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Medium Formulation

Prof. S.T. YangDept. Chemical & Biomolecular Eng.The Ohio State University

Overview

(limited)supplies

of substrate

SURVIVAL

METABOLITES

REPRODUCTION

environment

CatabolismAnabolism

BiosynthesisTransport

ENZYMES

Basic knowledge

Must satisfy the elemental requirements for cell growth and metabolites productionAdequate supply of energy for biosynthesis and cell maintenanceOptimal compositions might differ greatly depending on the aims:

cell growth vs. metabolites productionSporulation vs. fermentation (in molds and Acetomyces)

Basic knowledge (cont’d)

Might affect final fermentation products

Need to consider broth rheology:Affect oxygen transfer

Power consumption

Foaming – need anti-foaming agent?

Impurity – inhibition or toxic? affecting product recovery / purification?

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Type of Medium

DefinedPure chemical components known in exact amountExpensiveMostly for research studies

Complex (Undefined)Natural substratesUnknown compositionContains necessary growth factorsLess expensive

Medium Formulation

SubstratesCheapAvailable in large quantityEasy to handlingMeet nutrient requirementsNon-toxicBuffer capabilityEx: corn starch, molasses, whey, soybean meals

Medium Formulation (cont’d)

Nutrient SupplementsSupplement to the substrateCorn steep liquor, malt extract, yeast extract, casein hydrolysate, etc.

Minerals and SaltsBuffers:

Calcium carbonate, phosphateProteins, peptides, amino acids and salts

Medium Formulation (cont’d)

InducersFor induced enzymesEx: methanol for citric acid (help product diffuse out of cell)

InhibitorsTo accumulate the metabolic intermediateEx: glycerol

Precursors:Directly incorporated into the desired productsEx: Penicillin

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Industrial MediaNitrogen

BarleyBeet molasesCorn-steep liquorOat flourPharma mediaRye flourSoybean mealWhey powder

Carbohydrateo Glucose: - Glucose monohydrate

- Hydrolysed starch

o Lactose: - Pure lactose- Whey

o Starch: - Barley- Groundnut meal- Oat flour- Rye flour- Soybean meal

o Sucrose: - Beet molasses- Cane molasses- Crude brown sugar- Pure white sugar

Nutrient Requirements

Basic Growth:WaterEnergy sourceCarbon sourceNitrogen source (10% – 14% of cell weight)Minerals

Others:Growth factors; i.e. amino acids and vitaminsOxygen for aerobic growth

Energy Sources

Phototrophs:Energy from lightUse light for ATP formatione.g. Photosynthetic bacteria

Chemotrophs:Energy from oxidation of medium components:Two classes:

Autotrophs: from inorganic compoundsHeterotrophs: from organic compounds

Carbon Sources

Heterotrophs:Carbon for oxidationMostly carbohydrates (molasses, starch)Can be lipids or proteins

Autotrophs:CO2 as carbon source

Phototrophs:CO2 as carbon source

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Nitrogen Sources

Nitrogen for amino acids, purines, pyrimidinesand vitamins Most microorganisms can metabolize inorganic nitrogen, i.e. ammonia or ammonium saltsBut might grow faster with organic nitrogenSome require organic nitrogenOrganic nitrogen compounds:Yeast extract, casein hydrolysate, tryptophan, peptone, corn steep liquor, soybean meal

Mineral sources

Need to be added as distinct componentsPhosphorous for ATP: phosphate (as buffer)Sulfur: sulfate, H2S, cysteinMagnesium: MgSO4

Potassium: KH2PO4, K2HPO4 (as buffer)Calcium: CaCl2, CaSO4

Sodium: NaCl (halophilic bacteria)Chloride: NaCl (halophilic bacteria)

Trace metals

Essential - act as cofactors for enzymesIron (Fe), Copper (Cu), Cobalt (Co), Manganese (Mn), Zinc (Zn), Molybdenum (Mo)Some require Selenium (Se) and Nickel (Ni)

Present as impurities in major ingredientsSome present in (tap) water

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Growth Factors

Vitamins, amino acids, fatty acidsCannot be synthesized by some cellsNeed to be supplied from growth medium

OXYGENFrom O2 or airAct as electron receptor (for aerobic)20% of cell dry weight

Auxotrophy

AuxotrophsLack of one or more biosynthetic pathwayInability to synthesize some organic compounds

PrototrophsAbility to synthesize all the needed organic compounds from C sources and salts.e.g. E. coli

Fastidious Auxotrophs

Environmental Factors

TemperatureMesophiles: 25 – 40 ˚C; optimum: 30 – 37 ˚CPsychrophiles: -5 – 35 ˚C; optimum: 15 – 20 ˚CThermophiles: 40 – 75 ˚C; optimum: 45 – 60 ˚CExtremophiles: 60 – 110 ˚C

Gaseous Requirement (free oxygen)Aerobic (e.g. molds)Anaerobic (e.g., methanogens)Facultative anaerobic (can grow under both aerobic and anaerobic conditions; E. coli, LAB, Yeasts, etc.)

Environmental Factors (cont’d)

pHAcidophiles: pH 2 – 5.5

fungi

Alkalophiles: pH 9 - 12Neutrophiles: pH 5.5 – 8

Most bacteriaOptimum 6.5 – 7.5

Osmolality (Tonicity)Osmotic pressure, π = RT(W/Mw) = RTC

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Environmental Factors (cont’d)

Water activity (aw)Amount of free water in the systemAw = Ps / Pw

Ps = vapor pressure of water in solutionPw = vapor pressure of pure water

Aw = MW / (Ms + MW)Mw = molar concentration of water (55 M)Ms = molar concentration of solute

Bacteria: > 0.9, G(-) > G(+); Yeasts: 0.88 Molds: 0.8 halophilic bacteria (~0.75); xerophilic fungi (~0.65); osmophilicbacteria (~0.60)

Environmental Factors (cont’d)

Oxidation-Reduction (Redox) Potential (Eh)The tendency of a solution to give or take up e-

Aerobe: (+) mV of Eh

Anaerobe: (-) mV of Eh

)reductant()oxidant( cnebHa ⇔++ −+

[ ] [ ][ ]c

ba

hH

nFRTEE

reductantoxidantln0

+

+=

F: Faraday quantity of electricity

Environmental Factors (cont’d)

Ionic Strength (I)

NaCl Na+ + Cl-

NaClClNa mmmI =+= −+ )(21

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21

ii zmI Σ= mi: molal concentration of ion I

Zi: charge