Browsing by Author "De Long, Susan K., advisor"
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Item Open Access Anaerobic digestion of organic wastes: the impact of operating conditions on hydrolysis efficiency and microbial community composition(Colorado State University. Libraries, 2012) Griffin, Laura Paige, author; De Long, Susan K., advisor; Sharvelle, Sybil, committee member; Stromberger, Mary, committee memberAnaerobic digestion (AD) is an environmentally sustainable technology to manage organic waste (e.g., food, yard, agricultural, industrial wastes). Economic profitability, however, remains a key barrier to widespread implementation of AD for the conversion of specific feedstocks (e.g., manure, the organic fraction of municipal solid waste (OFMSW), and agricultural residue) to energy. Specifically, high capital and operating costs and reactor instability have continually deterred the use of AD. In order to develop AD systems that are highly efficient and more cost-effective, it is necessary to optimize the microbial activity that mediates the digestion process. Multi-stage AD systems are promising technologies because they allow for separate process optimization of each stage and can enable processing of high-solids content waste. Leachate is recycled through the system, which reduces heating and pumping costs, as well as conserving water. The leachate recycle, however, leads to an increase in ammonia and salinity concentrations. At this time, the impact of reactor conditions (ammonia and salinity concentrations) on hydrolysis is not well understood. As hydrolysis is one rate-limiting step of the process in the conversion of refractory wastes (e.g., lignocellulosic materials), optimization of hydrolysis has the potential to radically improve the economic profitability of AD. The specific objectives of this research were to: 1) determine the effects of operating conditions on hydrolysis efficiency for a variety of solid wastes (manure, food waste, and agricultural residue); 2) determine hydrolysis kinetic parameters as a function of the operating conditions; and 3) identify characteristics of microbial communities that perform well under elevated ammonia and salinity concentrations. To this end, small-scale batch reactors were used to determine hydrolysis efficiency and kinetic rates. Initially, the AD sludge inoculum was exposed directly to the high ammonia and salinity concentrations (1, 2.5, 5 g Total Ammonia Nitrogen (TAN)/L and 3.9, 7.9, 11.8 g sodium/L) as would occur in a reactor treating organic waste with leachate recycle. Results demonstrated a need to acclimate, or adapt, the microorganisms to high concentrations, as methane generation was significantly inhibited with high concentrations. Thus, the organisms were acclimated for two to four months to these testing conditions. The batch studies were repeated, and results demonstrated substantial improvement in hydrolysis efficiency and methane generation. However, although differences in kinetic rates were not statistically significant, general trends in hydrolysis rates suggested that hydrolysis efficiency decreases with increased ammonia and salinity concentrations for a variety of feedstocks (i.e., manure, food waste, agricultural residue). Additionally, results demonstrated that acclimation was necessary to achieve optimal hydrolysis rates. Furthermore, microbial community composition changes in the inocula post-acclimation indicated that reactor inoculation could help improve tolerance to elevated levels of ammonia and salinity to minimize reactor start-up times and improve economic viability.Item Open Access 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 memberMicrobially-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.Item Open Access Biotic control of LNAPL longevity - laboratory and field- scale studies(Colorado State University. Libraries, 2017) Emerson, Eric Douglas, author; De Long, Susan K., advisor; Sale, Thomas, advisor; Butters, Gregory, committee memberNatural source zone depletion (NSZD) is an emerging strategy for managing light nonaqueous phase liquids (LNAPLs). Unfortunately, little is known about NSZD rates over extended periods of time, where heterogeneous redox conditions and changing LNAPL saturations may influence processes governing losses. Understanding long-term rates is central to anticipating LNAPL longevity under both natural and engineered conditions. Herein, laboratory and field-scale modeling studies were conducted to evaluate LNAPL longevity. Laboratory studies evaluated loss rates as a function of total contaminant concentration under sulfate-reducing (SR) and methanogenic (MG) conditions. Biotic and abiotic loss rates were determined via tracking biodegradation products and hydrocarbons in column effluents and produced gasses over time. Furthermore, compositional weathering of LNAPL was evaluated. Loss rates with elevated sulfate averaged 39.8 mmole carbon/day/m3 (±9.1 mmole carbon/day/m3). Once sulfate in the soil was depleted to influent water sulfate concentrations of 20 mg/L, subsequent average loss rates were 39.7 mmole carbon/day/m3 (±19.6 mmole carbon/day/m3). Overall, loss rates with and without elevated sulfate were similar. Furthermore, results suggested that loss rates are independent of LNAPL concentration over the range of 9,000 to 37,000 mg/kg and redox conditions observed. Loss rates independent of LNAPL concentrations indicated that biologically mediated NSZD follows zero-order kinetics over the range of conditions evaluated. Column loss rates were compared to field-measured loss rates assuming an LNAPL thickness of three meters. Given this assumption, mean observed early- and late-loss rates are 1.38 and 1.41 μmole carbon/m2/sec, respectively. Assuming decane as a representative LNAPL, observed loss rates are equivalent to 7890 and 8060 L/hectare/year. A column was sacrificed at the completion of the study. Predicted mass losses of the study equate to approximately 1% total initial LNAPL mass lost. Total