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Most fermentations require liquid media, often referred to as broth, although some solid-substrate fermentations are operated.
Constituents of medium
Water Carbon source / Nitrogen source / Sources of phosphorous and sulfur / Minor and trace elements / Vitamins such as biotin and riboflavin Oxygen: even some anaerobic fermentations require initial aeration, e.g. beer fermentations Buffers or controlled by acid and alkali additions Antifoam agents Precursor, inducer or inhibitor compounds
Nutritional requirements
Nutritional requirements include elemental, specific nutrient, and energy requirements Elemental requirements : the stoichiometry for growth and product formation C-source + N-source + O2 + minerals + specific nutrients cell mass + product + CO2 + H2O + heat Specific nutrient requirements: Auxotroph: To use a complex medium or to identify the specific nutrient
Elemental requirements
Main elemental formula of microbial cells C4H7O2N (dry weight basis 48% C, 7% H, 32% O, 14% N), e.g. Bakers yeast C3.72H6.11O1.95N0.61S0.017P0.035K0.056 Average: C 45-55%, N 6-14%, K 0.5-2%, P 1-3%, Mg 0.1-1%, S 0.02-1%, minor minerals (mg /100g cell) Cu 0.1-1, Fe 1-10, Zn ~1, Mn 0-5 (e.g. 10g/L of cell mass containing 0.4% magnesium will require at least 0.04 g/L of Mg or 0.2 g/L of MgSO4 or 0.4 g/L of MgSO47H2O) Chemical composition of fermentation product Typical concentration of fermentation products in the broth (dry wt / vol, %): lactic acid (13), citric acid (12), glutamic acid (10), ethanol (8), bakers yeast (5), benzyl penicillin (3), riboflavin (1), vitamin B12 (0.002)
Energy requirements
dS/dt = X/Yx/s + mX + qpX/Yp/s Mass synthesis + Maintenance + Product synthesis Growth yield: YS = 0.4~1.0 g cell / g substrate 1.3 g cell / g C of substrate; YATP 10.5 g cell / mole ( X mole ATP / g substrate YS) Maintenance: m = 0.01 ~ 0.04 g / g / h ( m); m 4 m mole /g cell / h (X mole ATP / g substrate m); short-term fermentation long-term fermentation Product yield: direct stoichiometry and theoretical yield
Environmental requirements
Effect of growth temperature on cell yield / below optimal temperature for growth Effect of water activity (Aw = Ps/Pw) on growth rate, vapor pressure of water in solution (Ps) or in pure water(Pw) Combined effect of temperature and pH on growth / opt pH for growth and production is not always the same Environmental effect of substrate
Cost analysis
Raw materials (consumed in production or recovery) constitute a major part of the manufacturing cost 30 to 80% of the production cost for biologically based production system 10 to 50% of the production cost for conventional chemical production plants Nutrients: up to 60% of the production cost Examples
Example
Major C sources
Molasses
Byproduct of cane or beet sugar production / residues remaining after most of the sucrose has been crystallized from the plant extract Dark colored viscous syrup containing 50-60% (w/v) carbohydrate, primarily sucrose, with 2% (w/v) nitrogenous substances, along with some vitamins and minerals. Overall composition varies depending upon the plant source, the location of the crop, the climatic conditions under which it was grown, and the factory where it was processed The carbohydrate concentration may be reduced during storage by contaminating microorganisms Hydrol molasses, containing primarily glucose, is a byproduct of maize starch processing
Malt extract
Concentrated aqueous extracts of malted barley to form syrups / particularly useful for the cultivation of filamentous fungi, yeasts and actinomycetes App. 90% carbohydrate (w/w) and some vitamins and app. 5% nitrogenous substances, proteins, peptides and amino acids / carbohydrate comprising 20% hexoses (glucose and small amounts of fructose), 55% disaccharides (maltose and traces of sucrose), 10% maltotriose, and additionally contain 15-20% branched and unbranched dextrins, which may or may not be metabolized, depending upon the microorganisms Careful sterilization to prevent over-heating /Maillard reaction products (brown condensation products resulting from the reaction of amino groups and carbonyl groups) when heated at low pH / color change, loss of fermentable materials, some toxic products
Cellulose
Predominantly as lignocellulose (composed of cellulose, hemicellulose and lignin) Available from agricultural, forestry, industrial and domestic wastes Relatively few microorganisms can utilize it directly / The cellulose component is in part crystalline, encrusted with lignin, and provides little surface area for enzyme attack At present, mainly used in solid-substrate fermentations (e.g. mushrooms) Potentially a very valuable renewable source of fermentable sugars once hydrolyzed, particularly in the bioconversion to ethanol for fuel use
Whey
