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Fermentation
Fermentation is a metabolic process that converts sugar to acids , gases or alcohol. It occurs in yeast and bacteria, and also in oxygen-starved muscle cells, as in the case of lactic acid fermentation. Fermentation is also used more broadly to refer to the bulk growth of microorganisms on a growth medium, often with the goal of producing a specific chemical product like enzyme, vaccines, antibiotics, food product/additive etc. French microbiologist Louis Pasteur is often remembered for his insights into fermentation and its microbial causes. The science of fermentation is known as zymology.
Fermentation takes place in the lack of oxygen (when the electron transport chain is unusable) and becomes the cell’s primary means of ATP (energy) production. It turns NADH and pyruvate produced in the glycolysis step into NAD+^ and various small molecules depending on the type of fermentation. In the presence of O 2 , NADH and pyruvate are used to generate ATP in respiration. This is called oxidative phosphorylation, and it generates much more ATP than glycolysis alone. For that reason, cells generally benefit from avoiding fermentation when oxygen is available, the exception being obligate anaerobes which cannot tolerate oxygen.
The first step, glycolysis, is common to all fermentation pathways:
C 6 H 12 O 6 + 2 NAD+^ + 2 ADP + 2 Pi → 2 CH 3 COCOO−^ + 2 NADH + 2 ATP + 2 H 2 O + 2H+
Pyruvate is CH 3 COCOO−. Pi is inorganic phosphate. Two ADP molecules and two Pi are converted to two ATP and two water molecules via substrate-level phosphorylation. Two molecules of NAD+^ are also reduced to NADH. In oxidative phosphorylation the energy for ATP formation is derived from an electrochemical proton gradient generated across the inner mitochondrial membrane (or, in the case of bacteria, the plasma membrane) via the electron transport chain. Glycolysis has substrate-level phosphorylation (ATP generated directly at the point of reaction).
Humans have used fermentation to produce food and beverages since the Neolithic age. For example, fermentation is used for preservation in a process that produces lactic acid as found in such sour foods as pickled cucumbers, kimchi and yogurt, as well as for producing alcoholic
beverages such as wine and beer. Fermentation can even occur within the stomachs of animals, such as humans.
Definitions of Fermentation
To many people, fermentation simply means the production of alcohol: grains and fruits are fermented to produce beer and wine. If a food soured, one might say it was 'off' or fermented. Here are some definitions of fermentation. They range from informal, general usage to more scientific definitions.
- Preservation methods for food via microorganisms (general use).
- Any process that produces alcoholic beverages or acidic dairy products (general use).
- Any large-scale microbial process occurring with or without air (common definition used in industry).
- Any energy-releasing metabolic process that takes place only under anaerobic conditions (becoming more scientific).
- Any metabolic process that releases energy from a sugar or other organic molecules, does not require oxygen or an electron transport system, and uses an organic molecule as the final electron acceptor (most scientific).
Examples of Fermentation
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 aerobic respiration, as long as sugars are readily available for consumption (a phenomenon known as the Crabtree effect). The antibiotic activity of hops also inhibits aerobic metabolism in yeast. Fermentation react NADH with an endogenous, organic electron acceptor. Usually this is pyruvate formed from the sugar during the glycolysis step. During fermentation, pyruvate is metabolized to various compounds through several processes:
- Ethanol fermentation , aka alcoholic fermentation, is the production of ethanol and carbon dioxide
- Lactic acid fermentation refers to two means of producing lactic acid: Homolactic fermentation is the production of lactic acid exclusively Heterolactic fermentation is the production of lactic acid as well as other acids and alcohols.
