2. 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

3. 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)

4. Transition from Batch to Chemostat
1 Drop of Inflow of feed medium  increases X by a small amount.
1 Drop of reactor volume flows out  removes biomass (X).
If the Feed drop allows more X to grow than is taken out by the reactor drop  X in reactor will increase slightly
Repeat the loop and there will be gradual increase in X 
However, as the X increases each drop of outflow will contain more X which is removed from the reactor  rate of out put increases
as a consequence the biomass will reach a certain steady state level.
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)

5. Transition from Batch to Chemostat
However, as the X increases each drop of outflow will contain more X which is removed from the reactor  rate of out put increases.
as a consequence the biomass will reach a certain steady state level.
The flow rate can be varied but has little effect on the level of biomass concentration.
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)

6. Transition from Batch to Chemostat
1 Drop of Inflow of feed medium  increases X by a small amount
1 Drop of reactor volume flows out  removes biomass (X)
If the Feed drop allows more X to grow than is taken out by the reactor drop  X in reactor will increase slightly
Repeat the loop and there will be gradual increase in X 
However, as the X increases each drop of outflow will contain more X which is removed from the reactor  rate of out put increases
as a consequence the biomass will reach a certain steady state level.
The flow rate can be varied but has little effect on the level of biomass concentration.
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)

7. Effect of Increasing the Dilution rate
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.
If the dilution rate is doubled :
 More substrate per time is supplied
 bacteria grow faster
 bacteria are washed out faster
 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

8. Effect of loading rate increase (R = X * D)
Chemostat 4
Effect of loading rate increase (R = X * D) 1
Double F 
and D 
doubles μ
Almost doubles R
Affects X only a little
Double SR 
doubles X
doubles R
Affects μ only a little
SR =
2 g/L
SR =
2 g/L h h
SR =
2 g/L
SR =
4 g/L
SR =
2 g/L h h
X and μ can be set independently, X by SR and μ by D

9. Steady State Concentration
Key features of steady states 1:
Inflow rate = Outflow rate
Flowrate divided by Volume F/h /V L = Dilution rate D (h-1)
Dilution rate= specific growth rate u
S limitation of growth
X stays constant over wide range of D
If D approaches umax  washout (Dcrit)
Beyond (Dcrit) S = SR SR X
Steady State Concentration R S D
Dcrit
Effect of Dilution rate on chemostat steady state concentrations X= biomass, S= substrate, SR= substrate in Reservoir R=productivity

10. Key features of steady states 1:
F (L/h)/V(L) = D (h-1) =u
Dilution rate= specific growth rate u
S limitation of growth
X stays constant over wide range of D
If D approaches umax  washout (Dcrit)
Beyond (Dcrit) S = SR SR X
Steady State Concentration R S D
Dcrit
Effect of Dilution rate on chemostat steady state concentrations X= biomass, S= substrate, SR= substrate in Reservoir R=productivity

11. Key features of steady states 2:
Open system, time factor excluded
Allows to study microbial behaviour at constant growth rate
µ of culture can be controlled by changing D
D  S  µ but not X (because of washout)
How can X be controlled? SR X
Steady State Concentration R S D
Dcrit
Effect of Dilution rate on chemostat steady state concentrations X= biomass, S= substrate, SR= substrate in Reservoir R=productivity

12. Key features of steady states 3:
X can be controlled by SR (dotted line using more dilute feed)
Doubling SR doubling of X and of R (to a point)
The level of X also depends on Y SR X
Steady State Concentration R S D
Dcrit
Effect of Dilution rate on chemostat steady state concentrations X= biomass, S= substrate, SR= substrate in Reservoir R=productivity

13. Key features of steady states 4:
R = X * D
g/L/h = g/L * h-1
R is largely a function of D until washout occurs
R can be increased by:
D   µ but not X
SR  X but not  u
Such control does not exist in batch culture. SR X
Steady State Concentration R S D
Dcrit
Effect of Dilution rate on chemostat steady state concentrations X= biomass, S= substrate, SR= substrate in Reservoir R=productivity

14. Steady State ConcentrationX S D
Steady State Concentration R
Dcrit SR
Productivity R :
R = X * D
g/L/h = g/L * h-1
As R =D*X and u=D
R can be increased by
D, Y, SR
kS
note: ms is ignored for high D and Y is assumed to be ~Ymax
Where is the max chemostat productivity?
SR – kS.D
R = D.Y
µmax -D

15. Max chemostat productivy (Rm):
D at which R= Rm is called Dm
Dm can be calculated from growth constants:
Rm = X* Dm SR X Rm
Steady State Concentration R
Dm = µmax. ( kS/(SR- kS) S D
Dcrit
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 SX)
Dm is dangerously close to Dcrit (especially when ks is low)

16. Chemostat productivity no lag phase, no preparation phase
but only highly concentrated cell suspensions with close to exponential growth 
very high productivity compared to batch total or maximum productivity. SR X
(g/L)
chemo-
stat
batch
maximum
productivity
batch
total
productivity
Time (h)

17. Comparison of productivity batch vs chemostat:
Chemostats:
no lag phase,
no preparation phase
but only highly concentrated cell suspensions with close to exponential growth 
very high productivity compared to batch total or maximum productivity. SR X
(g/L)
chemo-
stat
batch
maximum
productivity
batch
total
productivity
Time (h)

