Solvent-forming species, including C. acetobutylicum and C. beijerinckii, are mesophilic, growing best between 30° and 40°C. The pH varies during the fermentation and can drop from an initial value of 6.8-7.0 to about 5.0-4.5 (acidogenesis) and can also rise up to 7.0 later in the fermentation (solventoge — nesis). It has been suggested that the switch to solvent production is an adaptive response of the cell to the low medium pH resulting from acid production (Bahl et al., 1982).

Solventogenic clostridia can be grown on simple media such as ground corn, molasses, whey permeate, or on semidefined and defined media. When semi — defined and defined media are used, a wide array of vitamins and minerals are required in addition to a carbohydrate source. Clostridia can utilize a wide range of carbohydrates. C. acetobutylicum and C. beijerinckii can utilize starch, hexoses, pentoses, and cellobiose. Currently, those clostridia that are able to utilize cellu­lose directly produce little or no solvents. Recently, attempts have been made to express cellulase genes in the solventogenic clostridia.

The uptake of carbohydrates in the solventogenic clostridia is achieved by a phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS). This mechanism involves simultaneous uptake and phosphorylation of substrate that results in the conversion of glucose to glucose-6-phosphate, which is subsequently metabolized to pyruvate via the Embden-Meyerhof-Parnas (EMP) pathway (Mitchell, 2001). Fructose is converted to fructose-1-phosphate and enters the EMP pathway upon conversion to fructose 1,6-bisphosphate. D-xylose is con­verted to D-xylulose by the xylose isomerase enzyme and the metabolism pro­ceeds by a phosphorylation reaction. The reaction is catalyzed by xylulokinase, which results in the formation of D-xylulose-5-phosphate. The pentose phosphate pathway utilizes enzymes transaldolase and transketolase to convert D-xylulose — 5-phosphate to glyceraldehyde-3-phosphate and fructose-6-phosphate (Singh and Mishra, 1995). The glyceraldehyde-3-phosphate and fructose-6-phosphate enter the EMP pathway leading to the formation of pyruvate. The ability of solvento — genic clostridia to metabolize these sugars is important when corn is considered as the starting material for fermentation, as all of these sugars can be derived from corn or corn coproducts.

Solvent producing clostridia metabolize substrates in a biphasic fermentation fashion. During the first phase, acid intermediates (acetic and butyric acids), hydrogen, and a large amount of ATP are produced. In the second phase, butanol, acetone, and ethanol are produced, and hydrogen and ATP production decrease (Jones and Woods, 1986). CO2 is produced during both phases of growth—two moles are produced from each mole of glucose metabolized to pyruvate—but CO2 production in the solventogenic phase is higher as an additional mole is produced for every mole of acetone produced. The simplified overall fermentation pathway is given in Figure 6.1.

During the acidogenic phase, cells typically grow exponentially due to the high amount of ATP (3.25 mol/mol of glucose) being produced (Jones and Woods, 1986). The enzymes phosphate acetyltransferase and acetate kinase convert acetyl-CoA to acetate and, analogously, phosphate butyltransferase and butyrate kinase convert butyryl-CoA to butyrate during this phase of growth. The pH of the fermentation broth decreases as butyric and acetic acids accumulate. The acetic and butyric acids produced during the fermentation may be freely perme­able to the cell membrane and these acids equilibrate the internal (bacterial) and fermentation broth pH. Both reduction of pH and accumulation of acetate and butyrate have been associated with triggering solventogenesis (Jones and Woods, 1986).

The solventogenic phase is typically associated with stationary phase. ATP production is reduced to 2 mol/mol of glucose during this phase. The fermentation intermediates (acetic and butyric acids) are reassimilated and converted into acetone and butanol. It has been suggested that butyric and acetic acids are reassimilated by the action of the enzyme acetoacetyl-CoA:acetate/butyrate:CoA transferase (Andersch et al., 1983). This enzyme catalyzes the reaction that transfers CoA from acetoacetyl-CoA to either acetate or butyrate. Acetate is converted to acetyl-CoA, which can be converted to acetone, butanol, or ethanol. Butyrate is converted to butyryl-CoA, which can only be used to produce butanol. This is because there is no metabolic pathway to regenerate acetyl-CoA from butyryl-CoA. When CoA is removed from acetoacetyl-CoA, acetoacetate is pro­duced, which can be transformed directly into acetone and CO2 by acetoacetate decarboxylase.

The central core of both the acidogenic and solventogenic pathways is the series of reactions that produces butyryl-CoA from acetyl-CoA. Thiolase con­denses two molecules of acetyl-CoA into one molecule of acetoacetyl-CoA. Acetoacetyl-CoA is reduced to 3-hydroxybutyryl-CoA by hydroxybutyryl-CoA dehydrogenase. From this, crotonyl-CoA is formed by dehydration, catalyzed by

crotonase. The carbon-carbon double bond in crotonyl-CoA is reduced with NADH to produce butyryl-CoA. This last step is catalyzed by butyryl-CoA dehydrogenase (Bennett and Rudolph, 1995).

The accumulation of both acids (butyrate and acetate) and solvents (acetone, butanol, and ethanol) in the fermentation broth is toxic to the microorganism and eventually causes cell death. The shift to solventogenesis is effective in extending the fermentation, but the butanol produced eventually reaches toxic levels. The presence of butanol in the membrane increases membrane fluidity and destabilizes the membrane and membrane-associated processes (Jones and Woods, 1986). The
maximum amount of solvents (total acetone, butanol, and ethanol) that the cell can tolerate is 20 gL-1 (Maddox, 1989). This limits the amount of glucose that can be fermented in batch culture to 60 gL-1 because using a higher concentration of glucose would result in incomplete substrate utilization due to butanol toxicity. Many studies today are focused on overcoming the butanol toxicity issue, whether by developing a more butanol tolerant microorganism or by selectively removing butanol from the fermentation broth.