Teaching Aids Service by KRRC Information Section

Fermenter - Basic Function The basic function of a fermenter is to provide a suitable environment in which an organism can efficiently produce a target product that may be - cell biomass, - a metabolite, - or bioconversion product.

INOCULUM DEVELOPMENT The inoculum is the starter culture that is injected into the fermenter It must be of sufficient size for optimal growth kinetics Since the production fermenter in industrial fermentations is so large, the inoculum volume has to be quite large

 The preparation of a population on microorganisms from a dormant stock culture to an active state of growth that is suitable for inoculation in the final production stage is called inoculum development.  As a first step in inoculum development, inoculum is taken from a working stock culture to initiate growth in a suitable liquid medium  It is usually done in a stepwise manner to increase the volume to the desired level.  Typically the inoculum volume used for production stage is about 5% of the medium volume.  Inoculum preparation media are designed for rapid microbial growth.  production process depend on inducible enzymes.  Contamination free during inoculum development.

Microbes brings about fermentation by secreting certain enzymes which have an optimum temperature or a temperature range. Temperature: Temperature affects the solubility and diffusivity of oxygen in the fermentation broth. The solubility of oxygen decreases but diffusivity increase with the rise in temperature.  Cooling water in cooling jacket or coil Temp. control methods

HEAT TRANSFER CONFIGURATIONS  The primary heat transfer configurations in fermentation vessels are: i. External jackets ii. Internal coils iii. External surface heat exchanger  The internal coils though provide better heat transfer capabilities, but they cause problems of microbial film growth on coil surfaces, alteration of mixing patterns and fluid velocities.  The external surface heat exchangers, the media is pumped through an external heat exchanger where the heat transfer takes place through the surface of exchanger tubes.

pH control  Most microorganisms have narrow pH growth ranges (pH )  The buffering in culture media is generally low  Metabolites which released into the medium can change the pH  Initial pH of the fermentation medium must be very well defined and optimized depending on the microorganism, substrate and production technique  Addition of base ( NaOH ) or acid ( HCl )  Addition of physiological acid substance ((NH 4 ) 2 SO 4 ) or physiological alkali substance (ammonium hydroxide)

Dissolved oxygen (DO ） Control  Most industrial fermentations are aerobic processes  Supplying oxygen to aerobic cells has a problem ： oxygen is poorly soluble in water  Oxygen supplying is the rate limiting step in an aerobic fermentation  The objective is to maintain the optimal dissolved oxygen concentration above a critical concentration to avoid inhibition of the cell growth rate due to lack of oxygen.

Factors affecting dissolved oxygen concentration  Temperature  Elevation  Salinity  Turbulence  Increase aeration and agitation rate maintain a constant dissolved oxygen concentration during the fermentation process

Important factor in a fermenters Provision for adequate mixing of its contents Mixing in fermentation  to disperse the air bubbles  to suspend the cells  to enhance heat and mass transfer in the medium

AERATION AND AGITATION  Aeration refers to the process of introducing air to increase oxygen concentration in liquids  Aeration may be performed by bubbling air through the liquid, spraying the liquid into the air or agitation of the liquid to increase surface absorption  Agitation – uniform suspension of microbial cells in homogeneous nutrient medium

STRUCTURAL COMPONENTS INVOLVED IN AERATION AND AGITATION  Agitator (impeller)  Baffles  Aeration system (sparger)

AGITATOR (impeller) Achieve mixing objectives – bulk fluid and gas-phase mixing, air dispersion, oxygen transfer, heat transfer, suspension of solid particles and maintaining uniform environment throughout vessel contents.

Radial flow impellers - Rushton turbine  The most commonly used agitator in microbial fermentations  Like all radial flow impellers, the Rushton turbine is designed to provide the high shear conditions required for breaking bubbles and thus increasing the oxygen transfer rate. Agitator design and operation

AERATION SYSTEM (SPARGER)  Introduces air into liquid of fermenter  Three basic types – porous sparger 1.Orifice sparger – a perforated pipe 2.Nozzle sparger – an open or partially closed pipe 3.Combined sparger-agitator

 Sparger structures can affect the overall transfer of oxygen into the medium, as it “influences the size of the gas bubbles produced.  Small bubbles are desirable because the smaller the bubble, the larger the surface area to volume ratio, which provides greater oxygen transfer.  However, spargers with small pores that are effective in producing small air bubbles are more prone to blockage and require a higher energy input.  The availability of the oxygen depends on:  · Solubility  · Mass transfer rate of oxygen in the fermentation broth  · Rate of utilization of DO by microbial biomass.