Heterolactic fermentation , where some lactate is further metabolized and results in ethanol and carbon dioxide (via the phosphoketolase pathway), acetate, or other metabolic products, e.g.: C 6 H 12 O 6 →CH 3 CHOHCOOH+C 2 H 5 OH+CO 2 If lactose is fermented (as in yogurts and cheeses), it is first converted into glucose and galactose (both six-carbon sugars with the same atomic formula): C 12 H 22 O 11 + H 2 O → 2 C 6 H 12 O 6 Heterolactic fermentation is in a sense intermediate between lactic acid fermentation, and other types, e.g. alcoholic fermentation. The reasons to go further and convert lactic acid into anything else are:
The acidity of lactic acid impedes biological processes; this can be beneficial to the fermenting organism as it drives out competitors who are unadapted to the acidity; as a result the food will have a longer shelf-life (part of the reason foods are purposely fermented in the first place); however, beyond a certain point, the acidity starts affecting the organism that produces it. The high concentration of lactic acid (the final product of fermentation) drives the equilibrium backwards (Le Chatelier's principle), decreasing the rate at which fermentation can occur, and slowing down growth Ethanol, that lactic acid can be easily converted to, is volatile and will readily escape, allowing the reaction to proceed easily. CO 2 is also produced, however it's only weakly acidic, and even more volatile than ethanol. Acetic acid (another conversion product) is acidic, and not as volatile as ethanol; however, in the presence of limited oxygen, its creation from lactic acid releases a lot of additional energy. It is a lighter molecule than lactic acid, that forms fewer hydrogen bonds with its surroundings (due to having fewer groups that can form such bonds), and thus more volatile and will also allow the reaction to move forward more quickly. If propionic acid, butyric acid and longer monocarboxylic acids are produced (see mixed acid fermentation), the amount of acidity produced per glucose consumed will decrease, as with ethanol, allowing faster growth.
Aerobic respiration
In aerobic respiration, the pyruvate produced by glycolysis is 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 is 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, caproate 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 H 2. Hydrogen gas is a substrate for methanogens and sulfate reducers, which keep the concentration of hydrogen low and favor the production of such an energy-rich compound, but hydrogen gas at a fairly high concentration can nevertheless be formed, as in flatus.
As an example of mixed acid fermentation, bacteria such as Clostridium pasteurianum ferment glucose producing butyrate, acetate, carbon dioxide and hydrogen gas: The reaction leading to acetate is:
C 6 H 12 O 6 + 4 H 2 O → 2 CH 3 COO−^ + 2 HCO 3 −^ + 4 H+^ + 4 H 2
Glucose could theoretically be converted into just CO 2 and H 2 , but the global reaction releases little energy.
Methane gas production in fermentation
Acetic acid can also undergo a dismutation reaction to produce methane and carbon dioxide:
CH 3 COO−^ + H+^ → CH 4 + CO 2 ΔG° = -36 kJ/reaction
This disproportionation reaction is catalysed by methanogen archaea in their fermentative metabolism. One electron is transferred from the carbonyl function (e−^ donor) of the carboxylic
Advances in microbiology and fermentation technology have continued steadily up until the present. For example, in the late 1970s, it was discovered that microorganisms could be mutated with physical and chemical treatments to be higher-yielding, faster-growing, tolerant of less oxygen, and able to use a more concentrated medium. Strain selection and hybridization developed as well, affecting most modern food fermentations. Other approach to advancing the fermentation industry has been done by companies such as BioTork, a biotechnology company that naturally evolves microorganisms to improve fermentation processes. This approach differs from the more popular genetic modification, which has become the current industry standard.
Industrial fermentation
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. Some commodity chemicals, such as acetic acid, citric acid, and ethanol are made by fermentation. The rate of fermentation depends on the concentration of microorganisms, cells, cellular components, and enzymes as well as temperature, pH and for aerobic fermentation oxygen. Product recovery frequently involves the concentration of the dilute solution. Nearly all commercially produced enzymes, such as lipase, invertase and rennet, are made by fermentation with genetically modified microbes. In some cases, production of biomass itself is the objective, as in the case of baker's yeast and lactic acid bacteria starter cultures for cheese making. In general, fermentations can be divided into four types:
Production of biomass (viable cellular material) Production of extracellular metabolites (chemical compounds) Production of intracellular components (enzymes and other proteins) Transformation of substrate (in which the transformed substrate is itself the product)
These types are not necessarily disjoint from each other, but provide a framework for understanding the differences in approach. The organisms used may be bacteria, yeasts, molds, animal cells, or plant cells. Special considerations are required for the specific organisms used in the fermentation, such as the dissolved oxygen level, nutrient levels, and temperature.
