Fermentation

Anaerobic Cellular Respiration

Some prokaryotes and eukaryotes use anaerobic respiration in which they can create energy for use in the absence of oxygen.

Learning Objectives

Describe the process of anaerobic cellular respiration.

Key Takeaways

Key Points

  • Anaerobic respiration is a type of respiration where oxygen is not used; instead, organic or inorganic molecules are used as final electron acceptors.
  • Fermentation includes processes that use an organic molecule to regenerate NAD+ from NADH.
  • Types of fermentation include lactic acid fermentation and alcohol fermentation, in which ethanol is produced.
  • All forms of fermentation except lactic acid fermentation produce gas, which plays a role in the laboratory identification of bacteria.
  • Some types of prokaryotes are facultatively anaerobic, which means that they can switch between aerobic respiration and fermentation, depending on the availability of oxygen.

Key Terms

  • archaea: A group of single-celled microorganisms. They have no cell nucleus or any other membrane-bound organelles within their cells.
  • anaerobic respiration: A form of respiration using electron acceptors other than oxygen.
  • fermentation: An anaerobic biochemical reaction. When this reaction occurs in yeast, enzymes catalyze the conversion of sugars to alcohol or acetic acid with the evolution of carbon dioxide.

Anaerobic Cellular Respiration

The production of energy requires oxygen. The electron transport chain, where the majority of ATP is formed, requires a large input of oxygen. However, many organisms have developed strategies to carry out metabolism without oxygen, or can switch from aerobic to anaerobic cell respiration when oxygen is scarce.

image

Anaerobic bacteria: The green color seen in these coastal waters is from an eruption of hydrogen sulfide-producing bacteria. These anaerobic, sulfate-reducing bacteria release hydrogen sulfide gas as they decompose algae in the water.

During cellular respiration, some living systems use an organic molecule as the final electron acceptor. Processes that use an organic molecule to regenerate NAD+ from NADH are collectively referred to as fermentation. In contrast, some living systems use an inorganic molecule as a final electron acceptor. Both methods are called anaerobic cellular respiration, where organisms convert energy for their use in the absence of oxygen.

Certain prokaryotes, including some species of bacteria and archaea, use anaerobic respiration. For example, the group of archaea called methanogens reduces carbon dioxide to methane to oxidize NADH. These microorganisms are found in soil and in the digestive tracts of ruminants, such as cows and sheep. Similarly, sulfate-reducing bacteria and archaea, most of which are anaerobic, reduce sulfate to hydrogen sulfide to regenerate NAD+ from NADH.

Eukaryotes can also undergo anaerobic respiration. Some examples include alcohol fermentation in yeast and lactic acid fermentation in mammals.

Lactic Acid Fermentation

The fermentation method used by animals and certain bacteria (like those in yogurt) is called lactic acid fermentation. This type of fermentation is used routinely in mammalian red blood cells and in skeletal muscle that has an insufficient oxygen supply to allow aerobic respiration to continue (that is, in muscles used to the point of fatigue). The excess amount of lactate in those muscles is what causes the burning sensation in your legs while running. This pain is a signal to rest the overworked muscles so they can recover. In these muscles, lactic acid accumulation must be removed by the blood circulation and the lactate brought to the liver for further metabolism. The chemical reactions of lactic acid fermentation are the following:

Pyruvic acid + NADH ↔ lactic acid + NAD+

image

Lactic acid fermentation: Lactic acid fermentation is common in muscle cells that have run out of oxygen.

The enzyme used in this reaction is lactate dehydrogenase (LDH). The reaction can proceed in either direction, but the reaction from left to right is inhibited by acidic conditions. Such lactic acid accumulation was once believed to cause muscle stiffness, fatigue, and soreness, although more recent research disputes this hypothesis. Once the lactic acid has been removed from the muscle and circulated to the liver, it can be reconverted into pyruvic acid and further catabolized for energy.

Alcohol Fermentation

Another familiar fermentation process is alcohol fermentation, which produces ethanol, an alcohol. The use of alcohol fermentation can be traced back in history for thousands of years. The chemical reactions of alcoholic fermentation are the following (Note: CO2 does not participate in the second reaction):

Pyruvic acid → CO2 + acetaldehyde + NADH → ethanol + NAD+

image

Alcohol Fermentation: Fermentation of grape juice into wine produces CO2 as a byproduct. Fermentation tanks have valves so that the pressure inside the tanks created by the carbon dioxide produced can be released.

