Methanogenic Degradation of Polysaccharides and the Characterization of Polysaccharolytic Clostridia from Anoxic Rice Field Soil

Abstract

Summary The anaerobic degradation of polysaccharides to methane via intermediary metabolites and the characteristics of some polysaccharolytic bacteria were investigated in Italian rice field soil. The addition of xylan, pectin or cellulose to slurries of anoxic rice field soil enhanced the production rates of CH 4 and CO 2 both at 15 and 30 °C (cellulose only after prolonged incubation), and resulted in the transient accumulation of H 2 , acetate, propionate and lactate. Counting of bacteria using the MPN technique indicated that xylan- and pectin-fermenting bacteria were more abundant than cellulolytic bacteria. Three gram-positive, strictly anaerobic, rod-shaped clostridia strains, RCel1, RXyl1 and RPec1 were isolated by enrichment cultures using as substrates cellulose, xylan and pectin, respectively, and characterized phylogenetically (16S rDNA sequences) and physiologically. The fermentation of cellulose by the cellulolytic strain RCell, closely related to Clostridium papyrosolvens , was investigated in more detail both at 30 °C and 15 °C, both in monoculture and in coculture with either Methanospirillum hungatei or Sporomusa ovata . The cocultures generally resulted in higher growth yields of strain RCel1 than in monoculture suggesting a better ATP yield per mol of anhydroglucose fermented. In monoculture, strain RCel1 fermented cellulose primarily to H 2 , acetate and ethanol and to smaller amounts of succinate, lactate and formate. Propionate was produced only at 15 °C. In coculture with M. hungatei , an increase in H 2 and acetate production and a decrease in lactate, ethanol, and formate production was observed. The H 2 partial pressures in the cocultures were lower than in the monoculture. In coculture with S. ovata , cellulose was fermented to acetate as main product. The steady state H 2 partial pressure in coculture with S. ovata was higher than with M. hungatei , and was lower at 15 °C than at 30 °C in both cocultures as expected from the theory. The levels of H 2 in coculture with M. hungatei were similar to those observed in rice field soil slurries. The other fermentation products of cellulose, xylan or pectin observed in bacterial cultures were also mostly the same as observed in slurries of anoxic rice field soil, but important quantitative differences remained.