General overview of Industrial fermentation
In most industrial fermentations, the organisms are submerged in a liquid medium; in others, such as the fermentation of cocoa beans, coffee cherries, and miso, fermentation takes place on the moist surface of the medium. There are also industrial considerations related to the fermentation process. For instance, to avoid biological process contamination, the fermentation medium, air, and equipment are sterilized. Foam control can be achieved by either mechanical foam destruction or chemical anti-foaming agents. Several other factors must be measured and controlled such as pressure, temperature, agitator shaft power, and viscosity. An important element for industrial fermentations is scale up. This is the conversion of a laboratory procedure to an industrial process. It is well established in the field of industrial microbiology that what works well at the laboratory scale may work poorly or not at all when first attempted at large
something else altogether. For large scale fermentations, such as those used for the production of ethanol, inexpensive sources of carbohydrates, such as molasses, corn steep liquor, sugar cane juice, or sugar beet juice are used to minimize costs. More sensitive fermentations may instead use purified glucose, sucrose, glycerol or other sugars, which reduces variation and helps ensure the purity of the final product. Organisms meant to produce enzymes such as beta galactosidase, invertase or other amylases may be fed starch to select for organisms that express the enzymes in large quantity.
Fixed nitrogen sources are required for most organisms to synthesize proteins, nucleic acids and other cellular components. Depending on the enzyme capabilities of the organism, nitrogen may be provided as bulk protein, such as soy meal; as pre-digested polypeptides, such as peptone or tryptone; or as ammonia or nitrate salts. Cost is also an important factor in the choice of a nitrogen source. Phosphorus is needed for production of phospholipids in cellular membranes and for the production of nucleic acids. The amount of phosphate which must be added depends upon the composition of the broth and the needs of the organism, as well as the objective of the fermentation. For instance, some cultures will not produce secondary metabolites in the presence of phosphate.
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 foam- reinforcing 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.
Production of biomass
Microbial cells or biomass is sometimes the intended product of fermentation. Examples include single cell protein, baker’s yeast, Lactobacillus, E. coli, and others. In the case of single-cell protein, algae are grown in large open ponds which allow photosynthesis to occur. If the biomass is to be used for inoculation of other fermentations, care must be taken to prevent mutations from occurring.
Production of extracellular metabolites
Microbial metabolites can be divided into two groups: those produced during the growth phase of the organism, called primary metabolites and those produced during the stationary phase, called secondary metabolites. Some examples of primary metabolites are ethanol, citric acid, glutamic acid, lysine, vitamins and polysaccharides. Some examples of secondary metabolites are penicillin, cyclosporin A, gibberellin, and lovastatin.
Primary metabolites
Primary metabolites are compounds made during the ordinary metabolism of the organism during the growth phase. A common example is ethanol or lactic acid, produced during glycolysis. Citric acid is produced by some strains of Aspergillus niger as part of the citric acid cycle to acidify their environment and prevent competitors from taking over. Glutamate is produced by some Micrococcus species, and some Corynebacterium species produce lysine, threonine, tryptophan and other amino acids. All of these compounds are produced during the normal "business" of the cell and released into the environment. There is therefore no need to rupture the cells for product recovery.
Secondary metabolites
Secondary metabolites are compounds made in the stationary phase; penicillin, for instance, prevents the growth of bacteria which could compete with Penicillium molds for resources. Some bacteria, such as Lactobacillus species, are able to produce bacteriocins which prevent the growth of bacterial competitors as well. These compounds are of obvious value to humans wishing to prevent the growth of bacteria, either as antibiotics or as antiseptics (such as
Sewage treatment
In the process of sewage treatment, 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 fertilizers. Digested solids, known also as sludge, is dried and used as fertilizer. Gaseous byproducts such as methane can be utilized as biogas to fuel electrical generators. One advantage of bacterial digestion is that it reduces the bulk and odor of sewage, thus reducing space needed for dumping. The main disadvantage of bacterial digestion in sewage disposal is that it is a very slow process.
