Relationships between cellobiose catabolism, enzyme levels, and metabolic intermediates inClostridium cellulolyticum grown in a synthetic medium

Abstract

Continuous cultures, under cellobiose sufficient concentrations (14.62 mM) using a chemically defined medium, were examined to determine the carbon regulation selected by Clostridium cellulolyticum. Using a synthetic medium, a qcellobiose of 2.57 mmol g cells−1 h−1 was attained whereas the highest value obtained on complex media was 0.68 mmol g cells−1 h−1 (Payot et al. 1998. Microbiology 144:375–384). On a synthetic medium at D = 0.035 h−1 under cellobiose excess, lactate and ethanol biosynthesis were able to use the reducing equivalents supplied by acetic acid formation and the H2/CO2 ratio was found equal to 1. At a higher dilution rate (D = 0.115 h−1), there was no lactate production and the pathways toward ethanol and NADH‐ferredoxin‐hydrogenase contributed to balance the reducing equivalents; in this case a H2/CO2 ratio of 1.54 was found. With increasing D, there was a progressive increase (i) in the steady‐state concentration of NADH and NAD+ pools from 11.8 to 22.1 μmol (g cells) −1, (ii) in the intracellular NADH/NAD+ ratios from 0.43 to 1.51. On synthetic media, under cellobiose excess the carbon flow was also equilibrated by three overflows: exopolysaccharide, extracellular protein, and amino acid excretions. At D = 0.115 h−1, 34% of the cellobiose consumed was converted into exopolysaccharides; this deviation of the carbon flow and the increase of the phosphoroclastic activity decreased dramatically the pyruvate excretion and explained the break in lactate production. Whatever the dilution rate, C. cellulolyticum, using ammonium and cellobiose excess, always spilled usual amino acids accompanied by other amino compounds. In vitro, GAPDH, phosphoroclastic reaction, alcohol dehydrogenase, and acetate kinase activities were high under conditions giving high in vivo specific production rates. There were also correlations between the in vitro lactate dehydrogenase activity and in vivo lactate production, but in contrast with the preceding activities, these two parameters decreased with D. All the results demonstrate that C. cellulolyticum was able to optimize carbon catabolism from cellulosic substrates in a synthetic medium. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 67: 327–335, 2000.

Isolation and Characterization of Shigella flexneri G3, Capable of Effective Cellulosic Saccharification under Mesophilic Conditions

“…This is similar to the rate of cellulose degradation observed for C. thermocellum at 60°C (33,36). However, no further cellulose degradation or cell growth was observed after 60 h. This could be due to either the depletion of a particular nutrient from the culture medium (21) or an accumulation of inhibitory intracellular compounds in the medium or in the cell (10,20). Various types of cellulase enzymes could be inhibited by soluble products (glucose, cellobiose, cellotriose, etc.)…”

Microbial Cellulose Utilization: Fundamentals and Biotechnology

“…During growth of C. cellulolyticum in complex medium, NADH/NAD ϩ ratios as high as 57 have been reported, suggesting that catabolism is limited by the rate of NADH reoxidation (226,519). Inability to regenerate NAD ϩ leads to metabolism of excess carbon via phosphoglucomutase, with the G-1-P ultimately used for cellodextrin synthesis and efflux, intracellular glycogen synthesis, and extracellular polysaccharide production (225,227). The difficulty in reoxidizing NADH may explain why this species is the only mesophilic cellulolytic organism reported to produce lactate in excess of 0.5 mol per mol of hexose consumed (156).…”

Kinetics and Metabolism of Cellulose Degradation at High Substrate Concentrations in Steady-State Continuous Cultures of Clostridium cellulolyticum on a Chemically Defined Medium

“…This excess of produced NADH correlated with increases of both the H 2 / CO 2 ratio, which was always higher than 1, and the q NADH-Fd . These results suggested that the intracellular NADH/NAD ϩ ratio was maintained by the NADH-Fd reductase and hydrogenase, since these interconnected enzymatic activities can oxidize NADH via H 2 production (18)(19)(20). The O/R, determined from the gas production ratio and fermentative end product concentration, was very close to 1 and indicated an efficient reoxidation of NADH via H 2 production in addition to carbon fermentative pathways (11).…”

“…Using cellobiose, which is one of the soluble cellodextrins released during cellulolysis, major differences in the regulation of the carbon flow between carbon-limited and carbon-sufficient continuous cultures have been reported (19,20). As the dilution rate increases, in cellobiose-limited chemostats the metabolic pathways towards ethanol and lactate contribute to balance the reducing equivalents supplied by acetate formation (19), while under cellobiose-saturated conditions (20) the redox balance is essentially maintained by NADH-ferredoxin (NADH-Fd) reductase-hydrogenase and ethanol dehydrogenase activities and the carbon flow is equilibrated by three overflows, i.e., exopolysaccharide, extracellular protein, and amino acid excretions.…”

“…, and all other nutrients appeared in excess (10,20). Growth under substrate excess generally results in oscillations and hysteresis (23,24,37,52), but such phenomena did not occur, since steady states of both residual cellulose concentration and biomass monitored during cell culture could a Carbon flows were calculated as specified in Materials and Methods and are diagrammed in Fig.…”