Principle of Fermentation Technology

					4. Media for Industrial Fermentation

      Design of Industrial Fermentation Media
               Basic requirements
                     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)
             Dry Mass Yield Coefficient =
                     (Cell Dry Mass)/(Carbon Utilization) (table)
        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
                      Local Brand protection
            Examples of Carbon Sources
                   Oils and Fats – Higher energy production
                          (2.4X than glucose)
                          Antifoam properties – better recovery
                          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

            Complex medium – may cause problem in recovery
      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

      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

      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

             “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. consistent / definable medium composition
            2. reduction of potential contamination
            3. potential cost saving
            4. 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
    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
                            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) Superior maintenance of medium quality
      2) Ease scale-up
      3) Easier automation
      4) The reduction of surge capacity for stream
      5) The reduction of sterilization cycle time
      6) (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
                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
                           1) short heating up time
                           2) better suspended solid
                           3) Lower capital invest
                           4) Easy operation
                           5) High efficiency of steam utilization
                           1) Forming
                           2) Condense steam – Dilution
                           3) “Clean Steam” due to corrosion

                           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

               Small particles – Brown movement
               More significant in the filtration of gases than in the
               filtration of liquids

               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
                       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
                       Second filter – 0.5um / Positively charged
                                      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
                    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
                     Residual Nutrient Concentration
                     Morphological form

              On-line monitoring as indicators of physiological condition
                     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

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