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Browsing by Author "Irianni Renno, Maria, author"

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    Biogeochemical characterization of a LNAPL body in support of STELA
    (Colorado State University. Libraries, 2013) Irianni Renno, Maria, author; De Long, Susan K., advisor; Sale, Thomas C., advisor; Borch, Thomas, committee member; Payne, Fred, committee member
    Microbially-mediated depletion of light non-aqueous phase liquids (LNAPL) has gained regulatory acceptance as a method for managing impacted sites. However, the fundamental microbiology of anaerobic hydrocarbon degradation, in source zones, remains poorly understood. Two site-specific studies (Zeman, 2012 & McCoy, 2012) performed at the Center for Contaminant Hydrology (CCH), Colorado State University (CSU) demonstrated that LNAPL biodegradation increases drastically when temperatures are maintained between 18°C and 30°C as compared to lower or higher temperatures. These results have supported the design of a Sustainable Thermally Enhanced LNAPL Attenuation (STELA) technology that is currently being tested at field scale at a former refinery in Wyoming. The focus of the present study was to perform a depth-resolved characterization of the mixed microbial communities present in LNAPL-impacted soils, as well as to characterize the site's geochemical parameters in order to establish a baseline data set to evaluate the STELA system performance. Seventeen soil cores were collected from the impacted site, frozen on dry ice and subsampled at 6-inch intervals for analysis of biogeochemical parameters. Multi-level sampling systems were installed at the core sites to monitor aqueous and gas phases. Diesel and gasoline range organics and benzene, toluene, ethylbenzene and xylenes (BTEX) present in the cores and in water samples were analyzed. Temperature, inorganic dissolved ions, pH, and oxidation reduction potential (ORP) were also measured. DNA was extracted in triplicate from each subsample corresponding to the study's center core (21 samples). Total Eubacteria and Archaea were quantified via 16S rRNA gene-targeted qPCR. Microorganisms present at selected depth intervals were identified via 454 pyrosequencing of both eubacterial and archaeal 16S rRNA genes. Results indicate that at the study site, the majority of the hydrocarbon contamination is found between 5 and 12 feet below ground surface (bgs). The average of the maximum total petroleum hydrocarbon (TPH) soil concentrations within each core was 17,800 mg/kg with a standard deviation of 8,280 mg/kg. The presence of methane in the vadose zone and depleted sulfate concentrations in water samples suggest that both methanogenesis and sulfate reduction are likely driving LNAPL depletion processes. Four distinct biogeochemical zones where identified within the surveyed aquifer region. Interestingly, the quantity of eubacterial 16S rRNA genes dominate the quantity of archaeal 16S rRNA genes at sampled depths within the aerobic aquifer region. In the strictly anaerobic aquifer regions, these quantities are approximately equal. The latter can be interpreted as evidence of syntrophism, which has been reported in other hydrocarbon biodegradation studies. Pyrosequencing results support these findings as well and contribute to further elucidating the spatial correlation between microbial communities and geochemical parameters. In-situ biodegradation rates are largely controlled by the quantity and activity of key microbes capable of mediating conversion of specific hydrocarbon constituents. Furthermore, it is anticipated that biodegradation rates are governed by complex interactions of diverse microbial communities that vary both in space and time. The overall vision of this initiative is that advancing a better understanding of processes controlling biologically mediated losses of LNAPL will support the development of more efficient treatment technologies for LNAPL releases. In particular, the site specific analysis produced through this study will support the development of STELA.
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    Tools for characterizing and monitoring natural source zone depletion
    (Colorado State University. Libraries, 2024) Irianni Renno, Maria, author; De Long, Susan K., advisor; Sale, Thomas C., advisor; Key, Trent A., committee member; Scalia, Joseph, committee member; Stromberger, Mary, committee member
    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.