A simplified central metabolic pathway for ethanol production in yeast and bacteria under anaerobic conditions is presented in Fig. 3.7 [15, 35-37].

Three major interrelated pathways that control catabolism of carbo­hydrate in most ethanol-producing organisms are

■ Embden-Meyerhof pathway (EMP) or glycolysis

■ Pentose phosphate pathway (PPP)

■ Krebs or tricarboxylic acid cycle (TCA)

In glycolysis, glucose is anaerobically converted to pyruvic acid and then to ethanol through acetaldehyde. This pathway provides energy in the form of ATP to the cells. The net yield in glycolysis is 2 moles of pyru­vate (or ethanol) and 2 moles of ATP from each mole of glucose. This pathway is also the entrance of other hexoses such as fructose, mannose, and galactose to metabolic pathways. With only 2 moles of ATP formed per glucose catabolized, large amounts of ethanol (at least 3.7 g of ethanol per gram of biomass) must be formed [15, 38].

The PPP handles pentoses and is important for nucleotide (ribose — 5-phosphate) and fatty acid biosynthesis. The PPP is mainly used to
reduce NADP+. In Saccharomyces cerevisiae, 6-8% of glucose passes through the PPP under anaerobic conditions [8, 15].

The TCA cycle functions to convert pyruvic and lactic acids and ethanol aerobically to the end products CO2 and H2O. It is also a common channel for the ultimate oxidation of fatty acids and the carbon skele­tons of many amino acids. In cells containing the additional aerobic pathways, the NADH that forms during glycolysis results in ATP gen­eration in the TCA cycle [8].

Ethanol production from hexoses is redox-neutral, i. e., no net forma­tion of NADH or NADPH occurs. However, biosynthesis of the cells results in net formation of NADH and consumption of NADPH. The PPP is mainly used to reduce NADP+ to NADPH. Oxidation of surplus NADH under anaerobic conditions in S. cerevisiae is carried out through the glycerol pathway. Furthermore, there are other by-products—mainly carboxylic acids: acetic acid, pyruvic acid, and succinic acid—that add to the surplus NADH. Consequently, glycerol is also formed to com­pensate the NADH formation coupled with these carboxylic acids. Thus, formation of glycerol is coupled with biomass and carboxylic acid for­mation in anaerobic growth of S. cerevisiae [15, 39].

We should keep in mind that growth of the cells and increasing their biomass is the ultimate goal of the cells. They produce ethanol under anaerobic conditions in order to provide energy through catabolic reac­tions. Glycerol is formed to keep the redox balance of the cells, and car­boxylic acids may leak from the cells to the medium. Therefore, the ethanol-producing microorganisms produce ethanol as the major product under anaerobic conditions, while biomass, glycerol, and some carboxylic acids are the by-products.