THE MEDIA IN

FERMENTATION
Nutritional Requirements
 Introduction
 Microorganisms obtain energy for support of biosynthesis and growth
from their environment in a variety of ways
 Need detailed investigation to establish the most suitable medium for
an industrial fermentation process, but certain basic requirements
must be met by any such medium.
 nutritional requirements are complex and varied
 Not only are the types of microorganisms diverse (bacteria, molds,
yeast), but the species and strains become very specific; the products
 All microorganism requires water, source of energy, C, N, Mineral,
vitamins, oxygen (aerobic).
 On a small scale its easy to maintain but on large scale very difficult
to maintain the satisfactory growth.
Medium criteria
 Produce max yield of prod. or biomass per g substrate used
 Produce max concentration of prod.
 permit max rate of prod formation
 Permit min yield of undesired prod
 Consistent quality & available throughout year
 Cause min problems during preparation
 Cause min problems during production & down stream process
Consideration
 availability of nutrients required by microorganisms: energy,
grow, production
 fermentation conditions and condition change during
fermentation (e.g. substrate pH, MC, incubation temperature)
 stage of growth of micro-organisms  product
 presence of other competing micro-organisms
 Type of reactor
 Preparation & downstream process (e.g. separation, recovery,
effluent treatment)
 Problems in upscale (e.g. agitation, viscosity, gas transfer)
Natural or formulated-defined media?
Natural Media:
 Cheaper
 High variation/batch
 Unpredictable
product/biomass
 More complex in down
stream process
 Difficult to detect small
yield improvement
Formulated-MEDIA
 Easily reproduce
 Specific to the strain
 More expensive
 More predictable result
 More simple process
cheaper
 Easier to detect process
improvement
Natural media
 MEDIA FORMULATION
 Essential stage in design of successful laboratory experiments, pilot-scale
development & manufacturing processes
 The constituents of a medium must satisfy:
 the elemental requirements for cell biomass and metabolite production

 must be an adequate supply of energy for biosynthesis and cell maintenance

 Some components need for product formation but not for biomass formation
 Stoichiometry for growth & product formation:

C(energy sources)+N source+othersbiomass+Product+CO2+H2O+heat

 This equation should be expressed in quantitative terms

 It should be possible to calculate:
~ the minimal quantities of nutrients which will be needed to produce biomass
~ the substrate concentration in order to produce required product yield
 Commercially, trade secret
 Microbial Nutrient
 Nutrient: combination of elements require for energy,
biosynthesis of cellular matter, products in cell operation,
maintenance and reproduction
 predominant atomic constituents of organisms, C, H, N, O, P,
and S,  40-50% C, 30-50% O2, 6-8% H2, 3-12 N
 All living cells contain water as their predominant constituent
 Cell: lipids, polysaccharides, nucleic acids, proteins and few
common salts
 Nutrients divided into 3 group (often overlapping): energy
supplier, C supplier and any & everything else supplier
 According to nutritional pattern, organisms are classified:
energy sources and C source
Water
 Major comp. require in fermentation use for nutrient
solvent and solvent within the cell
 it has some unusual properties ionizes into acid and
base, and has a propensity for hydrogen bonding
 May need treatments
 Consider: pH, salts dissolved, mineral, effluent
contaminant
 Base on water availability: Solid state fermentation
(SSF) & Submerged liquid fermentation (SmF)
 SSF  the media is absence of free water
 SSF
Advantage Disadvantage