The first reaction is catalyzed by pyruvate decarboxylase, a cytoplasmic enzyme, with a coenzyme of thiamine pyrophosphate (TPP, derived from vitamin B1 and also called thiamine). A carboxyl group is removed from pyruvic acid, releasing carbon dioxide as a gas. The loss of carbon dioxide reduces the size of the molecule by one carbon, making acetaldehyde. The second reaction is catalyzed by alcohol dehydrogenase to oxidize NADH to NAD+ and reduce acetaldehyde to ethanol.

The fermentation of pyruvic acid by yeast produces the ethanol found in alcoholic beverages. Ethanol tolerance of yeast is variable, ranging from about 5 percent to 21 percent, depending on the yeast strain and environmental conditions.

Other Types of Fermentation

Various methods of fermentation are used by assorted organisms to ensure an adequate supply of NAD+ for the sixth step in glycolysis. Without these pathways, that step would not occur and no ATP would be harvested from the breakdown of glucose.Other fermentation methods also occur in bacteria. Many prokaryotes are facultatively anaerobic. This means that they can switch between aerobic respiration and fermentation, depending on the availability of oxygen. Certain prokaryotes, like Clostridia, are obligate anaerobes. Obligate anaerobes live and grow in the absence of molecular oxygen. Oxygen is a poison to these microorganisms, killing them on exposure.

It should be noted that all forms of fermentation, except lactic acid fermentation, produce gas. The production of particular types of gas is used as an indicator of the fermentation of specific carbohydrates, which plays a role in the laboratory identification of the bacteria.

Clostridial and Propionic Acid Fermentation

Acetogenesis is a biological reaction wherein volatile fatty acids are converted into acetic acid, carbon dioxide, and hydrogen.

Learning Objectives

Discuss the process of acidogenesis and the production of propionate

Key Takeaways

Key Points

  • Acetogenesis is the third stage in the four stages of anaerobic digestion.
  • Acetogenesis end products are acetate, hydrogen, and carbonic gas.
  • Acetogenesis occurs in three main groups of bacteria: homoacetogens, syntrophes, and sulphoreductors.

Key Terms

  • acetogenesis: The anaerobic production of acetic acid or acetate by bacteria.
  • metabolite: Any substance produced by, or taking part in, a metabolic reaction.

Four Stages of Anaerobic Digestion

Acidogenesis is the second stage in the four stages of anaerobic digestion: hydrolysis, acidogenesis, acetogenesis, and methanogenesis. Hydrolysis is a chemical reaction wherein particulates are solubilized and large polymers are converted into simpler monomers. Acidogenesis is a biological reaction wherein simple monomers are converted into volatile fatty acids. Acetogenes is a biological reaction wherein volatile fatty acids are converted into acetic acid, carbon dioxide, and hydrogen. Finally, methanogenesis is a biological reaction wherein acetates are converted into methane and carbon dioxide, and hydrogen is consumed.

image

Biofuel production can come from plants, algae, and bacteria.: Biohydrogen is defined as hydrogen produced biologically, most commonly by algae, bacteria, and archaea. Species of the Clostridium genus allow hydrogen production, a potential biofuel, in mixed cultures.

Anaerobic digestion is a complex biochemical process of mediated reactions undertaken by a consortium of microorganisms to convert organic compounds into methane and carbon dioxide. It is a stabilization process, reducing odor, pathogens, and mass reduction. Hydrolytic bacteria form a variety of reduced end-products from the fermentation of a given substrate.

One fundamental question in anaerobic digestion concerns the metabolic features that control carbon and electron flow. This flow is directed toward a reduced end-product during pure culture and mixed methanogenic cultures of hydrolytic bacteria. Thermoanaerobium brockii is a representative thermophilic, hydrolytic bacterium, which ferments glucose, via the Embden–Meyerhof Parnas Pathway.

