Metabolism of cellobiose by Clostridiurn cellulolyticurn growing in continuous culture : evidence for decreased NADH reoxidation as a factor limiting growth

Laboratoire de Chimie Biologique I, Universitri Henri PoincarC, FacultC des Sciences, BP 239, 54506 Vandoeuvre-I&-Nancy cedex, France Previous results indicated that molar growth yields are reduced when Clostridium cellulolyticum is cultured in media containing cellobiose concentrations greater than 1 g I-'. Continuous cultures were examined to determine the physiological basis of these poor growth yields. Acetate was the main product of C. cellulolyticum metabolism, whereas the production of reduced compounds such as ethanol or lactate was low. Such patterns of product formation were accompanied by a 12-fold increase in intracellular NADH concentration when the cellobiose flow was increased. Catabolic enzymic activities were measured in vitro. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), acetate kinase and phosphoroclastic activities were found at similar levels as in cells metabolizing higher substrate concentrations. In contrast, lactate dehydrogenase activity was low and correlated with the rate of lactate production. Furthermore, an inhibition of GAPDH activity by high NADH/NAD+ ratios was established. These results suggested that a decreased NADH reoxidation could be responsible for limiting C. cellulolyticum growth. Lactate and ethanol production were not sufficient to balance out the NADH produced in the GAPDH step of glycolysis. One consequence of poor NADH reoxidation would be an increase in intracellular concentration of NADH, which in turn could inhibit GAPDH activity.