Cell Cultivation - Productivity
Introduction to chemostat culture
Preparation for chemostat simulation (BioProSim)
Obtaining growth constant from chemostat data
Biomass feedback
Pro and Con of Chemostat
And 11
Chemostat 1
How to increase growth of microbial culture?
1. Increase initial substrate concentration problem: substrate
inhibition
2. Add more substrate during growth (Fed-batch) problem:
endproduct inhibition
3. Replace old medium including endproducts by fresh
medium at given intervals (semi continuous)
4. Automate semicontinuous culture by applying constant
inflow and outflow (continuous culture, chemostat)
And 22
Transition from Batch to Chemostat
1. 1 Drop of Inflow of feed medium increases X by a small
amount.
2. 1 Drop of reactor volume flows out removes biomass
(X).
3. If the Feed drop allows more X to grow than is taken out
by the reactor drop X in reactor will increase slightly
4. Repeat the loop and there will be gradual increase in X
5. However, as the X increases each drop of outflow will
contain more X which is removed from the reactor rate
of out put increases
6. as a consequence the biomass will reach a certain steady
state level.
Assumptions: Reactor volume stays constant And 33
(Use learning activity on chemostat to visualise how it works)
Transition from Batch to Chemostat
1.However, as the X increases each drop of outflow will
contain more X which is removed from the reactor rate of
out put increases.
2.as a consequence the biomass will reach a certain steady
state level.
3.The flow rate can be varied but has little effect on the level
of biomass concentration.
4.The specific flowrate (i.e. flowrate (L/h) per reactor volume
(L) is called the Dilution rate
Assumptions: Reactor volume stays constant
(Use learning activity on chemostat to visualise how it works)
(use bioprosim simulation to demonstrate chemostat versus batch)
(Could use a modelling spreadsheet to demonstrate the steady state)
And 44
Transition from Batch to Chemostat
1. 1 Drop of Inflow of feed medium increases X by a small amount
2. 1 Drop of reactor volume flows out removes biomass (X)
3. If the Feed drop allows more X to grow than is taken out by the
reactor drop X in reactor will increase slightly
4. Repeat the loop and there will be gradual increase in X
5. However, as the X increases each drop of outflow will contain more X
which is removed from the reactor rate of out put increases
6. as a consequence the biomass will reach a certain steady state level.
7. The flow rate can be varied but has little effect on the level of
biomass concentration.
8. The specific flowrate (i.e. flowrate (L/h) per reactor volume (L) is
called the Dilution rate
Assumptions: Reactor volume stays constant
(Use learning activity on chemostat to visualise how it works)
(use bioprosim simulation to demonstrate chemostat versus batch) And 55
(Could use a modelling spreadsheet to demonstrate the steady state)
Effect of Increasing the Dilution rate
1. After a steady state is reached from continuously adding and
removing drops (continuous dilution rate) a steady state is reached
consisting of a constant high biomass level (X) and a very low
substrate level (S) because the biomass degrades the substrate
down to limiting concentrations.