18. Key features of steady states 2:X S
Key features of steady states 2:
R is largely a function of D until washout occurs
µ of culture can be controlled by changing D
X can be controlled by changing SR (
Steady State Concentration R D
Dcrit
Effect of Dilution rate on chemostat steady state concentrations X= biomass, S= substrate, R=productivity

19. Comparison Chemostat / Batch Culture
Productivity (R) –
Comparison Chemostat / Batch Culture
Batch culture
t prep = preparation time since last run (cleaning, sterilizing, lag phase etc.)
Xmax - Xo
R =
tprep + tlog
t log = running time under full growth
R = DX (1 - tprep / tlog)
Chemostat
for long runs R = DX
The productivity of chemostats can be several times higher than in batch culture

20. 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.
Chemostat under oxygen limitation
S-limited
O2-limited
S-limited
D ks
kLa
X = Y (SR )( )
μmax -D D 1
1) Low D  no O2 limitation 2 X
2)  D  O2 limitation  μ<D   X
3)  X   O2 limitation 3 4 D

21. The Critical Dilution Point
Chemostat
The Critical Dilution Point
The critical dilutino point Dc results in washout of biomass (X = 0, S = SR)
 μmax   Dc
 ks   Dc
SR little affect on Dc
μmax SR
Dc =
Ks + SR
Neglecting ms
Dilution rates > μmax can be used to determine μmax:
If D > μmax  logarithmic biomass washout
In Xt -In Xo
μmax = + D t X
μmax = (In Xt - In Xo) t + D
Washout kinetics
Fixed D
D > μmax
Time

22. Advantages and disadvantages compared to batch
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

23. How to obtain microbial growth constants from chemostat runs
Run a chemostat at equilibrium making sure that oxygen is not limiting
Note down the substrate in the reservoir (SR)
Calculate the Dilution rate from the Flowrate and the reactor Volume: D= F/V. check that units cancel to h-1
Note down the steady state values for S, X and DO
Determine the observed yield coefficient Y by dividing the biomass formed (X) by the amount of substrate degraded (SR-S)
Change the dilution rate by about 10% steps upward and downward. Wait for equilibrium each time.
Tabulate your values of X, S, SR, DO for each D in a spreadsheet

24. How to obtain microbial growth constants from chemostat runs
Tabulate your values of X, S, SR, DO for each D in a spreadsheet
Considering that at steady state u=D, produce additional columns that calculate 1/Y and 1/u
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
Obtain the ms and Ymax constants from the slope and the intercept respectively
Alternatively using simultaneous equations allows you to calculate the constants from the equation

25. How to obtain microbial growth constants from chemostat runs
Tabulate the inverse of the substrate concentration present in the chemostat (S that determines u)
Tabulate 1/(u +ms*Ymax)
Use the double inverse plot of the two items above. This is similar to the Lineweaver Burk Plot
Read 1/u from the Y axis intercept and -1/ks from the x axis intercept
All 4 of the growth constants are now obtained

26. Effect Double inverse plot of Y and u from chemostat runs (u=D)
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)

27. some activity will be used exclusively for maintenance without growth.
The relationship between the rate of substrate uptake (= OUR) and growth rate
some activity will be used exclusively for maintenance without growth.
If qS is > ms  growth will occur. qS
1/Ymax ms
Relationship between specific metabolic acitity (qS) and specific growth rate qS = v = substrate uptake rate / X

28. How to obtain microbial growth constants from chemostat runs
µ = µmax * mS *Ymax
S + kS
Formulae used to determine ms and Ymax ms
Y D Ymax
--- =
Growth- maintenenace

29. How to obtain microbial growth constants from chemostat runs
Formulae used to determine ms and Ymax ms
Y D Ymax
--- =
_____1_______ ms
D Ymax
Y =
Y = Ymax + u/ms
Ymax = Y - u/ms
Growth- maintenenace

30. 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
measured via the endogenous respiration rate.

31. Formulas not to be used:
R = X* µmax * mS *Ymax
S + kS

32. 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)

33. 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)

34. 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)

35. 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

36. Advantages and disadvantages compared to batch
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)

37. 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.

38. 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

39. 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.

40. 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

41. 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

42. 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

43. Chemostat Biomass Feedback 2
Biomass feedback by internal sedimentation of biomass
→ Flocculation
necessary (Stroke’s Law)

44. 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

45. 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)

46. Retaining biomass in a chemostat
How can biomass be retained?
In theory a filter that allows liquid outflow but no biomass outflow works
In practice: Filter fouling
Alternatives (Cross flow filtration,
Inflow
Outflow

47. Practical Biomass retention inside the reactor
Biofilm reactors
(a) fixed bed reactor (trickle reactor)
(b) fluidised bed reactor
(c) sludge blanket reactor (settling biomass flocs)
(e.g. sequencing batch reactor (Feed-React-Settle-Decant
Problems: mass transfer (e.g. oxygen), channeling
Inflow
Outflow

48. Practical Biomass retention via biomass feedback
Centrifuging of recycle liquid
Membrane filtration of recycle liquid
Flocculation
Gravity settling of flocculated biomass
Inflow
Outflow
Recycle
(Feedback)

49. Effect of biomass feedback (here 3 fold):
Dotted line no feedback:
Washout occuring early
3-fold Feedback approximately:
3*X 3*R  1/3* S
allows 1/3 reactor size to do same work
Feedback essential for pollutant removal. Can be used 100-fold  100-fold smaller treatment plant
Note: same assumed feed concentration (SR) SR X
Steady State Concentration R S D
Dcrit

50. Effect of increased μmax Effect of increased Y
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 μmax
Effect of increased Y