Methanogenic Paraffin Biodegradation: Alkylsuccinate Synthase Gene Quantification and Dicarboxylic Acid Production

Abstract

Paraffinic -alkanes (>C) 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 paraffin -octacosane (CH). Compared with that in controls, the consumption of -octacosane was coupled to methane production, demonstrating its biodegradation under these conditions. was postulated to be an important CH degrader in the culture on the basis of its high relative abundance as determined by 16S rRNA gene sequencing. An identified gene (known to encode the α subunit of alkylsuccinate synthase) aligned most closely with those from other organisms. Quantitative PCR (qPCR) and reverse transcription qPCR assays for demonstrated significant increases in the abundance and expression of this gene in CH-degrading cultures compared with that in controls, suggesting -octacosane activation by fumarate addition. A metabolite analysis revealed the presence of several long-chain α,ω-dicarboxylic acids only in the CH-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. Understanding 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 (<C) can be biodegraded anaerobically, but less is understood regarding the biodegradation of longer chain waxy alkanes (>C) that comprise many fuel mixtures. Using an enrichment culture derived from a freshwater fuel-contaminated site, we demonstrate that the model waxy alkane -octacosane can be biodegraded under methanogenic conditions by a presumed phylotype. Compared with that of controls, we show an increased abundance and expression of the gene, which is known to be important for anaerobic-alkane metabolism. Metabolite analyses revealed the presence of a range of α,ω-dicarboxylic acids found only in -octacosane-degrading cultures, a novel finding that lends insight as to how anaerobic communities may access waxes as growth substrates in anoxic environments.

Long-chain n-alkane biodegradation coupling to methane production in an enriched culture from production water of a high-temperature oil reservoir

“…This proposed mechanism is similar to that which has been proposed for hexadecane degradation (Embree et al 2014). Similar observation was reported in a methanogenic waxy hydrocarbon-degrading consortium from freshwater hydrocarbon-impacted aquifer sediments, where the consumption of n-octacosane (C28) was coupled with methane production and detection of several dicarboxylic acids (Oberding and Gieg 2018). Fig.…”

“…Although no alkylsuccinate-like metabolites were acquired from the samples, dicarboxylic acids were detected only in long-chain n-alkaneamended cultures. According to previously postulated mechanism (Oberding and Gieg 2018), in which both ends of paraffin can be activated by the fumarate addition pathway. Future studies such as the design of new and universal primers as well as SIP techniques (Cheng et al 2013;Jimenez et al 2016;Kleindienst et al 2014;Kunapuli et al 2007) are required to unravel the exact activation mechanisms of paraffin.…”

“…With respect to solid n-alkane (> C17) recent studies which used paraffin (n-octacosane) as the sole carbon and energy sources under methanogenic condition enriched from hydrocarbon-contaminated aquifer sediments of low temperature, has proved paraffin degradation coupled with methane generation. Metagenomic analysis proposed that the paraffin molecular was most likely activated via fumarate addition pathway by syntrophic members of Smithella (Oberding and Gieg 2018;Wawrik et al 2016). Furthermore, it was shown that putative alkylsuccinate synthase (assA) gene was found in paraffin-degrading enrichment cultures derived from hydrocarbon-impacted aquifer sediments, indicative of anaerobic long-chain n-alkane metabolism (Callaghan et al 2010).…”

“…Despite the fact that high molecular weight alkanes are solid at room temperature, they actually tend to be melted in the oil phase under high-temperature condition (Etoumi 2007;Sanjay et al 1995). Thus, longchain paraffin is theoretically expected to be metabolized by hydrocarbon-degrading consortia and subsequently converted into methane by syntrophic microorganisms and methanogenic archaea in deep subsurface of high temperature (Jones et al 2008;Oberding and Gieg 2018). Nevertheless, knowledge regarding the anaerobic biodegradation of long-chain paraffin and possible microbial populations in thermophilic cultures under methanogenic conditions remain unclear (Callaghan 2013;Wentzel et al 2007).…”

Second-generation stoichiometric mathematical model to predict methane emissions from oil sands tailings

“…showed excellent fit, ranging from 0.81 -0.98 for the three combinations of MFT and diluent. (Oberding and Gieg, 2018); and (6) organic additives used in ore processing and deposited with tailings, e.g., citrate that is used as an amendment in some OSTPs and is a potentially large source of unaccounted CH4 in CNUL MRM. Another explanation for larger masses of measured emissions is the delayed, stochastic release of methane produced years ago from labile HCs that is 'trapped' in lower strata of MFT (Guo, 2009) until (1) suitably-sized and -oriented channels are created (e.g., by microbial activity, Siddique et al, 2014) and/or (2) There is good agreement between the model predictions and measured field emissions despite the obvious reasons of discrepancy discussed above.…”