Upstream Processing

The upstream processing in a fermentation process includes preparation of the fermentation medium, sterilization of air and fermentation medium and inoculation of the fermentor.

3.5.1.1 Fermentation Medium

Metabolic activity can only be maintained if the necessary nutrients are available to the cell. A medium is defined as the substance surrounding the cells, which enables the microorganisms to grow and form products. While the single components of a medium are defined as substrates, generally, only the carbon source is called the substrate. Formulation of proper medium is an essential requirement for the success of a fermentation process.

Medium used for industrial fermentations should have the following criteria (78):

1. Maximum yield and concentration of product or biomass per unit mass of substrate

2. Minimum production of undesirable metabolites

3. Consistent quality of product

4. Minimal problems during sterilization and fermentation

The medium used for fermentation may be classified as defined, complex or technical medium. Defined medium consists only of precisely chemically defined substrates. At laboratory and pilot plant level where physical, chemical, and physiological parameters need to be standardized first, only defined media should be used for studies.

Complex medium is composed of substrates with undefined composition, such as extracts or hydrolysates from waste products, which are cheap substrates commonly used in industrial production. Relatively expensive substrates, such as yeast extract, brain heart infusion, peptone, casamio acids, and tryptone are often used for complex medium. In order to maximize the yield and selectivity of serine alkaline protease production by recombinant Bacillus subtilis, based on the amino acid compositions of the enzyme, the fermentation medium was designed with complex nutrients to supply the most needed amino acids to increase the yield (79).

Technical media are used on an industrial scale and are cheaper. Normally, but not necessarily, complex substrates are the main components. Culture medium formulated for the extracellular production of a hybrid 6-1,3-glucanase from Bacillus using a recombinant Escherichia coli contained lactose, yeast extract, and sodium chloride, and fermentation was carried out in shake flasks and a stirred tank bioreactor (80). The substrate sources can also be derived from industrial waste, and are often highly impure mixtures, requiring pretreatment before they could be used for a fermentation process. Examples are soy meal, whey, fishmeal, malt extract, and sulfite waste liquor. Wastewater from monosodium glutamate production, which contains high levels of chemical oxygen demand (COD), sulphate, and ammoniacal nitrogen at a low pH, has been used as the nitrogen and water source, with sugar beet pulp as the carbon source, for the production of pectinase by solid-state fermentation using Aspergillus niger (81).

3.5.1.2 Optimization of Fermentation Medium

Optimization of fermentation medium is done by using an experimental design considering the variables, mostly medium ingredients, that may have a significant influence on the fermentation process. Experimental design for the fermentation process involves the determination of the optimum medium composition, while simultaneously figuring out the best process parameters. Design of experiments (DOE) is a systematic approach to problem solving which is applied to data collection and analysis to obtain information rich data (82). DOE is concerned with carrying out experiments under the constraints of minimum expense of time, costs, and number of runs. A properly designed experiment is more important than detailed statistical analysis. The primary goal of designing an experiment is to obtain valid results using a minimum of effort, time, and resources (83). The limitation of single factor at a time optimization is overcome by taking into account all the significant variables collectively by statistical experimental design using Response Surface Methodology (RSM) (84), which is used to evaluate the relative significance of variables in the presence of complex interactions (85,86).

3.5.1.3 Components of Industrial Fermentation Medium

In industrial processes, the cheapest substrates are used to keep the production costs low. Some of the cheap substrates available include barley, barley malt, blood meal, cane molasses, corn gluten meal, corn meal, corn steep liquor, cotton seed meal, dried distillers' solubles, fish solubles, fishmeal, linseed meal, meat meal, bone meal, oat flour, peanut meal, rice bran, rice flour, soybean meal, wheat flour, whey powder, and yeast hydrolysate (87). The composition of the medium has to reflect the demands necessary for the growth and synthesis of products other than cells. The carbon to nitrogen ratio has been found to be a good indicator for studying the optimum requirements for growth and product formation (88). Generally, media consist of an aqueous solution containing dissolved nutrient salts and a gaseous component (O2). The different nutrient sources commonly used in fermentation media are shown in Table 3.3.

With the economics of fermentation playing an important role in a successful fermentation process, the efficient conversion of substrate to product is most crucial. In the present stage of advanced biotechnological development, this involves genetic engineering of the strain as well as a practical, focused approach in optimal use of the substrates with the aid of metabolic engineering (89). Metabolic engineering of S.cerevisiae for efficient xylose fermentation to ethanol has been reported (90).

Table 3.3

Nutritional sources used in fermentation media (91)

Table 3.3

Nutritional sources used in fermentation media (91)

Carbon Source

Nitrogen Source Organic Inorganic

Inorganic Elements

Vitamins

Medium Additives

Carbohydrates,

Urea,

Nitrates,

Phosphorus,

Thiamine,

Growth factors,

Alcohols,

Aminoacids,

Nitrites,

Sulphur,

Riboflavin,

Precursors,

Carboxylic acids,

Purines,

Ammonia,

Magnesium,

Pyridoxin,

Detergents,

Fats,

Pyrimidines,

Molecular

Potassium,

Biotin,

Antifoaming

Hydrocarbons,

Complex

Nitrogen.

Calcium,

Pantothenic

agents,

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