2. If the dilution rate is doubled :
3. More substrate per time is supplied
4. bacteria grow faster
5. bacteria are washed out faster
6. steady state is reached again with a
• slightly higher substrate steady state concentration,
• slightly lower biomass level
• bacteria growth rate being twice as high
• bacteria growth rate u compensating for the Dilution rate D
And 66
Chemostat 4
Effect of loading rate increase (R = X * D)
1
Double F Double SR
and D doubles X
doubles μ doubles R
Almost doubles R Affects μ only a little
Affects X only a little
SR = SR =
2 g/L h 2 g/L
h
SR =
SR = 2 g/L SR =
2 g/L 4 g/L
h h
X and μ can be set independently, X by SR and μ by And
D 77
Steady State Concentration Key features of steady states 1:
SR
•Inflow rate = Outflow rate
X
Flowrate divided by Volume F/h /V L =
Dilution rate D (h-1)
Dilution rate= specific growth rate u
•Dilution rate= specific growth rate u
R
•S limitation of growth
•X stays constant over wide range of
S D
•If D approaches umax washout
D Dcrit (Dcrit)
Effect of Dilution rate on chemostat
steady state concentrations
•Beyond (Dcrit) S = SR
X= biomass, S= substrate,
SR= substrate in Reservoir R=productivity
And 88
Steady State Concentration Key features of steady
SR states 1:
X
•F (L/h)/V(L) = D (h-1) =u
•Dilution rate= specific
growth rate u
R
•S limitation of growth
•X stays constant over
S
wide range of D
D Dcrit
Effect of Dilution rate on chemostat •If D approaches umax
steady state concentrations washout (Dcrit)
X= biomass, S= substrate,
SR= substrate in Reservoir R=productivity
•Beyond (Dcrit) S = SR
And 99
Steady State Concentration Key features of steady
SR states 2:
X
•Open system, time factor
excluded
•Allows to study microbial
R behaviour at constant
growth rate
•µ of culture can be
S
controlled by changing D
D Dcrit
Effect of Dilution rate on chemostat D S µ but not
steady state concentrations X (because of washout)
X= biomass, S= substrate,
SR= substrate in Reservoir R=productivity
•How can X be
controlled? And 1010
Steady State Concentration Key features of steady
SR states 3:
X
•X can be controlled by
SR (dotted line using
more dilute feed)
R •Doubling SR doubling
of X and of R (to a point)
•The level of X also
S
depends on Y
D Dcrit
Effect of Dilution rate on chemostat
steady state concentrations
X= biomass, S= substrate,
SR= substrate in Reservoir R=productivity
And 1111
Steady State Concentration Key features of steady
SR states 4:
X
•R =X *D
•g/L/h = g/L * h-1
R •R is largely a function of
D until washout occurs
S •R can be increased by:
D Dcrit D µ but not X
Effect of Dilution rate on chemostat
steady state concentrations SR X but not u
X= biomass, S= substrate,
SR= substrate in Reservoir R=productivity
•Such control does not
exist in batch culture. And 1212
Steady State Concentration
Productivity R :
SR
X •R =X *D
•g/L/h = g/L * h-1
•As R =D*X and u=D
R
S – k .
D
R = D.Y --------------
R S
µmax -D
S
•R can be increased by
D Dcrit D, Y, SR
kS
•note: ms is ignored for high D
and Y is assumed to be ~Ymax
•Where is the max chemostat
And 1313
productivity?
Steady State Concentration Max chemostat
SR productivy (Rm):
X Rm
•D at which R= Rm is
called Dm
•Dm can be calculated
R from growth constants:
Dm = µmax. (1- kS/(SR- kS)
S
D Dcrit •Rm = X* Dm
Note: at Dm some substrate wastage occurs
Less conversion efficiency than at lower D
Operator can aim for: high productivity or high conversion efficiency
(of SX)
Dm is dangerously close to Dcrit (especially when ks is low) And 1414
Chemostat productivity
X SR
(g/L) chemo- •no lag phase,
stat
•no preparation phase
batch
maximum batch
productivity total •but only highly
productivity concentrated cell
suspensions with close to
exponential growth
Time (h) •very high productivity
compared to batch total or
maximum productivity.
And 1515
Comparison of
X SR productivity batch vs
(g/L) chemo- chemostat:
stat •Chemostats:
batch •no lag phase,
maximum batch
productivity total
productivity •no preparation phase
•but only highly
concentrated cell
Time (h) suspensions with close to
exponential growth
•very high productivity
compared to batch total or
maximum productivity.
