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

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)

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)

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)

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)

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

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

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

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

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

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

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

Steady State Concentration X 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

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. (1- 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)

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)

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)

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

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

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 - )( +1 ) 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

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

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

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

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

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

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)

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 µ Relationship between specific metabolic acitity (qS) and specific growth rate qS = v = substrate uptake rate / X

How to obtain microbial growth constants from chemostat runs µ = µmax * -------- - mS *Ymax S + kS Formulae used to determine ms and Ymax 1 ms 1 Y D Ymax --- = --- + ------ 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_______ ms 1 D Ymax Y = --- + ------ Y = Ymax + u/ms Ymax = Y - u/ms 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 measured via the endogenous respiration rate.

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

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)

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)

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)

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

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)

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.

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

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.

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

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

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

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

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

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)

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

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

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)

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

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