MEDIA FOR INDUSTRIAL FERMENTATIONS The media is the feed solution  It must contain the essential nutrients needed for the microbe to grow Factors of consideration when choosing media -Quality consistence and availability -Ensure there are no problems with Media Prep or other aspects of production process Ex. Cane molasses, beet molasses, cereal grains

 Where biomass or primary metabolites are the target product, the objective is to provide a production medium that allows optimal growth of the m/o  Secondary metabolite production is not growth related. Consequently, media are designed to provide an initial period of cell growth, followed by conditions optimized for secondary metabolite production. At this point the supply of one or more nutrients (carbon, phosphorus or nitrogen source) may be limited and rapid growth ceases

 Compounds that are rapidly metabolize may repress product formation.  Certain media nutrients or environmental conditions may affect the physiology, biochemistry, and morphology of the microorganism.  In some yeasts the single cells may develop into pseudo-mycelium or flocculate, and filamentous fungi may form pellets. This is not desirable as it affects the product yield

Factors influencing the choice of C-source  The rate at which C-sources metabolized  Price & availability  Media sterilization  Carbohydrates: Starch from cereal, grains and maize; malt from barley; sucrose from sugar cane; impure form: cane molasses, corn steep liquor, whey from diary industry  Oil & Fats: Fatty acid contents (also antifoaming properties)) Hyrdocarbons and their derivatives: n-alkanes for production of organic acids, aminoacids, vitamins Molasses, a byproduct of sugar production, is one of the cheapest sources of carbohydrate. Malt extract, an aqueous extract of malted barley, is an excellent substrate for many fungi, yeasts, and actinomycetes. Nitrogrn sources: yeast extract, soyabean meal, corn steep liquor, peptone etc. Carbon Sources

FOAMING Foams consist of liquid lamellas filled with gas How is foam formed?  Surface-active media (peptides) and foaming components (proteins)  High aeration and agitation rates Foaming  Removal of cells from culture  Physical changes  Reduction in working volume  Lower mass & heat ratios  Decrease in sterility

FOAM CONTROL METHODS 1.Mechanical defoaming Foam-breaker （ Defoamers ）  Defoaming blades in fermentation tank  Separate rotor in the upper area of the culture tank 2. Chemical defoaming Addition of chemical antifoam agents in the beginning （ 1 ） Animal and vegetable oils, 0.1%  0.2% （ 2 ） Polyether surfactant （ Defoamer GPE-1 ）, 0.02%-0.03%

Ideal antifoam  Should disperse easily and have fast action on foam  Should be active at low concentrations  Should be long acting in preventing new foam formation  Should not be metabolized by m/o  Should be nontoxic to m/o  Should not cause any problem in the extraction step  Should be cheap  Should be sterilazable Examples: Silicones, sulphonates, esters, fatty acids, alcohols

Criteria in the choice of organism: 1.The nutrional characteristics of the organism 2.The optimum temperature of the organism 3.The toxicity of organism and the product 4.The suitability of the organism 5.The stability of organism 6.The productivity of organism 7.The ease of product recovery from culture STRAIN OPTIMIZATION

 In selecting strains or mutants for large-scale production, several important factors need consideration. These include stability of strains without undergoing physiological or biochemical degeneration upon subculture for mass propagation, non-utilization of the acid formed and non-formation of the other metabolic acids like gluconic, oxalic, and malic acid Isolation: Long + expensive procedure it is essential to – keep the desirable characteristics – eliminate genetic change – retain viability – protect against contamination Avoid subculturing : Small probability of having mutations at each cell division, repeated sub- culturing carries the risk of contamination..

STRAIN IMPROVEMENT Three approaches can be used  Mutant strain  Recombination  Recombination DNA technology  Many mutation bring about marked changes in a biochemical character of practical interest called as major mutants.  A mutant strain of Streptomyces aurofaciens produces 6- dimethyl tetracyline in prrsence of tetracyline, this demethylated form of tetracyline is the major commercial tetracyline.

 Most improvements in biochemical production have been due to the step wise accumulation of Minor genes.  Penicillium chrysogenum – increased peniciilin production  Each cycle of selection was preceeded by mutagen (chemical) treatment and gave only small increase in yield.  Several cycles (dozen) of selection, a strain (E15-1) was obtained that yielded 55% more penicillin than the original strain

Recombination As formation of new gene combinations among those present in different strains Once several different mutants have been isolated, recombination is use for both genetic analysis as well as strain Improvement. Recombination DNA technology  It involves the isolation and cloning of genes of interest, production of the necessary gene constructs using appropriate enzymes and then transfer and expression of these gens into an appropriate host organism.

Fermentation Basics (Kinetics) Four Phases of Bacterial Growth Curve  Lag phase  Log phase  Stationary phase  Death phase

Lag Phase  Period of adjustment to new conditions  Cell growth is minimal Log Phase  Cell growth rate and metabolic activity are the highest Number of cells produced ﹥ Number of cells dying  Cells are most susceptible to adverse environmental factors (e.g. radiation, antibiotics)

Stationary Phase  Population size begins to stabilize Number of cells produced = Number of cells dying  Factors that slow down microbial growth Accumulation of toxic waste materials Acidic pH of media Limited nutrients Insufficient oxygen supply

Death or Decline Phase  Population size begins to decrease Number of cells dying > Number of cells produced  Most of the nutrients in the medium have been consumed