media design and sterilization process during industrial fermentation

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(Bioprocess Technology)
Fermentation Media design and Sterilization

• Submitted to
• Dr. Bina Mishra
• Senior Scientist
• Division of Biological Products

• Dr. Arvind Sonwane
• Scientist
• Division of Biological Products

• Dr. C.L Patel
• Scientist
• Division of Biological
• Products

• Submitted by
• Dr. Alok khanduri
• ID- M-5584
• Division of Veterinary Biotechnology

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Outlines of the topic

• Significance of media design.
• Classification of media.
• Various components of media and their sources.
• Sterilization.
• Methods of sterilization.
• Measurement of sterilization.
• Evaluation of efficiency of sterilization.

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Significance of media design

• Maximize biomass or product yield for each gram
  of substrate used.
• Maximize concentration of biomass/product.
• Maximize rate of product formation.
• Minimize risk of undesirable products.
• Minimize problem in media preparation and
  sterilization.
• Minimize problem in safedisposal of effluents
  and waste treatment.

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Aspects of fermentation affected by desired media

• Fermenter design
• Power input for effective stirring(due to
  viscosity)
• pH variation
• Foam formation
• Oxidation-reduction potential
• Downstream processing
• Effluent treatment

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Contd..
.

The medium must
provide all the elements needed for cell biomass
formation and for metabolite production in the
required quantities, and it must provide enough
energy for cell metabolism and maintenance. An
idea of the medium composition is developed from
the equation based on the stoichiometry for
growth and product formation. For an aerobic
process, the equation is as followsC Energy
N2 O2 Other req. Biomass Product CO2
H2O Heat
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Classification of media

• Natural media/Crude media Natural media consist
  of naturally available components , that are of
  complex nature and their chemical composition is
  difficult to determine in precise terms.
• Ex. Media containing cereal grains, slaughter
  house waste, fermentation residues.

• Synthetic media Synthetic media consist of
  defined media components i.e. pure
  chemicals/compounds, therefore their chemical
  composition is precisely known.
• Ex. Media containing molasses, corn steep liquor,
  phenyl acetic acid and lard oil(antifoam) is
  used for production of penicillin.

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Substrates used for media design and their
sourcesCarbon

• Main product of fermentation process depend on
  the choice of Carbon source.
• Rate at which the carbon source is metabolized
  can often influence the formation of biomass or
  production of primary or secondary metabolites.
• Cereal grains , molasses,malt, starch, cellulose,
  sucrose, glucose, lactose and whey are the most
  commonly used sources of carbon.

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Energy source

• Energy comes either from oxidation of medium
  components or from light.
• Most industrial microorganism are chemo-
  organotrophs, therefore the commonest source of
  energy will be the carbon (CHO), lipids
  protein.
• Oils are richer in energy than carbohydrates.
• Some micro-organisms can also use hydrocarbons or
  methanol as energy sources.

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Nitrogen

• Most industrially used microorganisms can utilize
  inorganic or organic sources of nitrogen.
• Inorganic Nitrogen may be supplied as NH3 gas,
  NH4 salts or nitrates. NH3 has been used for pH
  control and as the major Nitrogen source in a
  defined medium.
• Organic nitrogen may be supplied as Amino acids,
  Protein or Urea. Other proteinaceous Nitrogen
  compounds serving as sources of amino acids
  include corn-steep liquor, soya meal, peanut
  meal, fermentation residues and cotton seeds.

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Minerals

• All micro-organism requires certain mineral
  elements for growth metabolism.
• K, Mg, Ca and Fe are normally required in
  relatively large amounts(Macronutrients) and
  should normally always be included as salts in
  media.
• In many media Co, Cu, Zn, Mn, Mb are essentially
  added in small quantities(Micronutrients).
• Phosphorus is used as a Buffer to minimize pH
  changes when external pH is not being used.
• The functions of each vary from serving as
  coenzyme to catalyze many reactions, some act as
  cofactors, vitamin synthesis, and cell wall
  transport.

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Growth Factors

• Vitamins, specific Amino acids, Fatty acids or
  Sterols.
• Many of the natural Carbon and nitrogen sources
  used in media formulation contain all or some of
  the required growth factors.
• If only one vitamins is required it may be more
  economical to add a pure vitamin instead of large
  bulk of cheaper multiple vitamins source.

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Buffers

• The control of pH may be extremely important if
  optimal productivity is to be achieved.
• PO4 which are the part of many media also play an
  important role in buffering. High PO4
  concentration is critical in the production of
  many secondary metabolites.
• Many media are buffered at about pH 7.0 by the
  incorporation of CaCO3.
• Carbon and nitrogen sources are also basis for pH
  control as buffering capacity can be provided by
  the protein, peptides and amino acids.
• The pH may also be controlled externally by
  addition of NH3 or NaOH H2SO4.

