lactate. This reaction is catalysed 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. This acetyl-CoA is then converted into acetate in E. coli, whilst producing ATP by substrate-level phosphorylation. 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 (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 (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. 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 + 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 and ATP synthase. 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 enzyme 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