What are the three main processes in an industrial scale fermentation?
- Upstream processing
- Fermentation process
- Downstream processing.
Describe the three types of fermentation medium.
- Defined medium: Medium composed of pure ingredients in measured concentrations.
- Complex medium: Composed of substrates with undefined composition. Rich in nutrients.
- Technical medium: Cheap, complex substrates such as barley, malt, molasses etc.
State the four criteria for choosing an industrial fermentation medium.
- Maximum yield
- Minimum undesirable metabolites.
- Consistent quality
- Minimal problems during sterilization and fermentation.
How can a medium be sterilized?
- High temperature
- Membrane filtration
- Microwave irradiation
- High voltage pulses
What is used as the indicator for complete sterilization?
- Endospores
Compare between batch sterilization and continuous sterilization.
Batch:
- Medium go through the heating, holding and cooling cycles as batches.
- Low capital, low risk of contamination, easier to control and suitable for media containing high amount of solids.
Continuous:
- Consist of heat exchangers and a holding section to maintain the medium at sterilization temperature.
- Energy efficient, low nutrient degradation.
- In efficient sterilization due to the presence of solids.
What type of filter is used for the sterilization of air?
- PTFE (Polytetrafluoroethylene, Teflon) membrane
- 0.2-0.01um pore size
- Hydrophobic and water resistant.
What are the differences between seed fermenter and production fermenter?
- Seed fermenter is for growing the starter culture (inoculum) while the production fermenter is for producing the product.
What are the factors to be considered for selecting a microorganism for industrial scale fermentation?
- Nutritional requirement
- Optimum temperature of microorganisms
- Equipment and process requirement of the microorganism
- Stability of organism
- Productivity of the organism
- Ease of product recovery
How can inoculum be added for fermentation?
- Spontaneous fermentation: Fermentation by indigenous microflora of pre-treated raw materials.
- Back-slop fermentation: Inoculation of part of a fermented product from previous batch
- Addition of starter culture: Indigenous microflora is inactivated prior to addition.
Compare the two types of fermentation
Submerged fermentation:
- Rheological problem at high substrate conc. (Difficult to flow)
- Slow mass transfer from gas to liquid.
- Better mixing.
- Prone to contamination
Solid state fermentation:
- Solid substrates, no processing of raw materials required.
- Less contamination, lower power consumption.
Compare the three modes of fermentation.
Batch fermentation:
- Fermenter is filled with substrates and added with inoculum. After some time, products are drawn off and fermenter is sterilized for the next batch.
Fed-batch fermentation:
- Cells are grown under batch regime, the reactor is fed with substrates without removing culture fluid.
- Balanced feed keeps the desired specific growth rate.
Continuous fermentation:
- Cells are maintained in a state of balanced growth where substrate are added and products are removed constantly.
- Rate of adding fresh medium = Rate of removing culture medium.
Compare the two types of continuous fermenter.
Chemostat:
- Fresh medium is continuously added while culture liquid are removed at the same rate. Culture volume is constant
Auxostat:
- Uses feedback from a measurement taken on the growth chamber to control the media flow rate. (e.g. pH auxostat)
Describe the cell productivity in a continuous fermentation system.
- Amount of cells increase when the amount of feed increases.
- When Dmax (Maximum dilution) is reached, cell will not grow faster because steady-state equilibrium is reached.
- After dilution rate exceeding Dmax, cell concentration decrease. There will be a steep increase in substrate concentration as there are fewer cells to utilize it.
- At some point, cells will be washed out, the feed will have diluted the reactor to the point where no cells are left.
Describe the parts of a fermenter and their uses.
“MIB WISPOR”
Motor: Powers the impeller
Impeller: Mixes the contents of the fermenter
Baffles: Prevents formation of vortex
Water jacket: Controls the temperature
Inlets: Addition of nutrients and other substances
Sparger: Adds oxygen to the liquid
Probes: Measure and monitor various parameters such as pH and temperature
Outlets: Allow for the removal of samples and the final product
Rakes: Suppress foam.
Compare the different types of bioreactors.
Stirred tank bioreactor:
- Agitator mixes the medium.
- High oxygen transfer rate and low cost.
Air lift bioreactor:
- No impeller, mixing is done by air.
- Low risk of contamination, lower shear stress, increased oxygen solubility,
- High power consumption, excessive foaming, cell damage from bubbles bursting.
Fluidized bed bioreactor:
- For cells that have been immobilized onto particulate matter
- Suitable for shear sensitive cells, good mixing, high density particles can be used
- Carrier abrasion and damage, high pumping costs and leakage.
What are the variables to be controlled during fermentation?
Physical variables:
Temp, impeller speed, aeration rate, pressure
Chemical variables:
pH, dissolved oxygen & CO2, redox potential
Biological variables:
Biomass, oxygen uptake rate, CO2 production rate and respiration quotient.
What is respiration quotient?
The ratio between CO2 production rate and oxygen uptake rate. (CPR/OUR)
What are the four processes of product (fermented) recovery and purification?
SDCC
- Cell separation
- Cell disruption
- Clarification
- Concentration
Describe cell disruption and provide some examples of the methods for cell disruption.
Cell disruption is a method to break open the cell to extract the intracellular metabolites/products. Some cell disruption methods include:
- Freeze-thaw: Cycling between dry ice and 37C water bath
- High pressure homogenization: High pressure forces cells through a small gap causes shear and cavitation which disrupts cell walls & membranes.
- Sonication: Use of ultrasound
- Detergent lysis: Use of non-ionic detergent such as tween, triton etc.
- Solid shear: French press and hughes press
What are some method of cell separation?
- Settling
- Centrifugation
- Dead-end filtration
- Cross-flow filtration
Compare between dead-end filtration and crossflow filtration.
Dead-end filtration:
- Suitable when amount of particles to be removed is low
- Low cost
- Susceptible to clogging
Crossflow filtration:
- Suitable when amount of particles to be removed is high
- Higher cost
- Less susceptible to clogging.
Based on size exclusion, the types of filtration process can be classified as?
- Reverse Osmosis
- Nanofiltration
- Ultrafiltration
- Microfiltration
