ABSTRACTParaffinicn-alkanes (>C17) that are solid at ambient temperature comprise a large fraction of many crude oils. The comparatively low water solubility and reactivity of these long-chain alkanes can lead to their persistence in the environment following fuel spills and pose serious problems for crude oil recovery operations by clogging oil production wells. However, the degradation of waxy paraffins under the anoxic conditions characterizing contaminated groundwater environments and deep subsurface energy reservoirs is poorly understood. Here, we assessed the ability of a methanogenic culture enriched from freshwater fuel-contaminated aquifer sediments to biodegrade the model paraffinn-octacosane (C28H58). Compared with that in controls, the consumption ofn-octacosane was coupled to methane production, demonstrating its biodegradation under these conditions.Smithellawas postulated to be an important C28H58degrader in the culture on the basis of its high relative abundance as determined by 16S rRNA gene sequencing. An identifiedassAgene (known to encode the α subunit of alkylsuccinate synthase) aligned most closely with those from otherSmithellaorganisms. Quantitative PCR (qPCR) and reverse transcription qPCR assays forassAdemonstrated significant increases in the abundance and expression of this gene in C28H58-degrading cultures compared with that in controls, suggestingn-octacosane activation by fumarate addition. A metabolite analysis revealed the presence of several long-chain α,ω-dicarboxylic acids only in the C28H58-degrading cultures, a novel observation providing clues as to how methanogenic consortia access waxy hydrocarbons. The results of this study broaden our understanding of how waxy paraffins can be biodegraded in anoxic environments with an application toward bioremediation and improved oil recovery.IMPORTANCEUnderstanding the methanogenic biodegradation of different classes of hydrocarbons has important applications for effective fuel-contaminated site remediation and for improved recovery from oil reservoirs. Previous studies have clearly demonstrated that short-chain alkanes (<C17) can be biodegraded anaerobically, but less is understood regarding the biodegradation of longer chain waxy alkanes (>C17) that comprise many fuel mixtures. Using an enrichment culture derived from a freshwater fuel-contaminated site, we demonstrate that the model waxy alkanen-octacosane can be biodegraded under methanogenic conditions by a presumedSmithellaphylotype. Compared with that of controls, we show an increased abundance and expression of theassAgene, which is known to be important for anaerobicn-alkane metabolism. Metabolite analyses revealed the presence of a range of α,ω-dicarboxylic acids found only inn-octacosane-degrading cultures, a novel finding that lends insight as to how anaerobic communities may access waxes as growth substrates in anoxic environments.
A novel exopolysaccharide-producing and long-chain n-alkane degrading bacterium Bacillus licheniformis strain DM-1 with potential application for in-situ enhanced oil recovery