The Molecular Microbial Ecology and Mechanisms of Biomarker Degradation in Crude Oil

Biomarkers are structurally complex components of petroleum derived from biological molecular precursors, such as chlorophyll, sterols, and hopanoids (Peters and Moldowan, 1993). The biomarker profile of a crude oil is distinctive and diagnostic, often allowing correlation of an oil to its source rock. Many biomarkers in crude oil are resistant to biodegradation and are used by petroleum geochemists to assess genetic relationships, thermal maturity, and biodegradation. However, the reliability of these biomarkers depends on this recalcitrance to biodegradation. The studies presented here used bacterial enrichment cultures to examine the distribution and extent of biomarker­ degrading activity in aerobic and anaerobic environments. The biodegradation of C30 17α,21β(H)-hopane within Bonny Light crude oil by laboratory enrichment cultures was demonstrated to be temperature sensitive through incubation at 4°C, 15°C, 30°C, and 37°C. Although degradation of the n-alkanes and the branched alkanes, pristane and phytane, was observed at 15°C, 30°C, and 37°C, the degradation of hopane was limited to 30°C. At 4°C, no degradation of any crude oil components was observed. Denaturing gradient gel electrophoresis (DGGE) analysis of the cultures at these different temperatures revealed no striking differences in the composition of the microbial communities. However, the enrichment cultures were each developed from the same soil, suggesting that temperature affects the activity of the enriched microbial community. This implies that, in situ, the degradation of larger molecular weight biomarkers will be dependent on temperature and will be limited to temperate climates. The aerobic microbial degradation of two Venezuelan crude oils enriched in 25- norhopanes was examined after a five-week aerobic incubation using the Light Crude (LC) enrichment culture, an enrichment culture developed from a creosote-contaminated soil and previously shown to degrade the C30 17α,21β(H)-hopane. Based on reservoir oil observations, petroleum geochemists consider the 25-norhopanes as the putative degradation products of the hopanes. After five weeks, analysis of the Venezuelan oils using gas chromatography-mass spectrometry revealed extensive biomarker degradation. The C28 tricyclic terpane, C29 - C34 17α(H),21β(H)-hopanes, and the C29 17α(H),21β(H)- 25-norhopane were all degraded. The C35 17α(H),21β(H)-hopane and 18α(H)-oleanane were conserved. Further and previously unreported, the C28 – C34 17α(H),21β(H)-25- norhopanes were degraded and no formation of 25-norhopanes was observed. Degradation caused preferential removal of the 22R versus the 22S isomer in both the extended hopanes and 25-norhopanes, implying that bacteria remove these compounds in aerobic environments. These data also demonstrated 25-norhopane degradation on a time scale similar to other biomarkers, suggesting that the 25-norhopanes and hopanes share a microbial mechanism of degradation. Typically, in aerobic environments the order of hydrocarbon degradation follows this order: n-alkanes \u3e branched alkanes \u3e low molecular weight aromatics \u3e cyclic alkanes. In anaerobic enrichment cultures initiated with sediment from the Charleston Harbor and Cooper River, the order of crude oil degradation was very similar to that observed under aerobic conditions. Although degradation of large molecular weight biomarkers was not observed, the n-alkanes and the C19 – C28 branched alkanes and several polycyclic aromatic hydrocarbon (PAH) components (the alkylated naphthalenes (C2 and C3), unsubstituted and alkylated fluorenes (C1), unsubstituted and C1 phenanthrene, and dibenzothiophene) of a Venezuelan crude oil were degraded under sulfate-reducing conditions. This is the first report of the degradation of the C21-C28 branched alkanes under anaerobic conditions. Degradation of the saturate compounds was not observed in molybdate-amended samples or in heat-killed controls. PAH degradation, however, was observed in the molybdate-amended controls. This suggests that saturate degradation was specific to the sulfate reducers and that other microbial population(s) exclusive of sulfate reducers contributed to PAH degradation. Cometabolism is the transformation of a secondary substrate during growth on a primary substrate. The potential cometabolic mechanisms of C30 17α,21β-hopane transformation were examined using the Degraded Light Crude (DLC) enrichment culture and a Hopane-Enriched Fraction (HEF) of Bonny Light crude oil. In preliminary studies with HEF, hopane degradation was not observed, which suggested that a component of the saturate fraction of Bonny Light crude was necessary to induce hopane degradation. For these studies, increasing amounts of the saturate fraction without n­ alkanes were incrementally added to HEF. These studies did demonstrate hopane degradation within HEF, suggesting that the degradation of C30 17α,21β(H)-hopane by the DLC culture is not cometabolic. DGGE analysis of the DLC culture on the various hydrocarbon additions indicated a band (H) whose intensity correlated with the onset and prevalence of hopane degradation in several of the fractions. Band H was excised from the DGGE gels and sequenced. Phylogenetic analysis of the band H sequence placed it within the Burkholderia subgroup. DGGE analysis of the 16S rDNA V9 region amplicons from culturable isolates from the DLC culture on HEF determined that one of the isolates, 14K, comigrated with this band. Although this isolate was capable of n­ alkane degradation, the degradation of hopane within HEF was not observed after 110 days. A general observation in the laboratory with regard to each enrichment culture capable of biomarker degradation is the conservation of 18α(H)-oleanane relative to the degradation of C30 17α,21β(H)-hopane. The pentacyclic structures of 18α(H)-oleanane and C30 17α,21β(H)-hopane only differ in their E rings. Growth on the E ring moieties corresponding to hopane and oleanane was not observed. Intermediates of hopane metabolism were not observed in HEF residues derivatized to the corresponding methyl esters with diazomethane. Further, the inclusion of a P-oxidation inhibitor, acrylic acid, into the DLC culture temporarily abated hopane degradation. DOGE analysis of the HEFDLC culture in the presence and absence of acrylic acid associated the presence of a single band to the onset of hopane degradation in the acrylic acid cultures. This band was identified by comigration as the isolate 14K. These data suggest that a common metabolic mechanism, P-oxidation, is involved in the initial attack on C30 17α,21β(H)­ hopane and that 14K is associated with the onset of hopane degradation. Based on these findings, a commensal scheme of hopane degradation by the DLC culture was proposed. The application of biomarkers in petroleum bioremediation and geochemistry depends on a complete understanding of the potential fates of these compounds in aerobic and anaerobic environments. The elucidation and description of aerobic and anaerobic mechanisms of biomarker transformation may reveal metabolically distinct routes that can be used as signatures of aerobic and anaerobic transformations. Of course, extrapolation of this knowledge to a geological setting will depend on the distribution and accumulation of these intermediates in reservoirs. If found in reservoir oils, these intermediates may provide petroleum geochemists, bioremediation specialists, and biodegradation scientists with better tools to predict, monitor, and describe the fate of crude oil in the environment