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Tuesday, August 7, 2012

Fermentation Media and its Ideal Characteristics

Characteristics of an Ideal Fermentation media

Fermentation media optimization

Chemical composition: the production medium must have a suitable chemical composition. Medium should contain a source of carbon, a source of Nitrogen, growth factors and mineral salts.

Precursors: In certain fermentation, the media should supply the required precursor for better yields of a desired product.

Buffering capacity: Maintenance of pH in the optimum range is necessary for making the process successful, since acidic and /or basic compounds depending on the nature of the fermentation process accumulate during the progress of the fermentation. To control the pH of the medium, buffers should be added to the medium (E.g. CaCO3). Media containing considerable quantities of proteins, peptides, and amino acids possess good buffering capacity in the pH range near neutrality. Additional buffering capacity in this pH range also provided by phosphates (Mono and di-hydrogen potassium or Sodium phosphates).

Avoidance of Foaming: Foaming is a serious problem in fermentation industry, Foaming can invite contamination the fermentation medium and also causes other problems for the fermentation. Hence defoamers (e.g. hard oil mixed with octadecanol for penicillin fermentation) should be used for controlling foam. These defoamers are added to the production medium before sterilization or incorporated after sterilization or added during the fermentation.

Toxicity: The ideal production medium free from any toxic effect on culture or product formation.

Consistency: In aerobic fermentation, it is necessary to supply sterile air into the medium. Under such circumstances, liquid media allow the diffusion of air throughout the medium under agitation. Fermentation media should not be viscous. Viscous nature of the medium creates difficulty in the penetration of the air interior of the medium. Air is not easily absorbed by the liquid medium.

Contamination: Certain conditions of the production medium are helpful to check the contamination. For example low pH values in citric acid production using Aspergillus niger avoids contamination.

Recovery: Recovery of the desired product is an important criteria. Components of the medium should be such that separation and extraction of the product becomes easy and cheaper.

Availability of raw materials: Raw materials required for designing of the production medium should be freely available in large quantities at a reasonable price.

Natural Media for Fermentation:
Different types of raw material are used in different types of industrial fermentation processes. Usually crude nutritive sources are preferred, since they are economical. Mostly agricultural products are utilized as a source of raw material in fermentation industries.

Carbon Source and Nitrogen Source
Carbon and Nitrogen sources are essential in fermentation media for the growth of microorganisms, These are certain carbon and nitrogen sources used in the fermentation media.

  • Saccharine Materials:
Sugar cane, sugar beets, molasses, and fruit juices may be included in this category.

Molasses: Molasses is a byproduct of the cane and beet sugar industry. It is recovered at any one of several stages in the sugar refining process. Chemical composition of sugarcane black strap is variable. It depends on the quality and variety of the cane but also on the process involved in the manufacture of sugar. About 95%of the total sugar in cane molasses is fermentable. It is particularly rich in biotin, pantothenic acid, thiamine, phosphorous and sulphur. The organic nitrogen content is less than beet molasses, since it does not contain betaine. But this substance is not assimilated by yeasts.

Beet molasses: Beet molasses are produced by the same process employed for cane molasses. Vitamins such as biotin, pyridoxine, thiamine, pantothenic acid and inositol are present in beet molasses also. Beet molasses have limited biotin. Therefore, in fermentation involving yeast culture, a small amount of cane black strap molasses or other biotin supplying material should be incorporated in the production medium. Because yeast require biotin for their growth. The largest utilization of cane black strap molasses in India is in the alcohol industry, which utilizes it for the manufacture of spirit, country liquors, rum, brandy, gin and whisky.

Fruit juices: Fruit juices contain soluble sugars. Grape juice contain glucose & fructose. Therefore, fruit juices can be used as a source of carbon in fermentation industries. Grapes are used in the production of wine.

Cheese whey: The straw coloured liquid produced as a byproduct of cheese making is called cheese whey. It is a major waste product for the cheese industry. It cannot be disposed of without proper treatment. Therefore, it is desirable to use it for useful products. It is also used as pig feed. For lactic acid production and SCP production it is served as raw material because it contains lactose, nitrogenous substances including vitamins (eg: vitamins) and inorganic salts.

  • Starchy Material:
There are two main sources of commercial starches
  1. Cereals (Wheat, rice, maize)
  2. Roots & tubers (potatoes, tapioca e.t.c)
The moisture content of the grain is low where as that of roots and tubers are very high. Starch require pretreatment to bring about the conversion to fermentable sugars. This is done either by enzymatic or chemical agents.

Cellulosic Materials:

Cellulosic materials are complex carbohydrate materials. The cellulosic molecule is made up of the repeating units of beta-glucose. The formation of beta-cellobiose requires two molecules of beta-glucose, which are linked through alpha 1, 4-linkage. 1000 to 10,000 units of cellobiose are required to form a simple linear polymer called cellulose. Units of cellobiose are joined end to end through 1, 4-beta-glucosidic linkages. For this reason cellulosic materials require some sort of pretreatment. Cellulosic materials are Sulfite waste liquor, wood molasses and Rice straw.

Sulfite waste liquor:

In the manufacture of paper pulp, wood is subjected to hydrolysis which is brought about with the help of Calcium bisulfite under heat and pressure. This operation is called digestion process. At the end of this process, the spent liquid is left and it is referred to as sulfite waste liquor. It cannot be disposed of unless it is properly treated. Sulfite waste liquor contains 10 to 12 percent solids, of which sugars make up about 20%. It contains sugars in the form of hexoses and pentoses. It is used in the industrial production of ethyl alcohol using Saccharomyces cerevisiae and in the growth of Torula utilis cells for animal feed. Saccharomyces cerevisiae requires hexoses where as Torula utilis requires both hexoses and pentoses. Sulfite waste liquor can not used directly as fermentation medium. It contains free sulfurdioxide or sulfurous acid which is toxic to microorganisms. These toxicants are removed by steam stripping or precipitation with lime.

