Increased levels of ATP and NADH are associated with increased solvent production in continuous cultures of Clostridium acetobutylicum

Meyer, Charles L.; Papoutsakis, Eleftherios T.

doi:10.1007/bf00263849

Cited by 86 publications

(51 citation statements)

References 27 publications

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“…Acetate formation is favored relative to lactate and ethanol formation for growth of C. cellulolyticum on cellobiose under carbonlimited compared to carbon-sufficient conditions (225,226). With respect to noncellulolytic anaerobes, limitation by a nutrient other than the carbon source is a powerful modulator of solvent formation in C. acetobutylicum (443,572), for which solvent formation is typically not growth associated. This, however, is not the case for T. thermosaccharolyticum (268), for which solvent formation is growth associated.…”

Section: Physiology Of Cellulolytic Microorganismsmentioning

confidence: 99%

Microbial Cellulose Utilization: Fundamentals and Biotechnology

Lynd

Weimer

Zyl³

et al. 2002

Microbiol Mol Biol Rev

4,006

2,868

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Fundamental features of microbial cellulose utilization are examined at successively higher levels of aggregation encompassing the structure and composition of cellulosic biomass, taxonomic diversity, cellulase enzyme systems, molecular biology of cellulase enzymes, physiology of cellulolytic microorganisms, ecological aspects of cellulase-degrading communities, and rate-limiting factors in nature. The methodological basis for studying microbial cellulose utilization is considered relative to quantification of cells and enzymes in the presence of solid substrates as well as apparatus and analysis for cellulose-grown continuous cultures. Quantitative description of cellulose hydrolysis is addressed with respect to adsorption of cellulase enzymes, rates of enzymatic hydrolysis, bioenergetics of microbial cellulose utilization, kinetics of microbial cellulose utilization, and contrasting features compared to soluble substrate kinetics. A biological perspective on processing cellulosic biomass is presented, including features of pretreated substrates and alternative process configurations. Organism development is considered for “consolidated bioprocessing” (CBP), in which the production of cellulolytic enzymes, hydrolysis of biomass, and fermentation of resulting sugars to desired products occur in one step. Two organism development strategies for CBP are examined: (i) improve product yield and tolerance in microorganisms able to utilize cellulose, or (ii) express a heterologous system for cellulose hydrolysis and utilization in microorganisms that exhibit high product yield and tolerance. A concluding discussion identifies unresolved issues pertaining to microbial cellulose utilization, suggests approaches by which such issues might be resolved, and contrasts a microbially oriented cellulose hydrolysis paradigm to the more conventional enzymatically oriented paradigm in both fundamental and applied contexts

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Section: Physiology Of Cellulolytic Microorganismsmentioning

confidence: 99%

Microbial Cellulose Utilization: Fundamentals and Biotechnology

Lynd

Weimer

Zyl³

et al. 2002

Microbiol Mol Biol Rev

4,006

2,868

View full text Add to dashboard Cite

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“…Those two genes were shown to be part of an operon (7), and our results suggest a lower induction or a partial repression of the genes in the glycerol-glucose culture. It is also possible that high intracellular ATP concentrations (Table 2) are important for the in vivo regulation at the enzyme level (17,26). Wiesenbom et al (43) showed that ATP is inhibitory to phosphotransbutyrylase.…”

Section: Discussionmentioning

confidence: 99%

“…Two distinct conditions exist under which solvent formation may be initiated in continuous cultures of C. acetobutylicum: (i) low pH or addition of butyric acid results in the formation of butanol, ethanol, and acetone and is associated under steady-state conditions with a normal level of NADH and a high level of ATP (12,26); and (ii) decreasing the in vivo activity of the hydrogenase by carbon monoxide, methyl viologen, or iron limitation leads to alcohol production alone (16,27) and is correlated, under steady-state conditions, with high levels of both NADH and ATP (16,26). The shift observed when glycerol is metabolized at neutral pH belongs to this second category, as both high intracellular NADH and ATP concentrations and low hydrogen production were observed.…”

