Temporal change of C-isotope signatures and methanogenic pathways in rice field soil incubated anoxically at different temperatures

Production of CH₄ and CO₂ was quantified in anoxically incubated soil samples taken from an Italian rice field. The rates increased with temperature between 10 and 37°C. The δ¹³C of the accumulated CO₂, CH₄ and acetate changed with time in a systematic way. The data were used in mass balance equations to constrain isotopic fractionation factors and pathways of CH₄ production. The calculations were further constrained by the determination of ¹⁴CH₄ production from ¹⁴CO₂ at steady state. At 50°C, CH₄ was exclusively produced from CO₂, indicating a fractionation factor of α_CO2/CH4 = 1.073. Between 10 and 37°C, the results showed a temporal change in the methanogenic pathway. A relatively high (40-60%) CO₂-derived fraction of CH₄ production in the beginning was followed by a phase in which contribution of CO₂-derived CH₄ decreased to low (<15%) values, and ultimately by the steady state phase in which values increased to <40% (the theoretically expected value). The rate of change from one phase to the next increased with temperature. Incubation temperature had a strong effect on the overall fractionation of ¹³C during the formation and consumption of acetate, with stronger fractionation at low than at high temperature. The results further showed that, especially at low temperatures, fractionation occurred during acetate turnover and acetoclastic methanogenesis, despite the fact that steady-state conditions caused (apparent) substrate-limitation.