Media Components - Water & Carbon Sources

The composition of fermentation media is dependent on a number of factors characteristic of the particular fermentation.  The major ingredients of a typical fermentation media are:

■       Water

■       Carbon source

■       Nitrogen source

■       Sources of P, S, minor and trace elements

■       Vitamins – Biotic, riboflavin etc

■       Buffers

■       Antifoam

■       Precursor

■       Inducer

■       Inhibitor

 

Water

Water is a major component of all fermentation media except solid-substrate fermentation. It also provides trace mineral elements. Water is also important for ancillary equipment and cleaning. Supply of large quantities of clean water, of consistent composition, is therefore essential. The quality of water in terms of pH, dissolved salts and effluent contamination is important.  The mineral content is important in brewing (mashing step) and has influenced the location of breweries and types of beer produced.  Before use, suspended solids, colloids and microorganisms should be removed. If the water is ‘hard’, it should be treated to remove salts such as calcium carbonate. Iron and chlorine should also be removed. For some fermentations, especially, plant and animal cell culture, the water must be highly purified. Since, water is becoming increasingly expensive, its recycle/reusage wherever possible should be encouraged. This minimizes water costs and reduces the volume requiring waste-water treatment.

Carbon Sources

A carbon source is required for all biosynthesis leading to reproduction, product formation and cell maintenance.  In most fermentations it also serves as the energy source. 

Carbon requirements may be determined from the biomass yield coefficient (Y), an index of the efficiency of conversion of a substrate into cellular material.

Y_carbon(g/g) =   Biomass produced (g)

                      carbon substrate utilized (g)

 

For commercial fermentations, the determination of yield coefficients for all nutrients is usually essential.  As most carbon substrates also serve as energy sources, the organism’s efficiency of adenosine triphosphate (ATP) generation and its utilization are key factors.  Carbohydrates are traditional carbon and energy sources for microbial fermentations, although other sources may be used, such as alcohols, alkanes and organic acids.  Animal fats and plant oils may also be incorporated into some media, often as supplements to the main carbon source.

Molasses Pure glucose and sucrose are rarely used for industrial-scale fermentations, primarily due to cost.  Molasses, a by-product of cane and beet sugar production, is a cheaper and more usual source of sucrose.  Molasses are concentrated syrups or mother liquors recovered at any one of several steps in the sugar refining process with different names depending on the step from which it is recovered.  Blackstrap molasses from sugar cane is the cheapest and most used sugar source for industrial fermentation.  This is the residue remaining after most of the sucrose has been crystallized from the plant extract.  It is a dark-coloured viscous syrup containing 50-60% (w/v) carbohydrates, primarily sucrose, with 2% 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. 

Refinery blackstrap molasses is a similar product obtained from the recrystallization refining of crude sucrose.  High test or invert molasses is produced after whole cane juice is partially inverted/partially hydrolyzed to monosaccharides to prevent sugar crystallization. It contains approximately 70-75% sugar and is preferable to blackstrap molasses as it has lower levels of non-fermentable solids. Beet molasses are produced from beetroot, in a similar process as for sugarcane.  However, it may be limiting in biotin for yeast growth and a small amount of cane molasses may need to be added in these fermentations. Hydrol molasses, a by-product of maize starch processing primarily contains glucose (60%) and a relatively high salt concentration.

Malt Extract Aqueous extracts of malted barley can be concentrated to form syrups that are useful carbon sources for the cultivation of filamentous fungi, yeast and actinomycetes. The composition of malt extracts varies to some extent, but they usually contain approximately 90% carbohydrate, on a dry weight basis.  This contains 20% hexoses (glucose and small amounts of fructose), 55% disaccharides (mainly maltose and traces of sucrose), along with 10% maltotriose, a trisaccharide.  These products contain a range of branched and unbranched dextrins (15-20%), which may or may not be metabolized, depending upon the microorganism.  Malt extracts also contain some vitamins and approximately 5% nitrogenous substances, proteins, peptides and amino acids.

Sterilization of media containing malt extract must be carefully controlled to prevent overheating which produce Maillard reaction products. These are brown condensation products due to the reaction of amino groups of amines, amino acids and proteins with the carboxyl groups of reducing sugars, ketones and aldehydes. This occurs when reducing sugars and amino acids are heated at low pH. Such reactions cause loss of fermentable materials, colour change and some reaction products may inhibit microbial growth.

