4. Media for Industrial Fermentation
Introduction
Design of Industrial Fermentation Media
Basic requirements
Water
Energy sources
Carbon / Nitrogen / Mineral Elements / Vitamin
Possible Oxygen for aerobes
Criteria for Media for Industrial Fermentation Process
1) Maximum yield of product / biomass
2) Maximum concentration of product / biomass
3) Maximum rate of product formation
4) Minimum yield of undesired products
5) Consistent quality (through years)
6) Minimal problem during media making and sterilization
7) Minimal problem during product processing
(aeration / agitation / extraction / purification / waste treatment)
Carbon Sources:
Cane Molasses / Beet Molasses / Cereal Grains / Starch
Glucose / Sucrose / Lactose
Nitrogen Sources:
Ammonium salts / Urea / Nitrates / Corn Steep Liquor
Soya Bean Meal / Slaughter-House waste / Fermentation residue
Media Creation – “Cheap” based on Cost
However, pure substrates – simpler processes
Media – Variation of Fermenter Design / Process
Consideration of Media formulation at Lab / Pilot / Industrial Scale
Gas transfer limitation
Viscosity – power input
pH variation (gradient)
Forming problem
Oxidation – Reduction potential (gradient)
Morphological variation of organisms
Precursors / Metabolic Inhibitors
Product recovery / Effluent treatment
In complex natural materials
Batch variation in Components / Impurity
Unpredictable Biomass / Product yield
Undetectable small improvement of fermentation process
Product recovery-Purification / Effluent treatment – High BOD
In molasses – high forming / difficult pH control
So, Industrial process – Simpler substrates for better process
Development – specially recombinant proteins
Typical Media
Experimental Design but, not necessary for best performance (table)
Medium Formulation
Medium formulation for successful process development
Media – support energy requirement for
Cell growth / Biosynthesis / Cell maintenance
In Aerobic fermentation
Carbon / Energy Source + Nitrogen Source + O2 + Others
= Biomass + Product + CO2 + H2O + Heat
Equation – Economical Design
(for minimal waste generation)
Theoretical value for Biomass / Product formation
But, difficult in real fermentation
Elemental Composition
Microorganisms – Element balance equation
(C, H, N, S, P, Mg, Na, Ca and K) Cl(bacteria?)
In media – Phosphate as buffering
Trace Elements
(Fe, Zn, Cu, Mn, Co, Mo, B)
Other nutrients – Based on growth requirement / Biosynthesis capability
Amino acids / Vitamins / Nucleotides
Growth factors
Carbon source – as Dual roles
Energy generation (Cell growth + Maintenance)
Biosynthesis
Dry Mass Yield Coefficient =
(Cell Dry Mass)/(Carbon Utilization) (table)
Water
As a major component of all fermentation
As ancillary service – heating / cooling / cleaning / rinsing
Water – Minerals / Salts
Energy Sources
For growth
Energy by Oxidation / Light
Carbohydrates / Lipids / Proteins
Carbon Sources
Factors Influencing the Choice of Carbon Source
“Rate of Carbon Metabolism” – Biomass / Product formation
Fast growth rate – by rapid carbon metabolisms
Often lower secondary metabolites
Carbon catabolic regulation
Carbon sources – relationship to products
By dissimilation
As Major cost association
So, Industry – develop alternative carbon sources
Based on cost / geographical locations
Carbon sources – Impurity problem
Fe++ concentration
Carbon source – sterilization process
“Maileard reaction” – reducing sugar + Amino
“Gelatinization” of starch
Separate sterilization
Government regulation
Subsidization
Local Brand protection
Examples of Carbon Sources
Carbohydrates
Oils and Fats – Higher energy production
(2.4X than glucose)
Antifoam properties – better recovery
Hydrocarbons
Lower cost
Nitrogen Sources
Inorganic Vs. Organic sources of Nitrogen
Inorganic Nitrogen Sources
Ammonia / Ammonium salts / Nitrates
Organic Nitrogen Sources
Amino acids / Proteins / Urea
Protein Hydrolysate – Cheaper sources
High proteins containing wastes – complex medium
Corn-steep liquor
Seeds meals
Factors Influencing the Choice of Nitrogen Source
Nitrogen Regulations
Nitrate – conversion of nitrate to ammonium ion
“Nitrate reductase”
Ammonia repression on amino acid uptaking
pH control as Salts
pH buffering by phosphate – reducing antibiotic
formation
Complex medium – may cause problem in recovery
