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FERMENTATION TECHNOLOGY

MCB 512

Industrial fermentation is the intentional use of fermentation by microorganisms such as bacteria and fungi to make products useful to humans. Fermented products have applications as food as well as in general industry.

Food fermentation

Ancient fermented food processes, such as making bread, wine, cheese, curds, idli, dosa, etc., can be dated to more than 6,000 years ago. They were developed long before man had any knowledge of the existence of the microorganisms involved. Fermentation is also a powerful economic incentive for semi-industrialized countries, in their willingness to produce bio-ethanol.

Pharmaceuticals and the biotechnology industry

There are 5 major groups of commercially important fermentation:

1. Microbial cells or biomass as the product, e.g. single cell protein, bakers yeast, lactobacillus, E. coli, etc.
2. Microbial enzymes: catalase, amylase, protease, pectinase, glucose isomerase, cellulase, hemicellulase, lipase, lactase, streptokinase, etc.
3. Microbial metabolites :
1. Primary metabolites – ethanol, citric acid, glutamic acid, lysine, vitamins, polysaccharides etc.
2. Secondary metabolites: all antibiotic fermentation
4. Recombinant products: insulin, HBV, interferon, GCSF, streptokinase
5. Biotransformations: phenyl acetyl carbinol, steroid biotransformation, etc.

 

Sewage disposal

In the process of sewage disposal, sewage is digested by enzymes secreted by bacteria. Solid organic matters are broken down into harmless, soluble substances and carbon dioxide. Liquids that result are disinfected to remove pathogens before being discharged into rivers or the sea or can be used as liquid fertilisers. Digested solids, known also as sludge, is dried and used as fertilisers. Gaseous by-products such as methane, can be utilised as biogas to fuel generators. One advantage of bacterial digestion is that it reduces the bulk and odour of sewage, thus reducing space needed for dumping, on the other hand, a major disadvantage of bacterial digestion in sewage disposal is that it is a very slow process.

Phases of microbial growth

When a particular organism is introduced into a selected growth medium, the medium is inoculated with the particular organism. Growth of the inoculum does not occur immediately, but takes a little while. This is the period of adaptation, called the lag phase. Following the lag phase, the rate of growth of the organism steadily increases, for a certain period--this period is the log or exponential phase. After a certain time of exponential phase, the rate of growth slows down, due to the continuously falling concentrations of nutrients and/or a continuously increasing (accumulating) concentrations of toxic substances. This phase, where the increase of the rate of growth is checked, is the deceleration (decline) phase. After the deceleration phase, growth ceases and the culture enters a stationary phase or a steady state. The biomass remains constant, except when certain accumulated chemicals in the culture lyse the cells (chemolysis). Unless other micro-organisms contaminate the culture, the chemical constitution remains unchanged. Mutation of the organism in the culture can also be a source of contamination, called internal contamination.

Fermentation (biochemistry)

Fermentation is the process of deriving energy from the oxidation of organic compounds, such as carbohydrates, and using an endogenous electron acceptor, which is usually an organic compound. In contrast, respiration is where electrons are donated to an exogenous electron acceptor, such as oxygen, via an electron transport chain. Fermentation is important in anaerobic conditions when there is no oxidative phosphorylation to maintain the production of ATP (Adenosine triphosphate) by glycolysis. During fermentation, pyruvate is metabolised to various different compounds. Homolactic fermentation is the production of lactic acid from pyruvate; alcoholic fermentation is the conversion of pyruvate into ethanol and carbon dioxide; and heterolactic fermentation is the production of lactic acid as well as other acids and alcohols. Fermentation does not necessarily have to be carried out in an anaerobic environment. For example, even in the presence of abundant oxygen, yeast cells greatly prefer fermentation to oxidative phosphorylation, as long as sugars are readily available for consumption.

Sugars are the most common substrate of fermentation, and typical examples of fermentation products are ethanol, lactic acid, and hydrogen. However, more exotic compounds can be produced by fermentation, such as butyric acid and acetone. Yeast carries out fermentation in the production of ethanol in beers, wines, and other alcoholic drinks, along with the production of large quantities of carbon dioxide. Fermentation occurs in mammalian muscle during periods of intense exercise where oxygen supply becomes limited, resulting in the creation of lactic acid.

Chemistry

Fermentation products contain chemical energy (they are not fully oxidized), but are considered waste products, since they cannot be metabolized further without the use of oxygen (or other more highly-oxidized electron acceptors). A consequence is that the production of adenosine triphosphate (ATP) by fermentation is less efficient than oxidative phosphorylation, whereby pyruvate is fully oxidized to carbon dioxide.

Ethanol fermentation

Ethanol fermentation (performed by yeast and some types of bacteria) breaks the pyruvate down into ethanol and carbon dioxide. It is important in bread-making, brewing, and wine-making. Usually only one of the products is desired; in bread-making, the alcohol is baked out, and, in alcohol production, the carbon dioxide is released into the atmosphere or used for carbonating the beverage. When the ferment has a high concentration of pectin, minute quantities of methanol can be produced.