Section: Discussionmentioning

confidence: 99%

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

1994

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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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“…High ATP concentration related to low ATP demand or high efficiency of ATP generation would lead to enhanced solvent production (i) for glucose-sufficient cultures at a low pH with biomass recycling (12,13); (ii) for iron-, nitrogen-, or phosphate-limited cultures (1,2,19); and (iii) during shifts induced on phosphatelimited cultures by lowering the pH or adding organic acids (6). Ethanol and butanol productions were associated with increased availability of reducing power (i) when the in vivo activity of the hydrogenase was decreased by CO gassing (5,10,13,14) or by adding methyl viologen (6); (ii) during a shift in solvent production induced by lowering the pH when acetyl coenzyme A (CoA) was first converted to acetone (a pathway consuming no reducing energy), creating a redox imbalance (6); (iii) when an NADH pressure was provided by culturing the microorganism on glucose and a more reduced substrate (21). Under such mixed substrate (glucose plus glycerol) growth, the culture was reported to be glucose but not glycerol limited.…”

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confidence: 99%

Regulation of Clostridium acetobutylicum metabolism as revealed by mixed-substrate steady-state continuous cultures: role of NADH/NAD ratio and ATP pool

1994

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Glycerol-glucose-fed (molar ratio of 2) chemostat cultures of Clostridium acetobutylicum were glucose limited but glycerol sufficient and had a high intracellular NADH/NAD ratio (I. Vasconcelos, L. Girbal, and P. Soucaille, J. Bacteriol. 176:1443-1450, 1994. We report here that the glyceraldehyde-3-phosphate dehydrogenase, one of the key enzymes of the glycolytic pathway, is inhibited by high NADH/NAD ratios. Partial substitution of glucose by pyruvate while maintaining glycerol concentration at a constant level allowed a higher consumption of glycerol in steady-state continuous cultures. However, glycerol-sufficient cultures had a constant flux through the glyceraldehyde-3-phosphate dehydrogenase and a constant NADH/NAD ratio. A high substitution of glucose by pyruvate [P/(G + P) value of 0.67 g/g] provided a carbon-limited culture with butanol and butyrate as the major end products. In this alcohologenic culture, the induction of the NADH-dependent butyraldehyde and the ferredoxin-NAD(P) reductases and the higher expression of alcohol dehydrogenases were related to a high NADH/NAD ratio and a low intracellular ATP concentration. In three different steady-state cultures, the in vitro phosphotransbutyrylase and butyrate-kinase activities decreased with the intracellular ATP concentration, suggesting a transcriptional regulation of these two genes, which are arranged in an operon (K. A. Walter, R V. Nair, R. V. Carry, G. N. Bennett, and E. T. Papoutsakis, Gene 134:107-111, 1993).Clostridium acetobutylicum, a strictly anaerobic spore-forming bacterium, usually shows a biphasic batch fermentation pattern. After producing acetate and butyrate during exponential growth, the organism switches to the formation of acetone, butanol, and ethanol shortly before entering the stationary phase. The mechanisms responsible for the onset of solventogenesis are currently the focus of much scientific research.In batch cultures, the initiation and sustained production of solvents are associated with a low extracellular and intracellular pH and a high undissociated butyric acid concentration (7,15,20). In continuous culture, ATP and NAD(P)H availabilities appear to play a key role in product selectivity. High ATP concentration related to low ATP demand or high efficiency of ATP generation would lead to enhanced solvent production (i) for glucose-sufficient cultures at a low pH with biomass recycling (12, 13); (ii) for iron-, nitrogen-, or phosphate-limited cultures (1, 2, 19); and (iii) during shifts induced on phosphatelimited cultures by lowering the pH or adding organic acids (6). Ethanol and butanol productions were associated with increased availability of reducing power (i) when the in vivo activity of the hydrogenase was decreased by CO gassing (5, 10, 13, 14) or by adding methyl viologen (6); (ii) during a shift in solvent production induced by lowering the pH when acetyl coenzyme A (CoA) was first converted to acetone (a pathway consuming no reducing energy), creating a redox imbalance (6); (iii) when an NADH pressure was ...

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Increased levels of ATP and NADH are associated with increased solvent production in continuous cultures of Clostridium acetobutylicum

Cited by 86 publications

References 27 publications

Microbial Cellulose Utilization: Fundamentals and Biotechnology

Microbial Cellulose Utilization: Fundamentals and Biotechnology

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

Regulation of Clostridium acetobutylicum metabolism as revealed by mixed-substrate steady-state continuous cultures: role of NADH/NAD ratio and ATP pool

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