And 1616
Steady State Concentration Key features of steady
states 2:
X S
•R is largely a function of
D until washout occurs
R •µ of culture can be
controlled by changing D
•X can be controlled by
changing SR (
D Dcrit
Effect of Dilution rate on chemostat
steady state concentrations
X= biomass, S= substrate, R=productivity
And 1717
Chemostat 8
Productivity (R) –
Comparison Chemostat / Batch Culture
Batch culture
t prep = preparation time since last run
R= Xmax - Xo (cleaning, sterilizing, lag phase etc.)
tprep + tlog
t log = running time under full growth
Chemostat
R = DX (1 - tprep / tlog)
• for long runs R = DX
The productivity of chemostats can be several times higher
than in batch culture
And 1818
Chemostat 10
Oxygen limitation
The classical chemostat theory is based on the concept
of substrate limitation.
In practice the oxygenation capacity of a bioreactor may
become limiting. This results in the deviation of the classical
chemostat.
S-limited O2-limited S-limited Chemostat under oxygen limitation
X = Y (SR - D ks )( kLa +1 )
μmax -D D
1
2 1) Low D no O2 limitation
X
2) D O2 limitation μ<D X
3 3) X O2 limitation
4 And 1919
D
Chemostat
The Critical Dilution Point
The critical dilutino point Dc results in washout of biomass
(X = 0, S = SR)
μmax SR μmax Dc
Dc = ks Dc
Ks + SR
SR little affect on Dc
Neglecting ms
Dilution rates > μmax can be used to determine μmax:
If D > μmax logarithmic biomass washout
In Xt -In Xo
μmax = t +D
X
μmax = (In Xt - In Xo) t + D
Washout kinetics
Fixed D
D > μmax And 2020
Time
Chemostat
Advantages and disadvantages compared to batch
Chemostat
+ prodctivity
+ constant requirement for cooling, O2 transfer, labour, etc
Heat requirements for batch cultures
Heat Cool Heat
And 2121
How to obtain microbial growth constants
from chemostat runs
1. Run a chemostat at equilibrium making sure that
oxygen is not limiting
2. Note down the substrate in the reservoir (SR)
3. Calculate the Dilution rate from the Flowrate and the
reactor Volume: D= F/V. check that units cancel to h-1
4. Note down the steady state values for S, X and DO
5. Determine the observed yield coefficient Y by dividing
the biomass formed (X) by the amount of substrate
degraded (SR-S)
6. Change the dilution rate by about 10% steps upward
and downward. Wait for equilibrium each time.
7. Tabulate your values of X, S, SR, DO for each D in a
spreadsheet And 2222
How to obtain microbial growth
constants from chemostat runs
7. Tabulate your values of X, S, SR, DO for each D in a
spreadsheet
8. Considering that at steady state u=D, produce
additional columns that calculate 1/Y and 1/u
9. Plot 1/Y against 1/u and obtain the linear equation with
the slope. Make sure you are clear about the units of
both axes and the slope
10. Obtain the ms and Ymax constants from the slope and
the intercept respectively
11. Alternatively using simultaneous equations allows you
to calculate the constants from the equation
And 2323
How to obtain microbial growth
constants from chemostat runs
12. Tabulate the inverse of the substrate
concentration present in the chemostat (S that
determines u)
13. Tabulate 1/(u +ms*Ymax)
14. Use the double inverse plot of the two items
above. This is similar to the Lineweaver Burk
Plot
15. Read 1/u from the Y axis intercept and -1/ks
from the x axis intercept
16. All 4 of the growth constants are now obtained
And 2424
Graphical determination
of 2 growth constants
from chemostat steady
state data
1/y
mS
1/Ymax
1/µ Dcrit
Effect Double inverse plot of Y and u
from chemostat runs (u=D)
And 2525
The relationship between
the rate of substrate
uptake (= OUR) and
growth rate
some activity will be used
qS
exclusively for
1/Ymax maintenance without
growth.
ms
If qS is > ms growth
will occur.
