Regulation of carbon and electron flow in Clostridium acetobutylicum grown in chemostat culture at neutral pH on mixtures of glucose and glycerol

Abstract

The metabolism of Clostridium acetobutylicum was manipulated, at neutral pH and in chemostat culture, by changing the overall degree of reduction of the substrate, using mixtures of glucose and glycerol. Cultures grown on glucose alone produced only acids, and the intracellular enzymatic pattern indicated the absence of butyraldehyde dehydrogenase activity and very low levels of coenzyme A-transferase, butanol, and ethanol dehydrogenase activities. In contrast, cultures grown on mixtures of glucose and glycerol produced mainly alcohols and low levels of hydrogen. The low production of hydrogen was not associated with a change in the hydrogenase level but was correlated with the induction of a ferredoxin-NAD reductase and a decreased level of NADH-ferredoxin reductase. The production of alcohols was related to the induction of a NAD-dependent butyraldehyde dehydrogenase and to higher expression of NAD-dependent ethanol and butanol dehydrogenases. The coenzyme A-transferase was poorly expressed, and thus no acetone was produced. These changes in the enzymatic pattern, obtained with cultures grown on a mixture of glucose and glycerol, were associated with a 7-fold increase of the intracellular level of NADH and a 2.5-fold increase of the level of ATP.The complex anaerobic metabolism of Clostridium acetobutylicum has been studied in considerable detail in recent years, but the factors involved in triggering the metabolic shift, and the physiological state associated with the transition from the acidogenic to solventogenic phase, are still not totally understood.In a number of studies, the effects of electron flow regulation, nutrient limitation, and end product accumulation on the onset and maintenance of solvent production have been investigated. In typical batch fermentations, initiation of solventogenesis is associated with a pH-acid effect. Lowering the intracellular pH and thereby increasing the concentration of undissociated butyric acid has a positive effect on the production of acetone and butanol (2,13,14,28,29,38). The alteration of the electron flow by carbon monoxide, a reversible inhibitor of the hydrogenase, can also induce the shift from an acidogenic fermentation to an alcohologenic one (9,20,25,27): alcohol (butanol and ethanol) and lactate production at very high specific rates is obtained, without acetone and little or no acetate and butyrate formation. The decrease of hydrogenase activity or concentration by iron limitation conditions (19) specifically yields butanol and ethanol as the major fermentation end products. It has also been shown, in continuous cultures at low pH, that phosphate limitation, nitrogen limitation, and glucose excess are conditions under which primarily solvents are produced by C. acetobutylicum (3,4,35).In this study, the metabolic flexibility of C. acetobutylicum was studied in chemostat culture by increasing the NAD(P)H pressure, using mixtures of glucose and a more reduced chemical like glycerol. The pH of the culture was maintained at 6.5 so that no changes could be r...

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