Methanogenic Degradation of Long n-Alkanes Requires Fumarate-Dependent Activation

Methanogenic degradation of n-alkanes is prevalent in n-alkane-impacted anoxic oil reservoirs and oil-polluted sites. However, little is known about the initial activation mechanism of the substrate, especially n-alkanes with a chain length above C₁₆ Here, a methanogenic C₁₆ to C₂₀n-alkane-degrading enrichment culture was established from production water of a low-temperature oil reservoir. At the end of the incubation (364 days), C₁₆ to C₂₀ (1-methylalkyl)succinates were detected in the n-alkane-amended enrichment culture, suggesting that fumarate addition had occurred in the degradation process. This evidence is supported further by the positive amplification of the assA gene encoding the alpha subunit of alkylsuccinate synthase. A phylogenetic analysis shows these assA amplicons to be affiliated with Smithella and Desulfatibacillum clades. Together with the high abundance of these clades in the bacterial community, these two species are postulated to be the key players in the degradation of C₁₆ to C₂₀n-alkanes in the present study. Our results provide evidence that long n-alkanes are activated via a fumarate addition mechanism under methanogenic conditions.IMPORTANCE Methanogenic hydrocarbon degradation is the major process for oil degradation in subsurface oil reservoirs and is blamed for the formation of heavy oil and oil sands. Addition of n-alkanes to fumarate yielding alkyl-substituted succinates is a well-characterized anaerobic activation mechanism for hydrocarbons and is the most common activation mechanism in the anaerobic biodegradation of n-alkanes with chain lengths less than C₁₆ However, the activation mechanism involved in the methanogenic biodegradation of n-alkanes longer than C₁₆ is still uncertain. In this study, we analyzed a methanogenic enrichment culture amended with a mixture of C₁₆ to C₂₀n-alkanes. These n-alkanes can be activated via fumarate addition by mixed cultures containing Smithella and Desulfatibacillum species under methanogenic conditions. These observations provide a fundamental understanding of long-n-alkane metabolism under methanogenic conditions and have important applications for the remediation of oil-contaminated sites and for energy recovery from oil reservoirs.