µ
Relationship between specific
metabolic acitity (qS) and specific
growth rate
qS = v = substrate uptake rate / X
And 2626
How to obtain microbial growth constants
from chemostat runs
S
µ = µmax * -------- - mS *Ymax
S + kS
Formulae used to determine ms and Ymax
1 ms 1
--- = --- + ------
Y D Ymax
And 2727
Growth- maintenenace
How to obtain microbial growth constants
from chemostat runs
Formulae used to determine ms and Ymax
1 ms 1
--- = --- + ------
Y D Ymax
_____1_______
Y = ms--- + 1------
D Ymax
Y = Ymax + u/ms
Ymax = Y - u/ms And 2828
Growth- maintenenace
Microbial Growth
Ms at [S] = 0
• Even if [S] = 0, some metabolism is required to stay alive.
• The substrate used from either energy stores (e.g. PßHB)
or biomass itself.
Thus:
Net growth = total growth – biomass consumed
Exogenous Respiration
+S
q
e.g.
SPOUR
Endogenous Respiration
Time
For aerobic organisms the maintenance coefficient can be
And 2929
measured via the endogenous respiration rate.
• Formulas not to be used:
S
R = X* µmax * -------- - mS *Ymax
S + kS
And 3030
A chemostat is the preferred culture
method …
• A chemostat is the preferred culturing method…
• …when the process wants to select for the fasted growing strain
(single cell protein, degradation of pollutants)
• …when substrate limitation (e.g. substrate toxicity) is desired
• …contamination or mutation does not play a role (e.g. extreme
conditions of temperature, pH, etc.)
• …for studying metabolic behavior at specified conditions (e.g. pH,
cell density, substrate concentration, product concentration, specific
growth rate) (remember u and X can be set constant separately by
selecting D and SR)
• … for studying effects of shocks (e.g. toxic substances) or minute
disturbances of equilibrium (pH change, DO change)
And 3131
Chemostats are not suitable for…
• production of recombinant products (tendency of
backmutation to the wild strain “contamination from
inside”)
• aseptic cultures with high tendency of contamination
(continuous sterile supply of feed and harvest is difficult)
• where traditional methods play a role
• where there is a need for changing conditions (e.g.
preventing respiratory deficient mutants in brewing, feast
and famine regime
• the production of secondary metobolites (produced after
growth)
And 3232
Chemostat productivity (R = X*D)
can be increased by
• production of recombinant products (tendency of back-
mutation to the wild strain “contamination from inside”)
• aseptic cultures with high tendency of contamination
(continuous sterile supply of feed and harvest is difficult)
• where traditional methods play a role
• where there is a need for changing conditions (e.g.
preventing respiratory deficient mutants in brewing, feast
and famine regime)
• the production of secondary metabolites (produced after
growth)
And 3333
Chemostat productivity (R = X*D)
can be increased by
• maximising the dilution rate (D)
• maximising the biomass concentration in the reactor (via
substrate in the feed (SR) or via organism with high
Ymax)
• Highest biomass productivity (g of cells produced L-1 h-
1)
highest product formation rate of primary metabolites
highest substrate degradation rate
highest oxygen uptake rate
And 3434
Chemostat
Advantages and disadvantages compared to batch
+ less labour
+ facilitates automation
+ constant output
+ easy for problem monitoring
+ - selects for best growing strain
- more susceptible to contamination from outside
longer running times higher probability
continuous sterilisation difficult
- contamination from inside (back mutation to wild type)
- breaking tradition of batch processes (e.g. beer)
And 3535
Chemostat
Main applications
1. Where the fastest growing strain is required, e.g. single
cell protein (SCP), effluent, strain selection (e.g. higher
affinity).
2. Generating functional understanding (not empirical
observation) e.g. process optimisation, research.
2.1. Time is excluded every part of a batch process can be
studied over extended periods. E.g. to investigate problem; set
chemostat to exactly these conditions.
2.2. Permits to vary μ by changing D under otherwise constant
conditions.