Minerals
Essential for Growth / Metabolisms
Magnesium / Phosphorus / Potassium / Sulphur
Calcium / Chlorine as Addition
Cobalt / Copper / Iron / Manganese / Molybdeum / Zinc
As usually impurities of complex media
Product composition – require higher concentration
Phosphate – as pH controlling
Secondary metabolites formation
Mineral toxicity – Lower tolerance ranges
Manganese / Iron / Zinc – Critical for secondary metabolite
Insufficient Vs. Toxic for cell growth
Chelators
During sterilization – Precipitation of Insoluble metal phosphates
“Chelator” – prevent precipitation
EDTA / Citric acid / Polyphosphate
Formation of Complex formation with metals
Slower use of metal ions by Microoganisms
In industrial media
Complex ingredient – Yeast extract / Peptone
Complex with metal ions
Gradual release during growth
Growth Factors
Limiting synthesis of full components of cell components
Vitamins / Amino Acids / Fatty acids / Sterols as growth factors
Complex ingredients – usually sufficient supply
Calcium pantothenate / Biotin / Thiamine
Nutrient Recycle
In continuous culture – Cost reduction
Buffers
pH control – relation with Biomass / Product formation
Calcium Carbonate / Phosphate salts
Balances of carbon / Nitrogen sources
The Addition of Precursors and Metabolic Regulators to Medium
Precursors / Inhibitors / Inducers
Precursors – side chain modification (phenylalanine)
Inhibitors – Product formation / Metabolism rearrange
Inducers – Enzyme inducers
Oxygen Requiements
Controlling growth rate / Metabolic production
Oxygen availability
a. Fast metabolism – higher oxygen demand
b. Rheology – Media Viscosity by Individual components
c. Antiform – surface active agents
Limiting oxygen transfer rate
Antifoams
“Foam” – mainly due to proteins in medium
Protein denaturing at air-broth interface
Foam cause cell removals – cause “autolysis”
Microbial proteins – worsen foaming problem
Foam – Physical / Biological problems
Exhausted gas circulation
Bubble size of air
Lower mass / heat transfer
Probe interference
Biological Consequence
Deposition of cells – wall formation
Sterilization problem – wet filter
Microbial infection
Siphoning – product loss
Foaming Patten in Fermentor
1. Constant foam level
Initially Media, then M/O activity
2. Steady falling foaming
Initially Media, no M/O acitivity
3. Early falling foam, but rising foaming
Slight effect by Medium
Major foaming by M/O activity
4. Lower initial foaming, then rising
Solely to M/O activity
5. Complex foaming pattern
Combination of Medium & M/O
Foam Control
1. Use Defined / Modification of Medium
pH / Temp / Aeration / Agitation
if medium components is major problem
2. Antifoam – as Standard approach
3. Use of Mechanical foam breaker
Antifoam – Surface agents
Destabilizing protein films by
Hydrophobic bridges between two surfaces
Displacement of absorbed protein
Rapid spreading on the surface of film
Ideal Antifoam Properties
1. Rapid dispersion – fast action
2. Active at lower concentration
3. Long acting in preventing foam formation
4. Not metabolized
5. Non-toxic to M/O
6. Non-toxic to human / animal
7. Not causing problem in Extraction / Purification
8. No Hazard in handling
9. Cheap
10.No effect on oxygen transfer
11.Heat sterilizable
Some Industrial Antifoam
1. Alcohols – longer fatty alcohols
2. Esters
3. Fatty acids
4. Silicones
5. Sulfonates
6. Miscellaneous – Polymers
In Industry,
Foaming Control – as an “Empirical Art”
Medium Optimization
Classical Approach – Changing one independent variable
Nutrient / Antifoam / pH / Temp
But, factorial combination – expensive / time consuming
Alternative strategies
Statistical approach
Relationship between independent variables
Animal Tissue Culture Medium
~40% Unit cost
Serum - ~80% of medium cost
Serum – Containing 1000 components
But, all components is not required cell growth or
cell differentiation
Should be free of bacterial / viral / BSE contamination
Serum-free media supplements
Components for cell growth / cell differentiation
1.
2.
3.
4.
consistent / definable medium composition
reduction of potential contamination
potential cost saving
Simplifying downstream processing – less protein
Serum replacements
Albumin / Insulin / Transferrin / Ethanolamine
Selenium / B-mercaptoethanol etc.
