Advanced methods for RNA recovery from petroleum impacted soils

Abstract

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.

Discovery of Oxygenated Hydrocarbon Biodegradation Products at a Late-Stage Petroleum Release Site

“…Samples from two intervals were selected for detailed differential characterization based on representing two distinct hydrogeologic zones and the presence of a substantial TPH concentration (Figure ). RNA was extracted from each soil sample with a Powerlyser Powersoil DNA Isolation Kit (MoBio, Carlsbad, CA) generally according to the manufacturer’s instructions but with a modified protocol that preserves intact RNA and removes molecules that interfere with downstream analysis of extracted RNA (i.e., polymerase chain reaction inhibitors) while maximizing yield. , RNA concentrations were quantified via optical density with a NanoDrop 2000 UV–vis spectrophotometer (Thermoscientific). RNA was stored at −80 °C prior to it being reverse transcribed into complementary DNA (cDNA) for downstream analysis.…”

“…First, we collected a cryogenic soil core for subsequent high-resolution stratigraphic and chemical characterization. In contrast to traditional soil coring methods, such as direct push or hollow-stem auger drilling, cryogenic coring with a liquid nitrogen cooling system improves core recovery and preserves critical soil attributes such as pore fluid distribution , and ribonucleic acid content . Second, we sought to define the prevailing redox conditions in the low-k and transmissive zones governing hydrocarbon biodegradation through determining the active microbial community composition via 16S rRNA gene transcript analysis as opposed to traditional DNA-based microbial community profiling.…”

“…In contrast to traditional soil coring methods, such as direct push or hollow-stem auger drilling, cryogenic coring with a liquid nitrogen cooling system improves core recovery and preserves critical soil attributes such as pore fluid distribution 22,23 and ribonucleic acid content. 24 Second, we sought to define the prevailing redox conditions in the low-k and transmissive zones governing hydrocarbon biodegradation through determining the active microbial community composition via 16S rRNA gene transcript analysis as opposed to traditional DNA-based microbial community profiling. Third, we applied Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS) to highlight compositional differences in acidic and aromatic species between soil samples originating from transmissive and low-k zones.…”