During the growth of Clostridium cellulolyticum in chemostat cultures with ammonia as the growth-limiting nutrient, as much as 30% of the original cellobiose consumed by C. cellulolyticum was converted to cellotriose, glycogen, and polysaccharides regardless of the specific growth rates. Whereas the specific consumption rate of cellobiose and of the carbon flux through glycolysis increased, the carbon flux through the phosphogluco-mutase slowed. The limitation of the path through the phosphoglucomutase had a great effect on the accumulation of glucose 1-phosphate (G1P), the precursor of cellotriose, exopolysaccharides, and glycogen. The specific rates of biosynthesis of these compounds are important since as much as 16.7, 16.0, and 21.4% of the specific rate of cellobiose consumed by the cells could be converted to cellotriose, exopolysaccharides, and glycogen, respectively. With the increase of the carbon flux through glycolysis, the glucose 6-phosphate (G6P) pool decreased, whereas the G1P pool increased. Continuous culture experiments showed that glycogen bio-synthesis was associated with rapid growth. The same result was obtained in batch culture, where glycogen biosyn-thesis reached a maximum during the exponential growth phase. Glycogen synthesis in C. cellulolyticum was also not subject to stimulation by nutrient limitation. Flux analyses demonstrate that G1P and G6P, connected by the phosphoglucomutase reaction, constitute important branch points for the distribution of carbon fluxes inside and outside cells. From this study it appears that the properties of the G1P-G6P branch points have been selected to control excretion of carbon surplus and to dissipate excess energy, whereas the pyruvate-acetyl coenzyme A branch points chiefly regulate the redox balance of the carbon catabolism as was shown previously (E. Guedon et al., J. Bacteriol. 181:3262-3269, 1999). Clostridium cellulolyticum, a strictly anaerobic cellulolytic bacterium, was isolated from decayed grass (18) and degrades cellulose by using a complex cellulolytic system called cellulo-some (11). The cellulosome, wherein the cellulases were found to be organized into a high-molecular-weight, cellulolytic complex , has been extensively investigated (2, 27), while few studies have focused on the carbon metabolic pathway, mainly in C. cellulolyticum (5). Most studies of the carbon metabolism in cellulolytic clostridias have been performed with cellobiose, the major end product of the degradation process, which is taken up and assimilated by the cells (5, 16, 28). Our recent study (7) demonstrated that when C. cellulolyticum was grown in continuous cellobiose-limited culture, there was a shift from an acetate-ethanol fermentation at low levels of carbon flow to a lactate-ethanol fermentation at high catabolic rates; in addition , increasing levels of pyruvate in the extracellular medium were detected as the dilution rate increased. The pyruvate overflow suggested that the carbon flow through glycolysis was higher than the rate of processing by pyruvate-ferredoxin oxi-doreductase (7) (Fig. 1). Consequently, under such conditions, the rates of energy production in the catabolic pathway were not correlated with the anabolic energy requirements, i.e., more ATP was produced than was needed by the biosynthetic and maintenance demands (25). Furthermore, because ligno-cellulosic compounds usually contain high levels of carbon and a low levels of nitrogen, growth of C. cellulolyticum on these compounds would lead to an excess of energy, and energy-spilling reactions must be utilized. In addition, it is also necessary to overcome the potentially deleterious osmotic effects of the accumulation of surplus intracellular metabolites (19). There are different types of energy-consuming reactions selected by the bacteria during the course of evolution; for instance , (i) an overflow metabolism, wherein bacteria excrete or leak partially oxidized metabolites (29); (ii) metabolic shifts, where bacteria can change their end products and alter ATP production (30); (iii) futile cycles (15); and (iv) the synthesis of intra-and extracellular polysaccharides, which are energy-consuming processes and limit the carbon flow toward glycolysis and ATP production (20-22). In addition to pyruvate, extra-cellular polysaccharides were previously found to be secreted by C. cellulolyticum at high rates of cellobiose consumption (7), leading to the belief that carbon flow was regulated at the branch point of the glucose phosphate pools (Fig. 1). This kind of regulation is further suggested by the fact that growth of C. cellulolyticum was inhibited in the presence of excess carbon (8). The aim of the present study was to investigate how changes in catabolic flux affected (i) the intracellular turnover of glucose 1-phosphate (G1P) and glucose 6-phosphate (G6P) and (ii) the distribution of the carbon fluxes inside and outside the cells.