Starch and Dextrins These polysaccharides are not as readily utilized as monosaccharides and disaccharides. It can be metabolized by amylase-producing microorganisms, particularly filamentous fungi which hydrolyze the substrate to a mixture of glucose, maltose or maltotriose. The product composition is similar to that of malt extracts.  Maize starch is most widely used, but may also be obtained from other cereal or root crops. Starch is usually converted into sugar syrup, containing mostly glucose before use in fermentations.  It is first gelatinized and then hydrolyzed by dilute acids or amylolytic enzymes, often microbial glucoamylases.

Sulphite Waste Liquor Sulphite waste liquor is the product of the paper pulping industry. It is obtained after wood for paper manufacture is digested to cellulose pulp.  It can be used as a dilute fermentation medium for ethanol production by S. cerevisiae and the growth of Torula utilis for feed.   Waste liquors from coniferous trees contain 2-3% (w/v) sugar, which is a mixture of hexoses (80%) and pentoses (20%).  Hexoses include glucose, mannose and galactose, whereas the pentose sugars are mainly xylose and arabinose.   The liquors derived from deciduous trees contain mainly pentoses.  Sulphite Waste Liquor requires processing before use as it contains sulphur dioxide or calcium hydroxide or calcium carbonate which need to be stripped or removed by precipitation with lime. Supplementation with sources of nitrogen and phosphorous is also required.

Cellulose Cellulose is mainly present as lignocellulose in plant cell walls, which has cellulose, hemicellulose and lignin.  Lignocellulose is available from agricultural, forestry, industrial and domestic wastes.  Very few microorganisms can utilize it directly, as it is difficult to hydrolyze.  The cellulose component is encrusted with lignin and provides little surface area for enzyme attack.  It is mainly used in solid-substrate fermentations to produce various mushrooms. Preliminary processing by hydrolysis may be required in case of cellulose to expose fermentable sugars.  It has potential as a valuable renewable source of fermentable sugars particularly in the bioconversion to ethanol for fuel use.  

Whey Whey is an aqueous by-product of the dairy industry.  The annual worldwide production is over 80 million tonnes, containing over 1 million tonnes of lactose and 0.2 million tonnes of milk protein.  It is expensive to store and transport.  Milk proteins can be removed from whey to be used as food supplements. Whey is then evaporated and lactose concentrates are obtained which can be used for later fermentation. Lactose is generally less useful as a fermentation foodstock than sucrose, as it is metabolized by fewer organisms.  S. cerevisiae, for example, does not ferment lactose.  It was used extensively in penicillin fermentations and is still employed for producing ethanol, single cell protein, lactic acid, xanthan gum, vitamin B12 and gibberellic acid.

Alkanes and Alcohols n-Alkanes of chain length C10-20 are readily metabolized by certain microorganisms.  Mixtures, rather than a specific compound, are usually most suitable for microbial fermentations.  However, their industrial use is dependent upon the prevailing price of petroleum.  Methane is utilized as a carbon source by a few microorganisms, but its conversion product methanol is often preferred for industrial fermentations as it presents fewer technical problems.  High purity methanol is readily obtained and it is completely miscible with water.  Methanol has a high percent carbon content and is relatively cheap, although only a limited number of organisms will metabolize it.  Also unlike many other carbon sources, only low concentrations, 0.1-1% (v/v), are tolerated by microorganisms, higher levels being toxic.  During fermentations on methanol, the oxygen demand and heat of the fermentations are high, but this is even more problematic when growing on alkanes.  Several companies used methanol in microbial protein production in the 1970s and early 1980s, but these processes are currently uneconomic.

 Ethanol is less toxic than methanol and is used as a sole or co-substrate by many organisms, but it is too expensive for general use as a carbon source.  However, its biotransformation to acetic acid by acetic acid bacteria remains a major fermentation process.

Fats and Oils

Hard animal fats that are mostly composed of glycerides of palmitic and stearic acids are rarely used in fermentations.  However, plant oils (primarily from cotton seed, linseed, maize, olive, palm, rape seed and soya) 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 soya oil also have a substantial amount of linolenic acid.  The oils contain more energy per unit weight than carbohydrates.  In addition, the carbohydrates occupy a greater volume because they are usually prepared as aqueous solutions of concentrations no greater than 50% (w/v).  Consequently, oils can be particularly useful in fed-batch operations as less spare capacity is needed to accommodate further additions of the carbon source.

References

1. Industrial Microbiology: An Introduction. Michael J. Waites, Neil L. Morgan, John S
2. Principles of Fermentation Technology- Peter Stanbury, Allan Whitaker, Stephen Hall