LESSON 17 MEDIA REQUIREMENTS FOR FERMENTATION PROCESSES INTRODUCTION

Introduction
Fermentation is an important part of industrial microbiology. Fermentation technology got its origins the first time someone made wine, was perfected in the 1940s with the production of antibiotics, and is now the primary method of production in the biotechnology industry. Fermentation has always been an important part of our lives: foods can be spoiled by microbial fermentations, foods can be made by microbial fermentations, and muscle cells use fermentation to provide us with quick responses. Fermentation could be called the staff of life because it gives us the basic food, bread. But how fermentation actually works was not understood until the work of Louis Pasteur in the latter part of the nineteenth century and the research which followed. Fermentation is the process that produces alcoholic beverages or acidic dairy products. For a cell, fermentation is a way of getting energy without using oxygen. In general, fermentation involves the breaking down of complex organic substances into simpler ones. The microbial or animal cell obtains energy through glycolysis, splitting a sugar molecule and removing electrons from the molecule. The electrons are then passed to an organic molecule such as pyruvic acid. This results in the formation of a waste product that is excreted from the cell. Waste products formed in this way include ethyl alcohol, butyl alcohol, lactic acid, and acetonethe substances vital to our utilization of fermentation. Within the last century, processes with few specific technological requirements were developed which resulted in the large-scale production of microbial biomass (e.g., bakers yeast) and primary metabolites (e.g., alcohols, organic acids). Scientific advances in the general principles of fermentation and engineering after World War II enabled the first antibiotic penicillin to be manufactured. Previous to this, fermentation processes were conducted without specifically excluding undesired microorganisms. Rather, the microbial population was controlled by the ecological and environmental conditions in the fermentation vessel. With the development of techniques for microbial strain selection, large-scale sterilization, aeration, and product separation, it became possible to cultivate a specific microorganism under more optimal conditions. This resulted in increased production and significantly improved product yields. Fermentation processes have now become more complicated and more expensive, and end products more valuable. Today, a variety of primary and secondary metabolites (e.g., antibiotics, steroids, vitamins, enzymes) are produced industrially using these technologies.

UNIT III MEDIA DESIGN AND STERILISATION FOR FERMENTATION PROCESSES

Antifungal agents Organic synthesis intermediates Antiprotozoal agents Pharmaceutical significant compounds Carbohydrates Plant growth factors Dyes and cosmetics Steroids Enzymes Toxins Foods Vitamins and coenzymes
Products Produced by Recombinant DNA Technology

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Human therapeutics Enzymes Amino acids Fermentations are divided into three broad classes based on the relationships between microbial growth, utilization of the energy source, and product formation. Within these classes, fermentation products can be classified according to where in the process they are produced. Products produced during the growth phase of the microorganism are referred to as primary metabolites. Those produced maximally after growth has stopped (or in some cases during restricted growth) are termed secondary metabolites. The third class focuses on production of cells or biomass. A growing cell normally uses its primary metabolites for growth, and does not export them into growth medium. However, in fermentation processes designed for the production of primary metabolites, the fermentation medium is developed so that the cells are encouraged to excrete the metabolite of interest into the medium. A primary metabolic product is derived in one of two ways: directly from the primary metabolism used for energy production; or indirectly in a secondary pathway, which is separate from primary metabolism. In the former instance, the product is produced maximally during primary metabolism of the energy source and coincident with the exponential phase of growth. The production of ethanol or biomass are common examples. In the second instance, the product is produced after growth has ceased, although its production remains dependent on continuing metabolism of the energy source. Here, the growth phase is accompanied by rapid substrate utilization with little or no product formation; while the production phase shows minimal growth but continued rapid consumption of the energy source. Citric acid is an example of a primary metabolite produced in this fashion.

Products Produced by Microbial Activity. Amino acids Lipids Antibacterial agents Nucleotides and precursors

