FOOD FERMENTATION

TECHNOLOGY
(FSI 26703)

TOPIC 6: FERMENTATION MEDIA

Nurhayati Yusof, PhD

nurhayatiyusof@unisza.edu.my
ext: 3587
CISCO WEBEX LINK
Lecture FSI26703 Topic 6: Fermentation Media
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Thursday, Jun 4, 2020 9:00 am | 2 hours | (UTC+08:00) Kuala Lumpur, Singapore
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Fermentation classified on the basis of
substrate used
 Submerged fermentation (SmF)/liquid fermentation
 Solid state fermentation (SSF)

Development of this fermentation techniques has leads to industrial level

production of bioactive compounds such as antibiotics, pigments, antioxidants,
antitumor agent, bio-surfactants, bioactive peptides etc.

The metabolism exhibited by microorganism is different in SSF and SMF. The

influx of nutrients and efflux of waste materials needs to be carried out based on
the metabolic parameters.
Submerged Fermentations (SmF)
 Submerged liquid fermentations are traditionally used for the production of
microbial derived enzymes. Submerged fermentations involves submersion
of the microorganism in an aqueous solution containing all the nutrients
needed for growth.
 Fermentation takes place in large vessels (fermenter) with volumes of up
1,000 cubic meters. The fermentation media sterilizes nutrients based on
renewable raw materials like maize, sugar and soya.
 Most industrial enzymes are secreted by microorganisms into the
fermentation medium in order to break down the carbon and nitrogen
sources, batch-fed and continuous fermentation process are common. In the
batch-fed process, sterilized nutrients are added to the fermenter during the
growth of the biomass.
 In the continuous process, sterilized liquid nutrients are fed into the fermenter
at the same flow rate as the fermentation broth leaving the system.
 Parameters like temperature, pH, oxygen consumption and carbon dioxide
formation are measured and controlled to optimize the fermentation process.
 In harvesting enzymes from the fermentation medium, insoluble products
must be removed, e.g. microbial cells. This is normally done by
centrifugation.
 As most industrial enzymes are extracellular (secreted by cells into the
external environment they remain in the fermented broth after the biomass
has been removed.
 The enzymes in the remaining broth are then concentrated by evaporation,
membrane filtration or crystallization depending on their intended application.
 If pure enzyme preparations are required, they are usually isolated by gel or
ion exchange chromatography.
Solid State Fermentation (SSF)
 SSF is used for the production of bioproducts from microorganisms under
conditions of low moisture content for growth. The medium used for SSF is
usually a solid substrate (e.g. rice bran, wheat bran, or grain), which
requires no processing.
 Fewer problems due to contamination are observed.
 The power requirements are lower than submerged fermentation
 Inadequate mixing, limitations of nutrients diffusion, metabolic heat
accumulation, and ineffective process control renders SSF generally
applicable for low value products with less monitoring and control.
Soild State Fermentation (SSF) and Submerged
Fermentation (SmF)
Growth medium
 Microorganisms used for fermentation process on or in growth medium
which satisfies the nutritional needs of microbes
 Complete analysis is needed to be done to establish the most favorable
medium for the growth of the microbe used for fermentation
 Formulating medium at lab scale can be done by adding main ingredients
like water, carbon source, nitrogen source, minerals ad other supplements
in pure and in required quantities is very easy which supported the
satisfactory growth of the same organisms at industrial level.
On a large scale one must normally use sources of nutrients to
create a medium which will meet as many as possible of the
following criteria:
•The use of sugarcan molasses, beet molasses, cereal grains, starch, glucose,
sucrose & lactose as carbon (C) source and ammonium salts (NH4), nitrates,
corn steep liquor, soya bean meal, slaughter house waste and fermentation
residues as nitrogen (N) source – meet the above criteria for production media
because they are cheap material.

•On contrary, some sensitive fermentation makes use of glucose, sucrose and
other carbohydrates in their pure form which ensures the purity and quality of
the final products.

•At lab level, peptone or tryptone beef extract which is partially digested
hydrolysate, which is utilized in synthesis of proteins, components of nucleic
acids and other essential cellular components.

