# Fermentation

Fermentation is a type of redox metabolism carried out in the absence of oxygen. During fermentation, organic molecules (e.g., glucose) are catabolized and donate electrons to other organic molecules. In the process, ATP and organic end products (e.g., lactate) are formed.
Because oxygen is not required, it is an alternative to aerobic respiration. Over 25% of bacteria and archaea carry out fermentation. They live in the gut, sediments, food, and other environments. Eukaryotes, including humans and other animals, also carry out fermentation.
Fermentation is important in several areas of human society. Humans have used fermentation in production of food for 13,000 years. Humans and their livestock have microbes in the gut that carry out fermentation, releasing products used by the host for energy. Fermentation is used at an industrial level to produce commodity chemicals, such as ethanol and lactate. In total, fermentation forms more than 50 metabolic end products This process highlights the power of microbial activity.

## Definition

The definition of fermentation has evolved over the years. The most modern definition is catabolism, where organic compounds are both the electron donor and acceptor. A common electron donor is glucose, and pyruvate is a common electron acceptor. This definition distinguishes fermentation from aerobic respiration, where oxygen is the acceptor and types of anaerobic respiration, where an inorganic species is the acceptor.
Fermentation had been defined differently in the past. In 1876, Louis Pasteur described it as "la vie sans air" (life without air). This definition came before the discovery of anaerobic respiration. Later, it had been defined as catabolism that forms ATP through only substrate-level phosphorylation. However, several pathways of fermentation have been discovered to form ATP through an electron transport chain and ATP synthase, also.
Some sources define fermentation loosely as any large-scale biological manufacturing process. See Industrial fermentation. This definition focuses on the process of manufacturing rather than metabolic details.

## Biological role and prevalence

Fermentation is used by organisms to generate ATP energy for metabolism. One advantage is that it requires no oxygen or other external electron acceptors, and thus it can be carried out when those electron acceptors are absent. A disadvantage is that it produces relatively little ATP, yielding only between 2 and 4.5 per glucose compared to 32 for aerobic respiration.
Over 25% of bacteria and archaea carry out fermentation. This type of metabolism is most common in the phylum Bacillota, and it is least common in Actinomycetota. Their most common habitat is host-associated ones, such as the gut.
Animals, including humans, also carry out fermentation. The product of fermentation in humans is lactate, and it is formed during anaerobic exercise or in cancerous cells. No animal is known to survive on fermentation alone, even as one parasitic animal (Henneguya zschokkei) is known to survive without oxygen.

## Substrates and products of fermentation

Fermentation uses a range of substrates and forms a variety of metabolic end products. Of the 55 end products formed, the most common are acetate and lactate. Of the 46 chemically-defined substrates that have been reported, the most common are glucose and other sugars.

## Biochemical overview

When an organic compound is fermented, it is broken down to a simpler molecule and releases electrons. The electrons are transferred to a redox cofactor, which in turn transfers them to an organic compound. ATP is generated in the process, and it can be formed by substrate-level phosphorylation or by ATP synthase.
When glucose is fermented, it enters glycolysis or the pentose phosphate pathway and is converted to pyruvate. From pyruvate, pathways branch out to form a number of end products (e.g. lactate). At several points, electrons are released and accepted by redox cofactors (NAD and ferredoxin). At later points, these cofactors donate electrons to their final acceptor and become oxidized. ATP is also formed at several points in the pathway.
While fermentation is simple in overview, its details are more complex. Across organisms, fermentation of glucose involves over 120 different biochemical reactions. Further, multiple pathways can be responsible for forming the same product. For forming acetate from its immediate precursor (pyruvate or acetyl-CoA), six separate pathways have been found.

## Biochemistry of individual products
### Ethanol

In ethanol fermentation, one glucose molecule is converted into two ethanol molecules and two carbon dioxide (CO$_2$) molecules. It is used to make bread dough rise: the carbon dioxide forms bubbles, expanding the dough into a foam. The ethanol is the intoxicating agent in alcoholic beverages such as wine, beer and liquor. Fermentation of feedstocks, including sugarcane, maize, and sugar beets, produces ethanol that is added to gasoline. In some species of fish, including goldfish and carp, it provides energy when oxygen is scarce (along with lactic acid fermentation).
Before fermentation, a glucose molecule breaks down into two pyruvate molecules (glycolysis). The energy from this exothermic reaction is used to bind inorganic phosphates to ADP, which converts it to ATP, and convert NAD$^+$ to NADH. The pyruvates break down into two acetaldehyde molecules and give off two carbon dioxide molecules as waste products. The acetaldehyde is reduced into ethanol using the energy and hydrogen from NADH, and the NADH is oxidized into NAD$^+$ so that the cycle may repeat. The reaction is catalyzed by the enzymes pyruvate decarboxylase and alcohol dehydrogenase.

