Fermentation
Fermentation is an ancient mode of metabolism, and it must have evolved with the
appearance of organic material on the planet. Fermentation is metabolism in which
energy is derived from the partial oxidation of an organic compound using organic
intermediates as electron donors and electron acceptors. No outside electron
acceptors are involved; no membrane or electron transport system is required; all
ATP is produced by substrate level phosphorylation.
By definition, fermentation may be as simple as two steps illustrated in the following
model. Indeed, some amino acid fermentations by the clostridia are this simple. But
the pathways of fermentation are a bit more complex, usually involving several
preliminary steps to prime the energy source for oxidation and substrate level
phosphorylations.
In biochemistry, for the sake of convenience, fermentation pathways start with
glucose. This is because it is the simplest molecule, requiring the fewest catalytic
steps, to enter into a pathway of glycolysis and central metabolism. In procaryotes
there exist three major pathways of glycolysis (the dissimilation of sugars):
The Embden-Meyerhof Pathway
This is the pathway of glycolysis most familiar to biochemists and eukaryotic
biologists, The pathway is used by the (homo)lactic acid bacteria to produce lactic
acid, and it is used by many other bacteria to produce a variety of fatty acids, alcohols
and gases. Diagnostic microbiologists use bacterial fermentation profiles (e.g. testing
an organism's ability to ferment certain sugars, or examining an organisms's array of
end products) in order to identify them, down to the genus level.
The first three steps of the pathway prime (phosphorylate) and rearrange the hexose
for cleavage into 2 trioses (glyceraldehyde-phosphate). Fructose 1,6-diphosphate
aldolase is the key (cleavage) enzyme in the E-M pathway. Each triose molecule is
oxidized and phosphorylated followed by two substrate level phosphorylations that
yield 4 ATP during the drive to pyruvate.
Lactic acid bacteria reduce the pyruvate to lactic acid; yeast reduce the pyruvate to
alcohol (ethanol) and CO2 as shown in Figure 9 below.
The oxidation of glucose to lactate yields a total of 56 kcal per mole of glucose. Since
the cells harvest 2 ATP (16 kcal) as useful energy, the efficiency of the lactate
fermentation is about 29 percent (16/56). Ethanol fermentations have a similar
efficiency.
Figure. The Embden Meyerhof pathway for glucose dissimilation. The overall reaction is the
oxidation of glucose to 2 pyruvic acid. The two branches of the pathway after the cleavage are
identical, drawn in this manner for comparison with other bacterial pathways of glycolysis.
Besides lactic acid, Embden-Meyerhof fermentations in bacteria can lead to a wide
array of end products depending on the pathways taken in the reductive steps after the
formation of pyruvic acid. Usually, these bacterial fermentations are distinguished by
their end products into the following groups.
1. Homolactic Fermentation. Lactic acid is the sole end product. Pathway of the
homolactic acid bacteria (Lactobacillus and most streptococci). The bacteria are used
to ferment milk.
2. Mixed Acid Fermentations. Mainly the pathway of the Enterobacteriaceae. End
products are a mixture of lactic acid, acetic acid, formic acid, succinate and
ethanol, with the possibility of gas formation (CO2 and H2) .
3. Butanediol Fermentation. Forms mixed acids and gases as above, but, in addition,
2,3 butanediol from the condensation of 2 pyruvate.
4. Butyric acid fermentations, as well as the butanol-acetone fermentation , are run
by the clostridia, the masters of fermentation. In addition to butyric acid, the clostridia
form acetic acid, CO2 and H2 from the fermentation of sugars.
5. Butanol-acetone fermentation. Butanol and acetone were discovered as the main
end products of fermentation by Clostridium acetobutylicum during the World War I.
This discovery solved a critical problem of explosives manufacture of gunpowder.
6. Propionic acid fermentation. This is an unusual fermentation carried out by the
propionic acid bacteria .
The Heterolactic (Phosphoketolase) Pathway
The phosphoketolase pathway is distinguished by the key cleavage enzyme,
phosphoketolase, which cleaves pentose phosphate into glyceraldehyde-3-phosphate
and acetyl phosphate. As a fermentation pathway, it is employed mainly by the
heterolactic acid bacteria, which include some species of Lactobacillus and
Leuconostoc. In this pathway, glucose-phosphate is oxidized to 6-phosphogluconic
acid, which becomes oxidized and decarboxylated to form pentose phosphate.
Pentose phosphate is subsequently cleaved to glyceraldehyde-3-phosphate (GAP) and
acetyl phosphate. GAP is converted to lactic acid by the same enzymes as the E-M
pathway. This branch of the pathway contains an oxidation coupled to a reduction
while 2 ATP are produced by substrate level phosphorylation. Acetyl phosphate is
reduced in two steps to ethanol, which balances the two oxidations before the
cleavage but does not yield ATP. The overall reaction is :
Glucose ---------->1 lactic acid + 1 ethanol +1 CO2 with a net gain of 1 ATP. The
efficiency is about half that of the E-M pathway.
The Entner-Doudoroff Pathway
Only a few bacteria, most notably Zymomonas, employ the Entner-Doudoroff
pathway as a fermentation path. However, many bacteria, especially those grouped
around the pseudomonads, use the pathway as a way to degrade carbohydrates for
respiratory metabolism. The E-D pathway yields 2 pyruvic acid from glucose (same
as the E-M pathway) but like the phosphoketolase pathway, oxidation occurs before
the cleavage, and the net energy yield per mole of glucose utilized is one mole of
ATP. In the E-D pathway, glucose phosphate is oxidized to 2-keto-3-deoxy-6phosphogluconic acid (KDPG) which is cleaved by KDPG aldolase to pyruvate and
GAP. The latter is oxidized to pyruvate by E-M enzymes wherein 2 ATP are
produced by substrate level phosphorylations. Pyruvic acid from either branch of the
pathway is reduced to ethanol and CO2, in the same manner as yeast, by the "yeastlike bacterium", Thus, the overall reaction is:
Glucose ---------->2 ethanol +2 CO2, and a net gain of 1 ATP.
Figure 11. The heterolactic (phosphoketolase) pathway of fermentation. Compare with the
Embden-Meyerhof pathway in Figure 9. This pathway differs in the early steps before the
cleavage of the molecule. The overall reaction in the fermentation of glucose is
Glucose -------> Lactic acid + ethanol + CO2 + 1 ATP (net).
Figure 12. The Entner-Doudoroff Pathway of Fermentation
Table 2. End product yields in microbial fermentations.
Pathway
Key enzyme
Ethanol Lactic Acid CO2 ATP
Embden-Meyerhof
fructose 1,6-diP aldolase 2
0
2
2
fructose 1,6-diP aldolase 0
2
0
2
phosphoketolase
1
1
1
1
KDPG aldolase
2
0
Saccharomyces
Embden-Meyerhof
Lactobacillus
Heterolactic
Streptococcus
Entner-Doudoroff
Zymomonas