Methanogenic biodegradation of cyclohexane by microbial consortia from oil sands tailings

Cyclohexane, a significant component of light oils and some refined fuels, can be introduced into the environment through petroleum production and fuel spills; it is also present in tailings from certain surface-mined oil sands ores. The environmental fate of cyclohexane is cryptic in oxygen-depleted methanogenic environments. Indigenous microorganisms in primary oil sands tailings cultures and cyclohexane-adapted- d 12 enrichments were used to test methanogenic biodegradation of 1 mM cyclohexane alone or with the potential co-substrate n -heptane in a 70:30 mixture. We assessed substrate depletion, metabolite production, methane accumulation, and changes in microbial community composition during incubation. Cyclohexane was depleted slowly but completely in primary cultures during 260 d when added alone or with n -heptane, and more quickly in enrichment cultures during 120 d incubation, yielding ∼66–75 % of theoretical maximum methane production. Analysis of derivatized extracts from cultures grown with unlabelled or fully deuterated cyclohexane‑ d 12 revealed fragment ions of m/z 329 and m/z 341 characteristic of cyclohexylsuccinic acid, the first intermediate in a fumarate addition pathway. Members of the bacterial and archaeal families Desulfotomaculaceae (previously classified in Peptococcaceae ), Methanosaetaceae , and Methanoregulaceae were associated with cyclohexane degradation. A major shift in Desulfotomaculaceae abundance (7.2 % of the total community) occurred with cyclohexane‑ d 12 enrichment by d 120, indicating a significant role in biodegradation. Additional contributions from Syntrophaceae and Coriobacteriaceae were observed in n -heptane degradation. This report of cyclohexane biodegradation under methanogenic conditions, likely via fumarate addition, provides insight into the fate of resistant compounds in hydrocarbon-impacted sites and helps predict greenhouse gas emissions from oil-contaminated methanogenic environments. • Cyclohexane is degraded methanogenically by oil sands tailings microbes, yielding 66–75 % CH₄. • Cyclohexane‑ d ₁₂ is degraded via fumarate addition, forming cyclohexylsuccinic acid. • Desulfotomaculaceae likely initiates cyclohexane ring activation under methanogenic conditions. • Syntrophaceae and Coriobacteriaceae degrade intermediates; methanogenic archaea produce CH₄. • Findings reveal anaerobic fate of cyclic hydrocarbons and methane emissions from tailings.