Type of energy sources and characteristic for a large scale media Presentation by: Mohd Raside Shafad Nur Helya Iman Tan Yan Ching Noraini Othman

INTRODUCTION TO THE MEDIA OF FERMENTATION All micro-organisms require water, sources of energy, carbon, nitrogen, mineral element and vitamin plus oxygen in their growth medium. On a small scale, it is simple to device a medium containing pure compounds, but the resulting medium although satisfy the growth, may be unsuitable for use in a large scale process.

On a large scale one must use sources of cheap nutrient to create a medium which will meet as many as possible of the following criteria:

It will produce a maximum yield of product at biomass per gram of substrate used. It will produce a maximum concentration of product or biomass. It will permit the maximum rate of product formation. It will be the minimum yield of undesired product. It will be cheap and of a consistent quality and is readily available throughout the year. It will cause minimal problem in other aspects of production and agitation, extraction, purification and waste treatment.

Use of molasses, cereal grain, glucose, sucrose and lactose as carbon sources and ammonium salts, urea, nitrates, soya bean meal, slaughter-house waste and fermentation residues as nitrogen source have tended to meet the above criteria for production media.

The medium selected will affect the design of the fermenter to be used. A laboratory medium may not ideal in a large fermenter with a low gas-transfer pattern. Media with a high viscosity will also need a higher power input for effective stirring. Besides the requirement for growth and product formation, medium may also influence pH variation, foam formation, oxidation-reduction potential and the morphological form of the organisms. It may also be necessary to provide precursors or metabolic inhibitors.

Typical Media

MEDIUM FORMULATION Medium formulation is an essential stage in the design of ~successful laboratory experiments ~pilot-scale development ~manufacturing processes The constituents of a medium must satisfy ~the elemental requirements for cell biomass and metabolite production ~must be an adequate supply of energy for biosynthesis and cell maintenance

Equation based on the stoichiometry for growth and product formation. Carbon and energy source + nitrogen source + other requirements cell biomass + products + CO2 + H2O + heat This equation should be expressed in quantitative terms, which is economical design of media if component wastage is to be minimal. It should be possible to calculate ~the minimal quantities of nutrients which will be needed to produce a specific amount of biomass ~the substrate concentration in order to produce required product yield

A knowledge of the elemental composition of a named micro-organisms, which should include the content of C, H, O, N, S, P, Mg and K, is required in the solution of the elemantal balance equation. Elemental composition of micro-organism in medium.

From Table 4.2: ~the absolute minimum quantities that needed to include in an initial medium recipe are N, S, P, Mg and K. ~trace elements may also be needed in smaller quantities such as Fe, Zn, Cu, Mn, Co, Mo, B. An analysis of relative concentrations of individual elements in bacterial cells shows ~ some nutrients are frequently added in substantial excess of that required e.g. P, K ~others are often near limiting values, e.g. Zn, Cu.

Some micro-organisms cannot synthesize specific nutrients such as amino acid, vitamins or nucleotides. Once a specific growth factor has been identified it can be incorporated into a medium in adequate amounts as a pure compound or as a component of a complex mixture. The role of carbon substrate involved in ~biosynthesis ~energy generation

The carbon requirement under aerobic condition may be estimated from the cellular yield coefficient (Y) which s defined as: = Quantity of cell dry matter produced Quantity of carbon substrate utilized Some value are given in Table 4.3

Thus, for bacteria Y of glucose = 0.5 = 0.5 g cells 1.0 g glucose-1 = the quantity of glucose neededto obtain 30 g dm-3 = 30/0.5 = 60 g dm-3 glucose thus, medium would also need to contain approximately 3.0 g dm-3N, 1.0 g dm-3P and 0.3 g dm-3S. An adequate supply of the carbon source is also essential for a product forming fermentation process. In a critical study, analyses are made to determine how the observed conversion of the carbon source to the product compares with the theoretical maximum yield.

Cooney (1979) has calculated theoretical yield for penicillin G biosynthesis on the basis of material and energy balances using a biosynthetic pathway based on reaction stoichiometry. The stoichiometry for the overall synthesis is:

Solution of this equation yields: In this instance it was calculated that the theoretical yield was 1.1 g penicillin G g-1 glucose. The other major nutrient which will be required is oxygen which will be provided by aerating the culture. The design of medium will influence the oxygen demand of a culture in that the more reduced carbon sources will result in higher oxygen demand.

Water Major component of all fermentation media Factors need to be considered pH Dissolve salt Effluent contamination Mineral water content of water is very important in brewing, and most critical in the mashing process, and historically influenced the siting of breweries and the type of beer produced.

Nowadays, the water may be treated by deionization or other techniques and salts added, or pH adjusted, to favour different beers so that breweries are not so depend on the local water sources. In large continuous-culture SCP plants it is common practice to recycle the water streams. It may even be possible to reuse all the water in the fermenter fed with appropriate adjustment of nutrient levels.

Energy Sources Energy for growth comes from either the oxidation of medium components or from light. Most industrial micro-organisms are chemo- organotrophs, therefore the commonest sources of energy will be the carbon source such as carbohydrates, lipids and proteins. Some micro-organisms can use methane or methanol as carbon and energy sources.

Carbon requirement for the medium is normally provided by: Carbon Source Carbon requirement for the medium is normally provided by: Nutrient Raw material Sucrose Sugarcane, sugar beet molasses Glucose Corn sugar, starch, cellulose Lactose Milk whey Fats Vegetable oil Starch Maize grains, cereals, potatoes and casava Hydrocarbons Petroleum fractions

Cont.. Barley grains It may be partially germinated and heat treated to give material known as malt. Malt: Contains a variety of sugar besides starch Is the main substrate for brewing in many countries. Malt extract may also be prepared from malted grain.

Lactose and crude lactose(milk whey) Sucrose Obtained from sugar cane and sugar beet Commonly used in fermentation media in very impure form as beet or cane molasses Lactose and crude lactose(milk whey) Now is extremely limited in media formulation since the introduction of continuous-feeding process. Whey is used as a substrate for biomass production

Example: olive, maize, cotton seed, linseed, soya bean, etc. Vegetable oil Example: olive, maize, cotton seed, linseed, soya bean, etc. Source of carbon : oleic, linoleic and linolenic acid. As an antifoam in association with a surface active agent.

Other carbon sources: Example: Alcohols Simple organic acids with alkanes -more expensive than equivalent quantities of crude carbohydrate. These compound can be obtained in a pure form -by simplify subsequent recovery and purification process For methane, methanol and n-alkalines: - Utilized as a substrates for biomass production.

Vitamins Biotin also known as vitamin H or B7 chemical formula :