#### History of bioethanol fermentation

The history of ethanol as a fuel spans several centuries and is marked by a series of significant milestones. Samuel Morey, an American inventor, was the first to produce ethanol by fermenting corn in 1826. However, it was not until the California Gold Rush in the 1850s that ethanol was first used as a fuel in the United States. Rudolf Diesel demonstrated his engine, which could run on vegetable oils and ethanol, in 1895, but the widespread use of petroleum-based diesel engines made ethanol less popular as a fuel. In the 1970s, the oil crisis reignited interest in ethanol, and Brazil became a leader in ethanol production and use. The United States began producing ethanol on a large scale in the 1980s and 1990s as a fuel additive to gasoline, due to government regulations. Today, ethanol continues to be explored as a sustainable and renewable fuel source, with researchers developing new technologies and biomass sources for its production.
* 1826: Samuel Morey, an American inventor, was the first to produce ethanol by fermenting corn. However, ethanol was not widely used as a fuel until many years later. (1)
* 1850s: Ethanol was first used as a fuel in the United States during the California gold rush. Miners used ethanol as a fuel for lamps and stoves because it was cheaper than whale oil. (2)
* 1895: German engineer Rudolf Diesel demonstrated his engine, which was designed to run on vegetable oils, including ethanol. However, the widespread use of diesel engines fueled by petroleum made ethanol less popular as a fuel. (3)
* 1970s: The oil crisis of the 1970s led to renewed interest in ethanol as a fuel. Brazil became a leader in ethanol production and use, due in part to government policies that encouraged the use of biofuels. (4)
* 1980s–1990s: The United States began to produce ethanol on a large scale as a fuel additive to gasoline. This was due to the passage of the Clean Air Act in 1990, which required the use of oxygenates, such as ethanol, to reduce emissions. (5)
* 2000s–present: There has been continued interest in ethanol as a renewable and sustainable fuel. Researchers are exploring new sources of biomass for ethanol production, such as switchgrass and algae, and developing new technologies to improve the efficiency of the fermentation process. (6)

### Lactic acid

Homolactic fermentation (producing only lactic acid) is the simplest type of fermentation. Pyruvate from glycolysis undergoes a simple redox reaction, forming lactic acid. Overall, one molecule of glucose (or any six-carbon sugar) is converted to two molecules of lactic acid:
    C$_6$H$_{12}$O$_6$ → 2 CH$_3$CHOHCOOH
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 the type of bacteria that convert lactose into lactic acid in yogurt, giving it its sour taste. These lactic acid bacteria can carry out either homolactic fermentation, where the end-product is mostly lactic acid, or heterolactic fermentation, where some lactate is further metabolized to 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. Reasons to go further and convert lactic acid into something else include:
* The acidity of lactic acid impedes biological processes. This can be beneficial to the fermenting organism as it drives out competitors that are unadapted to the acidity. As a result, the food will have a longer shelf life (one 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, into which lactic acid can be easily converted, is volatile and will readily escape, allowing the reaction to proceed easily. CO$_2$ is also produced, but it is 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 additional energy. It is a lighter molecule than lactic acid, forming fewer hydrogen bonds with its surroundings (due to having fewer groups that can form such bonds), thus is more volatile and will also allow the reaction to proceed more quickly.
* If propionic acid, butyric acid, and longer monocarboxylic acids are produced, the amount of acidity produced per glucose consumed will decrease, as with ethanol, allowing faster growth.

### Hydrogen gas

Hydrogen gas is produced in many types of 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.
For example, Clostridium pasteurianum ferments glucose to 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$_2$

### Glyoxylate

Glyoxylate fermentation is a type of fermentation used by microbes that are able to utilize glyoxylate as a nitrogen source.

