Based on the presence and absence of enzyme activities, the biochemical pathways for the fermentation of inulin by Clostridium thermosuccinogenes DSM 5809 are proposed. Activities of nine enzymes (lactate dehydrogenase, phosphoenolpyruvate carboxylase, malate dehydrogenase, fumarase, fumarate reductase, phosphotransacetylase, acetate kinase, pyruvate kinase, and alcohol dehydrogenase) were measured at four temperatures (37, 47, 58, and 70°C). Each of the enzymes increased 1.5 to 2.0-fold in activity between 37 and 58°C, but only lactate dehydrogenase, fumarate reductase, malate dehydrogenase, and fumarase increased at a similar rate between 58 and 70°C. No acetate kinase activity was observed at 70°C. Arrhenius energies were calculated for each of these nine enzymes and were in the range of 9.8 to 25.6 kcal/mol. To determine if a relationship existed between product formation and enzyme activity, serum bottle fermentations were completed at the four temperatures. Maximum yields (in moles per mole hexose unit) for succinate (0.23) and acetate (0.79) and for biomass (29.5 g/mol hexose unit) occurred at 58°C, whereas the maximum yields for lactate (0.19) and hydrogen (0.25) and the lowest yields for acetate (0.03) and biomass (19.2 g/mol hexose unit) were observed at 70°C. The ratio of oxidized products to reduced products changed significantly, from 0.52 to 0.65, with an increase in temperature from 58 to 70°C, and there was an unexplained detection of increased reduced products (ethanol, lactate, and hydrogen) with a concomitant decrease in oxidized-product formation at the higher temperature.Clostridium thermosuccinogenes is a strictly anaerobic sporeforming gram-positive bacterium that can ferment inulin to succinate and acetate as major products and to lactate, ethanol, formate, hydrogen, and carbon dioxide as minor products (7). Inulin is a carbohydrate found in the roots or tubers of some plants, consisting of 20 to 30 fructose molecules connected to a terminal glucose residue by a  (231) linkage. High concentrations of inulin are found in the roots of Jerusalem artichoke (80% [weight/dry weight]), chicory (75 % [wt/ wt]), and dahlia (72% [wt/wt]) (16).Succinic acid is a four-carbon aliphatic dicarboxylic acid having a pK a1 of 4.2 and a pK a2 of 5.6 which has applications in the manufacture of specialty chemicals and in agriculture, food, medicine, textiles, plating, and waste gas scrubbing (41). Industrially, succinic acid is currently produced by hydrogenation of maleic anhydride to succinic anhydride followed by hydration to succinic acid (5, 41). Succinic acid can be produced by many anaerobic microorganisms, usually near neutral pH (5). For example, one method to produce succinic acid microbially employs the strict anaerobe Anaerobiospirillum succiniciproducens (5, 10, 11, 24). Under optimal conditions, these bacteria produce succinic acid from glucose with a yield of 87% and a final concentration of 35 g/liter (11, 24). Recently, Guettler et al. (15) isolated the facultatively anaerobic gram-negativ...
Clostridium thermosuccinogenes are anaerobic thermophilic bacteria that ferment various carbohydrates to succinate and acetate as major products and formate, lactate, and ethanol as minor products. Metabolic carbon flux analysis was used to evaluate the effect of pH and redox potential on the batch fermentation of C. thermosuccinogenes. In a first study, the effects of four pH values (6.50, 6.75, 7.00, and 7.25) on intracellular carbon flux at a constant redox potential of -275 mV were compared. The flux of carbon toward succinate and formate increased whereas the flux to lactate decreased significantly with a pH increase from 6.50 to 7.25. Both specific growth rate and specific rate of glucose consumption were unaffected by changes in pH. The fraction of carbon flux at the phosphoenolpyruvate (PEP) node flowing to oxaloacetate increased with an increase in pH. At the pyruvate node, the fraction of flux to formate increased with increasing pH. At the acetyl CoA node, the fraction of flux to acetate increased significantly with an increase in pH. A second study elucidated the effect of four controlled culture redox potentials (-225, -250, -275, and -310 mV) on metabolic carbon flux at a constant pH of 7.25. Lower values of culture redox potential were correlated with increased succinate, acetate, and formate fluxes and decreased ethanol and hydrogen fluxes in C. thermosuccinogenes. Lactate formation was not significantly influenced by redox potential. At the PEP node, the fraction of carbon to oxaloacetate increased with a decrease in redox potential. At the pyruvate node, the fraction of carbon to formate increased, while at the acetyl CoA node, the fraction of carbon flux to acetate increased with reduced redox potential. The presence of hydrogen in the headspace or the addition of nicotinic acid to the growth media resulted in increased hydrogen and ethanol fluxes and decreased succinate, acetate, formate, and lactate fluxes.
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