Mixed acid fermentation (MAF) is a specialized type of anaerobic metabolism employed by certain bacteria to generate energy when oxygen is unavailable. This process is distinct from simpler fermentations, such as homolactic fermentation, because it results in a complex and variable mixture of end products. The characteristic products include organic acids—lactate, acetate, formate, and succinate—along with neutral compounds like ethanol and the gases hydrogen and carbon dioxide. This metabolic flexibility allows organisms to maintain energy production and balance their cellular redox state under oxygen-deprived conditions.
The Microbial Performers of Mixed Acid Fermentation
The ability to perform mixed acid fermentation is a defining metabolic trait of the Enterobacteriaceae family, a large group of Gram-negative bacteria that primarily inhabit the intestines of animals and humans. Prominent members, such as Escherichia coli, Salmonella, and Shigella, rely on this pathway for survival and growth in the low-oxygen environment of the gut. This metabolic strategy provides a mechanism for generating adenosine triphosphate (ATP) through substrate-level phosphorylation, enabling rapid proliferation even without respiration.
The specific profile of acidic end products generated by MAF is used in microbiology laboratories to identify different bacterial species within the Enterobacteriaceae family. For instance, the Methyl Red (MR) test detects the stable production of a high concentration of mixed acids, causing a significant drop in the growth medium’s pH. Species that are “MR-positive,” such as E. coli, produce a larger quantity of these strong acids compared to related organisms utilizing variations like butanediol fermentation.
The Common Central Route to Pyruvate
The entire process of mixed acid fermentation begins with the conversion of a sugar, typically glucose, into a three-carbon molecule called pyruvate. This initial, shared metabolic sequence is known as the Embden-Meyerhof-Parnas (EMP) pathway, or more commonly, glycolysis. This pathway is highly conserved across many life forms and acts as the central hub for carbohydrate breakdown.
The EMP pathway proceeds in two general phases: an energy investment phase and an energy payoff phase. In the initial steps, the cell must expend two molecules of ATP to activate the glucose molecule, modifying it to fructose-1,6-bisphosphate. This phosphorylation prepares the six-carbon sugar for cleavage into two three-carbon molecules of glyceraldehyde-3-phosphate.
The subsequent payoff phase extracts energy from these three-carbon intermediates. Key reactions generate high-energy phosphate bonds and produce two molecules of the reduced electron carrier NADH. Through substrate-level phosphorylation, four molecules of ATP are generated, resulting in a net gain of two ATP molecules per molecule of glucose processed. The pathway concludes with the formation of two molecules of pyruvate, which serves as the branching point for mixed acid fermentation.
Enzymatic Branching and Acidic Product Formation
The pyruvate molecules generated by glycolysis are immediately channeled into several distinct metabolic fates, a process that determines the final, characteristic mixture of products. This metabolic divergence is controlled by a suite of specific enzymes that activate only under anaerobic conditions. A major portion of the pyruvate is directed through the enzyme Pyruvate Formate Lyase (PFL), which is the most defining enzyme of this pathway.
PFL catalyzes the cleavage of pyruvate into acetyl-Coenzyme A (acetyl-CoA) and formate. This reaction is highly sensitive to oxygen and is the primary route for pyruvate breakdown in many anaerobic Enterobacteriaceae. The acetyl-CoA product then follows two paths: it can be converted to acetate, yielding an additional ATP, or it can be reduced to ethanol through a two-step reaction involving alcohol dehydrogenase. The reduction to ethanol helps regenerate the NAD+ needed to keep glycolysis running.
Another significant branch involves the enzyme Lactate Dehydrogenase (LDH), which converts pyruvate directly into lactate. This reaction is crucial for maintaining the cell’s redox balance, as it consumes NADH and regenerates NAD+. The lactate produced is an acid that contributes to the overall drop in pH observed in the fermentation broth.
A third branch of the mixed acid pathway starts not from pyruvate, but from phosphoenolpyruvate (PEP), a precursor molecule in glycolysis. PEP is carboxylated by PEP carboxylase to form oxaloacetate, which then enters a sequence of reactions that ultimately produce succinate. This pathway involves enzymes like malate dehydrogenase and fumarate reductase, and its final reduction step can sometimes be linked to an anaerobic respiratory chain.
Finally, the formate produced by PFL must be addressed to prevent its accumulation, which would over-acidify the cell’s environment. Many organisms possessing MAF, such as E. coli, employ the Formate Hydrogen Lyase (FHL) complex. This enzyme complex breaks down formate into the gaseous products carbon dioxide (CO2) and hydrogen gas (H2). This reaction helps to buffer the cell’s internal pH and contributes to the characteristic gas production of the fermentation.