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In
biochemistry Biochemistry or biological chemistry is the study of chemical processes within and relating to living organisms. A sub-discipline of both chemistry and biology, biochemistry may be divided into three fields: structural biology, enzymology and ...
, mixed acid fermentation is the
metabolic process Metabolism (, from el, μεταβολή ''metabolē'', "change") is the set of life-sustaining chemical reactions in organisms. The three main functions of metabolism are: the conversion of the energy in food to energy available to run ce ...
by which a six-carbon sugar (e.g. glucose, ) is converted into a complex and variable mixture of
acid In computer science, ACID ( atomicity, consistency, isolation, durability) is a set of properties of database transactions intended to guarantee data validity despite errors, power failures, and other mishaps. In the context of databases, a sequ ...
s. It is an
anaerobic Anaerobic means "living, active, occurring, or existing in the absence of free oxygen", as opposed to aerobic which means "living, active, or occurring only in the presence of oxygen." Anaerobic may also refer to: * Anaerobic adhesive, a bonding a ...
(non-oxygen-requiring) fermentation reaction that is common in bacteria. It is characteristic for members of the Enterobacteriaceae, a large family of
Gram-negative bacteria Gram-negative bacteria are bacteria that do not retain the crystal violet stain used in the Gram staining method of bacterial differentiation. They are characterized by their cell envelopes, which are composed of a thin peptidoglycan cell wall ...
that includes '' E. coli''. The mixture of end products produced by mixed acid fermentation includes
lactate Lactate may refer to: * Lactation, the secretion of milk from the mammary glands * Lactate, the conjugate base of lactic acid Lactic acid is an organic acid. It has a molecular formula . It is white in the solid state and it is miscible with w ...
,
acetate An acetate is a salt formed by the combination of acetic acid with a base (e.g. alkaline, earthy, metallic, nonmetallic or radical base). "Acetate" also describes the conjugate base or ion (specifically, the negatively charged ion called an ...
, succinate, formate, ethanol and the gases and . The formation of these end products depends on the presence of certain key enzymes in the bacterium. The proportion in which they are formed varies between different bacterial species. The mixed acid fermentation pathway differs from other fermentation pathways, which produce fewer end products in fixed amounts. The end products of mixed acid fermentation can have many useful applications in
biotechnology Biotechnology is the integration of natural sciences and engineering sciences in order to achieve the application of organisms, cells, parts thereof and molecular analogues for products and services. The term ''biotechnology'' was first used by ...
and
industry Industry may refer to: Economics * Industry (economics), a generally categorized branch of economic activity * Industry (manufacturing), a specific branch of economic activity, typically in factories with machinery * The wider industrial sector ...
. For instance, ethanol is widely used as a
biofuel Biofuel is a fuel that is produced over a short time span from biomass, rather than by the very slow natural processes involved in the formation of fossil fuels, such as oil. According to the United States Energy Information Administration (EIA ...
. Therefore, multiple bacterial strains have been metabolically engineered in the laboratory to increase the individual yields of certain end products. This research has been carried out primarily in ''E. coli'' and is ongoing. Variations of mixed acid fermentation occur in a number of bacterial species, including bacterial pathogens such as '' Haemophilus influenzae''l where mostly acetate and succinate are produced and lactate can serve as a growth substrate.


Mixed acid fermentation in ''E. coli''

''E. coli'' use fermentation pathways as a final option for energy metabolism, as they produce very little energy in comparison to respiration. Mixed acid fermentation in ''E. coli'' occurs in two stages. These stages are outlined by the biological database for ''E. coli'', EcoCyc. The first of these two stages is a glycolysis reaction. Under anaerobic conditions, a glycolysis reaction takes place where glucose is converted into pyruvate:       glucose → 2 pyruvate There is a net production of 2 ATP and 2
NADH Nicotinamide adenine dinucleotide (NAD) is a coenzyme central to metabolism. Found in all living cells, NAD is called a dinucleotide because it consists of two nucleotides joined through their phosphate groups. One nucleotide contains an aden ...
molecules per molecule of glucose converted. ATP is generated by
substrate-level phosphorylation Substrate-level phosphorylation is a metabolism reaction that results in the production of ATP or GTP by the transfer of a phosphate group from a substrate directly to ADP or GDP. Transferring from a higher energy (whether phosphate group att ...
. NADH is formed from the reduction of NAD. In the second stage, pyruvate produced by glycolysis is converted to one or more end products via the following reactions. In each case, both of the NADH molecules generated by glycolysis are reoxidized to NAD+. Each alternative pathway requires a different key enzyme in ''E. coli''. After the variable amounts of different end products are formed by these pathways, they are secreted from the cell.


