Cellulose Catabolism by Clostridium cellulolyticum Growing in Batch Culture on Defined Medium Downloaded from
Résumé
A reinvestigation of cellulose degradation by Clostridium cellulolyticum in a bioreactor with pH control of the batch culture and using a defined medium was performed. Depending on cellulose concentration, the carbon flow distribution was affected, showing the high flexibility of the metabolism. With less than 6.7 g of cellulose liter ؊1 , acetate, ethanol, H 2 , and CO 2 were the main end products of the fermentation and cellulose degradation reached more than 85% in 5 days. The electron flow from the glycolysis was balanced by the production of H 2 and ethanol, the latter increasing with increasing initial cellulose concentration. From 6.7 to 29.1 g of cellulose liter ؊1 , the percentage of cellulose degradation declined; most of the cellulase activity remained on the cellulose fibers, the maximum cell density leveled off, and the carbon flow was reoriented from ethanol to acetate. In addition to that of previously indicated end products, lactate production rose, and, surprisingly enough, pyruvate overflow occurred. Concomitantly the molar growth yield and the energetic yield of the biomass decreased. Growth arrest may be linked to sufficiently high carbon flow, leading to the accumulation of an intracellular inhibitory compound(s), as observed on cellobiose (E. Guedon, M. Desvaux, S. Payot, and H. Petitdemange, Microbiology 145:1831-1838, 1999). These results indicated that bacterial metabolism exhibited on cellobiose was distorted compared to that exhibited on a substrate more closely related to the natural ecosystem of C. cellulolyticum. To overcome growth arrest and to improve degradation at high cellulose concentrations (29.1 g liter ؊1), a reinoculation mode was evaluated. This procedure resulted in an increase in the maximum dry weight of cells (2,175 mg liter ؊1), cellulose solubilization (95%), and end product concentrations compared to a classical batch fermentation with a final dry weight of cells of 580 mg liter ؊1 and 45% cellulose degradation within 18 days. Cellulolytic clostridia play a major role in cellulose decomposition , which is a key process in carbon cycling (29). Clos-tridium cellulolyticum is a nonruminal cellulolytic mesophilic bacterium isolated from decayed grass and capable of degrading crystalline cellulose (36). The biotechnological exploitation of this microorganism as well as the understanding of the role it plays in its own ecosystem requires knowledge of its metabolism and of its behavior when developed on cellulose. C. cellulolyticum is a low-GϩC gram-positive anaerobe belonging to clostridial group III (39, 40); it is also placed in family 4, genus 2, in a new proposed-hierarchical structure for clostridia (7). Recent metabolic investigations with this bacterium indicated that (i) compared to a complex medium previously used, mineral salt medium clearly produced a different regulatory response and permitted better control of the carbon flow (19, 34), (ii) early growth inhibition was associated with a carbon excess (18), and (iii) carbon-limited and carbon-saturated chemostats displayed major discrepancies in the regulation of carbon flow (20). These studies were performed using cellobiose, which is considered the most important end product of the enzymatic cellulose hydrolysis (31, 44, 45), and it was assumed that growth on cellulosic material was rather difficult to monitor and that metabolism changes would be more observable with soluble sugar (14, 17). Concerning the behavior of C. cellulolyticum towards cellulose substrate, earlier studies (i) suggested that the release of soluble sugars inhibited both cell growth and cellulase production (37) and (ii) described the growth of the bacteria on cellulose in terms of adhesion, colonization, release, and read-hesion processes (13). These experiments, however, were conducted systematically in complex media without pH regulation, and many of the effects observed may have been due to a decrease in the pH of these cultures (47). In fact, compared with other low-GϩC gram-positive anaerobes, particularly with bacteria defined as lactic acid bacteria, the bacteria of the clostridial type are generally considered to be restricted to a less acidic ecological niche due to their particular pattern of intracellular pH regulation (21, 41). Bacterial growth on cellulose differs from that on cellobiose by the necessity for bacteria first to adhere on the substrate and second to degrade it into soluble sugars. Thus both substrate limitation and substrate-sufficient periods could be encountered by bacteria, and so the carbon flow on a cellulose batch culture could be somewhere between that associated with carbon limitation when bacteria are released and that associated with carbon-sufficient conditions when bacteria have adhered to cellulose fibers. The aim of the present work was to investigate how C. cellulolyticum managed the abundance of insoluble substrate and to see whether or not the previous observations of cello-biose (15, 17-20, 34) were typical of bacterial behavior on cellulose. Taking into account previous considerations, this investigation on the metabolism and cellulolytic performance of C. cellulolyticum in batch fermentations was performed with controlled pH and using a mineral salt-based medium, which is more representative of the natural ecosystem of the bacterium.
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