1. Overview
Medium Formulation SURVIVAL
ENZYMES
(limited)
supplies METABOLITES
of substrate Catabolism
Anabolism
Biosynthesis
Transport
Prof. S.T. Yang
Dept. Chemical & Biomolecular Eng. REPRODUCTION
The Ohio State University environment
Basic knowledge Basic knowledge (cont’d)
Must satisfy the elemental requirements for Might affect final fermentation products
cell growth and metabolites production
Need to consider broth rheology:
Adequate supply of energy for biosynthesis
and cell maintenance Affect oxygen transfer
Optimal compositions might differ greatly Power consumption
depending on the aims:
Foaming – need anti-foaming agent?
cell growth vs. metabolites production
Sporulation vs. fermentation (in molds and Impurity – inhibition or toxic? affecting
Acetomyces) product recovery / purification?
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2. Type of Medium Medium Formulation
Defined Substrates
Pure chemical components known in exact amount Cheap
Expensive Available in large quantity
Mostly for research studies Easy to handling
Complex (Undefined) Meet nutrient requirements
Natural substrates Non-toxic
Unknown composition
Buffer capability
Contains necessary growth factors
Ex: corn starch, molasses, whey, soybean meals
Less expensive
Medium Formulation (cont’d) Medium Formulation (cont’d)
Nutrient Supplements Inducers
Supplement to the substrate For induced enzymes
Ex: methanol for citric acid (help product diffuse
Corn steep liquor, malt extract, yeast extract, out of cell)
casein hydrolysate, etc.
Inhibitors
Minerals and Salts To accumulate the metabolic intermediate
Buffers: Ex: glycerol
Calcium carbonate, phosphate Precursors:
Directly incorporated into the desired products
Proteins, peptides, amino acids and salts
Ex: Penicillin
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3. Industrial Media Nutrient Requirements
Carbohydrate Nitrogen
o Glucose: - Glucose monohydrate Barley Basic Growth:
- Hydrolysed starch Beet molases Water
o Lactose: - Pure lactose Corn-steep liquor
- Whey Oat flour
Energy source
o Starch: - Barley Pharma media Carbon source
- Groundnut meal Rye flour Nitrogen source (10% – 14% of cell weight)
- Oat flour Soybean meal
- Rye flour Whey powder
Minerals
- Soybean meal
o Sucrose: - Beet molasses
Others:
- Cane molasses Growth factors; i.e. amino acids and vitamins
- Crude brown sugar
- Pure white sugar
Oxygen for aerobic growth
Energy Sources Carbon Sources
Phototrophs: Heterotrophs:
Energy from light Carbon for oxidation
Use light for ATP formation Mostly carbohydrates (molasses, starch)
e.g. Photosynthetic bacteria Can be lipids or proteins
Chemotrophs: Autotrophs:
Energy from oxidation of medium components: CO2 as carbon source
Two classes: Phototrophs:
Autotrophs: from inorganic compounds CO2 as carbon source
Heterotrophs: from organic compounds
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4. Nitrogen Sources
Nitrogen for amino acids, purines, pyrimidines
and vitamins
Most microorganisms can metabolize
inorganic nitrogen, i.e. ammonia or
ammonium salts
But might grow faster with organic nitrogen
Some require organic nitrogen
Organic nitrogen compounds:
Yeast extract, casein hydrolysate, tryptophan, peptone, corn
steep liquor, soybean meal
Mineral sources Trace metals
Need to be added as distinct components Essential - act as cofactors for enzymes
Phosphorous for ATP: phosphate (as buffer) Iron (Fe), Copper (Cu), Cobalt (Co),
Sulfur: sulfate, H2S, cystein Manganese (Mn), Zinc (Zn), Molybdenum
Magnesium: MgSO4 (Mo)
Potassium: KH2PO4, K2HPO4 (as buffer) Some require Selenium (Se) and Nickel (Ni)
Calcium: CaCl2, CaSO4
Sodium: NaCl (halophilic bacteria) Present as impurities in major ingredients
Chloride: NaCl (halophilic bacteria) Some present in (tap) water
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5. Growth Factors Auxotrophy
Vitamins, amino acids, fatty acids Auxotrophs
Cannot be synthesized by some cells Lack of one or more biosynthetic pathway
Need to be supplied from growth medium Inability to synthesize some organic compounds
Prototrophs
Ability to synthesize all the needed organic
OXYGEN
compounds from C sources and salts.
From O2 or air e.g. E. coli
Act as electron receptor (for aerobic) Fastidious Auxotrophs
20% of cell dry weight
Environmental Factors Environmental Factors (cont’d)
pH
Temperature
Acidophiles: pH 2 – 5.5
Mesophiles: 25 – 40 ˚C; optimum: 30 – 37 ˚C
fungi
Psychrophiles: -5 – 35 ˚C; optimum: 15 – 20 ˚C
Thermophiles: 40 – 75 ˚C; optimum: 45 – 60 ˚C Alkalophiles: pH 9 - 12
Extremophiles: 60 – 110 ˚C Neutrophiles: pH 5.5 – 8
Gaseous Requirement (free oxygen) Most bacteria
Aerobic (e.g. molds) Optimum 6.5 – 7.5
Anaerobic (e.g., methanogens) Osmolality (Tonicity)
Facultative anaerobic (can grow under both aerobic and
Osmotic pressure, π = RT(W/Mw) = RTC
anaerobic conditions; E. coli, LAB, Yeasts, etc.)
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6. Environmental Factors (cont’d) Environmental Factors (cont’d)
Water activity (aw)
Amount of free water in the system
Oxidation-Reduction (Redox) Potential (Eh)
Aw = Ps / Pw The tendency of a solution to give or take up e-
Ps = vapor pressure of water in solution
Pw = vapor pressure of pure water a (oxidant ) + bH + + ne − ⇔ c(reductant)
Aw = MW / (Ms + MW )
Mw = molar concentration of water (55 M) E h = E0 +
RT [oxidant ] H +
ln
a
[ ]b
Ms = molar concentration of solute nF [reductant ]c
Bacteria: > 0.9, G(-) > G(+); F: Faraday quantity of electricity
Yeasts: 0.88
Molds: 0.8
halophilic bacteria (~0.75); xerophilic fungi (~0.65); osmophilic
Aerobe: (+) mV of Eh
bacteria (~0.60) Anaerobe: (-) mV of Eh
Environmental Factors (cont’d)
Ionic Strength (I)
1 mi: molal concentration of ion I
I= Σm i z i
2
Zi: charge
2
NaCl Na+ + Cl-
1
I= ( m + + mCl − ) = m NaCl
2 Na
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