Advances in our understanding of the microbial ecology at sites impacted by light non-aqueous phase liquids (LNAPLs) are needed to drive development of optimized bioremediation technologies, support longevity models, and develop culture-independent molecular tools. In this study, depth-resolved characterization of geochemical parameters and microbial communities was conducted for a shallow hydrocarbon-impacted aquifer. Four distinct zones were identified based on microbial community structure and geochemical data: (i) an aerobic, low-contaminant mass zone at the top of the vadose zone; (ii) a moderate to high-contaminant mass, low-oxygen to anaerobic transition zone in the middle of the vadose zone; (iii) an anaerobic, high-contaminant mass zone spanning the bottom of the vadose zone and saturated zone; and (iv) an anaerobic, low-contaminant mass zone below the LNAPL body. Evidence suggested that hydrocarbon degradation is mediated by syntrophic fermenters and methanogens in zone III. Upward flux of methane likely contributes to promoting anaerobic conditions in zone II by limiting downward flux of oxygen as methane and oxygen fronts converge at the top of this zone. Observed sulfate gradients and microbial communities suggested that sulfate reduction and methanogenesis both contribute to hydrocarbon degradation in zone IV. Pyrosequencing revealed that Syntrophus- and Methanosaeta-related species dominate bacterial and archaeal communities, respectively, in the LNAPL body below the water table. Observed phylotypes were linked with in situ anaerobic hydrocarbon degradation in LNAPL-impacted soils.

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Microbially-mediated hydrocarbon degradation is well documented. However, how these microbial processes occur in complex subsurface petroleum impacted systems remains unclear, and this knowledge is needed to guide technologies to enhance microbial degradation effectively. Analysis of RNA derived from soils impacted by petroleum liquids would allow for analysis of active microbial communities, and a deeper understanding of the dynamic biochemistry occurring during site remediation. However, RNA analysis in soils impacted with petroleum liquids is challenging due to: (A) RNA being inherently unstable, and (B) petroleum impacted soils containing problematic levels of polymerase chain reaction (PCR) inhibitors that must be removed to yield high-purity RNA for downstream analysis. A previously published soil wash pretreatment step and a commercially available DNA extraction kit protocol were combined and modified to be able to purify RNA from soils containing petroleum liquids. A key modification involved reformulation of the pretreatment solution via replacing water as the diluent with a commercially-available RNA preservation solution. Methods were developed and demonstrated using cryogenically preserved soils from three former petroleum refineries. Results showed the new soil washing approach had no adverse effects on RNA recovery but did improve RNA quality, by PCR inhibitor removal, which in turn allows for characterization of active microbial communities present in petroleum impacted soils. In summary, our method for extracting RNA from petroleum-impacted soils provides a promising new tool for resolving metabolic processes at sites as they progress toward restoration via natural and/or engineered remediation.

The late stage of petroleum hydrocarbon releases to the subsurface is an evolving but largely unexplored concept. Herein, transmissive aquifer zones with little remaining petroleum liquids become attenuation zones for dissolved organic species released from low-permeability (low-k) zones via back diffusion. To address the knowledge gaps surrounding these subsurface zones, we explored a 40-year-old depleted petroleum body at a former refinery through high-resolution chemical and biomolecular analyses of a cryogenically collected soil core. 16S rRNA gene transcript-based analyses of active microbial communities uncovered predominately aerobic bacteria in the transmissive zone in contrast to anaerobic fermenting bacteria and methanogenic archaea in the low-k zone. Unexpectedly, Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS) analyses revealed a substantially higher degree of oxygenation in the petroleum biodegradation metabolites from the anoxic low-k zone compared to species in the oxic transmissive zone. Likely, a small diffusive influx of molecular oxygen enables limited aerobic metabolism in the low-k zone, while more abundant O 2 in the transmissive zone promotes rapid aerobic biodegradation of petroleum hydrocarbons without the accumulation of highly oxygenated intermediates. While much remains to be uncovered, our work is a critical first step toward enabling better-informed decision making regarding best management practices for late-stage petroleum hydrocarbonimpacted sites.