
cells
* =
- anaerobic
max
anaerobic
- max
aerobic
max
David R. Shonnard
17
David R. Shonnard
18
Growth Yield
X
S
into
extracellular
products
energy
David R. Shonnard
19
YX/ S = -
X
S
YX/ O2 = -
X
O2
YX/O2, reduced S (0.15 to 0.3 ) < YX/ O2, oxidized S (0.3 to 1.5)
David R. Shonnard
20
10
YX/ ATP =
X
g dry cells
(
)
ATP mole ATP generated
David R. Shonnard
21
1 dP
= YP/ X
X dt
qP =
David R. Shonnard
qP = +
22
11
1. qP =
1 dP
= YP/ X
X dt
3. qP = +
2. qP =
David R. Shonnard
23
Substrate + O2 I
1/YH
(kJ/g dcw)
Energy Released
Total Available
=
by Growth
Energy of Substrate
Combustion
HS (kJ/g S)
II
Energy Available
+
in Biomass
III
CO2 + H2O
II
David R. Shonnard
III
Combustion of Cells
HC (kJ/g dcw)
24
12
YH =
YX / S
(HS - YX / S HC )
David R. Shonnard
25
HS (kJ/g S)
YH (g dcw/kJ)
15.64
22.68
29.67
47.64
55.51
0.072
0.029
0.043
0.038
0.015
David R. Shonnard
26
13
Q Gr = VL X
1 kJ
( )
YH hr
Liquid Volume
27
Unstructured Models:
Model assumes balanced growth where cell components do not
change with time. Much less complex and much more commonly
used. Valid for batch growth during exponential growth stage and
also for continuous culture during steady-state operation.
David R. Shonnard
28
14
Monod Equation
Similar to Michaelis-Menten Kinetics
Assumes that a single enzyme system is responsible for the uptake of
substrate (S), and that S is limited (growth-dependent variable).
This is the most common kinetic model for cell growth.
m S
KS + S
David R. Shonnard
29
1 dX
m S
=
X dt
KS + S
m (SoYX /S + X o - X)
dX
=
X
dt
(K SYX /S + SoYX / S + Xo - X)
David R. Shonnard
at t = 0, X = Xo
30
15
Bioprocess Engineering: Basic Concepts, Shuler and Kargi, Prentice Hall, 2002
David R. Shonnard
31
KS is determined differently.
KS is equal to S when = 1/2 max
X
ln
Xo
o
o
Slope = max
o o
max
David R. Shonnard
1 dX
=
X dt
= 1/2 max
KS
32
16
David R. Shonnard
33
Chemostat as a Tool
evaluate KS, max, YX/S and other system parameters
study changes in environment and effects on cell physiology
select for cells with desired metabolic capabilities (e.g. selection
for cells capable of degrading a toxic compound)
David R. Shonnard
34
17
David R. Shonnard
35
David R. Shonnard
36
18
metabolism
or
mass rate
mass rate
of cell loss
of cells
-
=
by endogenous
accumulation
metabolism
in bioreactor
FXo - FX + VR X - VR kd X = VR dX
dt
F = in and out volumetric flow rate (L / hr)
X = bioreactor and outlet cell mass concentration (g / L)
Xo = inlet cell mass concentration (g / L)
= specific cell growth rate neglecting endogenous metabolism (hr-1)
k d = endogenous cell loss rate constant (hr-1)
David R. Shonnard
37
= endogenous
of bioreactor
metabolism
or
FX = VRX
David R. Shonnard
F
= or D =
VR
F
where
= D, dilution rate
VR
38
19
Substrate Concentration
Using the Monod Equation, we can predict the bioreactor and
outlet stream concentration of Substrate.
= max S = D
KS + S
rearranging,
David R. Shonnard
S =
KS D
max - D
39
David R. Shonnard
max So
K S + So
40
20
D = - kd
and
S =
KS (D + kd )
max - D - kd
David R. Shonnard
Answer: X is lower!
41
Cell Concentration
How is X affected by D? A similar mass balance equation for S
in the absence of endogenous metabolism is written to answer
this question.
FSo - FS - VR X 1M - VR q p X 1 = VR dS
YX / S
YP /S
dt
S = bioreactor and outlet substrate concentration (g / L)
So = inlet substrate concentration (g/ L)
YXM/S = maximum cell yield coefficient (g cells / g substrate)
YP/ S = product yield coefficient (g product / g substrate)
gP
g cells hr
42
21
D(So - S) = X
YXM/S
at steady -state, = D, and solving for X,
M (S - S)
X = YX/S
o
or
KS D
M (S X = YX/S
)
o
max - D
David R. Shonnard
43
D (So - S) X
M
YX/S
=0
kd
mS =
M
YX /S
maintenance coefficient
based on S.
David R. Shonnard
D - D - kd = 0
M
YXM/S
YXAP/S YX/S
1 = 1 + mS = 0
D
Y AP
YM
X /S
X/S
44
22
kd
M
YX /S
M
kd = mSYX /S
David R. Shonnard
45
D = kd
o
o
max S
D =
kd
KS + S
1
D + kd
rearranging
1
D + kd
1
max
David R. Shonnard
slope =
KS
max
intercept =
1
max
o
o
KS 1
max S
Michigan Technological University
1
S
46
23
Productivity of a Chemostat
PrX = productivity for cell production = DX
PrP = productivity for product formation = DP
The dilution rate (D) which maximizes productivity is found by
taking dPr/dD = 0 and solving for D (Doptimum).
For example, Doptimum for X with kd = 0 and qP = 0
X = YXM/ S (So
KS D
)
max - D
DX = YXM/S D(So
KS D
)
max - D
Dopt = max (1 -
KS
)
K S + So
David R. Shonnard
KS is usually << S
so Dopt ~ max (washout point)
47
KS D
from X mass balance
max - D
X = YXM/S (So - S)
D
(D + q P
David R. Shonnard
YXM/ S
)
YP/ S
48
24
David R. Shonnard
Dopt
DX (g/Lhr)
D (hr-1)
David R. Shonnard
washout at
max So
D =
KS + So
50
25