Protein-Free medium – Attractive objective
Trace Elements / Osmolality / pH buffering
Non-Nutritional media supplements
Sodium carboxy methyl cellulose – mechanical damage
Pluronic F-68 (polyglycol) – stirring / sparging
Chapter 5 Sterilization
If foreign microorganisms exist in Fermenter,
i) Medium supports both organisms – loss productivity
i) In Continuous fermentation – contaminant outgrowth
ii) In single cell protein fermentation – a part of product
iii) Contaminant products – difficult to recovery
iv) Contaminant – product degradation
v) Phage contamination – cell lysis
To avoid contamination
i) Using pure inoculum
ii) Sterilizing the medium to be employed
iii)
Sterilizing the fermenter vessel
iv)
Sterilizing all materials to be added to the fermentation during
process
v) Maintaining aseptic conditions during the fermentation
Probability of contamination & the Nature of consequences
New procedure development – “Protected”
Medium – limited Microorganisms can grow
Medium – cell growth as selective force
(pH reduction as cell grows)
Beer fermentation
Hop resin – inhibition of M/O growth
Brewing – lowering pH
So, May not need to be sterilization
Medium Sterilization
Sterilization by Filtration / Radiation / Ultrasonic treatment
Chemical Treatment / Heat
But, Universal method for sterilization by “Steam”
A few animal cell cultures – filtration due “heat labile”
Steam sterilization
Kinetic of Microbial destruction
-dN/dt = kN (k= specific death rate)
Consideration of “Total number” not “concentration
Bacillus “Endospore” as “Reference”
As heat resistant nature of spores
“Bacillus stearothermophilus”
Media compositions
Fat / oil – less humidity
Longer sterilization time
During sterilization
Heat – Reduction of Nutritive quality
Initially “Cooking effect”
“availability of nutrition”
Heat detrimental effect on medium
1) Interaction between nutrient
components of medium
“ Maillard Reaction”
Reducing sugar + amino acids
Separate sterilization
2) Degradation of heat labile
components
Vitamins / Amino acids / Proteins
So, High Heat / Short time process for better results
Time / Temperature dependent of sterilization
Better destruction of M/O
Better preservation of Nutrients
Advantage of Continuous sterilization over Batch sterilization
1)
2)
3)
4)
5)
6)
Superior maintenance of medium quality
Ease scale-up
Easier automation
The reduction of surge capacity for stream
The reduction of sterilization cycle time
(Some) the reduction of fermentor corrosion
Advantage of Batch sterilization over continuous sterilization
1) Lower capital investment
2) Lower risk of contamination
3) Easier manual control
4) Easier control of high solid content medium
Continuous Sterilization – Using “Heat Exchanger”
1) Possible failure of gaskets
2) Particulate components – blocking
The Design of Batch Sterilization Process
Preferred by Industry because of easy operation
Sterility Vs. Minimum Nutrition damage
Batch sterilization at 121oC
Heating / Cooling period should be considered
Based on Temperature – Time Profile
Overall = Heating + Holding + Cooling
Nutritional damage
As scale-up increased, nutritional damage increased
By longer Heating / Cooling periods
Methods of Batch Sterilization
In situ Medium sterilization vs. Special Vessel (Mash cooker)
Sterilization vessel
1) Saving time : Fermenter clean-up during sterilization
2) High concentrated medium sterilization – smaller
cooker (less heating / cooling time)
3) Medium viscosity increased during sterilization
High power for sterilization vessel for multiple
fermenters
4) Less corrosion of fermenter at high temperature
But, disadvantage
1) Capital cost
2) Transfer line as inherent danger of contamination
3) Mechanical failure – multiple fermenters affected
In Industry
Large scale – “longer “Down Time” of in situ fermentation
Continuous system is preferred
The Design of Continuous Sterilization Process
Time / Temperature dependent like Batch system
But, Higher temperature / Short time
Less heating / Cooling time
2 Heat exchangers – Heat / Cooling
Direct vs. Indirect Heat Exchangers
Direct heat exchanger – Steam Injector
Advantages
1) short heating up time
2) better suspended solid
3) Lower capital invest
4) Easy operation
5) High efficiency of steam utilization
Disadvantages
1) Forming
2) Condense steam – Dilution
3) “Clean Steam” due to corrosion
Cooling
Flash cooling through expansion valve to
steam chamber (Instant cooling)
Indirect Heat exchanger
Double-spiral type
Countercurrent stream
Holding coil – used steam for partial heat
Less contamination as use end-gaskets
Higher clearance for suspended solids
Plate heat exchanger
Countercurrent – Between gaskets
May cross-contamination as gasket failure
Higher capacity as additional plates
Combination of Direct / Indirect Heat Exchanger
Starch – Rapid heating by steam injection
Prevent gelatinization at pre-heating
Industrial Sterilization Process : ‘Over-design”
Specially, high solid particle medium
Particle protection of M/O
Not practice for steam injection
Small scale vs. Large scale
During sterilization process – Ingredient interaction
Sterilization of the Fermenter
When separate system, fermenter sterilization
By Steam injection
Sparging steam
15 psi for 20 min
Following Sterilization – “Positive Pressure” to avoid vaccum
Sterilization of the Feeds
Sterilization of various feeds – dependent upon nature of additives
Sterilization of Liquid Wastes
Specially “Recombinant Strains” – Strict contamination regulation
Sterilization under contained conditions
Discharge at below 60oC
Sterilization kinetics based on Organisms (not spore)
Should be validated!