Lactate formation

Pyruvate produced by glycolysis is converted to
lactate Lactate may refer to: * Lactation, the secretion of milk from the mammary glands * Lactate, the conjugate base of lactic acid Lactic acid is an organic acid. It has a molecular formula . It is white in the solid state and it is miscible with w ...
. This reaction is
catalysed Catalysis () is the process of increasing the rate of a chemical reaction by adding a substance known as a catalyst (). Catalysts are not consumed in the reaction and remain unchanged after it. If the reaction is rapid and the catalyst recyc ...
by the enzyme lactate dehydrogenase (LDHA).       pyruvate + NADH + H+ → lactate + NAD+


Acetate formation

Pyruvate is converted into acetyl-coenzyme A (acetyl-CoA) by the enzyme
pyruvate dehydrogenase Pyruvate dehydrogenase is an enzyme that catalyzes the reaction of pyruvate and a lipoamide to give the acetylated dihydrolipoamide and carbon dioxide. The conversion requires the coenzyme thiamine pyrophosphate. Pyruvate dehydrogenase is u ...
. This acetyl-CoA is then converted into
acetate An acetate is a salt formed by the combination of acetic acid with a base (e.g. alkaline, earthy, metallic, nonmetallic or radical base). "Acetate" also describes the conjugate base or ion (specifically, the negatively charged ion called an ...
in ''E. coli'', whilst producing ATP by
substrate-level phosphorylation Substrate-level phosphorylation is a metabolism reaction that results in the production of ATP or GTP by the transfer of a phosphate group from a substrate directly to ADP or GDP. Transferring from a higher energy (whether phosphate group att ...
. Acetate formation requires two enzymes: phosphate acetyltransferase and acetate kinase.       acetyl-CoA + phosphate → acetyl-phosphate + CoA       acetyl-phosphate + ADP → acetate + ATP


Ethanol formation

Ethanol is formed in ''E. coli'' by the reduction of acetyl coenzyme A using NADH. This two-step reaction requires the enzyme
alcohol dehydrogenase Alcohol dehydrogenases (ADH) () are a group of dehydrogenase enzymes that occur in many organisms and facilitate the interconversion between alcohols and aldehydes or ketones with the reduction of nicotinamide adenine dinucleotide (NAD+) to NADH ...
(ADHE).       acetyl-CoA + NADH + H+ → acetaldehyde + NAD+ + CoA       acetaldehyde + NADH + H+ → ethanol + NAD+


Formate formation

Formate is produced by the cleavage of pyruvate. This reaction is catalysed by the enzyme pyruvate-formate lyase (PFL), which plays an important role in regulating anaerobic fermentation in ''E. coli''.       pyruvate + CoA → acetyl-CoA + formate


Succinate formation

Succinate is formed in ''E. coli'' in several steps.
Phosphoenolpyruvate Phosphoenolpyruvate (2-phosphoenolpyruvate, PEP) is the ester derived from the enol of pyruvate and phosphate. It exists as an anion. PEP is an important intermediate in biochemistry. It has the highest-energy phosphate bond found (−61.9 kJ/m ...
(PEP), a glycolysis pathway intermediate, is carboxylated by the enzyme PEP carboxylase to form oxaloacetate. This is followed by the conversion of oxaloacetate to malate by the enzyme
malate dehydrogenase Malate dehydrogenase () (MDH) is an enzyme that reversibly catalyzes the oxidation of malate to oxaloacetate using the reduction of NAD+ to NADH. This reaction is part of many metabolic pathways, including the citric acid cycle. Other malate ...
. Fumarate hydratase then catalyses the dehydration of malate to produce fumarate.       phosphoenolpyruvate + HCO3 → oxaloacetate + phosphate       oxaloacetate + NADH + H+ → malate + NAD+       malate → fumarate + H2O The final reaction in the formation of succinate is the reduction of fumarate. It is catalysed by the enzyme
fumarate reductase Fumarate reductase is the enzyme that converts fumarate to succinate, and is important in microbial metabolism as a part of anaerobic respiration. Succinate + acceptor fumarate + reduced acceptor Fumarate reductases can be divided into two classe ...
.       fumarate + NADH + H+ → succinate + NAD+ This reduction is an anaerobic respiration reaction in ''E. coli'', as it uses electrons associated with NADH dehydrogenase and the electron transport chain. ATP is generated by using an
electrochemical gradient An electrochemical gradient is a gradient of electrochemical potential, usually for an ion that can move across a membrane. The gradient consists of two parts, the chemical gradient, or difference in solute concentration across a membrane, and ...
and
ATP synthase ATP synthase is a protein that catalyzes the formation of the energy storage molecule adenosine triphosphate (ATP) using adenosine diphosphate (ADP) and inorganic phosphate (Pi). It is classified under ligases as it changes ADP by the formation ...
. This is the only case in the mixed acid fermentation pathway where ATP is not produced via substrate-level phosphorylation. Vitamin K2, also known as menaquinone, is very important for electron transport to fumarate in ''E. coli''.


