Elucidation of Enzymes in Fermentation Pathways Used by Clostridium thermosuccinogenes Growing on Inulin

Abstract

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...

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“…However, useful suggestions about how a major fraction of the dipeptides was converted to ethanol, acetate, and succinate are described below. On the basis of known catabolic enzymes of clostridia (for example, Clostridium thermosuccinogenes [48] or C. botulinum [12]), the following pathways could be involved in amino acid utilization. (48).…”

“…On the basis of known catabolic enzymes of clostridia (for example, Clostridium thermosuccinogenes [48] or C. botulinum [12]), the following pathways could be involved in amino acid utilization. (48). One molecule of ethanol would be produced from one molecule of aspartate (see equation 3 below).…”

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