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Summary

Carbon flow in a model methanogenic consortium capable of hydrocarbon degradation was investigated using a combination of stable isotope fractionation, protein-based stable isotope probing, and metaproteomics. Overall δ13C enrichment for methane and CO2 in the presence and absence of oil suggests that complex microbial interactions occur during methanogenic hydrocarbon mineralization. Specifically, the Δδ13C of CO2 was statistically identical in all incubations irrespective of oil presence, but the Δδ13C for methane was greater in the presence of oil compared with fatty acids alone. In addition, carbon from uniformly (13C) labelled n-fatty acids was distributed evenly among consortium members in the presence of oil, but used by relatively few community members when provided alone. In all incubations, aceticlastic and hydrogenotrophic methanogens were labelled to an equal extent, suggesting that no pathway is overwhelmingly dominant during methane production by the model consortium. Protein-based stable isotope probing identified key enzymes responsible for methanogenesis from CO2 and acetate labelled with 78.0 ± 4.4% and 73.3 ± 1.0% 13C respectively. Results suggest that acetate was used directly by methanogens in the presence of n-fatty acids alone, and that methanogenesis from CO2 was a secondary process. Proteins capable of catalysing hydrocarbon activation by addition to fumarate were not found. Collectively, this study demonstrates that significant microbial cooperation is required to recover hydrocarbons as methane.