Fermentation Medium

1. Fermentation Medium Most fermentations require liquid media, often referred to as broth, although some solid-substrate fermentations are operated.

3. Medium improvement to what degree • Medium designed for the initial production of antibioticusually does not have to be developed very skillfully since the potential for antibiotic production is quite low with wild-type strains. • Media for ultra-high antibiotic-producing strains, which have been developed through repeated genetic manipulations, must be formulated with utmost care. • In the past, strain improvement and media development were the responsibilities of different research groups. Today we know that each higher-producing clone, after mutation and screening, requires a medium optimized for its performance.

4. The importance of medium improvement • Only small to moderate increases in the level of production can result in an actual reduction in production cost so that it can economically be sold in competition with others. • There is little published literature on the complex substrates that have been developed for the production of various products. Most fermentation processes, which include the production of fermentation media, are closely guarded trade secrets.

5. Different technical objectives of media formulation • Inoculum (starter culture) propagation steps / pilot-scale fermentations / main production fermentation • Biomass or primary metabolites production / secondary metabolite production

6. Considerations in seed culture media formulation • The seed stages are designed to give rapid and reproducible growth without nutrient depletion, autolysis, or an adverse change in pH. • There is less concern with the cost of ingredients in the seed stages since the volume is usually only 5% of the fermentation volume and excellent uniformity of medium ingredients is highly desirable. • Some specially prepared dairy products have been used quite extensively in primary and secondary seeds.

7. 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

8. Considerations in media design Nutritional Requirements Environmental Requirements Techno-economic Factors

9. 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

11. Elemental requirements • Main elemental formula of microbial cells C4H7O2N (dry weight basis 48% C, 7% H, 32% O, 14% N), e.g. Baker’s 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 MgSO4‧7H2O) • 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), baker’s yeast (5), benzyl penicillin (3), riboflavin (1), vitamin B12 (0.002)

13. Energy requirements • dS/dt = μX/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 (可由X mole ATP / g substrate 推算YS) • Maintenance: m = 0.01 ~ 0.04 g / g / h (生長條件越差則m越大); m ≈ 4 m mole /g cell / h (可由X mole ATP / g substrate 推算m); short-term fermentation 不重要，long-term fermentation所佔比例增大 • Product yield: direct stoichiometry and theoretical yield

18. 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

20. Environmental effect of substrate • Substrate concentration: Monod equation, μ = μm S / (Ks + S) Ks for C-source 1 ~ 10 mg/L, when S = 10 ~ 100 mg/L, μ ≈ μm; Ks for amino acid 0.003 ~ 0.2 mg/L; Ks for ammonia 0.1 ~ 1.0 mg/L • Substrate inhibition: carbohydrate 50 to 100 ~ 150 g/L (osmotic pressure); phenol, toluene, butanol a few g/L (damage cell membrane); ammonia 3 ~ 5 g/L • Catabolite repression • NO3- → NO2- toxic effect • Phosphate repression and sulfate repression

21. Techno-economic factors that affect the choice of individual raw materials: • Cost: transport and storage, e.g. temperature control • Availability: consistent quality and year round availability • Ease of handling: solid or liquid forms • Sterilization: thermal damage and inhibitory byproduct • Operational characteristics: formulation, mixing, complexing and viscosity characteristics that may influence agitation, aeration, foaming and recovery • Supply: the concentration of target product attained, its rate of formation and yield per gram of substrate utilized • Purification: levels and range of impurities, potential for generating undesired products • Pollution control • Health and safety implications

22. 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

23. Example

27. Major C sources

28. 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

30. 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

32. Starch and dextrins • Can be directly metabolized by amylase-producing microorganisms, particularly filamentous fungi • Maize starch is most widely used • To allow use in a wide range of fermentations, the starch is usually converted into sugar syrup, containing mostly glucose. It is first gelatinized and then hydrolyzed by dilute acids or amylolytic enzymes, often microbial glucoamylases that operate at elevated temperatures

33. Sulfite waste liquor • Sugar containing wastes derived from the paper pulping industry are primarily used for the cultivation of yeasts • Waste liquors from coniferous trees contain 2-3% (w/v) sugar, 80% hexoses (glucose, mannose and galactose) and 20% pentoses (mostly xylose and arabinose) / Liquors derived from deciduous trees contain mainly pentoses • Usually the liquor requires processing before use as it contains sulfur dioxide / The low pH is adjusted with calcium hydroxide or calcium carbonate, and these liquors are supplemented with sources of nitrogen and phosphorus

34. 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

35. 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

36. Alkanes and alcohols • n-Alkanes (C10-C20): readily metabolized by certain microorganisms / industrial use is dependent upon the prevailing price of petroleum • Methane: utilized by a few microorganism, but its conversion product methanol is often preferred for industrial fermentations • High purity methanol is readily obtained / completely miscible with water / has a high per cent carbon content and is relatively cheap / only limited organisms will metabolize methanol / only low conc., 0.1-1% (v/v) are tolerated by microorganisms / oxygen demand and heat of fermentation are high, but this is even more problematic when growing on alkanes • Ethanol is less toxic than methanol / used as a sole or cosubstrate / too expensive for general use as a carbon source / its biotransformation to acetic acid remains a major fermentation process

37. Fats and oils • Hard animal fats (composed mainly of glycerides of palmitic and stearic acids) are rarely used in fermentation • Plant oils (primarily from cotton seed, linseed, maize, olive, palm, rape seed and soy) and occasionally fish oil, may be used as the primary or supplementary carbon source, especially in antibiotic production / Plant oils are mostly composed of oleic and linoleic acids, but linseed and soy oil also have a substantial amount of linolenic acid • Oils contain more energy per unit weight than carbohydrates / Oils can be particularly useful in fed-batch operations than carbohydrates (aqueous solutions less than 50%, w/v; occupy a greater volume)

38. Major N sources

39. Corn steep liquor • Byproduct of starch extraction from maize / first use in fermentations for penicillin production in the 1940s • Exact composition varies depending on the quality of maize and the processing conditions / Concentrated extracts generally contain about 4% (w/v) nitrogen, including a wide range of amino acids, along with vitamins and minerals / Any residual sugars are usually converted to lactic acid (9-20%, w/v) by contaminating bacteria • Can sometimes be replaced by liquor derived from potato starch production

40. Yeast extract - 1 • Produced from waste baker’s and brewer’s 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

41. 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)

43. 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

45. Soya bean meal • Residuals after extraction of soy oil • Composed of 50% protein, 7% non-protein nitrogenous compounds, 30% carbohydrates and 1% oil • Often used in antibiotic fermentation because the components are only slowly metabolized, thereby eliminating the possibility of repression of product formation

46. 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

47. 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

48. 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 (獸脂) • Synthetic antifoams are mostly silicon oils, poly alcohols and alkylated glycols

49. Special compounds -1 • Precursors: phenylacetic acid or phenylacetamide as side-chain precursors in penicillin production / D-threonine 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

50. 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