Batch, fedbatch and continuous fermentation

Batch, fedbatch and continuous fermentation

1. Batch, Fed-Batch and Continuous Fermentation Dr. Dhanya KC Assistant Professor, Department of Microbiology St. Mary’s College, Thrissur-680020, Kerala

2. Batch, Fed Batch and Continuous Fermentations Continuous - Open system Medium continuously fed Spent medium and cells removed continuously Volume remains constant Batch - Closed system Nutrients in a fixed volume Further additions - for pH control, aeration, etc . Fed-batch Medium or components fed continuously or intermittently Volume increases with time

3. Batch fermentation - Microbial cells - pass through a number of phases Lag phase L og or exponential phase Stationary phase D eath or decline phase

4. Lag Phase Immediately after inoculation N o apparent growth T ime of adaptation Length - reduced – economical commercial Batch fermentation

5. Logarithmic or log or exponential phase Lag and log phase - trophophase Production of primary metabolites Amino acids, nucleotides, vitamins, citric acid, acetic acid, ethanol, etc. Growth rate of cells increases - Constant , Maximum rate dx/ dt = µx x - Concentration of microbial biomass µ - Specific growth rate t - Time in hours Batch fermentation

6. Stationary Phase - Growth rate reduce Due to substrate limitation and toxin limitation Plot - biomass and initial substrate concentration Growth in different concentrations of substrates Initial increase in substrate - increase in biomass x = Y(S-S R ) x - concentration of biomass Y - yield factor (g biomass / substrate) S - initial substrate concentration S R - residual substrate concentration Yield factor - efficiency of substrate conversion to biomass Batch fermentation

7. Death or decline phase - Cessation of growth - depletion of substrate Stationary and death phase – idiophase Secondary metabolites are formed Examples - antibiotics , pigments, toxins, etc Monod equation µ = µ max s/(K s + s) s -Residual substrate concentration K s -substrate utilization constant substrate concentration when µ is half µ max A measure of the affinity for substrate µ - Specific growth rate µ max – Maximum Specific growth rate Batch fermentation

8. Death or decline phase - Cessation of growth - depletion of substrate Stationary and death phase – idiophase Secondary metabolites are formed Examples - antibiotics , pigments, toxins, etc Monod equation µ = µ max s/(K s + s) s -Residual substrate concentration K s -substrate utilization constant substrate concentration when µ is half µ max A measure of the affinity for substrate µ - Specific growth rate µ max – Maximum Specific growth rate Batch fermentation

9. Application of batch fermentation Various growth conditions used for production of Biomass - fastest growth rate and maximum population 1 o metabolite - extend exponential phase 2 o metabolite - short exponential phase and extended production phase Filling fermenter with medium Sterilization of fermenter and medium Inoculation Production phase Harvesting Cleaning of vessel Disadvantages - High down time - non-productive period

10. Fed batch fermentation Established initially in batch mode Fed continuously or sequentially with medium W ithout removal of culture fluid i ) Same medium - increase in volume ii ) Limiting substrate - same concentration - increase in volume iii) Limiting substrate - concentrated - increase in volume iv) Limiting substrate – very concentrated - no increase in volume i ), ii ) - variable volume fed batch system - fixed volume fed batch system ( iii) - intermediate between the variable and fixed volume systems

11. Continuous fermentation Fresh medium continuously added, Spent broth and cells are removed Exponential growth - prolonged Steady state achieved - new biomass balanced by loss of cells Dilution rate, D=F/V F is flow rate and V is the volume of vessel Change in cell mass over time, dx/ dt = growth - output dx/ dt = µx - Dx Under steady state concentrations, dx/ dt will be zero, then µx = Dx µ = D

12. Continuous fermentation Under steady state conditions S pecific growth rate is controlled by dilution rate Under steady state concentrations, dx/ dt will be zero, then µx = Dx µ = D If dilution rate increase above µmax, complete washout of cells Dilution rate at which washout is just avoided is critical dilution rate ( D crit )

13. Monod equation, µ = µ max s/( K s +s ) Chemostat - Substrate concentration is kept constant Turbidostat - Biomass concentration is kept constant At steady state, µ = D, so D = µ max ŝ/( K s +ŝ ) ŝ - steady state concentration of substrate ŝ = Ks D / (µ max - D) Substrate concentration is influenced by dilution rate Biomass depends upon substrate concentration and thereby on dilution rate Continuous fermentation

14. The concentration of cells in a chemostat at steady state ẍ = Y (S R - S r ) ẍ - steady-state cell concentration in chemostat S R - substrate concentration of inflowing medium Sr - steady-state residual substrate concentration Y - yield factor Biomass concentration under steady state conditions - controlled by S ubstrate feed concentration and Dilution rate Continuous fermentation

15. Advantages and disadvantages of continuous fermentation Down time - much less - more economic Easily automated - require less labour Chances of contamination and strain deterioration – more Control operations – complicated Problems in licensing of product - can’t trace product consignment to raw material batch      

16. References Industrial Microbiology: An Introduction, M J. Waites , N L. Morgan, J S. Rockey , G Higton Principles of fermentation technology, PF Stanbury , A Whittakker , SJ Hall, 1995, Butterworth-Heinemann publications Thank You….