Fermentation is a process used by cells to generate energy where a suitable substrate is metabolized to make ATP by Substrate Level Phosphorylation (SLP). Fermentation pathways operate under anaerobic cell growth conditions when electron acceptors are unavailable to support cellular respiration (e.g., without O2, nitrate, nitrite, TMAO, or DMSO present).  Fermentation energy yields are low and as a result, cells grow more slowly than when they respire.

Key Concepts 

  • Fermentation pathways vary among microorganisms.
  • These pathways differ in their key enzymes and end products.
  • ATP is always made by substrate level phosphorylation.
  • NADH which is generated during the oxidation of sugars (and other reduced organic compounds) must be re-oxidized to NAD+ during the late steps in the fermentation process.
  • E. coli employs the “mixed acid” fermentation pathway.
  • The “mixed acid” pathway makes alternative end products and in variable amounts (e.g., lactate, acetate, formate, succinate, ethanol, carbon dioxide, and hydrogen).
  • In contrast, other fermentation pathways give fewer products and in fixed amounts.


There are many fermentation pathways known in bacteria. They differ in the type of substrate used, their key enzymes, energy yields, and in the end products made. However, all fermentation pathways operate by the following principles:

  • A fermentable compound (e.g. glucose) is metabolized to generate one or more phosphorylated carbon intermediates plus NADH.
  • The phosphate moiety from a suitable phosphorylated carbon compound is transferred to ADP to make ATP (i.e., substrate level phosphorylation).
  • The newly generated NADH is re-oxidized to NAD+ in the late stages of the fermentation pathway where one or more of the intermediates are reduced. The resulting end products are excreted from the cell.

Examples of several well known fermentation pathways in bacteria and in several lower eukaryotes are described below.


E. coli is a metabolically versatile microbe that can ferment sugars besides growing aerobically or anaerobically by respiration. Since fermentation pathways yield very little energy, cells generally use this metabolic process as a last resort.  




Glucose fermentation by E. coli proceeds in two stages involving the glycolysis reactions plus the NADH recycling reactions.


Details of the E. coli Mixed Acid Fermentation

Stage 1 Reactions

During anaerobic conditions, glucose is initially metabolized to pyruvate via the glycolysis pathway.

            glucose → 2 pyruvate

These reactions also generate 2 molecules of NADH and 4 molecules of ATP. Since two ATP are consumed in early steps of the pathway, a net total of 2 ATP are produced per molecule of glucose consumed. Details of these SLP reactions are described in EcoCyc.

Stage 2 Reactions

Pyruvate is subsequently converted to one or more of the following end products: lactate, acetate, ethanol, succinate, formate, CO2 and H2 by one or more alternative pathways.  The two molecules of NADH formed in Stage 1 are recycled back to NAD+.

  • Lactate formation.       
  • Hydrogen and carbon dioxide formation.       
  • Acetic acid formation.       
  • Ethanol formation.         
  • Succinate formation.       

Key enzymes of the mixed acid fermentation pathway.

Key enzymes are diagnostic of an organism’s ability to perform a type of fermentation. For E coli  mixed acid fermentation, the key enzymes are pyruvate-formate lyase, formate-hydrogen lyase, and the anaerobic respiration pathway enzyme fumarate reductase.


Fermentation pathways in other microorganisms

A variety of different fermentation pathways exist in microbes.  They are often named by the end products made (e.g., lactic acid fermentation, propionic acid fermentation, butyric acid fermentation, ethanol fermentation). Some other more complex pathways include butanediol fermentation, butanol-acetone fermentation, glycine fermentation, and malo-lactic acid fermentation.

Brief descriptions of several of the commonly encountered fermentation pathways are presented below. BioCyc can be used to explore these pathways and the bacteria that utilize them.

  • Lactic acid fermentations       
  • Ethanol fermentations       
  • The Butyric acid fermentation       
  • Butanol-acetone fermentations       
  • Butanediol fermentation       
  • Propionic acid fermentation       



E. coli performs a mixed acid fermentation that operates in two stages.

In the first stage, glucose is first converted to pyruvate by the glycolysis pathway. This results in the net production of two ATPs which are made by SLP reactions. Two molecules of NADH are also formed. 

In the second stage, the two molecules of NADH are recycled back to NAD+ by a series of reactions that consume the pyruvate and generate end products.  There are several alternative routes to accomplish this task where variable amounts of the end products are made.

  • Substrate level phosphorylation reactions generates the ATP (with one exception).
  • Expression of the mixed acid fermentation pathway genes in E. coli is regulated. Key signals are anaerobiosis, NO3-, glucose, pyruvate and formate.
  • Since the types and amounts of end products made can vary with the cell growth conditions, this affects the ATP yield per glucose consumed!



Authored by Robert Gunsalus and Imke Schröder
©The Escherichia coli Student Portal

This project acknowledges support from:
Peter Karp and coworkers at EcoCyc.org
NIH Grant Award GM077678 to SRI, International
The UCLA Department of MIMG