Filter Sterilization
For Liquid sterilization
Removal of suspended particles from Liquid
a) Inertial impaction
b) Diffusion
c) Electrostatic attraction
d) Interception
Inertial Impaction
Particles remain in fibre
More significant in the filtration of gases than in the
filtration of liquids
Diffusion
Small particles – Brown movement
More significant in the filtration of gases than in the
filtration of liquids
Interception
Larger particles than pore size – direct interception
Smaller particles – retained by interception
Trapping by irregularity of particles
Equally important for gas and liquid
Two types of filter
Absolute filter – smaller pore size
Depth filter – “non-fixed pore filters”
Superior results, but flow resistance – pressure drop
But, filter should be sterilized before use
Filter Sterilization of Fermentation Media
Heat-labile proteins – specially animal tissue culture media
Filter sterilization of Animal Tissue Culture Medium
Criteria
1) Free of fungal / Bacterial / Mycoplasma
2) Minimal adsorption of protein on filter
3) Free of virus
4) Free of Endotoxins
Absolute Filter System
Steam Sterilizable Hydrophobic Materials
Membrane coating – Prevent protein adsorption
Ex) multiple filtration systems to meet criteria
Prefilter – 5um / Positively charged
Polypropylene
Second filter – 0.5um / Positively charged
Polypropylene
M/O removal
Endotoxin reduction
Third filter – 0.1um ; Nylon / Polyester
M/O removal
Endotoxin Reduction
Fourth filter – similar to third filter
Mycoplasma removal
Endotoxin removal – final
For viral removal – 0.04um Nylon / Polyester
Filter Sterilization of Air
Fixed pore filters – Absolute filter
Usually PTFE – Hydrophobic : prevent “Wetting”
Prefilter to remove particles / corrosives / oils
Sterilization of Fermenter Exhaust Air
Awareness of safety
Emission level
Exhaust air – water saturation
Foam over flow
Prefilter or Mechanical separator
The Theory of Depth Filter
Collection Efficiency
Characteristics of filter
It’s components
Glass wool
Glass fibre
Norite
Activated Carbon
Chapter 6
Development of Inocular
Inoculum Criteria
1) Healthy / Active to minimize ‘Lag period”
2) Sufficient large volume – optimum size
3) Suitable morphological form – Physiological condition
4) Free of Contamination
5) Retaining its product-forming capabilities
Inoculum Development
Effective inoculum development to minimize batch variation
Critical factor for inoculum – Choice of the Culture Medium
Sufficient similar to production medium for less lag time
Reduce pH / Osmotic pressure / Ion balance shock – Viability
Ex) Penicillin production – repressed inoculum development
As Selection pressure for non-producing organisms
cf) Production medium – maximum product formation
Inoculum Level
3-10% of medium volume
Serial Development from stock – multiple stage
Possible Contamination / Strain Degeneration
Higher volume of inoculum – Higher cost
Inoculum Development Program
Master culture
Sub-master culture – for production run
Shake flask culture – (for check productivity)
Large flask / Lab fermenter
Pilot-scale fermenter
At each stage – Culture Purity Check for contamination
Criteria for the Transfer of Inoculum
Optimum transfer time to Keep Physiological condition – “Age”
Standardization of culture conditions & Monitoring culture
Biomass as a key parameter
Packed cell volume
Dry weight
Wet weight
Turbidity
Respiration
Residual Nutrient Concentration
Morphological form
On-line monitoring as indicators of physiological condition
pH
Oxygen / Carbon Dioxide in effluent gas
Biomass sensor
Dielectric permittivity of viable yeast cells
The Aseptic Inoculation of Plant Fermentors
Inoculum as suspended vegetable culture
Spore suspension
Inoculum transfer – Containment of microorganisms
Based on safety level