Effect of Substrate Concentration on Carbon Isotope Fractionation during Acetoclastic Methanogenesis by Methanosarcina barkeri and M. acetivorans and in Rice Field Soil

Methanosarcina is the only acetate-consuming genus of methanogenic archaea other than Methanosaeta and thus is important in methanogenic environments for the formation of the greenhouse gases methane and carbon dioxide. However, little is known about isotopic discrimination during acetoclastic CH4 production. Therefore, we studied two species of the Methanosarcinaceae family, Methanosarcina barkeri and Methanosarcina acetivorans, and a methanogenic rice field soil amended with acetate. The values of the isotope enrichment factor (epsilon) associated with consumption of total acetate (epsilon(ac)), consumption of acetate-methyl (epsilon(ac-methyl)) and production of CH4 (epsilon(CH4)) were an epsilon(ac) of -30.5 parts per thousand, an epsilon(ac-methyl) of -25.6 parts per thousand, and an epsilon(CH4) of -27.4 parts per thousand for M. barkeri and an epsilon(ac) of -35.3 parts per thousand, an epsilon(ac-methyl) of -24.8 parts per thousand, and an epsilon(CH4) of -23.8 parts per thousand for M. acetivorans. Terminal restriction fragment length polymorphism of archaeal 16S rRNA genes indicated that acetoclastic methanogenic populations in rice field soil were dominated by Methanosarcina spp. Isotope fractionation determined during acetoclastic methanogenesis in rice field soil resulted in an epsilon(ac) of -18.7 parts per thousand, an epsilon(ac-methyl) of -16.9 parts per thousand, and an epsilon(CH4) of -20.8 parts per thousand. However, in rice field soil as well as in the pure cultures, values of epsilon(ac) and epsilon(ac-methyl) decreased as acetate concentrations decreased, eventually approaching zero. Thus, isotope fractionation of acetate carbon was apparently affected by substrate concentration. The epsilon values determined in pure cultures were consistent with those in rice field soil if the concentration of acetate was taken into account.