The chemical equation below summarizes the fermentation of glucose, whose chemical formula is C₆H₁₂O₆. One glucose molecule is converted into two ethanol molecules and two carbon dioxide molecules:

C₆H₁₂O₆ → 2 C₂H₅OH + 2 CO₂

C₂H₅OH is the chemical formula for ethanol.

Before fermentation takes place, one glucose molecule is broken down into two pyruvate molecules. This is known as glycolysis.

Lactic acid fermentation

Lactic acid fermentation is the simplest type of fermentation. In essence, it is a redox reaction. In anaerobic conditions, the cell’s primary mechanism of ATP production is glycolysis. Glycolysis reduces – transfers electrons to – NAD⁺, forming NADH. However, there is only a limited supply of NAD⁺ available in a cell. For glycolysis to continue, NADH must be oxidized – have electrons taken away – to regenerate the NAD⁺. This is usually done through an electron transport chain in a process called oxidative phosphorylation; however, this mechanism is not available without oxygen.

Instead, the NADH donates its extra electrons to the pyruvate molecules formed during glycolysis. Since the NADH has lost electrons, NAD⁺ regenerates and is again available for glycolysis. Lactic acid, for which this process is named, is formed by the reduction of pyruvate.

In heterolactic acid fermentation, one molecule of pyruvate is converted to lactate; the other is converted to ethanol and carbon dioxide. In homolactic acid fermentation, both molecules of pyruvate are converted to lactate. Homolactic acid fermentation is unique because it is one of the only respiration processes to not produce a gas as a byproduct.

Homolactic fermentation breaks down the pyruvate into lactate. It occurs in the muscles of animals when they need energy faster than the blood can supply oxygen. It also occurs in some kinds of bacteria (such as lactobacilli) and some fungi. It is this type of bacteria that converts lactose into lactic acid in yogurt, giving it its sour taste. These lactic acid bacteria can be classed as homofermentative, where the end-product is mostly lactate, or heterofermentative, where some lactate is further metabolized and results in carbon dioxide, acetate, or other metabolic products.

The process of lactic acid fermentation using glucose is summarized below. In homolactic fermentation, one molecule of glucose is converted to two molecules of lactic acid:

C₆H₁₂O₆ → 2 CH₃CHOHCOOH.

or one molecule of lactose and one molecule of water make four molecules of lactate (as in some yogurts and cheeses):

C₁₂H₂₂O₁₁ + H₂O → 4 CH₃CHOHCOOH.

In heterolactic fermentation, the reaction proceeds as follows, with one molecule of glucose being converted to one molecule of lactic acid, one molecule of ethanol, and one molecule of carbon dioxide:

C₆H₁₂O₆ → CH₃CHOHCOOH + C₂H₅OH + CO₂

Before lactic acid fermentation can occur, the molecule of glucose must be split into two molecules of pyruvate. This process is called glycolysis.

Glycolysis

To extract chemical energy from glucose, the glucose molecule must be split into two molecules of pyruvate. This process also generates two molecules of adenosine triphosphate as an immediate energy yield and two molecules of NADH.

C₆H₁₂O₆ + 2 ADP + 2 P_i + 2 NAD⁺ → 2 CH₃COCOO⁻ + 2 ATP + 2 NADH + 2 H₂O + 2H⁺

The chemical formula of pyruvate is CH₃COCOO⁻. P_i stands for the inorganic phosphate. As shown by the reaction equation, glycolysis causes the reduction of two molecules of NAD⁺ to NADH. Two ADP molecules are also converted to two ATP and two water molecules via substrate-level phosphorylation.

Aerobic respiration

In aerobic respiration, the pyruvate produced by glycolysis is further oxidized completely, generating additional ATP and NADH in the citric acid cycle and by oxidative phosphorylation. However, this can occur only in the presence of oxygen. Oxygen is toxic to organisms that are obligate anaerobes, and are not required by facultative anaerobic organisms. In the absence of oxygen, one of the fermentation pathways occurs in order to regenerate NAD⁺; lactic acid fermentation is one of these pathways.

Hydrogen gas production in fermentation

Hydrogen gas is produced in many types of fermentation (mixed acid fermentation, butyric acid fermentation, cdc  aproate fermentation, butanol fermentation, glyoxylate fermentation), as a way to regenerate NAD⁺ from NADH. Electrons are transferred to ferredoxin, which in turn is oxidized by hydrogenase, producing H₂. Hydrogen gas is a substrate for methanogens and sulfate reducers, which keep the concentration of hydrogen sufficiently low to allow the production of such an energy-rich compound.

Etymology

The word fermentation is derived from the Latin verb fervere, which means to boil. It is thought to have been first used in the late fourteenth century in alchemy, but only in a broad sense. It was not used in the modern scientific sense until around 1600.