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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.

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Biotechnology

Biotechnology is the use of living systems and organisms to develop or make useful products, or "any technological application that uses biological systems, living organisms or derivatives thereof, to make or modify products or processes for specific use". Depending on the tools and applications, it often overlaps with the (related) fields of bioengineering and biomedical engineering.

For thousands of years, humankind has used biotechnology in agriculture, food production, and medicine. The term itself is largely believed to have been coined in 1919 by Hungarian engineer Károly Ereky. In the late 20th and early 21st century, biotechnology has expanded to include new and diverse sciences such as genomics, recombinant gene technologies, applied immunology, and development of pharmaceutical therapies and diagnostic tests.

Definitions

The wide concept of "biotech" or "biotechnology" a wide range of procedures for modifying living organisms according to human purposes, going back to domestication of animals, cultivation of plants, and "improvements" to these through breeding programs that employ artificial selection and hybridization. Modern usage also includes genetic engineering as well as cell and tissue culture technologies. The American Chemical Society defines biotechnology as the application of biological organisms, systems, or processes by various industries to learning about the science of life and the improvement of the value of materials and organisms such as pharmaceuticals, crops, and livestock. As per European Federation of Biotechnology, Biotechnology is the integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services. Biotechnology also writes on the pure biological sciences (animal cell culture, biochemistry, cell biology, embryology, genetics, microbiology, and molecular biology). In many instances, it is also dependent on knowledge and methods from outside the sphere of biology including:

• bioinformatics, a new brand of computer science
• bioprocess engineering
• biorobotics
• chemical engineering

Conversely, modern biological sciences (including even concepts such as molecular ecology) are intimately entwined and heavily dependent on the methods developed through biotechnology and what is commonly thought of as the life sciences industry. Biotechnology is the research and development in the laboratory using bioinformatics for exploration, extraction, exploitation and production from any living organisms and any source of biomass by means of biochemical engineering where high value-added products could be planned (reproduced by biosynthesis, for example), forecasted, formulated, developed, manufactured and marketed for the purpose of sustainable operations (for the return from bottomless initial investment on R & D) and gaining durable patents rights (for exclusives rights for sales, and prior to this to receive national and international approval from the results on animal experiment and human experiment, especially on the pharmaceutical branch of biotechnology to prevent any undetected side-effects or safety concerns by using the products).

By contrast, bioengineering is generally thought of as a related field that more heavily emphasizes higher systems approaches (not necessarily the altering or using of biological materials directly) for interfacing with and utilizing living things. Bioengineering is the application of the principles of engineering and natural sciences to tissues, cells and molecules. This can be considered as the use of knowledge from working with and manipulating biology to achieve a result that can improve functions in plants and animals. Relatedly, biomedical engineering is an overlapping field that often draws upon and applies biotechnology (by various definitions), especially in certain sub-fields of biomedical and/or chemical engineering such astissue engineering, biopharmaceutical engineering, and genetic engineering.

History

Brewing was an early application of biotechnology

Although not normally what first comes to mind, many forms of human-derived agriculture clearly fit the broad definition of "'using a biotechnological system to make products". Indeed, the cultivation of plants may be viewed as the earliest biotechnological enterprise.

Agriculture has been theorized to have become the dominant way of producing food since the Neolithic Revolution. Through early biotechnology, the earliest farmers selected and bred the best suited crops, having the highest yields, to produce enough food to support a growing population. As crops and fields became increasingly large and difficult to maintain, it was discovered that specific organisms and their by-products could effectively fertilize, restore nitrogen, and control pests. Throughout the history of agriculture, farmers have inadvertently altered the genetics of their crops through introducing them to new environments and breeding them with other plants — one of the first forms of biotechnology.

These processes also were included in early fermentation of beer. These processes were introduced in early Mesopotamia, Egypt, China and India, and still use the same basic biological methods. In brewing, malted grains (containing enzymes) convert starch from grains into sugar and then adding specific yeasts to produce beer. In this process, carbohydrates in the grains were broken down into alcohols such as ethanol. Later other cultures produced the process of lactic acid fermentation which allowed the fermentation and preservation of other forms of food, such as soy sauce. Fermentation was also used in this time period to produce leavened bread. Although the process of fermentation was not fully understood until Louis Pasteur's work in 1857, it is still the first use of biotechnology to convert a food source into another form.

Before the time of Charles Darwin's work and life, animal and plant scientists had already used selective breeding. Darwin added to that body of work with his scientific observations about the ability of science to change species. These accounts contributed to Darwin's theory of natural selection.

For thousands of years, humans have used selective breeding to improve production of crops and livestock to use them for food. In selective breeding, organisms with desirable characteristics are mated to produce offspring with the same characteristics. For example, this technique was used with corn to produce the largest and sweetest crops.

In the early twentieth century scientists gained a greater understanding of microbiology and explored ways of manufacturing specific products. In 1917, Chaim Weizmann first used a pure microbiological culture in an industrial process, that of manufacturing corn starch using Clostridium acetobutylicum, to produce acetone, which the United Kingdom desperately needed to manufacture explosives during World War I.

