Mixed Acid Fermentation: Types and Products

Fermentation is an anaerobic process of breaking down molecules like glucose and other carbohydrates. The fermentation process is usually helpful in alcohol production. Bacteria follow different fermentation pathways. 

Among them, mixed acid fermentation is a characteristic feature of the family Enterobacteriaceae, especially the genera Citrobacter, Proteus, Shigella, Salmonella, Escherichia, Aeromonas, Yersinia, Vibrio, and some species of Aeromonas. Some anaerobic fungi also follow this pathway. 

These microorganisms ferment monosaccharides, disaccharides, polyalcohol, and frequently polysaccharides. The glycolytic pathway of this type of fermentation produces lactic acid, succinic acid, formic acids, acetic acids, and ethanol. 

Reactions Involved in Mixed Acid Fermentation

In mixed acid fermentation reactions, two stages are present. The first stage of mixed acid fermentation is glycolysis, converting glucose to pyruvate. Here two NADH molecules are produced.

The second stage of mixed acid fermentation follows the following reactions the conversion of the pyruvate produced after glycolysis to one or more end products. The two NADH molecules produced in the first stage are reoxidized to NAD+. The reactions in the second stage are discussed below.

Lactate Production

The enzyme lactate dehydrogenase catalyzes the formation of lactate/lactic acid. Here, glycolysis generates two molecules of pyruvate. Each molecule converts to lactate in the presence of a NADH+H+ molecule. 

The overall reaction of lactate production is as follows:

Pyruvate → Lactate; in presence of lactate dehydrogenase and NADH+H+, which converts to NAD+

Acetate Production

In this reaction, pyruvate converts to acetyl CoA with the enzyme pyruvate dehydrogenase and NADH catalysts. The acetyl CoA now converts to acetate, which produces ATP by substrate-level phosphorylation. The conversion of acetyl CoA to acetate is a two-step process requiring two separate enzymes; phosphate acetyltransferase and acetate kinase. 

The overall reaction of this reaction is as follows;

  1. Pyruvate → Acetyl CoA in the presence of pyruvate dehydrogenase
  2. Acetyl CoA + Phosphate → Acetyl phosphate + CoA in the presence of phosphate acetyltransferase.
  3. Acetyl phosphate + ADP → Acetate + ATP. 

Ethanol Production

The reduction of Acetyl CoA with the help of NADH forms the third end product of mixed acid fermentation; ethanol. This ethanol production is a two-step reaction and requires the enzyme alcohol dehydrogenase. 

The overall reaction of this step of fermentation is;

  1. Acetyl CoA + NADH +H+ → Acetaldehyde + NAD+ + CoA
  2. Acetaldehyde + NADH + H+ →  Ethanol + NAD+ 

Formate Production

The cleavage of pyruvate helps in the production of Formate. The enzyme pyruvate-formate-lyase catalyzes this production reaction. The enzyme pyruvate-formate-lyase also plays an essential role in regulating anaerobic fermentation in Enterobacteriaceae. 

The overall reaction of the formate production is as follows;

Pyruvate + CoA  → Acetyl CoA + Formate; catalyzed by pyruvate-formate-lyase. 

Succinate Production

The production of succinate is a multi-step process. The glycolytic pathway intermediate, phosphoenol pyruvate, is the first substrate for carboxylation to form oxaloacetate, with the enzyme phosphoenol pyruvate carboxylase catalyzing this step. 

In the second step, the oxaloacetate converts to malate in the presence of malate dehydrogenase. The third step is the formation of fumarate from the dehydration of malate in the fact of fumarate hydratase. 

Phosphoenol pyruvate + HCO3 → Oxaloacetate +Phosphate

Oxaloacetate + NADH + H+ → Malate + NAD+ 

Malate → Fumarate + H2O

The final step is succinate production by reducing formate catalyzed by the enzyme fumarate reductase. 

Fumarate + NADH + NAD+ →  Succinate + NAD+ 

The reduction is the anaerobic respiration reaction that uses electrons in NADH dehydrogenase and the electron transport chain. ATP is produced using electrochemical balance and ATP synthetase—this step of fermentation produces ATP, not through substrate-level phosphorylation. 

Hydrogen and Carbon Dioxide Production

The enzyme formate hydrogen lyase catalyzes the conversion of formate to hydrogen carbon dioxide gas. The production of these gases helps in preventing acidic condition inside the cells.  

End Products of Mixed Acid Fermentation

The end products of mixed acid fermentation are as follows:

  1. Lactate
  2. Acetate
  3. Formate
  4. Succinate
  5. Ethanol 

The concentration of these end products of fermentation vary depending on the environment and microorganism but it may be at the 4:1 ratio of neutral to acid products. Hydrogen and carbon dioxide is formed in bacteria with formate hydrogen lyase complex.

Application of Mixed Acid Fermentation

Mixed Acid fermentation is applicable in various fields of science, especially for producing many helpful end products. The applications of mixed acid fermentation are as follows:

  1. The use of single bacteria can help produce various products in biotechnology and the food industry. 
  2. Likewise, many different strains of bacteria have been metabolically engineered in the laboratory to increase the yield of the specific end product. 
  3. This fermentation method is applied to identify bacteria in the laboratory. Methyl red test is standard for detecting the bacteria following the mixed acid fermentation reaction. Here, the test solution turns red if the pH drops below 4.4 in the presence of those microorganisms that follows the mixed acid fermentation pathway.


  1. Thakker, C., Martínez, I., San, K. Y., & Bennett, G. N. (2012). Succinate production in Escherichia coli. Biotechnology journal, 7(2), 213–224. https://doi.org/10.1002/biot.201100061 
  2. Vuoristo, K. S., Mars, A. E., Sangra, J. V., Springer, J., Eggink, G., Sanders, J. P., & Weusthuis, R. A. (2015). Metabolic engineering of the mixed-acid fermentation pathway of Escherichia coli for anaerobic production of glutamate and itaconate. (thrivehomebuilders.com) AMB Express, 5(1), 61. https://doi.org/10.1186/s13568-015-0147-y 
  3. Böck, A. (2009). Fermentation. Encyclopedia of Microbiology, 132–144. https://doi.org/10.1016/b978-012373944-5.00074-2 

Ashma Shrestha

Hello, I am Ashma Shrestha. I had recently completed my Masters degree in Medical Microbiology. Passionate about writing and blogging. Key interest in virology and molecular biology.

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