Tools for characterizing and monitoring natural source zone depletion
Date
2024
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Abstract
Although natural source zone depletion (NSZD) has gained acceptance by practitioners as a remediation technology for mid- to late-stage sites containing light non-aqueous phase liquids (LNAPL), challenges remain for broader regulatory adoption of NSZD as the sole remedy. Adoption of NSZD as a remedy requires verifying that it is occurring. NSZD can be an efficient and cost-effective solution for LNAPL zones, but acceptance of this bioremediation technology relies on a multiple-lines-of-evidence approach that requires a solid understanding of baseline conditions and effective monitoring. Emerging use of in situ oxidation-reduction potential (ORP) sensors shows promise to resolve spatial and temporal redox dynamics during NSZD processes. Further, next generation sequencing (NGS) of present and active microbial communities can provide insights regarding subsurface biogeochemistry, associated elemental cycling utilized in electron transport (e.g., N, Mn, Fe, S), and the potential for biodegradation. 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 biodegradation effectively. Analysis of RNA derived from soils impacted by petroleum liquids allows 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: 1) RNA being inherently unstable, and 2) petroleum impacted soils containing problematic levels of polymerase chain reaction (PCR) inhibitors (e.g., aqueous phase metals and humic acids) that must be removed to yield high-purity RNA for downstream analysis. Herein, a new RNA purification method that allows for extracting RNA from petroleum impacted soils was developed and successfully implemented to discriminate between active (RNA) and present (DNA) microbes in soils containing LNAPL. A key modification involved reformulation of the sample pretreatment solution by replacing water as the diluent with a commercially available RNA preservation solution consisting of LifeGuard™ (Qiagen) 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 removing PCR inhibitors, which in turn allows for characterization of active microbial communities present in petroleum impacted soils. To optimally employ NSZD and enhanced NSZD (ENSZD) at sites impacted by LNAPL, monitoring strategies are required. Emerging use of subsurface Soil redox sensors shows promise for tracking redox evolution, which reflects ongoing biogeochemical processes. However, further understanding of how soil redox dynamics relate to subsurface microbial activity and LNAPL biodegradation pathways is needed. In this work, soil redox sensors and DNA and RNA sequencing-based microbiome analysis were combined to elucidate NSZD and ENSZD (biostimulation via periodic sulfate addition and air sparging) processes in columns containing LNAPL impacted soils from a former petroleum refinery. Herein, microbial activity was directly correlated to continuous soil-ORP readings. Results show expected relationships between continuous soil redox and active microbial communities. Continuous data revealed spatial and temporal detail that informed interpretation of the hydrocarbon biodegradation data. Redox increases were transient for sulfate addition, and DNA and RNA sequencing revealed how hydrocarbon concentration and composition impacted microbiome structure and naphthalene biodegradation. When alkanes were present, naphthalene degradation was not observed, likely because naphthalene degraders were outcompeted. Further, the results of the sulfate addition experiment indicated a direct correlation of Desulfovibrio spp. with naphthalene biodegradation and showed that Smithella spp. were enriched in sulfate enhanced soils containing alkanes. Periodic air sparging did not result in fully aerobic conditions suggesting observed increased rates of biodegradation could be explained by stimulating alternative anaerobic metabolisms that were more energetically favorable compared to baseline/control conditions (e.g., iron reduction due to air oxidizing reduced iron). Methods developed and emerging continuous monitoring tools that were tested in lab soil columns were also applied to a mid- and late-stage LNAPL site. Herein, a case study is presented that advances integration of multiple nascent technologies for characterizing mid- and late-stage LNAPL sites including: 1) cryogenic coring, 2) multiple level internet of things (IoT) soil redox and temperature sensors in soil, and 3) application of RNA- and DNA-based molecular biological tools (MBTs) for site characterization. The integration of the data sets produced by these tools allowed for progress of NSZD to be evaluated in parallel under LNAPL site-relevant biogeochemical conditions. Collectively, the research presented in this dissertation support combining cryogenic coring sampling, continuous redox and temperature sensing and microbiome analysis to provide insights beyond those possible with each monitoring tool alone. The synergy achieved between microbiome characterization and soil continuous sensing illustrates how the integration of new characterization tools can provide insight into complex biogeochemical systems. Further understanding of these technologies will lead to improved predictions on remediation outcomes. The modern tools tested for middle- and late-stage LNAPL sites offer opportunities to more effectively and efficiently manage legacy LNAPL sites.
Description
Rights Access
Embargo expires: 05/20/2026.
Subject
bioremediation
microbiome
natural source zone depletion
LNAPL
biogeochemistry
molecular biology