Hydrogen and carbon dioxide formation

Formate can be converted to hydrogen gas and carbon dioxide in ''E. coli''. This reaction requires the enzym
formate-hydrogen lyase
It can be used to prevent the conditions inside the cell becoming too acidic.       formate → H2 and CO2


Methyl red test

The methyl red (MR) test can detect whether the mixed acid fermentation pathway occurs in microbes when given glucose. A pH indicator is used that turns the test solution red if the pH drops below 4.4. If the fermentation pathway has taken place, the mixture of acids it has produced will make the solution very acidic and cause a red colour change. The methyl red test belongs to a group known as the
IMViC The IMViC tests are a group of individual tests used in microbiology lab testing to identify an organism in the coliform group. A coliform is a gram negative, aerobic, or facultative anaerobic rod, which produces gas from lactose within 48 hour ...
tests.


Metabolic engineering

Multiple bacterial strains have been metabolically engineered to increase the individual yields of end products formed by mixed acid fermentation. For instance, strains for the increased production of ethanol, lactate, succinate and acetate have been developed due to the usefulness of these products in
biotechnology Biotechnology is the integration of natural sciences and engineering sciences in order to achieve the application of organisms, cells, parts thereof and molecular analogues for products and services. The term ''biotechnology'' was first used by ...
. The major limiting factor for this engineering is the need to maintain a redox balance in the mixture of acids produced by the fermentation pathway.


For ethanol production

Ethanol is the most commonly used
biofuel Biofuel is a fuel that is produced over a short time span from biomass, rather than by the very slow natural processes involved in the formation of fossil fuels, such as oil. According to the United States Energy Information Administration (EIA ...
and can be produced on large scale via fermentation. The maximum theoretical yield for the production of ethanol was achieved around 20 years. A plasmid that carried the pyruvate decarboxylase and alcohol dehydrogenase genes from the bacteria '' Z. mobilis'' was used by scientists. This was inserted into ''E. coli'' and resulted in an increased yield of ethanol. The genome of this ''E. coli'' strain, KO11, has more recently been sequenced and mapped.


For acetate production

The ''E. coli'' strain W3110 was
genetically engineered Genetic engineering, also called genetic modification or genetic manipulation, is the modification and manipulation of an organism's genes using technology. It is a set of technologies used to change the genetic makeup of cells, including ...
to generate 2 moles of acetate for every 1 mole of glucose that undergoes fermentation. This is known as a homoacetate pathway.


For lactate production

Lactate can be used to produce a
bioplastic Bioplastics are plastic materials produced from renewable biomass sources, such as vegetable fats and oils, corn starch, straw, woodchips, sawdust, recycled food waste, etc. Some bioplastics are obtained by processing directly from natural bi ...
called polylactic acid (PLA). The properties of PLA depend on the ratio of the two
optical isomers In chemistry, a molecule or ion is called chiral () if it cannot be superposed on its mirror image by any combination of rotations, translations, and some conformational changes. This geometric property is called chirality (). The terms are ...
of lactate (D-lactate and L-lactate). D-lactate is produced by mixed acid fermentation in ''E. coli''. Early experiments engineered the ''E. coli'' strain RR1 to produce either one of the two optical isomers of lactate. Later experiments modified the ''E. coli'' strain KO11, originally developed to enhance ethanol production. Scientists were able to increase the yield of D-lactate from fermentation by performing several deletions.


For succinate production

Increasing the yield of succinate from mixed acid fermentation was first done by overexpressing the enzyme PEP carboxylase. This produced a succinate yield that was approximately 3 times greater than normal. Several experiments using a similar approach have followed. Alternative approaches have altered the redox and ATP balance to optimize the succinate yield.


Related fermentation pathways

There are a number of other fermentation pathways that occur in microbes. All these pathways begin by converting pyruvate, but their end products and the key enzymes they require are different. These pathways include: * Ethanol fermentation * Lactic acid fermentation *
Propionic acid Propionic acid (, from the Greek words πρῶτος : ''prōtos'', meaning "first", and πίων : ''píōn'', meaning "fat"; also known as propanoic acid) is a naturally occurring carboxylic acid with chemical formula CH3CH2CO2H. It is a liqu ...
fermentation * Butanol fermentation *
Butanediol fermentation 2,3-Butanediol fermentation is anaerobic fermentation of glucose with 2,3-butanediol as one of the end products. The overall stoichiometry of the reaction is :2 pyruvate + NADH --> 2 CO2 + 2,3-butanediol. Butanediol fermentation is typical for t ...


External links


Mixed acid fermentationEcoCyc Summary of Fermentation


References

{{DEFAULTSORT:Mixed Acid Fermentation Anaerobic digestion Fermentation