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.

Figure 7. Model fermentation. L. The substrate is oxidized to an organic intermediate; the usual oxidizing agent is NAD. Some of the energy released by the oxidation is conserved during the synthesis of ATP by the process of substrate level phosphorylation. Finally, the oxidized intermediate is reduced to end products. Note that NADH₂ is the reducing agent, thereby balancing its redox ability to drive the energy-producing reactions. R. In lactic fermentation by Lactobacillus, the substrate (glucose) is oxidized to pyruvate, and pyruvate becomes reduced to lactic acid. Redox balance is maintained by coupling oxidations to reductions within the pathway. For example, in lactic acid fermentation via the Embden-Meyerhof pathway, the oxidation of glyceraldehyde phosphate to phosphoglyceric acid is coupled to the reduction of pyruvic acid to lactic acid.

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 classic Embden-Meyerhof pathway, which is also used by most eucaryotes, including yeast (Saccharomyces): the phosphoketolase or heterolactic pathway related to the hexose-pentose shunt; and the Entner-Doudoroff pathway. Whether or not a bacterium is a fermenter, it will likely dissimilate sugars through one or more of these pathways (See Table 1 below).

Figure 8. 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.

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 pathway to pyruvate.

Lactic acid bacteria reduce the pyruvate to lactic acid (lactate); 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). Alcohol fermentations have a similar efficiency.

Figure 9. (a) The Embden Meyerhof pathway of lactic acid fermentation in lactic acid bacteria (Lactobacillus) and (b) the Embden Meyerhof pathway of alcohol fermentation in yeast (Saccharomyces). The pathways yield two moles of end products and two moles of ATP per mole of glucose fermented. The steps in the breakdown of glucose to pyruvate are identical. The difference between the pathways is the manner of reducing pyruvic acid, thereby giving rise to different end products.

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. Figure 10 below shows some of the pathways proceeding from pyruvic acid in certain bacteria. 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, Lactococcus and most streptococci). The bacteria are used to ferment milk and milk products in the manufacture of yogurt, buttermilk, sour cream, cottage cheese, cheddar cheese, and most fermented dairy products.

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) if the bacterium possesses the enzyme formate dehydrogenase, which cleaves formate to the gases.

2a. Butanediol Fermentation. Forms mixed acids and gases as above, but, in addition, 2,3 butanediol from the condensation of 2 pyruvate. The use of the pathway decreases acid formation (butanediol is neutral) and causes the formation of a distinctive intermediate, acetoin. Water microbiologists have specific tests to detect low acid and acetoin in order to distinguish non fecal enteric bacteria (butanediol formers, such as Klebsiella and Enterobacter) from fecal enterics (mixed acid fermenters, such as E. coli, Salmonella and Shigella).

3. Butyric acid fermentations, as well as the butanol-acetone fermentation (below), 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. Small amounts of ethanol and isopropanol may also be formed.

3a. 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 (acetone is required in the manufacture gunpowder) and is said to have affected the outcome of the War. Acetone was distilled from the fermentation liquor of Clostridium acetobutylicum, which worked out pretty good if you were on our side, because organic chemists hadn't figured out how to synthesize it chemically. You can't run a war without gunpowder, at least you couldn't in those days.

4. Propionic acid fermentation. This is an unusual fermentation carried out by the propionic acid bacteria which include corynebacteria, Propionibacterium and Bifidobacterium. Although sugars can be fermented straight through to propionate, propionic acid bacteria will ferment lactate (the end product of lactic acid fermentation) to acetic acid, CO₂ and propionic acid. The formation of propionate is a complex and indirect process involving 5 or 6 reactions. Overall, 3 moles of lactate are converted to 2 moles of propionate + 1 mole of acetate + 1 mole of CO₂, and 1 mole of ATP is squeezed out in the process. The propionic acid bacteria are used in the manufacture of Swiss cheese, which is distinguished by the distinct flavor of propionate and acetate, and holes caused by entrapment of CO₂.

Figure 10. Fermentations in bacteria that proceed through the Embden-Meyerhof pathway. Representive bacteria that utilize these pathways are in shown in BLUE.

The Embden-Meyerhof pathway for glucose dissimilation (Figure 8), as well as the TCA cycle discussed below (Figure 14),  are two pathways that are at the center of metabolism in nearly all bacteria and eucaryotes. Not only do these pathways dissimilate organic compounds and provide energy, they also provide the precursors for biosynthesis of macromolecules that make up living systems (see Figure 25 below). These are rightfully called amphibolic pathways since the have both an anabolic and a catabolic function.

Heterolactic species of bacteria are occasionally used in the fermentation industry. For example, kefir, a type of fermented milk to yogurt, is produced by is produced using a heterolactic Lactobacillus species. Likewise, sauerkraut fermentations use Leuconostoc, a heterolactic bacterium, to complete the fermentation.

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).
 

In the E-D pathway, glucose phosphate is oxidized to 2-keto-3-deoxy-6-phosphogluconic 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 "yeast-like bacterium", Zymomonas (Figure 12 below). Thus, the overall reaction is Glucose ---------->2 ethanol +2 CO₂, and a net gain of 1 ATP.

Zymomonas is a bacterium that lives on the surfaces of plants, including the succulent Maguey cactus which is indigenous to Mexico. Just as grapes are crushed and fermented by resident yeast to wine, so may the Maguey flesh be crushed and allowed to ferment with Zymomonas, which gives rise to "cactus beer" or "pulque", as it is known in Mexico. Distilled pulque yields tequila in the state of Jalisco, or mescal in the state of Oaxaca. Many cultures around the world prepare their native fermented brews with Zymomonas in deference to the yeast, Saccharomyces, although they may not have a choice in the matter. Zymomonas has potential advantageous over yeast for the industrial production of alcohol, but the industry is geared to do what it can do, and no change in organisms is forthcoming.
Figure 12. The Entner-Doudoroff Pathway of Fermentation. The overall reaction is Glucose -------> 2 ethanol + 2 CO₂ + 1 ATP (net).

Table 1. Oxidative pathways of glycolysis employed by various bacteria.

Bacterium	Embden-Meyerhof pathway	Phosphoketolase (heterolactic) pathway	Entner Doudoroff pathway
Acetobacter aceti	-	+	-
Agrobacterium tumefaciens	-	-	+
Azotobacter vinelandii	-	-	+
Bacillus subtilis	major	minor	-
Escherichia coli	+	-	-
Lactobacillus acidophilus	+	-	-
Leuconostoc mesenteroides	-	+	-
Pseudomonas aeruginosa	-	-	+
Vibrio cholerae	minor	-	major
Zymomonas mobilis	-	-	+

Table 2. End product yields in microbial fermentations.

Pathway	Key enzyme	Ethanol	Lactic Acid	CO2	ATP
Embden-Meyerhof Saccharomyces	fructose 1,6-diP aldolase	2	0	2	2
Embden-Meyerhof Lactobacillus	fructose 1,6-diP aldolase	0	2	0	2
Heterolactic Streptococcus	phosphoketolase	1	1	1	1
Entner-Doudoroff Zymomonas	KDPG aldolase	2	0	2	1