Biotechnology has also led to the development of antibiotics. In 1928, Alexander Fleming discovered the mold Penicillium. His work led to the purification of the antibiotic compound formed by the mold by Howard Florey, Ernst Boris Chain and Norman Heatley - to form what we today know as penicillin. In 1940, penicillin became available for medicinal use to treat bacterial infections in humans.

The field of modern biotechnology is generally thought of as having been born in 1971 when Paul Berg's (Stanford) experiments in gene splicing had early success. Herbert W. Boyer (Univ. Calif. at San Francisco) and Stanley N. Cohen (Stanford) significantly advanced the new technology in 1972 by transferring genetic material into a bacterium, such that the imported material would be reproduced. The commercial viability of a biotechnology industry was significantly expanded on June 16, 1980, when the United States Supreme Court ruled that a genetically modified microorganism could be patented in the case of Diamond v. Chakrabarty. Indian-born Ananda Chakrabarty, working for General Electric, had modified a bacterium (of the Pseudomonas genus) capable of breaking down crude oil, which he proposed to use in treating oil spills. (Chakrabarty's work did not involve gene manipulation but rather the transfer of entire organelles between strains of the Pseudomonas bacterium.

Revenue in the industry is expected to grow by 12.9% in 2008. Another factor influencing the biotechnology sector's success is improved intellectual property rights legislation—and enforcement—worldwide, as well as strengthened demand for medical and pharmaceutical products to cope with an ageing, and ailing, U.S. population.

Rising demand for biofuels is expected to be good news for the biotechnology sector, with the Department of Energy estimating ethanol usage could reduce U.S. petroleum-derived fuel consumption by up to 30% by 2030. The biotechnology sector has allowed the U.S. farming industry to rapidly increase its supply of corn and soybeans—the main inputs into biofuels—by developing genetically modified seeds which are resistant to pests and drought. By boosting farm productivity, biotechnology plays a crucial role in ensuring that biofuel production targets are met.

Examples

A rose plant that began as cells grown in a tissue culture

Biotechnology has applications in four major industrial areas, including health care (medical), crop production and agriculture, non-food (industrial) uses of crops and other products (e.g. biodegradable plastics, vegetable oil, biofuels), and environmental uses.

For example, one application of biotechnology is the directed use of organisms for the manufacture of organic products (examples include beer andmilk products). Another example is using naturally present bacteria by the mining industry in bioleaching. Biotechnology is also used to recycle, treat waste, cleanup sites contaminated by industrial activities (bioremediation), and also to produce biological weapons.

A series of derived terms have been coined to identify several branches of biotechnology; for example:

• Bioinformatics is an interdisciplinary field which addresses biological problems using computational techniques, and makes the rapid organization as well as analysis of biological data possible. The field may also be referred to as computational biology, and can be defined as, "conceptualizing biology in terms of molecules and then applying informatics techniques to understand and organize the information associated with these molecules, on a large scale." Bioinformatics plays a key role in various areas, such as functional genomics, structural genomics, and proteomics, and forms a key component in the biotechnology and pharmaceutical sector.
• Blue biotechnology is a term that has been used to describe the marine and aquatic applications of biotechnology, but its use is relatively rare.
• Green biotechnology is biotechnology applied to agricultural processes. An example would be the selection and domestication of plants via micro-propagation. Another example is the designing of transgenic plants to grow under specific environments in the presence (or absence) of chemicals. One hope is that green biotechnology might produce more environmentally friendly solutions than traditional industrial agriculture. An example of this is the engineering of a plant to express a pesticide, thereby ending the need of external application of pesticides. An example of this would be Bt corn. Whether or not green biotechnology products such as this are ultimately more environmentally friendly is a topic of considerable debate.
• Red biotechnology is applied to medical processes. Some examples are the designing of organisms to produce antibiotics, and the engineering of genetic cures through genetic manipulation.
• White biotechnology, also known as industrial biotechnology, is biotechnology applied to industrial processes. An example is the designing of an organism to produce a useful chemical. Another example is the using of enzymes as industrial catalysts to either produce valuable chemicals or destroy hazardous/polluting chemicals. White biotechnology tends to consume less in resources than traditional processes used to produce industrial goods. The investment and economic output of all of these types of applied biotechnologies is termed as "bioeconomy".

Medicine

In medicine, modern biotechnology finds applications in areas such as pharmaceutical drug discovery and production, pharmacogenomics, and genetic testing (or genetic screening).

Pharmacogenomics (a combination of pharmacology and genomics) is the technology that analyses how genetic makeup affects an individual's response to drugs. It deals with the influence of genetic variation on drug response in patients by correlating gene expression or single-nucleotide polymorphisms with a drug's efficacy or toxicity. By doing so, pharmacogenomics aims to develop rational means to optimize drug therapy, with respect to the patients' genotype, to ensure maximum efficacy with minimal adverse effects. Such approaches promise the advent of "personalized medicine"; in which drugs and drug combinations are optimized for each individual's unique genetic makeup.

