From technological viewpoint, fermentation can be divided into two well separable phases: acid formation phase and, after reaching an autoinhibition limit value of the acids, solvent formation phase. These steps can be performed in separated technological environments as well [70].

Hydrogen formation takes place in the acidogenic phase, so the composition of the gases (CO2 H2) changes during the fermentation process. The larger part of the carbon dioxide is formed in the pathway of acetone formation. Presence of hydrogen and carbon dioxide has large influence on each metabolic step. The effect of H2 and CO2 as product gases on solvent production was studied in a continuous culture of alginate-immobilized C. acetobutylicum. Fermentations were carried out at various dilution rates. With 10% H2 and 10% CO2 in the sparging gas, a dilu­tion rate of 0.07 h-1 was found to maximize volumetric productivity (0.58 g*L-1xh-1), while maxi­mal specific productivity of 0.27 g-1*h-1 occurred at 0.12 h-1. Continuous cultures with vigorous sparging of N2 produced only acids. It was concluded that in the case of continuous fermenta­tion H2 is essential for good solvent production, although good solvent production is possible in an H2-absent environment in case of batch fermentations. When the fermentation was carried out at atmospheric pressure under H2-enriched conditions, presence of CO2 in the sparging gas did not slow down glucose metabolism; rather it changed the direction of the phosphoroclastic reaction and, as a result, increased the butanol/acetone ratio [71].

Klei et al. [72] studied the effect of pure CO2 on the second phase of ABE fermentation. CO2 pressures up to 100 psig were used in a batch fermentor using glucose as substrate. Maximal solvent production occurred near 25 psig CO2 at the expense of cell growth. In addition, the BuOH:Me2CO ratio changed sharply at 40 psig from 5:1 to 20:1 and EtOH production was eliminated at >50 psig. As the pressure increased, both conversion rates of organic acids to solvents and the utilization rate of substrate glucose decreased.

Pressurization of the fermentation vessel with H2 appeared to decrease, rather than increase, the formation of neutral solvents in batch fermentations [73]. However, increasing H2 partial pressure increased BuOH and EtOH yields from glucose by an average of 18% and 13%, respectively, and the yields of acetone and of endogenous H2 decreased by an average of 40% and 30%, respectively, and almost no effect was observed on the growth of the culture. The BuOH-to-acetone ratio and the fraction of BuOH in the total solvents also increased with H2 partial pressure. There were no major differences in the observed pattern of change with pressurization at either t = 0 or t = 18 h [74].

Redox active additives such as carbon monoxide have important influence on the ABE fermentation processes. Addition of CO inhibited the hydrogenase activity of cell extracts and viable metabolizing cells. Increasing the partial pressure of CO (2 to 10%) in unshaken anaerobic culture tube headspaces significantly inhibited (90% inhibition at 10% CO) both growth and H2 production. The growth was not sensitive to low partial pressures of CO (~15%) in pH-controlled fermentors (pH 4.5). CO addition dramatically altered the glucose fermen­tation balance of C. acetobutylicum by diverting carbon and electrons away from H2, CO2, acetate and butyrate production and towards production of EtOH and BuOH. The BuOH concentration increased from 65 to 106 mM and the BuOH productivity (the ratio of BuOH produced/total acids and solvents produced) increased by 31% when glucose fermentation was maintained at pH 4.5 in presence of 85% N2-15% CO vs. N2 alone [75]. Carbon monoxide sparged into batch fermentations of C. acetobutylicum inhibited production of H2 and enhanced production of solvents by making available larger amounts of NAD(P)H2 to the cells. CO also inhibited biomass growth and acid formation as well. Its effect was mostly pronounced under fermentation conditions of excess carbon — and nitrogen-source supply [76]. When continuous, steady-state, glucose-limited cultures of Clostridium acetobutylicum were sparged with CO, complete or almost complete acidogenic fermentations became solvento — genic. Alcohol (butanol and ethanol) and lactate production at very high specific production rates were initiated and sustained without acetone, and little or no acetate and butyrate formation. In one fermentation strong butyrate uptake without acetone formation was observed. Growth could be sustained even with 100% inhibition of H2 formation. Although CO gasing inhibited growth up to 50%, and H2 formation up to 100%, it enhanced the rate of glucose uptake up to 300%. These results support the hypothesis that solvent formation is triggered by an altered electron flow [77]. The metabolic modulation by CO was particularly effective when organic acids such as acetic and butyric acid were added to the fermentation as electron sinks. The uptake of organic acids was enhanced, and increase in butyric acid uptake by 50-200% over control was observed. H2 production could be reduced by 50% and the ratio of solvent could be controlled by CO modulation and organic acid addition. Acetone production could be eliminated if desired. BuOH yield could be increased by 10-15%. Total solvent yield could be increased by 1-3% and the electron efficiency to acetone-BuOH-EtOH solvents could be increased from 73% for controls to 80-85% for CO — and organic acid — modulated fermentations. The dynamic nature of electron flow in this fermentation was elucidated and mechanisms for metabolic control were hypothesized [78].