Wood molasses:
It is produced by acid hydrolysis of wood cellulose itself. This may produce 65-85% fermentable sugars. Sulphuric acid of about 0.5% concentration is used at a temperature range of 150 to 185oC. Using a continuous process a syrup may be obtained from saw dust. This syrup may contain 4 to 5% reducing sugars (a mixture of glucose and pentoses) with an overall yield of 45 to 55%. It may be subjected to concentration to give a kind of wood molasses.

Rice Straw:
Rice straw and related agricultural materials can serve as a good source of cellulose. It is a poor quality animal feed in its natural state because of its bulkiness, poor palatability, low protein content and low digestibility. Numerous microorganisms are capable of using cellulose for their growth. Rice straw has been used as a fermentation medium in the production of silage and single cell protein (SCP), mushroom cultivation etc.
  • Hydrocarbons & Vegetable Oils
Hydrocarbons used as fermentation substrates are usually mixtures of various hydrocarbon components. These fermentation raw materials are relatively cheap. However, purified hydrocarbon fractions or hydrocarbon compounds are more expensive.

Hydrocarbon substrates (e.g. gas oil and n-paraffins) are used to produce single cell protein (SCP) products. In this way biomass of yeasts (e.g. Candida lipolytica, Candida kofuensis, Candida tropicalis) can be produced on a significant scale under aerobic conditions.

Vegetable oils:
Oils obtained by deoiling of vegetable seeds are called vegetable oils.On the basis of their degree of unsaturation, they may be grouped into following three major classes:
  1. Oleic (or 'non drying' type): These include olive and groundnut oils.
  2. Linoleic (or 'semi drying' type): These have a higher content of the double unsaturated fatty acid found in maize, sunflower and cotton seed oils.
  3. Linolenic acid (or 'drying type): These include linseed and soya bean oils containing a fatty acid with three double bonds.
These oils may undergo drying if exposed to the atmosphere due to the oxidation of the unsaturated components. Commercial vegetable oils (e.g. maize oil) may be used in conjunction with surface active agent as anti foams or alone as a nutrient source of carbon.

  • Nitrogenous Materials:
Corn steep liquor(CSL): The used steep water results from the steeping of corn during the manufacture of starch, gluten and other corn products this by product is subjected to concentration to approximately 50% solids and this concentrate is called corn steep liquor. Corn steep liquor was originally found to be useful for penicillin production specifically. But, it is now recognized as valuable in many fungal antibiotic fermentation media. In addition to this, it is also used in the manufacture of food stuffs.

Soya bean oil: the material left after removing oil from the soya bean seeds are called as soya bean meal. Soya bean meal contains approximately 8% w/w nitrogen. This differs from corn steep liquor, since soya bean meal is a much more complex nitrogenous source than corn steep liquor, and therefore not readily available to microbes. This is used as a ingredient for fermentation media in the production of streptomycin.

Pharmamedia: Pharmamedia is a clean, yellow, finely ground powder prepared from the embryo of cotton seed. It contains 56% w/w protein, 24% carbohydrate, 5% oil, and 5% ash. Ash, in turn, contains calcium, iron, chloride, phosphorous and sulfate. It is used as an ingredient for production media (Eg: Tetracycline production).

Distillers Solubles: In the manufacture of alcohol using grain or maize, alcohol is distilled from fermented grain or maize, leaving the residue (containing 6 to 8% w/v total solids). The suspended solids from the residue are eliminated by screening, leaving the effluent. Thereafter effluent is subjected to concentration, until the solid content reaches 35% w/v giving 'evaporator syrup'. This syrup is then drum dried to yield 'distillers solubles'. This may be used as a production medium component, since it supplies nitrogen, together with many accessory food factors (e.g. vitamin B complex).

Precursors & Inducers:

Certain substances, which generally improves the yield or quality of the product. These substances are known as the precursors. These precursors are incorporated without any major change in to the molecule of the fermentation product. Eg: Phenyl acetic acid and Cobalt are being added for penicillin G and Vitamin B12. Corn steep liquor yields various pencillins but addition of phenyl acetic acid determines the penicillin G production. Proteases for various proteins, alpha-amylases for starch, cellualse for cellulose, pectinase for pectin and penicillin acylase for phenyl acetic acid.

Repressors: The substances which are being employed for the repression of the industrial cultures are known as repressors.
  1. Media allowing restricted growth provides high product yield as major portion of carbon and other components of the medium are shunted to product formation rather than to growth.
  2. Nitrogen sources such as soyabean meal and praline for streptomycin; production is probably due to their slow utilization, thus avoiding nitrogen metabolite repression.
  3. Aspergillus niger for gluconic acid production is first grown on a medium that supports a rich growth as well as product formation, then the mycelium is separated and placed in a fresh medium high in carbon substrate (sugar) but lacking combined nitrogen so that additional growth cannot occur.
Antifoams: Antifoams are surface active agents, reducing the surface tension in the foams and destabilizing protein films by hydrophobic bridges between two surface, displacement of absorbed protein and rapid spreading on the surface of the film. An ideal antifoam should have a fast action on the existing foam but should not be metabolized by the microorganisms. It should be cheap, heat sterilizable, non toxic, long acting and active at low concentrations.

Examples: Stearyl alcohol and octyl decanol, esters, fatty acids, cotton seed oil, linseed oil, castor oil, cod liver oil etc, silicones, sulphonates.

If the oxygen transfer rate is severely affected by antifoam addition, then mechanical foam breakers may have to be considered as a possible alternative.

Food Biotechnology Course material,by K V Anand Raj