### Other

Other types of fermentation include mixed acid fermentation, butanediol fermentation, butyrate fermentation, caproate fermentation, and acetone–butanol–ethanol fermentation.

### In the broader sense

In food and industrial contexts, any chemical modification performed by a living being in a controlled container can be termed "fermentation". The following do not fall into the biochemical sense, but are called fermentation in the larger sense:

#### Alternative protein

Fermentation can be used to make alternative protein sources. It is commonly used to modify existing protein foods, including plant-based ones such as soy, into more flavorful forms such as tempeh and fermented tofu.
More modern "fermentation" makes recombinant protein to help produce meat analogue, milk substitute, cheese analogues, and egg substitutes. Some examples are:
* Recombinant myoglobin for faux meat (Motif Foodworks)
* Recombinant leghemoglobin for faux meat (Impossible Foods)
* Recombinant whey protein for dairy replacement (Perfect Day)
* Recombinant casein protein for dairy replacements (Those Vegan Cowboys)
* Recombinant egg white (EVERY)
Heme proteins such as myoglobin and hemoglobin give meat its characteristic texture, flavor, color, and aroma. The myoglobin and leghemoglobin ingredients can be used to replicate this property, despite them coming from a vat instead of meat.

#### Enzymes

Industrial fermentation can be used for enzyme production, where proteins with catalytic activity are produced and secreted by microorganisms. The development of fermentation processes, microbial strain engineering and recombinant gene technologies has enabled the commercialization of a wide range of enzymes. Enzymes are used in all kinds of industrial segments, such as food (lactose removal, cheese flavor), beverage (juice treatment), baking (bread softness, dough conditioning), animal feed, detergents (protein, starch and lipid stain removal), textile, personal care and pulp and paper industries.

## Modes of industrial operation

Most industrial fermentation uses batch or fed-batch procedures, although continuous fermentation can be more economical if various challenges, particularly the difficulty of maintaining sterility, can be met.

### Batch

In a batch process, all the ingredients are combined and the reactions proceed without any further input. Batch fermentation has been used for millennia to make bread and alcoholic beverages, and it is still a common method, especially when the process is not well understood.$^{:1}$ However, it can be expensive because the fermentor must be sterilized using high pressure steam between batches. Strictly speaking, there is often addition of small quantities of chemicals to control the pH or suppress foaming.$^{:25}$
Batch fermentation goes through a series of phases. There is a lag phase in which cells adjust to their environment; then a phase in which exponential growth occurs. Once many of the nutrients have been consumed, the growth slows and becomes non-exponential, but production of secondary metabolites (including commercially important antibiotics and enzymes) accelerates. This continues through a stationary phase after most of the nutrients have been consumed, and then the cells die.$^{:25}$

### Fed-batch

Fed-batch fermentation is a variation of batch fermentation where some of the ingredients are added during the fermentation. This allows greater control over the stages of the process. In particular, production of secondary metabolites can be increased by adding a limited quantity of nutrients during the non-exponential growth phase. Fed-batch operations are often sandwiched between batch operations.$^{:1}$

### Open

The high cost of sterilizing the fermentor between batches can be avoided using various open fermentation approaches that are able to resist contamination. One is to use a naturally evolved mixed culture. This is particularly favored in wastewater treatment, since mixed populations can adapt to a wide variety of wastes. Thermophilic bacteria can produce lactic acid at temperatures of around 50 °Celsius, sufficient to discourage microbial contamination; and ethanol has been produced at a temperature of 70 °C. This is just below its boiling point (78 °C), making it easy to extract. Halophilic bacteria can produce bioplastics in hypersaline conditions. Solid-state fermentation adds a small amount of water to a solid substrate; it is widely used in the food industry to produce flavors, enzymes and organic acids.

### Continuous

In continuous fermentation, substrates are added and final products removed continuously. There are three varieties: chemostats, which hold nutrient levels constant; turbidostats, which keep cell mass constant; and plug flow reactors in which the culture medium flows steadily through a tube while the cells are recycled from the outlet to the inlet. If the process works well, there is a steady flow of feed and effluent and the costs of repeatedly setting up a batch are avoided. Also, it can prolong the exponential growth phase and avoid byproducts that inhibit the reactions by continuously removing them. However, it is difficult to maintain a steady state and avoid contamination, and the design tends to be complex. Typically the fermentor must run for over 500 hours to be more economical than batch processors.

