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Journal of Bacteriology, January 2001, p. 119-130, Vol. 183, No. 1
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.1.119-130.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Carbon Flux Distribution and Kinetics of Cellulose Fermentation in Steady-State Continuous Cultures of Clostridium cellulolyticum on a Chemically Defined Medium

Mickaël Desvaux, Emmanuel Guedon, and Henri Petitdemange*

Laboratoire de Biochimie des Bactéries Gram +, Domaine Scientifique Victor Grignard, Faculté des Sciences, Université Henri Poincaré, 54506 Vandoeuvre-lès-Nancy Cédex, France

Received 22 May 2000/Accepted 6 October 2000

The metabolic characteristics of Clostridium cellulolyticum, a mesophilic cellulolytic nonruminal bacterium, were investigated and characterized kinetically for the fermentation of cellulose by using chemostat culture analysis. Since with C. cellulolyticum (i) the ATP/ADP ratio is lower than 1, (ii) the production of lactate at low specific growth rate (µ) is low, and (iii) there is a decrease of the NADH/NAD+ ratio and qNADH produced/ qNADH used ratio as the dilution rate (D) increases in carbon-limited conditions, the chemostats used were cellulose-limited continuously fed cultures. Under all conditions, ethanol and acetate were the main end products of catabolism. There was no shift from an acetate-ethanol fermentation to a lactate-ethanol fermentation as previously observed on cellobiose as µ increased (E. Guedon, S. Payot, M. Desvaux, and H. Petitdemange, J. Bacteriol. 181:3262-3269, 1999). The acetate/ethanol ratio was always higher than 1 but decreased with D. On cellulose, glucose 6-phosphate and glucose 1-phosphate are important branch points since the longer the soluble beta -glucan uptake is, the more glucose 1-phosphate will be generated. The proportion of carbon flowing toward phosphoglucomutase remained constant (around 59.0%), while the carbon surplus was dissipated through exopolysaccharide and glycogen synthesis. The percentage of carbon metabolized via pyruvate-ferredoxin oxidoreductase decreased with D. Acetyl coenzyme A was mainly directed toward the acetate formation pathway, which represented a minimum of 27.1% of the carbon substrate. Yet the proportion of carbon directed through biosynthesis (i.e., biomass, extracellular proteins, and free amino acids) and ethanol increased with D, reaching 27.3 and 16.8%, respectively, at 0.083 h-1. Lactate and extracellular pyruvate remained low, representing up to 1.5 and 0.2%, respectively, of the original carbon uptake. The true growth yield obtained on cellulose was higher, [50.5 g of cells (mol of hexose eq)-1] than on cellobiose, a soluble cellodextrin [36.2 g of cells (mol of hexose eq)-1]. The rate of cellulose utilization depended on the solid retention time and was first order, with a rate constant of 0.05 h-1. Compared to cellobiose, substrate hydrolysis by cellulosome when bacteria are grown on cellulose fibers introduces an extra means for regulation of the entering carbon flow. This led to a lower µ, and so metabolism was not as distorted as previously observed with a soluble substrate. From these results, C. cellulolyticum appeared well adapted and even restricted to a cellulolytic lifestyle.

* Corresponding author. Mailing address: Laboratoire de Biochimie des Bactéries Gram +, Domaine Scientifique Victor Grignard, Université Henri Poincaré, Faculté des Sciences, BP 239, 54506 Vandoeuvre-lès-Nancy Cédex, France. Phone: 33 3 83 91 20 53. Fax: 33 3 83 91 25 50. E-mail: hpetitde{at}lcb.uhp-nancy.fr.

Journal of Bacteriology, January 2001, p. 119-130, Vol. 183, No. 1
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.1.119-130.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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