•Where growth factors, vitamins, precursors, inducers and trace elements

directly supports the growth of microbe and anti-foaming agents are added to
prevent the foam formulation, in case of presence of higher concentrations of
metal ions which is not preferable chelating agents are added.
 Medium Formulation
 Medium formulation is an essential stage in the design of successful laboratory
experiments, pilot – scale development and manufacturing process.
 The constituent of the medium must satisfy the elemental requirements for cell biomass
and metabolite production and there must be an adequate supply of energy for
biosynthesis.
C + N+ O2 +other req (energy+ sources+ nutrients) ------- biomass + product + CO 2 + H2O + Heat

 This equation should be expressed in quantitative terms, which is important in the

economical design of media if component wastage is to be minimal.
 Thus, to calculate the minimal quantities of nutrients which will be needed to produce a
specific amount of biomass, it should calculate substrate concentration necessary to
produce required product yields.
 Carbon (C) requirement for biomass production under aerobic condition may be estimated the
cellular yield Y which is defined as
Quantity of cell dry matter produced/quantity of C substrate utilized.

 Oxygen – provided by aerating the culture, the design of medium will influence the oxygen
demand of a culture in that the more reduced C sources will result in a higher O2 demand.
 Carbon Sources
Factor influencing 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.
 Fast growth due to high concentrations of rapidly metabolized sugars is often associated with low
productivity of secondary metabolites.
 A carbon source is required for all biosynthesis leading to reproduction,
product formation and cell maintenance. In most fermentations it also serve
as the energy source.
 Molasses
 Malted barley
 Starch and dextrins
 Sulphite waste liquor
 Alkanes and alcohols n-alkanes
 Oils and fats
 Factors influencing the carbon source
 Cost of the product
 Rate at which it is metabolized
 Geographical locations
 Government regulations
 Cellular yield coeffificient
 Carbohydrates (CHO)
 Its common practice to use carbohydrate in microbial fermentation e.g starch from
maize grain
 It may also obtained from others cereals, potatoes and cassava.
 Hydrolyzed cassava starch is used as a major carbon source for glutamic acid
production.
 Syrups produced by acid hydrolysis may also contain toxic products which may
make them unsuitable for particular processes.
 Sucrose is obtained from sugar cane and sugar beet. It is commonly used in
fermentation media in a very impure form as beet or cane molasses which are the
residues left after crystallization of sugar solution in sugar refining.
 Oils and Fats
 Oils were first used as carriers for antifoams in antibiotics processes.
 Vegetables oils may also be used as carbon substrates, particularly for their content
of the fatty acid, oleic, linoleic and linolenic acid because of cost are competitive
with those of carbohydrates.
 Oil based fed-batch fermenter permit this procedure to operate more successfully.
 Oil also has antifoam properties which may make downstream processing simpler.
 Glycerol trioleat is known to be used in some fermentation where substrate purity is
an important consideration.
 Methly oleate has been used as the sole carbon substrate in cephalosporin
production
 Nitrogen sources (N)
 Most industrially used microorganism can utilize inorganic or organic sources of
nitrogen.
 Inorganic nitrogen may be supplied as ammonia (NH3) gas, ammonium (NH4) salts
or nitrates. NH3 has been used for pH control and as the major N source in a
defined medium for the commercial production of human serum albumin by yeast.
 Ammonia can also be used to adjust pH of the fermentation. NH4 salts i.e
(NH4)2SO4 will produce acid conditions & NH4NO3 normally cause an alkaline
drift.
 Organic nitrogen sources include amino acid, proteins and urea.
 E.g. organic nitrogen source corn steep liquor, yeast extracts, peptones, soy bean
meal
 Minerals
 All micro-organism requires certain mineral elements for growth & metabolism.
 In many media, magnesium (Mg), phosphorus (P), potassium (K), Sulphur (S),
calcium (Ca) and chlorine (Cl) are essential components, cobalt (Co), copper (Cu),
zinc (Zn), ferrous (Fe) and manganese (Mn) are also essential
 Phosphorus (P) is used as a Buffer to minimize pH changes when external pH is not
being used.
 Chelators
 Many media cannot be prepared or autoclaved without the formation of a visible
precipitation of insoluble metal (PO4).
 The problem of insoluble metal PO4 may be eliminated by incorporating low
concentration of chelating agents i.e EDTA, citric acid,polyphosphate etc
 These chelating agents form complexes with the metal ions in a medium. The metal
ions then my be gradually utilized by the micro-organism.
 Chelating agent does not cause inhibition of growth.
 Vitamis and Growth Factors
 Vitamins, specific arachidonic acid (AA), 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 as minor contaminants.
 If only one vitamins is required it may be move economical to add a pure vitamin
instead of large bulk of cheaper multiple vitamins source. E.g Calcium
pantothenate has been used in one medium formulation for vinegar production.
 In glutamic acid, limited concentration of biotin must be present in the medium,
some requires thiamin.
 Buffers
 The control of pH may be extremely important if optimal productivity is to be
achieved.
 Many media are buffered at about pH 7.0 by the incorporation of calcium carbonate
(CaCO3), if pH decreases the CO3 is decomposed.
 PO4 which are the part of many media also play an important role in buffering.
High PO4 concentration are critical in the production of many secondary
metabolites.
 Carbon and nitrogen sources will also a basis for pH control as buffering capacity
can be provided by the protein, peptides & AA such as in corn steep liquor.
 The pH may also be controlled externally by addition of NH3 or NaOH and H2SO4.
 The addition of precursors & metabolic regulators to media

