Media Preparation Abira Khan

Media Medium selected will affect the design of fermenter to be used Water, sources of energy, carbon, nitrogen, mineral elements and possibly vitamins plus oxygen if aerobic

Criteria of Ideal Medium It will produce the maximum yield of product or biomass per gram of substrate used It will produce the maximum concentration of product or biomass It will permit the maximum rate of product formation There will be the minimum yield of undesired products It will be of a consistent quality and be readily available throughout the year It will cause minimal problems during media making and sterilization It will cause minimal problems in other aspects of the production process particularly aeration and agitation, extraction, purification and waste treatment

Medium Formulation Carbon and energy source+ Nitrogen source + Oxygen + Other requirements = Biomass + Products + CO2 + Water + Heat The carbon substrate has a dual role in biosythesis and energy generation The carbon requirement for biomass production under aerobic conditions may be estimated from the cellular yield coefficient (Y) which is defined as Quantity of cell dry matter produced/Quantity of carbon substrate utilized

Water and Energy Sources Most industrial micro-organisms are chemo- organotrophs, therefore the commonest source of energy will be the carbon source such as carbohydrates, lipids and proteins Factors influencing the choice of carbon source: The main product of a fermentation process The purity of the carbon source The method of media preparation, particularly sterilization Government legislation

Commonly Used Carbon Sources Carbohydrates- Maize, Barley grains, sugar cane, sugar beet, Molasses Oils and fats- olive, maize, cotton seed, linseed, soya bean oil Hydrocarbons and their derivatives-

Nitrogen Sources Ammonia gas, ammonium salts or nitrates, amino acid, protein or urea, corn-steep liquor, soya meal, peanut meal, cotton-seed meal Factors influencing the choice of nitrogen source: In mixtures of nitrogen sources, individual nitrogen components may influence metabolic regulation so that there is preferential assimilation of one component until its concentration has diminished Antibiotic production by many micro-organisms is influenced by concentration of N source

Others Minerals- In many media, magnesium, phosphorus, potassium, sulphur, calcium and chlorine are essential components Chelators- Ethylene diamine tetra acetic acid (EDTA), citric acid, polyphosphates Growth factors- Vitamins, specific amino acids, fatty acids, sterols Buffers- Calcium carbonate Precursors Inhibitors Inducers

Oxygen Requirements Fast metabolism- The culture may become oxygen limited because sufficient oxygen cannot be made available in the fermenter if certain substrates, such as rapidly metabolized sugars which lead to a high oxygen demand, are available in high concentrations Rheology- The individual components of the medium can influence the viscosity of the final medium and its subsequent behaviour with respect to aeration and agitation Antifoams- Many of the antifoams in use will as surface active agents and reduce the transfer rate

Foaming The most common cause of foaming is due to proteins in the medium, such as corn-steep liquor 5 patterns of foaming in fermentations: Foaming remains at a constant level through-out the fermentation. Initially it is due to the medium and later due to microbial activity. A steady fall in foaming during the early part of the fermentation, after which it remains constant. Initially it is due to the medium but there are no later effects caused by the micro-organism. The foaming falls slightly in the early stages of the fermentation then rises. There are very slight effects caused by the medium but the major effects are due to microbial activity. The fermentation has a low initial foaming capacity which rises. These effects are due solely to microbial activity. A more complex foaming pattern during the fermentation which may be a combination of two or more of the previously described patterns.

Anti-Foaming To try and avoid foam formation by using a defined medium and a modification of some of the physical parameters (pH, temperature, aeration and agitation). This assumes that the foam is due to a component in the medium and not a metabolite. The foam is unavoidable and antifoam should be used. This is the more standard approach. To use a mechanical foam breaker

Anti-Foams Antifoams are surface active agents, reducing the surface tension in the foams and destabilizing protein films by (a) hydrophobic bridges between two surfaces, (b) displacement of the absorbed protein, and (c) rapid spreading on the surface of the film An ideal antifoam should have the following properties: Should disperse readily and have fast action on an existing foam. Should be active at low concentrations. Should be long acting in preventing new foam formation. Should not be metabolized by the microorganism. Should be non-toxic to the micro-organism. Should be non-toxic to humans and animals. Should not cause any problems in the extraction and purification of the product. Should not cause any handling hazards. Should be cheap. Should have no effect on oxygen transfer. Should be heat sterilizable Alcohols; stearyl and octyl decanol. Esters. Fatty acids and derivatives, particularly glycerides, which include cottonseed oil, linseed oil, soy-bean oil, olive oil, castor oil, sunflower oil, rapeseed oil and cod liver oil. Silicones. Sulphonates. Miscellaneous; Alkaterge C, oxazaline, poly-propylene glycol

Other Medium Animal cell media Serum- Very complex mixture contammg approximately 1000 components including inorganic salts, amino acids, vitamins, carbon sources, hormones, growth factors, haemoglobin, albumin and other compounds Serum-free media supplements Protein-free media Trace elements Osmolality pH Non-nutritional media supplements- Sodium carboxy methyl cellulose

Serum Free Media Advantages of removing serum from media More consistent and definable medium composition to reduce batch variation Reduction in potential contamination to make sterility easier to achieve Potential cost savings because of cheaper replacement components Simplifying downstream processing because the total protein content of the medium has been reduced