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Of the major fermentation products, many are metabolites, which the cell does not need directly for growth, but which are important for some other reason. In nature, they are produced at low concentration. But through laboratory mutation and selection, cells have been engineered to overproduce these metabolites. Many antibiotics and vitamins are secondary metabolites. During batch fermentation processes, product formation is sequential to primary metabolism and cell growth. As such, optimum medium composition for cell growth may differ from the medium composition required to maximize product yield. Nutrients in a fermentation medium are consumed by the microorganism for incorporation into biomass, and production of metabolic products. As a first approximation of the minimum nutrient metabolism of a fermenting organism: C-source + N-source + minerals + any specific nutrients + O2 (or other redox source) -cell mass + product + CO2 (or other carbon waste) + H2O In fermentation media, the carbon source is the most abundant nutrient. Carbohydrates are the usual carbon source, and are typically provided at a concentration of 0.2-20% (w/v). Carbohydrates have the general formulas of (CH2O)n and provide carbon, hydrogen, oxygen, and a source of energy to the microorganism. Nitrogen is the next most abundant, and its concentration is dependent on the type of microorganism and the end-product specific to the fermentation process. Eg. Soy as a nitrogen source Beyond this, it is necessary to optimally balance the nutrients for sufficient cell growth and maximum product formation. Generalizations regarding fermentation medium development are rather difficult a priori. There is no established method to calculate a medium composition that will be optimal for a specific fermentation process. Rather, it is often a combination of general principles and trial and error. The composition of each fermentation medium must be adapted to the process, and new batches of substrate must be evaluated in small-scale fermentations before they are used in production. As such, industrial fermentation media formulations arise from much research and development, and are often regarded as proprietary. In formulating a fermentation medium not only are the relative amounts of each ingredient important, but the characteristics, the availability, and the cost of each also must be considered. Nutrients account for 10% to 60% of the manufacturing cost of a fermentation product. To be effective sources of nutrients, the microorganism must have the appropriate metabolic (catabolic) capability. The form in which a particular nutrient is provided may significantly affect the extent of cell growth and the product yield. For example, proteins are only utilizable as a nitrogen source by microorganisms that excrete extracellular proteases. Proteases hydrolyze the proteins to peptides and amino acids, which then can be absorbed by the microorganism. Microorganisms, which are not proteolytic, require nitrogen predigested in the form of small peptides or amino acids. Other factors of importance in the design of a growth medium are the downstream processing and waste treatment requirements or restrictions. Media components that increase the cost of product recovery or waste treatment are undesirable.
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All microorganisms need for their microbial activity the presence of several nutrients.
Carbohydrates

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Carbohydrates are capable of being used by all microorganisms, although in no case is there an absolute requirement for this group of organic compounds. Glucose is the most readily metabolized sugar. Most fungi can use disaccharides.
Lipids

Microbial requirements for steroids, and long-chain fatty acids can be summarized as follows. Long-chain fatty acids like linoleic acid and oleic acid are required for bacteria and fungi. Generally, steroids, other than cholesterol, are not required or utilized by microorganisms. In all fungi, and including yeast, ergosterol is a nutritional requirement.
Purines and Pyrimidines

It is generally only in bacteria that cases of purine and pyrimidine metabolism have been reported. Algae do not utilize these compounds at all.
Vitamins and Growth Factors

There is considerable species variation in the requirements of vitamins and related factors by other microorganisms. Generally, vitamins A, C, D, and K are not necessary for growth.
Amino Acids

Amino acids are not generally required by algae, although several algae species are capable of utilizing them. Species of other microorganisms are capable of utilizing all amino acids, except for yeasts, where there is no evidence of critrulline being used. It is usually the L-form of the acids that are biologically active but, unlike higher animals, some bacteria can also utilize the Damino acids.
Nitrogen Sources

It should be stressed that not all species require or utilize these compounds but rather that some species have been identified that are able to utilize these compounds. Fungi require ammonia, nitrate and nitrite.
Sulfur Sources

Some species of yeasts can utilize elemental sulfur and sulfate. Generally yeasts do not require or utilize sulfur containing organic compounds. Bacteria require glutathione and thio-acetic acid while yeasts require sulphonic acid amides, thioacetate, thiocarbonate, thioglycolate and glutathione.
Chemical Elements and Inorganic Ions

Mineral nutrients required by microorganisms are species dependent but consists generally of Fe, K, Mg, Mn. Sometimes S, N, Ca, Co, Cu, P, Zn is required. A microbial fermentation can be viewed as a three-phase system, involving liquid-solid, gas-solid, and gas-liquid reactions. The liquid phase contains dissolved nutrients, dissolved substrates and dissolved metabolites.

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The solid phase consists of individual cells, pellets, insoluble substrates, or precipitated metabolic products. The gaseous phase provides a reservoir for oxygen supply and for CO2 removal.
Review Question

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Q1 What are the different Products produced by microbial activity?. Q2 All microorganisms need for their microbial activity the presence of several nutrients.,Explain them. Notes:

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