 Low water availability  reduce

 Substrate requires
contamination by bacteria &
pretreatment (e.g SR, sieving)
yeast
 Poor grow of high moisture
 Higher level of aeration 
microbial
porous
 Difficult in biomass
 Similar with natural mold
determination (mixed up)
environment
 Difficult in control process (e.g
 Higher yiled/product  low
pH, O2, MC)
nutrient dilution
 Static condition, no agitaion
 Lower in production cost  low
energy for agitation & aeration  Difficult in scale up (e.g
aeration)
SSF-microbial
 Filamentous fungi are the
best
 Good tolerance in low
water activity
 the hyphal features give
the power to penetrate into
solid substrate
 Penetration increase
accessibility of all nutrient
within substrate
Cell nutrients
 Nutrients required by cell: macronutrient & micronutrient
 Macronutrients needed > 10-4 M
 make up about 95% of dw
 e.g C, N, O, P, S
 Micronutrient  needed < 10-4 M
 involve in enzyme function, maintain in protein structure
 e.g Zn, Cu, Mn, Ca, Na
 Carbon
 Biomass 50% C ; skeleton of all cellular molecules
 for building cell mass and forming product, as well as acting as an
energy source
 Energy sources; secreted as CO2 & organic compounds
 Single comp for energy & C sources; add nutrients
 Energy used for metabolism-biosynthetic reaction & cell maintaining
 The main product of fermentation process will often determine the
choice of C source, particularly if the product results from the direct
dissimilation
 The impurity of C source should be considered
 sterilization may affect suitability of C source (e.g. sugar separated
from amino acid  black N comp. which may inhibit microbial grown
C sources
1- Carbohydrates: C, O, H & energy  0.2-25% of media
3 classes: mono-, di-, & polysacharides
Breakdown of poly- & di- to simpler sugars with enzyme help
major source of energy-rich comp
Lab media  glucose, sucrose & fructose
Industrial media  starch (glucose, dextrin), mollases (sucrose),
whey (lactose)
2 - Oils & Fats
Vegetable oil contains fatty acid, oleic, linoleic & linolenic acid
Typical oil contain~2.4x energy of glucose/weight
Less volume space smaller fermenter (10 kcal  1,24 l of oil; 5 l
of glucose
 act as antifoam too
3 - Hydrocaarbon & their derivatives
N-alkanes for organic acid, amino acid, vitamin etc
Provide energy ~ 2x C, 3x sugar
Nitrogen
 10-14% of cell
 N serve to build structural proteins, functional enzymes & nucleic acids
 N presents in cell as amino groups
 Supplied as organic or inorganic sources
 Organic acid faster growth & fulfill microorganism requirement (e.g.
Protein, peptide, amino acids etc)
 N inorganic supplied as ammonia salts (NH4Cl, NH4NO3), amonia
gas, nitrate
 In industries: soya meal, yeast extract, corn steep liquor
 Oxygen
 key nutrient for aerobic microbes
 20% of cell
 microorganisms that are dependent on respiration for generating
energy require molecular oxygen as the final hydrogen or electron
 commonly found as a constituent of cellular water and organic
compounds (e.g. carbohydrate)
 low solubility of oxygen in water
 Oxygen transfer achieved by shaking, sparging, aeration and
agitation.
 Trace elements

 In mM level essential comp, added as distinct comp in many

media : P, S, K and Mg
 In mM level  sometimes supplied from quantities occurring in
water or impurities of other comp: Fe, Na, Ca, Cu, Co, Zn, Mn
etc.
 Supplied during cell cultivation
 Various functions: serve in coenzyme, catalyse reactions, vitamin
synthesis, cell transport
 Primary metabolite production is usually not very sensitive to
trace element concentration, but not for secondary metabolite
production
 metallic elements can be supplied as nutrients in the form of
the cations of inorganic salts
 K, Mg, Ca and Fe are normally required in relatively large
amounts and should normally always be included as salts in
culture media
 Not possible to generalize ionic requirements
 Oxygen is always provided in water
 Aerobic organisms require molecular oxygen as terminal
oxidizing agents to fulfill their energetic needs through aerobic
respiration.
 For obligate anaerobes oxygen is a toxic substance
Growth Factors
 organic comp, require in very low concentration, perform specific
catalytic/structural roles
 vitamin, sterol, amino acids, fatty acids etc
 Specific for certain microorganism
 Required to stimulate microbial growth
 Many natural C & N contain some growth factor; not vital
 Vitamins are growth factors which fulfill specific catalytic needs in
biosynthesis
 Vitamin  function as (part of ) coenzymes to catalyze many
reactions
 The vitamins most frequently required are thiamin and biotin.
 Required in the greatest amounts are usually niacin, pantothenate,
riboflavin, and some folic derivatives, biotin, vitamin B, and lipoic
acid are required in smaller amounts