 Some components of a fermentation medium help to regulate the production of the

product rather than support the growth of the micro-organism. Such additive
include, precursors, inhibitors,
Precursors:
 Some chemicals when added to certain fermentation are directly incorporated into
the desired product.
 E.g Pinicillin yields
 A range of different side chain can be incorporated into the penicillin molecule, eg
corn steep liquor increased the yield of pinicillin form 20 units to 100 units.
 Corn steep liquor contain phenylethlamine when incorporated, it yield benzyl
penicillin.
 Phenylacetic acid is widely used precursor in penicillin production.
 Inhibitors
 When certain inhibitors are added to fermentations more of specific product may be
produced.
 Glycerol production depends on modifying the ethanol fermentation by removing
acetaldehyde.
 Inhibitors have also been used to affect cell wall structure and increase the
permeability for release of metabolites.
 The best example is the use of penicillin and surfactants in glutamic acid production.
 Inducers
 The majority of enzymes which are industrial interest are inducible.
 Induced enzymes are synthesized only in response to the presence in the
environment of an inducer.
 Inducers are often substrate such as starch or dextrins for amylase, maltose for
pullulanase and pectin for pectinase.
 Most inducers which are included in microbial enzyme media are substrate or
substrate analogues but intermediates and products may sometime be used as
inducers.
 E.g maltodextrins will induce amylase and fatty acids induce lipase. (use depend
upon the cost)
 Oxygen requirement

 Very important in controlling growth rate and metabolic production. Medium may
influence:
Fast metabolism: culture may become oxygen limited because sufficient oxygen
cannot be made available in the fermenter if certain substrate such as rapidly
metabolized sugars which lead to a high oxygen demand are available in high
concentration.
Rheology: the individual components of the medium can influence the viscosity of the
final medium and its subsequent behavior with respect to aeration and agitation.
Antifoam: Many of the antifoams in use will act as surface active agents and reduce
the oxygen transfer rate.
Rheology:
 There can be considerable variation in the viscosity of compounds that may be included in
fermentation media, eg. Polymer in solution ie polysaccharides
 As polysaccharides degraded, the effect on rhelogical properties will change.
Antifoam:
 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
corn steep liquor, pharmamedia, peanut, soybean, yeast extract or meat extract
 These proteins may denature at the air broth interface & form a skin which does not rupture
readily.
 The foaming can cause remove of cells from the medium which will lead to autolysis & the
further release of microbial cell proteins will probably increase the stability of the foam.
 If uncontrolled, then numerous changes may occur and physical and biological problems may
be created.
 These include reduction in the working volume of the fermenter due to exhausted gas
bubbles circulating in the system, lower mass and heat transfer rates, invalid process data due
to interference at sensing electrode & incorrect monitoring and control.
 Biological problem include deposition of cells in upper parts of the fermenter, problems of
sterile operation with the air filter exist of the fermenter become wet, so danger of microbial
infections, so loss of product.
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