Agricultural Feed
A wide variety of agroindustrial waste products can be fermented to use as food for animals, especially ruminants. Fungi have been employed to break down cellulosic wastes to increase protein content and improve in vitro digestibility.
BIOASSAY
Bioassay (commonly used shorthand for biological assay or assessment ), or biological standardization is a type of scientific experiment. A bioassay involves the use of live animal or plant ( in vivo ) or tissue or cell ( in vitro ) to determine the biological activity of a substance, such as a hormone or drug. Bioassays are typically conducted to measure the effects of a substance on a living organism and are essential in the development of new drugs and in monitoring environmental pollutants. Both are procedures by which the potency or the nature of a substance is estimated by studying its effects on living matter. A bioassay can also be used to determine the concentration of a particular constitution of a mixture that may cause harmful effects on organisms or the environment.
Use of bioassays
Bioassays are procedures that can determine the concentration or purity or biological activity of a substance such as vitamin, hormone or plant growth factor by measuring the effect on an organism, tissue, cells, enzyme or receptor. Bioassays may be qualitative or quantitative. Qualitative bioassays are used for assessing the physical effects of a substance that may not be quantified, such as seeds fail to germinate or develop abnormally deformity. An example of a qualitative bioassay includes Arnold Adolph Berthold's famous experiment on castrated chickens. This analysis found that by removing the testicles of a chicken, it would not develop into a rooster because the endocrine signals necessary for this process were not available. Quantitative bioassays involve estimation of the dose-response curve, how the response changes with increasing dose. That dose-response relation allows estimation of the dose or concentration of a substance associated with a specific biological response, such as the LC50 (concentration killing 50% of the exposed organisms). Quantitative bioassays are typically analyzed using the methods of biostatistics.
Purpose of bioassays
- Measurement of the pharmacological activity of new or chemically undefined substances
- Investigation of the function of endogenous mediators
- Determination of the side-effect profile, including the degree of drug toxicity
- Measurement of the concentration of known substances (alternatives to the use of whole animals have made this use obsolete)
Interpolation bioassay: Bioassays are conducted by determining the amount of preparation of unknown potency required to produce a definite effect on suitable test animals or organs or tissue under standard conditions. This effect is compared with that of a standard. Thus the amount of the test substance required to produce the same biological effect as a given quantity the unit of a standard preparation is compared and the potency of the unknown is expressed as a % of that of the standard by employing a simple formula.
Many times, a reliable result cannot be obtained using this calculation. Therefore it may be necessary to adopt more precise methods of calculating potency based upon observations of relative, but not necessarily equal effects, likewise, statistical methods may also be employed. The data (obtained from either of assay techniques used) on which bioassay are based may be classified as quantal or graded response. Both these depend ultimately on plotting or making assumption concerning the form of DRC.
Environmental bioassays
Environmental bioassays are generally a broad-range survey of toxicity. A toxicity identification evaluation is conducted to determine what the relevant toxicants are. Although bioassays are beneficial in determining the biological activity within an organism, they can often be time- consuming and laborious. Organism-specific factors may result in data that are not applicable to others in that species. For these reasons, other biological techniques are often employed, including radio-immunoassays.
QUALITY CONTROL
Quality control, or QC for short, is a process by which entities review the quality of all factors involved in production. ISO 9000 defines quality control as "A part of quality management focused on fulfilling quality requirements".
This approach places an emphasis on three aspects:
- Elements such as controls, job management, defined and well managed processes, performance and integrity criteria, and identification of records
- Competence, such as knowledge, skills, experience, and qualifications
- Soft elements, such as personnel, integrity, confidence, organizational culture, motivation, team spirit, and quality relationships.
Controls include product inspection, where every product is examined visually, and often using a stereo microscope for fine detail before the product is sold into the external market. Inspectors will be provided with lists and descriptions of unacceptable product defects such as cracks or surface blemishes for example. The quality of the outputs is at risk if any of these three aspects is deficient in any way.
Quality control emphasizes testing of products to uncover defects and reporting to management who make the decision to allow or deny product release, whereas quality assurance attempts to improve and stabilize production (and associated processes) to avoid, or at least minimize, issues which led to the defect(s) in the first place. For contract work, particularly work awarded by government agencies, quality control issues are among the top reasons for not renewing a contract.
