Carbon and hydrogen isotope fractionation during methanogenesis: A laboratory study using coal and formation water

Abstract

Carbon and hydrogen isotope compositions of CH 4 generated via methanogenesis in cultures of South Sumatra Basin (SSB) coalbed methane (CBM) formation waters grown on coal, acetate and H 2 +CO 2 were investigated. CH 4 production and molecular analysis confirmed the presence of active microbial communities that are able to convert coal into CH 4 using both acetoclastic and hydrogenotrophic pathways. The representative bacterial sequences were dominated by Bacteroidetes, Firmicutes and Deltaproteobacteria, while Methanosaeta and Methanosarcina were the most prevalent archaeal methanogens present in the cultures.CH 4 produced in this study's culturing experiments has δ 13 C values in the range of -50 ‰ to -20 ‰, with most values falling outside the current understanding of the carbon isotopic boundaries for biogenic CH 4 (-110 ‰ to -30 ‰). However, the corresponding apparent carbon isotopic α factor (α c = 1.02±0.006), and isotopic effect (ε c = -20.1 ‰ ±15.3) showed that CH 4 in SSB cultures was predominantly produced by acetoclastic methanogenesis, which is consistent with the results of molecular DNA analysis. In addition, the calculated contribution of CO 2 reduction from the δ 13 C values of coaltreated cultures was overall < 50 %, further confirming the high contribution of the acetoclastic pathway to CH 4 production in the SSB cultures. The outcome of this experimental study also suggests that δ 2 H-CH 4 values may not provide a reliable basis for distinguishing methanogenic pathways, while apparent carbon isotopic fractionation factor (α c ) and isotope effect (ε c ) are considered more useful indicators of the methanogenic pathway.The high δ 13 C-CH 4 values (>-30 ‰) and the dominance of Methanosaeta over Methanosarcina indicate that methanogens within the SSB cultures were operating at low substrate concentrations. An unusually positive δ 13 C-CH 4 suggests a substrate depletion effect, which is thought to be related to a decrease in the relative abundance of key bacterial coal degraders with formation water inoculum storage time. Closer observation of δ 13 C-CH 4 values during the growth of cultures within a single experiment also showed a 13 C-enrichment trend over time. At log phase of growth, the CH 4 produced was 13 C-depleted when compared to the stationary phase that also indicates substrate depletion effects. Finally, the δ 13 C-CH 4 values encountered in this study (as high as -20 ‰) highlight the possible positive extension of δ 13 C-CH 4 values of acetoclastic methanogenesis from those currently reported in the literature for natural and experimental samples (as high as -30 ‰).Keywords: Biogenic gas, coal, culture experiment, isotopic composition INTRODUCTIONArchaeal methanogens are the only known microorganisms capable of forming CH 4 . These strictly anaerobic microbes are restricted to utilizing simple compounds that contain no more than two carbon atoms. As such, complex organic compounds in anoxic environments (e.g., marine sediments, rice paddies, subsurface shales and coalbeds) ...

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