Pathway of CH4 formation in anoxic rice field soil and rice roots determined by 13C-stable isotope fractionation.

In anoxic rice fields methane is produced by either reduction of CO2 or cleavage of acetate. We measured the delta 13C-values of CH4 and CO2, acetate and organic carbon during time course experiments with anoxic methanogenic soil and root samples and used these values to calculate the fractions of CH4 (and acetate) produced from CO2 reduction. Comparison with radiotracer and/or inhibitor studies constrained the kinetic fractionation factors used for calculation. The fractionation factors for the conversion of CO2 to CH4 and of acetate to CH4 were on the order of alpha = 1.07 (epsilon = -70%) and epsilon > or = - 20%, respectively. The pathway of CH4 production changed with time of anoxic incubation. Anoxic slurries of rice field soil first produced CH4 predominantly (>50%) from CO2, then predominantly (>80%) from acetate and finally (after about one month) according to the theoretically expected ratio (33% CO2 and 67% acetate). Anoxic rice roots, on the other hand, initially produced CH4 exclusively from CO2, followed by contribution of acetate of about 40-60%. Rice roots also produced acetate that partially originated (< or = 1 30%) from reduction of CO2 as determined by calculation of isotopic fractionation using fractionation factors from the literature. The results demonstrate that there is quite some variability in pathways of CH4 production, and also indicate that isotopic fractionation factors may be different in different habitats and change with time.