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- 1. Batch, Fed-Batch and ContinuousFermentation Dr. Dhanya KC Assistant Professor, Department of Microbiology St. Mary’s College, Thrissur-680020, Kerala
- 2. Batch, Fed Batchand 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 • Log or exponential phase • Stationary phase • Death or decline phase
- 4. Lag Phase Immediately afterinoculation No apparent growth Time of adaptation Length - reduced – economical commercial Batch fermentation
- 5. Logarithmic or logor 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-SR) x - concentration of biomass Y - yield factor (g biomass / substrate) S - initial substrate concentration SR - residual substrate concentration Yield factor - efficiency of substrate conversion to biomass Batch fermentation
- 7. Death or declinephase - Cessation of growth - depletion of substrate Stationary and death phase – idiophase Secondary metabolites are formed Examples - antibiotics, pigments, toxins, etc Monod equation µ = µmax s/(Ks+ s) s -Residual substrate concentration Ks -substrate utilization constant substrate concentration when µ is half µmax A measure of the affinity for substrate µ - Specific growth rate µmax – Maximum Specific growth rate Batch fermentation
- 8. Death or declinephase - Cessation of growth - depletion of substrate Stationary and death phase – idiophase Secondary metabolites are formed Examples - antibiotics, pigments, toxins, etc Monod equation µ = µmax s/(Ks+ s) s -Residual substrate concentration Ks -substrate utilization constant substrate concentration when µ is half µmax A measure of the affinity for substrate µ - Specific growth rate µmax – Maximum Specific growth rate Batch fermentation
- 9. Application of batchfermentation Various growth conditions used for production of Biomass - fastest growth rate and maximum population 1o metabolite - extend exponential phase 2o metabolite - short exponential phase and extended production phase 1. Filling fermenter with medium 2. Sterilization of fermenter and medium 3. Inoculation 4. Production phase 5. Harvesting 6. 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 • Without 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 iv) - fixed volume fed batch system (iii) - intermediate between the variable and fixed volume systems
- 11. Continuous fermentation Fresh mediumcontinuously 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 steadystate conditions Specific 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 (Dcrit)
- 13. Monod equation, µ =µmax s/(Ks+s) Chemostat - Substrate concentration is kept constant Turbidostat- Biomass concentration is kept constant At steady state, µ = D, so D = µmax ŝ/(Ks+ŝ) ŝ - steady state concentration of substrate ŝ = Ks D / (µmax - D) Substrate concentration is influenced by dilution rate Biomass depends upon substrate concentration and thereby on dilution rate Continuous fermentation
- 14. The concentration ofcells in a chemostat at steady state ẍ = Y (SR- Sr) ẍ - steady-state cell concentration in chemostat SR - substrate concentration of inflowing medium Sr - steady-state residual substrate concentration Y - yield factor Biomass concentration under steady state conditions - controlled by Substrate feed concentration and Dilution rate Continuous fermentation
- 15. Advantages and disadvantagesof 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 1. Industrial Microbiology:An Introduction, M J. Waites, N L. Morgan, J S. Rockey, G Higton 2. Principles of fermentation technology, PF Stanbury, A Whittakker, SJ Hall, 1995, Butterworth-Heinemann publications