An aqueous byproduct of the dairy industry / Annual worldwide production is over 80 million tons, containing over 1 million tons of lactose and 0.2 million tons of milk proteins Expensive to store and transport / Lactose concentrates are often prepared for later fermentation by evaporation of the whey, following removal of milk proteins for use as food supplements Lactose is less useful than sucrose / e.g. S. cerevisiae does not ferment lactose Formerly used extensively in penicillin fermentation / Still employed for producing ethanol, single cell protein, lactic acid, xanthan gum, vitamin B12 and gibberellic acid
Major N sources
Yeast extract - 1
Produced from waste bakers and brewers yeast, or other strains of S. cerevisiae / Or Kluyveromyces marxianus (formerly K. fragilis) grown on whey and Candida utilis cultivated using ethanol, or wastes from wood and paper processing Extracts used in the formulation of fermentation media are normally salt-free concentrates of soluble components of hydrolyzed yeast cells / Extracts with sodium chloride concentrations greater than 0.05% (w/v) cannot be used in fermentation processes due to potential corrosion problems Yeast cell hydrolysis is often achieved by autolysis, which can be initiated by temperature or osmotic shock, causing cells to die but without inactivating their endogenous enzymes
Yeast extract - 2
Temperature and pH are controlled throughout an optimal and standardized autolysis process / Temperature control is particularly important to prevent loss of vitamins Autolysis (50-55oC for several hours before the temperature is raised to 75oC to inactivate enzymes), plasmolysis or mechanical disruption of cells / filtration or centrifugation to remove cell wall materials and other debris / rapid concentration Extracts are available as liquids containing 50-65% solids, viscous pastes or dry powders They contain amino acids (35-40%, w/v), peptides (30-45%, w/v), water-soluble vitamins and some glucose derived from the yeast storage carbohydrates (trehalose and glycogen)
Peptones
Peptones are usually too expensive for large-scale industrial fermentations Prepared by acid or enzyme hydrolysis of high protein materials: meat, casein, gelatin, keratin, peanuts, soy meal, cotton seed, etc. Amino acids compositions vary depending upon the original protein source / Gelatin-derived peptones are rich in proline and hydroxyproline, but almost devoid of sulfur-containing amino acids / Keratin peptone is rich in both proline and cystine, but lacks lysine Plant peptones invariably contain relatively large quantities of carbohydrates
Water
Use for media, cleaning, cooling ? A reliable source of large quantities of clean water, of consistent composition, is essential Before use, removal of suspended solids, colloids and microorganisms is usually required Hard water is treated to remove salts such as calcium carbonate Iron and chlorine may also require removal Water is becoming increasingly expensive / recycle / reuse wherever possible / minimizes water costs and reduces the volume requiring waste-water treatment
Antifoams -1
Foaming is largely due to media proteins that become attached to the air-broth interface where they denature to form a stable foam If foaming is minimized, then throughputs can be increased Three approaches to controlling foam production: modification of medium composition, use of mechanical foam breakers and addition of chemical antifoams Chemical antifoams are surface-active agents which reduce the surface tension that binds the foam together
Antifoams -2
Ideal antifoam: 1. readily and rapidly dispersed with rapid action; 2. high activity at low concentration; 3. prolonged action; 4. non-toxic to fermentation microorganisms, humans or animals; 5. low cost; 6. thermostable; 7. compatibility with other media components and the process , i.e. having no effect on oxygen transfer rates or downstream processing operations (e.g. some may adversely affect membrane filtration) Natural antifoams include plant oils (e.g. from soy, sunflower and rapeseed), deodorized fish oil, mineral oils and tallow () Synthetic antifoams are mostly silicon oils, poly alcohols and alkylated glycols
Special compounds -1
Precursors: phenylacetic acid or phenylacetamide as side-chain precursors in penicillin production / Dthreonine in L-isoleucine production by Serratia marsescens / anthranillic acid for L-tryptophan production by yeast Hansenula anomala Inducers and elicitors: Inducers are often necessary for genetically modified microorganisms (GMMs) / Production of secondary metabolites, such as flavonoids and terpenoids, in plant cell culture can be triggered by adding elicitors, which may be isolated from various microorganism, particularly plant pathogens
Special compounds -2
Inhibitors: 1. Used to redirect metabolism towards the target product and reduce formation of other metabolic intermediates (e. g. sodium bisulfite in production of glycerol by S. cerevisiae) 2. Antibiotics for some GMMS containing plasmids bearing an antibiotic resistance gene Cell permeability modifiers: e.g. penicillins and surfactants added to amino acid fermentations, including processes for producing L-glutamic acid by members of the genera Corynebacterium and Brevibacterium
BrothCorn steep liquor ElicitorGelatinizationGMMsMaintenance energy MolassesWater activityWhey