## History of the use of fermentation

The use of fermentation, particularly for beverages, has existed since the Neolithic and has been documented dating from 7000 to 6600 BCE in Jiahu, China, 5000 BCE in India, Ayurveda mentions many Medicated Wines, 6000 BCE in Georgia, 3150 BCE in ancient Egypt, 3000 BCE in Babylon, 2000 BCE in pre-Hispanic Mexico, and 1500 BC in Sudan. Fermented foods have a religious significance in Judaism and Christianity. The Baltic god Rugutis was worshiped as the agent of fermentation. In alchemy, fermentation ("putrefaction") was symbolized by Capricorn ♑︎.
In 1837, Charles Cagniard de la Tour, Theodor Schwann and Friedrich Traugott Kützing independently published papers concluding, as a result of microscopic investigations, that yeast is a living organism that reproduces by budding.$^{:6}$ Schwann boiled grape juice to kill the yeast and found that no fermentation would occur until new yeast was added. However, a lot of chemists, including Antoine Lavoisier, continued to view fermentation as a simple chemical reaction and rejected the notion that living organisms could be involved. This was seen as a reversion to vitalism and was lampooned in an anonymous publication by Justus von Liebig and Friedrich Wöhler.$^{:108–109}$
The turning point came when Louis Pasteur (1822–1895), during the 1850s and 1860s, repeated Schwann's experiments and showed fermentation is initiated by living organisms in a series of investigations.$^{:6}$ In 1857, Pasteur showed lactic acid fermentation is caused by living organisms. In 1860, he demonstrated how bacteria cause souring in milk, a process formerly thought to be merely a chemical change. His work in identifying the role of microorganisms in food spoilage led to the process of pasteurization.
In 1877, working to improve the French brewing industry, Pasteur published his famous paper on fermentation, "Etudes sur la Bière", which was translated into English in 1879 as "Studies on fermentation". He defined fermentation (incorrectly) as "Life without air", yet he correctly showed how specific types of microorganisms cause specific types of fermentations and specific end-products.
Although showing fermentation resulted from the action of living microorganisms was a breakthrough, it did not explain the basic nature of fermentation; nor did it prove it is caused by microorganisms which appear to be always present. Many scientists, including Pasteur, had unsuccessfully attempted to extract the fermentation enzyme from yeast.
Success came in 1897 when the German chemist Eduard Buechner ground up yeast, extracted a juice from them, then found to his amazement this "dead" liquid would ferment a sugar solution, forming carbon dioxide and alcohol much like living yeasts.
Buechner's results are considered to mark the birth of biochemistry. The "unorganized ferments" behaved just like the organized ones. From that time on, the term enzyme came to be applied to all ferments. It was then understood fermentation is caused by enzymes produced by microorganisms. In 1907, Buechner won the Nobel Prize in chemistry for his work.
Advances in microbiology and fermentation technology have continued steadily up until the present. For example, in the 1930s, it was discovered 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.

### Post 1930s

The field of fermentation has been critical to producing a wide range of consumer goods, from food and drink to industrial chemicals and pharmaceuticals. Since its early beginnings in ancient civilizations, fermentation has continued to evolve and expand, with new techniques and technologies driving advances in product quality, yield, and efficiency. The period from the 1930s onward saw a number of significant advancements in fermentation technology, including the development of new processes for producing high-value products like antibiotics and enzymes, the increasing importance of fermentation in the production of bulk chemicals, and a growing interest in the use of fermentation for the production of functional foods and nutraceuticals.
The 1950s and 1960s saw the development of new fermentation technologies, such as immobilized cells and enzymes, which allowed for more precise control over fermentation processes and increased the production of high-value products like antibiotics and enzymes. In the 1970s and 1980s, fermentation became increasingly important in producing bulk chemicals like ethanol, lactic acid, and citric acid. This led to developing new fermentation techniques and genetically engineered microorganisms to improve yields and reduce production costs. In the 1990s and 2000s, there was a growing interest in fermentation to produce functional foods and nutraceuticals, which have potential health benefits beyond basic nutrition. This led to new fermentation processes, probiotics, and other functional ingredients.
Overall, the period from 1930 onward saw significant advancements in the use of fermentation for industrial purposes, leading to the production of a wide range of fermented products that are now consumed worldwide.