And 3636
Chemostat
Main applications
2.3. Study of environmental changes (pH, temperature,
salinity, [S] etc.) at constant μ
2.4. Allows to study growth and metabolism under substrate
limitation (ecological studies, pollution, waste treatment)
2.5. Allows to study effect of 1 parameter only
3. Batteries of small research chemostats to complement to
sophisticated industrial batch reactor
And 3737
Why not continuous beer brewing?
1. Contamination from inside and outside (e.g. respiratory
deficient mutants (RMD))
2. Start up time until state gives low quality beer
3. Although HRT is 5 to 10 times less than for batch
process, only small economic benefit is achieved in
comparison with the cleaning condition and packaging of
the product.
And 3838
Chemostat
Biomass Feedback
Justification for biomass feedback
• High [X] is essential for high metabolic activity
-ds/dt = μ X/Y
• Usually X can be controlled by SR in reaction
• For waste water treatment (e.g. activated sludge)
SR can not be controlled and is very low
• Biomass feedback can keep X high, S extremely low
and achieve high R
Idea
Prevent X from being washed out
And 3939
Technique
Physically retain or return biomass resulting in longer
biomass (solid) retention time SRT than liquid retentiontime
(HRT)
Options
• Immobilization of cells (e.g. fixed bed reactors, fluidised
bed reactors, rotating disk contractor (RDC))
• Internal recycle
• Eternal recycle
And 4040
Chemostat
Biomass Feedback 2
The most simple idea to retain biomass: Filter
Feed
Chemostat
Filter
Outflow
Because the filter clogging (fouling)
simple bacterial filters cannot be
used
Cross flow filtration, and filter
capillaries are used for the
separation of expensive products And 4141
Chemostat
Biomass Feedback 2
Biomass feedback by internal sedimentation of biomass
→ Flocculation
necessary (Stroke’s Law)
And 4242
Chemostat
Biomass Feedback 3
External biomass feedback in activated sludge treatment
Feed
Outflow
Settler
Reactor
Biomass Recycle
Biomass feed back: Ú X, Ú R, Ú Dc, Æ S
proportionally to the efficiency of feedback
And 4343
Retaining biomass in a chemostat
• Preventing biomass from washout
• allows buildup of higher biomass concentration
• when is this useful?
• When feed concentration (SR) can not be concentrated
(eg. feed toxicity or fixed feed (e.g. waste water))
• When only very little biomass growth will be obtained
(e.g. mineral media, autotrophs, extreme conditions such
a bioleaching)
And 4444
Retaining biomass in a chemostat
• How can biomass be
retained?
Inflow
• In theory a filter that
allows liquid outflow
but no biomass outflow
works
• In practice: Filter
Outflow
fouling
• Alternatives (Cross
flow filtration,
And 4545
Practical Biomass retention
inside the reactor
• Biofilm reactors
• (a) fixed bed reactor
Inflow
(trickle reactor)
• (b) fluidised bed reactor
• (c) sludge blanket reactor
(settling biomass flocs)
• (e.g. sequencing batch
reactor (Feed-React-
Settle-Decant Outflow
• Problems: mass transfer
(e.g. oxygen), channeling
And 4646
Practical Biomass retention
via biomass feedback
• Centrifuging of recycle
liquid Inflow
• Membrane filtration of
recycle liquid
• Flocculation Outflow
• Gravity settling of
flocculated biomass
Recycle
(Feedback)
And 4747
Effect of biomass
feedback (here 3 fold):
Dotted line no feedback:
Steady State Concentration
SR •Washout occuring early
X
•3-fold Feedback
approximately:
•3*X 3*R 1/3* S
•allows 1/3 reactor size to
R do same work
•Feedback essential for
pollutant removal. Can be
S used 100-fold 100-fold
smaller treatment plant
D Dcrit
•Note: same assumed
feed concentration (SR)
And 4848
Effects of growth constants on steady state concentrations
of biomass and substrate in a chemostat as a function of dilution rate (x-
axis)
Effect of ms Effect of decrease ks
Effect of increased Y Effect of increased μmax
And 4949
