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.
- 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.
Fermentable substrates include many simple sugars, sugars derived from complex polysaccharides, certain amino acids, plus some purine and pyrimidine compounds. Microorganisms utilize distinct fermentation pathways to accomplish this and produce characteristic end products.
For many obligate anaerobes, fermentation is the sole mode of energy generation. This is in contrast to many facultative microbes like E. coli that can also respire either aerobically or anaerobically.
Some common modes of fermentation in bacteria other than E. coli include lactic acid fermentation, butyric acid fermentation, propionic acid fermentation, ethanol fermentation, and mixed acid fermentation. More complex fermentation pathways also exist for the conversion of amino acids plus other fermentable substrates to end products.
Examples of several well known fermentation pathways in bacteria and in several lower eukaryotes are described below.
E. coli PERFORMS A “MIXED ACID” FERMENTATION
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.
E. coli performs a sugar based mixed acid fermentation that generates a mixture of end products that can include lactate, acetate, ethanol, succinate, formate, carbon dioxide, and hydrogen. The process is atypical of most other types of microbial fermentations in that variable amounts of the end products are made. E. coli also incorporates an anaerobic respiration reaction to reduce fumarate to succinate.
Glucose fermentation by E. coli proceeds in two stages involving the glycolysis reactions plus the NADH recycling reactions.
In the first stage, glucose is metabolized to pyruvate via the glycolysis pathway reactions. This generates 2 molecules of NADH and 4 molecules of ATP. Since 2 ATP are needed in the early steps of the pathway, a total of 2 ATP are produced for each glucose molecule consumed.
In the second stage the 2 NADH molecules generated by the glycolysis pathway must be re-oxidized to NAD+ to support subsequent rounds of glucose metabolism by the cell. These NADH oxidation steps are accomplished by reducing several of the intermediates (e.g., pyruvate or a product derived from it). This results in the formation of one or more of the “mixed acid” fermentation products.
The ATP is generated by substrate level phosphorylation (SLP) reactions using suitable glycolysis pathway intermediates to donate a phosphate group to ADP.
Details of the E. coli Mixed Acid Fermentation
Stage 1 Reactions
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+.
- Hydrogen and carbon dioxide formation.
Like many other fermenting bacteria, E. coli can convert formate to hydrogen gas and carbon dioxide. This prevents acidification of the cell’s environment. The key enzyme is formate-hydrogen lyase.
Formate → H2 and CO2
- Acetic acid formation.
Many fermenting bacteria including E. coli can convert acetyl-CoA to acetate to generate ATP. The two key enzymes are phosphate acetyltransferase and acetate kinase. This mini-pathway generates 1 mole of acetate and 1 mole of ATP (by SLP) per acetyl-CoA consumed.
acetyl-CoA + Pi → acetyl-P + CoA
acetyl-P + ADP → acetate + ATP
- Ethanol formation.
E. coli can generate ethanol from acetyl-CoA in two steps using NADH as a reductant. In the first reaction CoA is released and acetaldehyde is formed. The same enzyme then catalyzes the second reactionthat reduces acetaldehyde to ethanol. The key enzyme in E. coli key is alcohol dehydrogenase.
acetyl-CoA + NADH + H+ → acetaldehyde + NAD+ + CoA
acetaldehyde + NADH + H+ → ethanol + NAD+
- Succinate formation.
The conversion of the glycolysis pathway intermediate, phosphoenolpyruvate (PEP), to succinate is performed in several steps. The final reaction is catalyzed by the membrane-bound electron transport enzyme, fumarate reductase. This step generates a proton gradient across the cytoplasmic enzyme which in turn can be used to generate ATP via the membrane-bound ATP synthase. Reduction of fumarate by fumarate reductase uses electrons derived from NADH via the respiratory chain associated NADH dehydrogenase. Menaquinone (MQ) shuttles the electrons between the two enzymes.
NADH + H+ + fumarate → succinate + NAD+
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
The homo-lactic acid fermentation is performed by the homo-lactic acid bacteria. The process generates 2 moles of lactate per mol of glucose consumed. This is accomplished by the glycolysis pathway plus one additional enzyme, lactate dehydrogenase. From these reactions the cell generates 2 molecules of ATP by SLP reactions per one molecule of glucose consumed.
glucose → 2 lactate
The Heterolactic acid fermentation is performed by the heterolactic acid bacteria. The process generates 1 mole of lactate, 1 mole of ethanol, and 1 mole of carbon dioxide per glucose consumed. Only 1 ATP is made since glucose is oxidized via the pentose phosphate pathway.
glucose → lactate + ethanol + CO2
- Ethanol fermentations
are performed by several bacterial species and by many types of yeasts. Here, glucose is converted to 2 moles of ethanol plus 2 moles of carbon dioxide. The amount of ATP made differs with the pathway used for glucose metabolism.
glucose → 2 ethanol + 2 CO2
Sacchromyces cerevisiae and Zymomonas mobilis have two key enzymes called pyruvate decarboxylase and alcohol dehydrogenase. In yeast the glycolysis pathway is used to generate 2 moles of ATP per mole of glucose consumed. In Zymomonas mobilis only 1 ATP is produced per glucose since this microbe employs the Entner-Doudoroff pathway rather than the glycolysis pathway.
- The Butyric acid fermentation
pathway is present in many obligate anaerobes including Clostridium sp., Fusobacterium sp. and Butyrovibrio sp.. Under neutral pH growth conditions, these bacteria produce butyrate, CO2 and H2 and make 3 ATP per mole of glucose consumed.
glucose → butyrate + CO2 + H2
Key enzymes of the pathway include butyrate kinase, which couples the formation of butyrate with the generation of ATP via substrate level phosphorylation (SLP).
Pyruvate:ferredoxin oxidoreductase oxidizes pyruvate to acetyl-CoA using ferredoxin as electron acceptor. Hydrogenase then re-oxidizes reduced ferredoxin to form H2 gas.
The butyrate fermentation pathway genes are regulated by pH.
- Butanol-acetone fermentations
are often performed by the same bacteria that do the butyric acid fermentation. When the growth medium pH decreases below pH 5 due to butyrate secretion, cells switch to another pathway and form butanol and acetate. This alternative pathway generates 2 ATP per mol of glucose.
2 glucose → butanol + acetone + 5 CO2 + 4 H2
- Butanediol fermentation
This fermentation is performed by Enterobacter and Klebsiella species. Production of the neutral fermentation product, 2,3 butanediol prevents acidification of the cell environment due to accumulation of mixed acid fermentation products via alternative reactions. A key intermediate is acetoin.
2 pyruvate → 2 CO2 + 2,3 butanediol
- Propionic acid fermentation
This complex fermentation pathway is a specialty of a obligate anaerobic group of microbes called Propionibacteria. These convert lactate to propionate, acetate and CO2 and generate 1 mole of ATP per 3 moles of lactate consumed.
3 lactate → 2 propionate + acetate + CO2
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).
ATP formed: Overall, the mixed acid fermentation pathway generates ~2.3 moles of ATP per mol of glucose by A) substrate level phosphorylation, and B) a respiratory step leading to succinate.
- 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