Acidogenisis

Acidogenic activity was found in the early 20th century, but it was not until mid-1960s that the engineering of phases separation was assumed in order to improve the stability and waste digester treatment. In this phase, complex molecules (carbohydrates, lipids, and proteins) are depolymerized into soluble compounds by hydrolytic enzymes (cellulases, hemicellulases, amylases, lipases and proteases). The hydrolyzed compounds are fermented into volatile fatty acids (acetate, propionate, butyrate, and lactate), neutral compounds (ethanol, methanol), ammonia, hydrogen and carbon dioxide. Acetogenesis is one of the main reactions of this stage. In this reaction, the intermediary metabolites produced are metabolized to acetate, hydrogen, and carbonic gas by the three main groups of bacteria—homoacetogens, syntrophes, and sulphoreductors. For the acetic acid production are considered three kind of bacteria: Clostridium aceticum, Acetobacter woodii, and Clostridium termoautotrophicum.

In 1979, Winter and Wolfe demonstrated that A. wodii in syntrophic association with Methanosarcina produce methane and carbon dioxide from fructose, instead of three molecules of acetate. C. thermoaceticum and C. formiaceticum are able to reduce the carbonic gas to acetate, but they do not have hydrogenases to inhabilite the hydrogen use, so they can produce three molecules of acetate from fructose. Acetic acid is equally a co-metabolite of the organic substrates’ fermentation (sugars, glycerol, lactic acid, etc.) by diverse groups of microorganisms, which produce different acids:

  • propionic bacteria (propionate + acetate)
  • Clostridium (butyrate + acetate)
  • Enterobacteria (acetate + lactate)
  • Hetero-fermentative bacteria (acetate, propionate, butyrate, valerate, etc.)

Fermentation Without Substrate-Level Phosphorylation

Fermentation is the process of extracting energy from the oxidation of organic compounds such as carbohydrates.

Learning Objectives

Give examples of various types of fermentation: homolactic, heterolactic and alcoholic

Key Takeaways

Key Points

  • Fermentation without substrate level phosphorylation uses an endogenous electron acceptor, which is usually an organic compound.
  • 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 compounds such as lactic acid, ethanol and carbon dioxide or other acids.

Key Terms

  • fermentation: Any of many anaerobic biochemical reactions in which an enzyme (or several enzymes produced by a microorganism) catalyses the conversion of one substance into another; especially the conversion (using yeast) of sugars to alcohol or acetic acid with the evolution of carbon dioxide.
  • substrate: a surface on which an organism grows or to which it is attached
  • oxidative phosphorylation: Oxidative phosphorylation (or OXPHOS in short) is a metabolic pathway that uses energy released by the oxidation of nutrients to produce adenosine triphosphate (ATP).
  • electron acceptor: An electron acceptor is a chemical entity that accepts electrons transferred to it from another compound. It is an oxidizing agent that, by virtue of its accepting electrons, is itself reduced in the process.
image

Pyruvic acid: Pyruvic acid can be made from glucose through glycolysis, converted back to carbohydrates (such as glucose) via gluconeogenesis, or to fatty acids through acetyl-CoA. It can also be used to construct the amino acid alanine and be converted into ethanol. Pyruvic acid supplies energy to living cells through the citric acid cycle (also known as the Krebs cycle) when oxygen is present (aerobic respiration), and alternatively ferments to produce lactic acid when oxygen is lacking (fermentation).

Fermentation is the process of extracting energy from the oxidation of organic compounds, such as carbohydrates, 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 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 (a phenomenon known as the Crabtree effect). The antibiotic activity of Hops also inhibits aerobic metabolism in Yeast.

Sugars are the most common substrate of fermentation, and typical examples of fermentation products are ethanol, lactic acid, lactose, 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.

Syntrophy

Syntrophy is the phenomenon where one species lives off the products of another species.

Learning Objectives

Give examples of syntrophy in microbial metabolism

Key Takeaways

Key Points

  • Anaerobic fermentation / methanogenesis is an example of a syntrophic relationship between different groups of microorganisms.
  • Fermentation is a specific type of heterotrophic metabolism that uses organic carbon instead of oxygen as a terminal electron acceptor.
  • The best studied example of syntrophy in microbial metabolism is the oxidation of fermentative end products (such as acetate, ethanol and butyrate) by organisms such as Syntrophomonas.

Key Terms

  • syntrophy: The relationship between the individuals of different species (especially of bacteria) in which one or both benefit nutritionally from the presence of the other.
  • symbiosis: A close, prolonged association between two or more organisms of different species, regardless of benefit to the members.
  • fermentation: Any of many anaerobic biochemical reactions in which an enzyme (or several enzymes produced by a microorganism) catalyses the conversion of one substance into another; especially the conversion (using yeast) of sugars to alcohol or acetic acid with the evolution of carbon dioxide.