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Inducers

• The majority of enzymes which are of industrial
  interest are inducible.
• Induced enzymes are synthesized only in response
  to the presence of an inducer in the environment.
• Inducers are generally used in case of
  genetically modified microorganisms.
• Most inducers which are included in microbial
  enzyme media are substrate or substrate analogues
  but intermediates and products may sometime be
  used as inducers. Examples include starch and
  dextrin for amylase, pectin for pectinase,
  cellulose foe cellulase.

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Inhibitors

• When certain inhibitors are added to fermentation
  more of specific product may be produced.
• Inhibitors have also been used to affect cell
  wall structure and increase the permeability for
  release of metabolites.
• Ex. Penicillin used in production of glutamic
  acid and sodium bisulphate used in the production
  of glycerol by Saccharomyces cerivisiae.

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Antifoaming agents

• In most microbiological process, foaming is a
  problem. It may be due to a component in the
  medium or some factor produced by the
  microorganism. E.g. most common due to protein
  i.e. peanut, soybean, yeast extract or meat
  extract.
• These proteins may denature at the air broth
  interface form a skin which does not rupture
  readily.
• Chemical anti-foam agents (AFA) are surface
  active substances, which decrease the surface
  elasticity of liquids and prevent metastable foam
  formation.
• Ex. Natural- Plant oils(sunflower and rapeseed
  oil), deodorized fish oil, mineral oil and
  tallow.Synthetic- Silicon oil and poly alcohols.

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Sterilization Sterilization  is the process
that eliminates (removes) or kills (deactivates)
all forms of  life and other biological
agents including transmissible agents (such
as bacteria, fungus, viruses) and their spore
forms, present in a specified region, such as a
surface, a volume of fluid, medication, or in a
compound such as biological culture media.
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Methods of sterilization

• Sterilization by heat
• Sterilization by radiation
• Sterilization by chemicals

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Sterilization by heat

• Heating causes denaturation of all the proteins
  and associated structures present in the media
• Heat sterilization are of two types
• a. Dry heat sterilization
• b. Steam sterilization -1. Direct steam
  injection
• 
  2.Indirect steam injection
• Bacterial spores are more resistant to dry heat
  than the wet heat.

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Sterilization by radiation

• Irradiation by ultraviolet light of wavelength
  250-280 nm leads to DNA damage. The main damage
  is the formation of pyrimidine dimers between
  adjacent bases.
• Gama rays and X rays are more useful because of
  their high penetration.
• Ultraviolet irradiation is not very penetrating
  and cannot be relied upon as a sterilizing agent
  unless direct exposure of the contaminating
  organism can be guaranteed.

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Sterilization by chemicals

• Chemical sterilizing agents may kill as a result
  of oxidizing or alkalizing ability.
• Although a number of chemical sterilizing agents
  are known, they cannot be used to sterilize
  nutrient media because there is a risk that
  inhibition of the fermentation organisms could
  occur from the residual chemicals.
• Commonly used chemicals for sterilization include
  formaldehyde gas, hydrogen peroxide, ethylene
  oxide gas and propylene oxide gas.

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Measurement of sterilization

• To kill organisms, by whatever means, a lethal
  dose will be required, which will depend on the
  duration of injurious treatment.
• Thus in heat process the energy transmission-time
  relationship will be paramount, in radiation the
  energy transmission-time and in chemical
  sterilization, the concentration-time
  relationships.

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Contd..

• Thermal death time Shortest time taken to
  destroy the organisms at a stated temperature.
• Thermal death point It is the lowest temperature
  required to kill the organisms in 10 minutes.
• Decimal reduction time(D value) Time in
  minutes(at a given temperature) required to
  reduce the viable population to 10 of its
  previous value.

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Filtration sterilization

• Filtration differs from other
  sterilization methods because organisms are not
  killed but physically removed.
• There are two types of filters used
• a. Depth filters These filters are made up of
  fibrous powdered materials in relatively thick
  layer pressed or bonded together to form a weft
  of multidirectional interconnected channels or
  varying size.

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Contd.. b. Screen filters These filters are
based on simple sieve principle made from
cellulose esters or other polymers.
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Evaluation of sterilization efficiency

• Thermocouplers
• Brownes tubes
• Spore strips
• Thermalog S indicator strips
• Autoclave tapes

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Thanks for the patience..