Shelf life depends on the degradation mechanism of the specific product. Most can be influenced by several factors: exposure to light, heat, moisture, transmission of gases, mechanical stress and contamination by things such as micro-organisms. Product quality is often mathematically modelled around a parameter (concentration of a chemical compound, a microbiological index, or moisture content).
For some foods, health issues are important in determining shelf life. Bacterial contaminants are ubiquitous, and foods left unused too long will often be contaminated by substantial amounts of bacterial colonies and become dangerous to eat, leading to food poisoning. However, shelf life alone is not an accurate indicator of how long the food can safely be stored. For example, pasteurized milk can remain fresh for five days after its sell-by date if it is refrigerated properly. In contrast, if milk already has harmful bacteria, the use-by dates become irrelevant.
The expiration date of pharmaceuticals specifies the date the manufacturer guarantees the full potency and safety of a drug. Most medications continue to be effective and safe for a time after the expiration date. A rare exception is a case of renal tubular acidosis purportedly caused by expired tetracycline. A study conducted by the U.S. Food and Drug Administration covered over 100 drugs, prescription and over-the-counter. The study showed that about 90% of them were safe and effective as long as 15 years past their expiration dates. Joel Davis, a former FDA expiration-date compliance chief, said that with a handful of exceptions - notably nitroglycerin, insulin and some liquid antibiotics - most expired drugs are probably effective.
Shelf life is not significantly studied during drug development, and drug manufacturers have economic and liability incentives to specify shorter shelf lives so that consumers are encouraged to discard and repurchase products. One major exception is the Shelf Life Extension Program (SLEP) of the U.S. Department of Defense (DoD), which commissioned a major study of drug efficacy from the FDA starting in the mid-1980s. One criticism is that the U.S. Food and Drug Administration (FDA) refused to issue guidelines based on SLEP research for normal marketing of pharmaceuticals even though the FDA performed the study. The SLEP and FDA signed a memorandum that scientific data could not be shared with the public, public health departments, other government agencies, and drug manufacturers. State and local programs are not permitted to participate. The failure to share data has caused foreign governments to refuse donations of
expired medications. One exception occurred during the 2010 Swine Flu Epidemic when the FDA authorized expired Tamiflu based on SLEP Data. The SLEP discovered that drugs such as Cipro remained effective nine years after their shelf life, and, as a cost-saving measure, the US military routinely uses a wide range of SLEP tested products past their official shelf life if drugs have been stored properly.
Preservatives and antioxidants may be incorporated into some food and drug products to extend their shelf life. Some companies use induction sealing and vacuum/oxygen-barrier pouches to assist in the extension of the shelf life of their products where oxygen causes the loss.
The DoD Shelf-Life Program defines shelf-life as,
The total period of time beginning with the date of manufacture, date of cure (for elastomeric and rubber products only), date of assembly, or date of pack (subsistence only), and terminated by the date by which an item must be used (expiration date) or subjected to inspection, test, restoration, or disposal action; or after inspection/laboratory test/restorative action that an item may remain in the combined wholesale (including manufacture's) and retail storage systems and still be suitable for issue or use by the end user. Shelf-life is not to be confused with service-life (defined as, A general term used to quantify the average or standard life expectancy of an item or equipment while in use. When a shelf-life item is unpacked and introduced to mission requirements, installed into intended application, or merely left in storage, placed in pre- expended bins, or held as bench stock, shelf-life management stops and service life begins).
Shelf life is often specified in conjunction with a specific product, package, and distribution system. For example, an MRE field ration is designed to have a shelf life of three years at 80 °F (27 °C) and six months at 100 °F (38 °C).
Temperature control
Nearly all chemical reactions can occur at normal temperatures (although different reactions proceed at different rates). However most reactions are accelerated by high temperatures and the degradation of foods and pharmaceuticals is no exception. The same applies to the breakdown of many chemical explosives into more unstable compounds. Nitroglycerine is notorious. Old
