BT603 Handouts by VUBuddy

1. Overview of Fermentation Technology

The word fermentation comes from the LATIN term ‘ferver’ which means ‘to boil’. It is actually referred as the physical state of boiling process. The bubbling appearance in fermentation is the indication of production of gases during fermentation process that is particularly CO2 which is produced by anaerobic catabolism of sugars present in the extract by the action of microorganisms.

Definition of Fermentation: Fermentation applies to the growth of microorganisms in media under either condition (aerobic and anaerobic) to produce economically useful products.

Fermentation in Terms of Biochemist: Biochemists consider fermentation as 'an energy-generating process in which organic compounds act both as electron donors and acceptors'; hence fermentation is ‘an anaerobic process where energy is produced without the participation of oxygen or other inorganic electron acceptors’.

Fermentation in Terms of Microbiologist: Microbiologists consider fermentation as 'any process for the production of a product by means of mass culture of micro-organisms'.

Fermentation in Terms of Biotechnologist: In biotechnology, the microbiological concept is widely used. Fermentation in biotechnology can be referred to the “Use of Biological system for the sake of development / improvement / facilitation in a process by mass culture of cells”.

As the Fermentation is a process of mass culturing of the cells for the sake of desired product so according to this definition fermentation involve following:

i. Mass culturing techniques Classical techniques for mass culture of microorganisms require the use of liquid culture in large vessels with the accompanying requirements for aeration and agitation etc. Automated fermenters, which are bulky and expensive, can be used in this technique.

ii. Types of cells and organisms A wide range of cells and organisms can be culture depending upon the type of process and product that is being produce.

iii. Fermentation Products A range of capacity of fermentation products can be produced depend upon the requirement and type of process as well as microorganisms used. Fermentation products include:

• Food products: from milk (yogurt, kefir, fresh and ripened cheeses), fruits (wine, vinegar), Vegetables (pickles, sauerkraut, soy sauce), meat (fermented sausages: salami)
• Industrial chemicals: (solvents: acetone, butanol, ethanol; enzymes; amino acids)
• Specialty chemicals (Vitamins, Pharmaceuticals)

Important Products of Fermentation Process: ➢ Acids ➢ Enzymes ➢ Gasses ➢ Alcohols ➢ Hormones ➢ Single Cell Protein ➢ Yogurt ➢ Cheese ➢ Wines ➢ Bread ➢ Pickles

2. Media for Industrial Fermentations

Growth Factors

Specific nutritional requirements of microorganisms used in industrial fermentation processes are as complex and varied as the microorganisms in question. Not only are the types of microorganisms diverse (bacteria, molds and yeast, normally), but the species and strains become very specific as to their requirements. Microorganisms obtain energy for support of biosynthesis and growth from their environment in a variety of ways. Besides a source of energy, organisms require a source of materials for biosynthesis of cellular matter and products in cell operation, maintenance and reproduction. These materials must supply all the elements necessary to accomplish this. Some microorganisms utilize elements in the form of simple compounds; others require more complex compounds, usually related to the form in which they ultimately will be incorporated in the cellular material. The four predominant types of polymeric cell compounds are the lipids (fats), the polysaccharides (starch, cellulose, etc.), the informationencoded poly-deoxyribonucleic acid and poly-ribonucleic acids (DNA and RNA), and proteins.

Lipids are essentially insoluble in water and can thus be found in the non-aqueous biological phases, especially the plasma and organelle membranes. Lipids also constitute portions of more complex molecules, such as lipoproteins and liposaccharides. Lipids also serve as the polymeric biological fuel storage. Natural membranes are normally impermeable to highly charged chemical species such as phosphorylated compounds. This allows the cell to contain a reservoir of charged nutrients and metabolic intermediates, as well as maintaining a considerable difference between the internal and external concentrations of small cations, such as H', Kf and Na'. Vitamins A, E, K and D are fat-soluble and water-insoluble. Sometimes they are also classified as lipids. Typically 30-70% of the cell's dry weight is protein. All proteins contain C, H, N, and 0. Sulfur contributes to the three-dimensional stabilization of almost all proteins. Proteins show great diversity of biological functions. The building blocks of proteins are the amino acids. The predominant chemical elements in living matter are: C, H, O, and N, and they constitute approximately 99% of the atoms in most organisms. Carbon, an element of prehistoric discovery, is widely distributed in nature. Carbon is unique among the elements in the vast number and variety of compounds it can form. It has been estimated that hydrogen makes up more than 90% of all the atoms or three quarters of the mass of the universe. Oxygen makes up 21% and nitrogen 78% volume percent of the air. These elements are the smallest ones in the periodic system that can achieve stable electronic configurations by adding one, two, three or four electrons respectively. This ability to add electrons, by sharing them with other atoms, is the first step in forming chemical bonds, and thus, molecules.

Some microorganisms need preformed compounds popularly known as "growth factors", as every microorganism cannot synthesize all most all the basic requirements to build up the cell components The commonly required growth factors are vitamins, selected amino acids, fatty acids, sterols etc. The need of some or many required growth factors can be satisfied by natural carbon and nitrogen sourced added in the media formulation. The vitamin deficiency can often be satisfied by careful blending of materials added in the medium. But, in case when there is a need of only one type of vitamin, it is very costly to add in pure form. Calcium pantothenate can be used in one medium formulation for vinegar production.

Growth factors and trace nutrients are included in the fermentation broth for organisms incapable of producing all of the vitamins they require. Yeast extract is a common source of micronutrients and vitamins for fermentation media. Inorganic nutrients, including trace elements such as iron, zinc, copper, manganese, molybdenum and cobalt are typically present in unrefined carbon and nitrogen sources, but may have to be added when purified carbon and nitrogen sources are used. Fermentations which produce large amounts of gas (or which require the addition of gas) will tend to form a layer of foam, since fermentation broth typically contains a variety of foamreinforcing proteins, peptides or starches. To prevent this foam from occurring or accumulating, antifoaming agents may be added. Mineral buffering salts, such as carbonates and phosphates, may be used to stabilize pH near optimum. When metal ions are present in high concentrations, use of a chelating agent may be necessary.

Buffers

Microbial growth is pH specific and thus the control of pH is very important if optimal yield is to be achieved. Certain compounds can be added to the medium to serve exclusively as a buffer. Many media are buffered by the incorporation of calcium carbonate at about pH 7.0 and during the process, if the pH decreases the carbonate is decomposed. Phosphate is an important part of several media. Many time, phosphate play an important role in buffering during production of secondary metabolites. The pH of the medium can also be controlled by balance use of the carbon and nitrogen sources, because buffering capacity can be provided by the amino acid,