Syntrophy, or symbiosis, is the phenomenon involving one species living off the products of another species. For example, house dust mites live off human skin flakes. A healthy human being produces about 1 gram of skin flakes per day. These mites can also produce chemicals that stimulate the production of skin flakes. People can become allergic to these compounds. Another example are the many organisms that feast on feces or dung. A cow eats a lot of grass, the cellulose of which is transformed into lipids by micro-organisms in the cow’s large intestine.

image

House dust mite: The house dust mite (sometimes referred to by allergists as HDM) is a cosmopolitan guest in human habitation. Dust mites feed on organic detritus such as flakes of shed human skin and flourish in the stable environment of dwellings.

These microorganisms cannot use the lipids because of a lack of dioxygen in the intestine, so the cow does not take up all the lipids produced. When the processed grass leaves the intestine as dung and comes into open air, many organisms, such as the dung beetle, feast on it. Yet another example is the community of micro-organisms in soil that live off leaf litter. Leaves typically last one year and are then replaced by new ones. These microorganisms mineralize the discarded leaves and release nutrients that are taken up by the plant. Such relationships are called reciprocal syntrophy because the plant lives off the products of micro-organisms. Many symbiotic relationships are based on syntrophy. Finally, anaerobic fermentation/methanogenesis is an example of a syntrophic relationship between different groups of microorganisms. Although fermentative bacteria are not strictly dependent on syntrophyic relationships, they still gain profit from the activities of the hydrogen-scavenging organisms. The fermentative bacteria gain maximum energy yield when protons are used as electron acceptor with concurrent H2 production.

Fermentation is a specific type of heterotrophic metabolism that uses organic carbon instead of oxygen as a terminal electron acceptor. This means that these organisms do not use an electron transport chain to oxidize NADH to NAD+ and therefore must have an alternative method of using this reducing power and maintaining a supply of NAD+ for the proper functioning of normal metabolic pathways (e.g. glycolysis ). As oxygen is not required, fermentative organisms are anaerobic. Many organisms can use fermentation under anaerobic conditions and aerobic respiration when oxygen is present. These organisms are facultative anaerobes. To avoid the overproduction of NADH, obligately fermentative organisms usually do not have a complete citric acid cycle. Instead of using an ATP synthase as in respiration, ATP in fermentative organisms is produced by substrate-level phosphorylation where a phosphate group is transferred from a high-energy organic compound to ADP to form ATP. As a result of the need to produce high energy phosphate-containing organic compounds (generally in the form of CoA-esters) fermentative organisms use NADH and other cofactors to produce many different reduced metabolic by-products, often including hydrogen gas (H2). These reduced organic compounds are generally small organic acids and alcohols derived from pyruvate, the end product of glycolysis. Examples include ethanol, acetate, lactate, and butyrate. Fermentative organisms are very important industrially and are used to make many different types of food products. The different metabolic end products produced by each specific bacterial species are responsible for the different tastes and properties of each food.

The best studied example of syntrophy in microbial metabolism is the oxidation of fermentative end products (such as acetate, ethanol and butyrate) by organisms such as Syntrophomonas. Alone, the oxidation of butyrate to acetate and hydrogen gas is energetically unfavorable. However, when a hydrogenotrophic (hydrogen-using) methanogen is present the use of the hydrogen gas will significantly lower the concentration of hydrogen (down to 10−5 atm) and thereby shift the equilibrium of the butyrate oxidation reaction under standard conditions (ΔGº) to non-standard conditions (ΔG’). Because the concentration of one product is lowered, the reaction is “pulled” towards the products and shifted towards net energetically favorable conditions (for butyrate oxidation: ΔGº= +48.2 kJ/mol, but ΔG’ = -8.9 kJ/mol at 10−5 atm hydrogen and even lower if also the initially produced acetate is further metabolized by methanogens). Conversely, the available free energy from methanogenesis is lowered from ΔGº= -131 kJ/mol under standard conditions to ΔG’ = -17 kJ/mol at 10−5 atm hydrogen. This is an example of intraspecies hydrogen transfer. In this way, low energy-yielding carbon sources can be used by a consortium of organisms to achieve further degradation and eventual mineralization of these compounds. These reactions help prevent the excess sequestration of carbon over geologic time scales, releasing it back to the biosphere in usable forms such as methane and CO2.