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 () is low, and (iii) there is a decrease of the NADH/NAD ؉ ratio and q NADH produced / q NADH 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 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 ␤-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 , 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. Cellulose is of cardinal importance in the global carbon cycle: it accumulates in the environment due to its durable nature (5), and the main final products released during its fermentation are CH 4 and CO 2 (76). Bacteria are the major cellulose hydrolyzers in anaerobic cellulosic microbiota (35, 67), where cellulolytic clostridia play a key role (34). The cellulose degradation process which occurs through cel-lulases has been studied extensively on cellulolytic clostridia, leading to the cellulosome concept (4, 6). The multienzymatic complexes found at the surface of the cells are responsible for adhesion of bacteria to cellulose fibers and allow a very efficient synergism of action of the different enzyme components (8). Genes encoding cellulases as well as the mechanism of action of the cellulosome are the subject of considerable research , while few studies have focused on the metabolic aspects of cellulose digestion by clostridia (27, 40). Recent characterization of the carbohydrate catabolism of Clostridium cellulolyticum, a nonruminal mesophilic bacterium able to degrade crystalline cellulose, showed that (i) better control of catabolism occurred on a mineral salt-based medium (24, 48), (ii) carbon-limited and carbon-sufficient chemostats displayed major differences in regulatory responses of the carbon flow (25), and (iii) in nitrogen-limited conditions, glucose 6-phosphate (G6P) and glucose 1-phosphate (G1P) branch points play an important role in carbon flux divergence (22). These investigations, however, were performed with cellobi-ose, which is one of the soluble cellodextrins released during cellulolysis (56). In such investigations, the use of soluble sugars obviated the bacterial metabolic analysis on cellulose that was assumed difficult to undertake. Metabolic regulation processes found using cellobiose could differ or even be distorted from those with insoluble substrates. While the first studies of cellulose focused mainly on C. cellulolyticum behavior, such as colonization or degradation with an insoluble substrate (19-21), recent investigations of cellulose fermentation in batch culture (12) have indicated that (i) metabolite yields depend strongly on the initial cellulose concentration and (ii) early growth arrest is linked to pyruvate overflow as in cellobiose batch culture (23). In the last decade, efficient continuous-culture devices for * Corresponding author. Mailing address: Laboratoire de Biochimie des Bactéries Gram ϩ, Domaine Scientifique Victor Grignard, Uni-versité Henri Poincaré, Faculté des Sciences, BP 239, 54506 Vandoeu-vre-lès-Nancy Cédex, France. Phone: 33 3 83 91 20 53. Fax: 33 3 83 91 25 50.