Biotechnology has contributed to the discovery and manufacturing of traditional small molecule pharmaceutical drugs as well as drugs that are the product of biotechnology - biopharmaceutics. Modern biotechnology can be used to manufacture existing medicines relatively easily and cheaply. The first genetically engineered products were medicines designed to treat human diseases. To cite one example, in 1978 Genentech developed synthetic humanized insulin by joining its gene with a plasmid vector inserted into the bacterium Escherichia coli. Insulin, widely used for the treatment of diabetes, was previously extracted from the pancreas of abattoir animals (cattle and/or pigs). The resulting genetically engineered bacterium enabled the production of vast quantities of synthetic human insulin at relatively low cost. Biotechnology has also enabled emerging therapeutics like gene therapy. The application of biotechnology to basic science (for example through the Human Genome Project) has also dramatically improved our understanding of biology and as our scientific knowledge of normal and disease biology has increased, our ability to develop new medicines to treat previously untreatable diseases has increased as well.

Genetic testing allows the genetic diagnosis of vulnerabilities to inherited diseases, and can also be used to determine a child's parentage (genetic mother and father) or in general a person's ancestry. In addition to studying chromosomes to the level of individual genes, genetic testing in a broader sense includes biochemical tests for the possible presence of genetic diseases, or mutant forms of genes associated with increased risk of developing genetic disorders. Genetic testing identifies changes in chromosomes, genes, or proteins. Most of the time, testing is used to find changes that are associated with inherited disorders. The results of a genetic test can confirm or rule out a suspected genetic condition or help determine a person's chance of developing or passing on a genetic disorder. As of 2011 several hundred genetic tests were in use. Since genetic testing may open up ethical or psychological problems, genetic testing is often accompanied by genetic counseling.

Agriculture

Genetically modified crops ("GM crops", or "biotech crops") are plants used in agriculture, the DNA of which has been modified using genetic engineering techniques. In most cases the aim is to introduce a new trait to the plant which does not occur naturally in the species.

Examples in food crops include resistance to certain pests, diseases, stressful environmental conditions, resistance to chemical treatments (e.g. resistance to a herbicide), reduction of spoilage, or improving the nutrient profile of the crop. Examples in non-food crops include production of pharmaceutical agents, biofuels, and other industrially useful goods, as well as for bioremediation.

Farmers have widely adopted GM technology. Between 1996 and 2011, the total surface area of land cultivated with GM crops had increased by a factor of 94, from 17,000 square kilometers (4,200,000 acres) to 1,600,000 km² (395 million acres). 10% of the world's crop lands were planted with GM crops in 2010. As of 2011, 11 different transgenic crops were grown commercially on 395 million acres (160 million hectares) in 29 countries such as the USA, Brazil, Argentina, India, Canada, China, Paraguay, Pakistan, South Africa, Uruguay, Bolivia, Australia, Philippines, Myanmar, Burkina Faso, Mexico and Spain.

Genetically modified foods are foods produced from organisms that have had specific changes introduced into their DNA using the methods of genetic engineering. These techniques have allowed for the introduction of new crop traits as well as a far greater control over a food's genetic structure than previously afforded by methods such as selective breeding and mutation breeding. Commercial sale of genetically modified foods began in 1994, when Calgene first marketed its Flavr Savr delayed ripening tomato. To date most genetic modification of foods have primarily focused on cash crops in high demand by farmers such as soybean, corn, canola, and cotton seed oil. These have been engineered for resistance to pathogens and herbicides and better nutrient profiles. GM livestock have also been experimentally developed, although as of November 2013 none are currently on the market.

There is broad scientific consensus that food on the market derived from GM crops poses no greater risk to human health than conventional food. GM crops also provide a number of ecological benefits, if not used in excess. However, opponents have objected to GM crops per se on several grounds, including environmental concerns, whether food produced from GM crops is safe, whether GM crops are needed to address the world's food needs, and economic concerns raised by the fact these organisms are subject to intellectual property law.

Industrial biotechnology

Industrial biotechnology (known mainly in Europe as white biotechnology) is the application of biotechnology for industrial purposes, including industrial fermentation. It includes the practice of using cells such as micro-organisms, or components of cells like enzymes, to generate industrially useful products in sectors such as chemicals, food and feed, detergents, paper and pulp, textiles and biofuels. In doing so, biotechnology uses renewable raw materials and may contribute to lowering greenhouse gas emissions and moving away from a petrochemical-based economy.

Regulation

The regulation of genetic engineering concerns approaches taken by governments to assess and manage the risks associated with the use of genetic engineering technology, and the development and release of genetically modified organisms (GMO), including genetically modified crops and genetically modified fish. There are differences in the regulation of GMOs between countries, with some of the most marked differences occurring between the USA and Europe. Regulation varies in a given country depending on the intended use of the products of the genetic engineering. For example, a crop not intended for food use is generally not reviewed by authorities responsible for food safety. The European Union differentiates between approval for cultivation within the EU and approval for import and processing. While only a few GMOs have been approved for cultivation in the EU a number of GMOs have been approved for import and processing. The cultivation of GMOs has triggered a debate about coexistence of GM and non GM crops. Depending on the coexistence regulations incentives for cultivation of GM crops differ.