petroleum hydrocarbons (TPH) soil analysis of initial and final grab samples of column soil did not detect significant mass losses. Moreover, no significant shifts in the LNAPL composition were seen during the course of the study. Mass losses in this range are difficult to accurately quantify via soil-phase hydrocarbon analyses, thus highlighting the utility of the approach used herein. An LNAPL longevity model (The Glide Path Model) was applied at a field site using a zero-order rate model for biological NSZD. LNAPL Longevity ranged from 35 to 105 years using a mean NSZD rate, plus or minus factors of 2 and ½, respectively. Active recovery was shown to have little effect on the longevity of LNAPL.Item Open Access Determining the effect of primer mismatches on quantitative PCR accuracy and developing guidance for design of primers with sequence variations(Colorado State University. Libraries, 2012) Ledeker, Brett Michael, author; De Long, Susan K., advisor; Omur-Ozbek, Pinar, committee member; Reardon, Kenneth F., committee memberAlthough quantitative PCR (qPCR) is a powerful tool for investigating environmental systems, target gene sequences for organisms of interest often are not well known, which has resulted in few reliable primers for many applications. Additionally, the sequences of target genes found in diverse strains often contain sequence variations, and therefore, primer sets containing single or multiple primer-template mismatches are common. However, the detrimental impact of these mismatches on quantification accuracy and amplification efficiency has not been investigated thoroughly. Thus, the research objectives of this study were to elucidate the relationships between primer mismatches and the accuracy of qPCR assays and to develop guidance for designing primers targeting genes displaying sequence variations. The pcrA gene (encoding perchlorate reductase) from Dechloromonas agitata was used as a model system for this study, and a linearized plasmid containing the cloned pcrA gene was used as the qPCR template. A large number of pcrA primers (16 forward and 16 reverse) were designed containing from zero to three mismatches at various locations. Combinations of primers were tested to determine the impact of mismatches on the amplification efficiency, the threshold cycle (CT), and the quantification accuracy. Quantification accuracy was calculated as the percent detected by dividing the quantity measured with mismatch primers by the quantity measured with perfect match primers and multiplying by 100. Single mismatches at the 3' end resulted in quantification accuracies as low as ~3%, and single mismatches at the 5' end resulted in quantification accuracies as low as ~33%. Double and triple mismatches at the 5' resulted in quantification accuracies as low as ~17% and ~2%, respectively. Reductions in quantification accuracy correlated with increases in CT induced by mismatches but not with changes in amplification efficiency. Combining mismatched forward and reverse primers had an impact equivalent to the combined effect of the individual mismatch primers. Analogous qPCR tests were run with three other model genes: celS (encoding family 48 cellulase), C23O (encoding catechol dioxygenase, involved in toluene degradation), and hydA (encoding periplasmic hydrogenase, involved in fermentation). Primers were artificially designed to contain mismatches with these target genes, and results demonstrated that single or double mismatches can have a substantial detrimental impact on quantification accuracy in a broad range of systems. The results of this study indicate that caution must be taken to avoid mismatches when designing qPCR primers targeting genes with sequence variations and the findings serve to guide future design of primers for accurately quantifying genes in environmentally relevant systems.Item Open Access Development of advanced microbial communities for enhancing waste hydrolysis processes: insights from the application of molecular biology tools(Colorado State University. Libraries, 2016) Wilson, Laura Paige, author; De Long, Susan K., advisor; Sharvelle, Sybil, committee member; Bareither, Christopher, committee member; Weir, Tiffany, committee memberAnaerobic digestion (AD) is an environmentally attractive technology for conversion of various solid wastes to energy. However, despite numerous benefits, AD applications to OFMSW remain limited in North America due to economic barriers with existing technologies. Suboptimal conditions in anaerobic digesters (e.g., presence of common inhibitors ammonia and salinity) limit waste hydrolysis in AD and lead to unstable performance and process failures compromising economic viability. To guide development of microbial management strategies to avoid process upsets and failures due to inhibitors, hydrolysis rates were determined in batch, single-stage digesters seeded with unacclimated or acclimated inocula under a range of ammonia and salinity concentrations for two model feedstocks (food waste and manure). Using unacclimated inocula, hydrolysis was found to be severely inhibited for elevated ammonia (decrease of nearly 4-fold relative to baseline) and salinity (decrease of up to 10-fold relative to baseline). However, for inocula acclimated over 2 to 4 months, statistically significant inhibition was not detectable except in the case of food waste subjected to elevated ammonia concentrations (p-value = 0.01). Inhibitors and feedstock were found to have a major influence on bacterial community structure. Next, a more detailed analysis of the acclimation process revealed that microbial communities under stressed conditions (elevated ammonia) adapt more slowly (weeks) to feedstock changes (from wastewater sludge to manure or filter paper) than under non-stressed conditions (days). Molecular tools were utilized to separate temporal effects on hydrolyzers from temporal effects on methanogens. Bacterial and archaeal sequencing results identified multiple organisms (e.g., Clostridiales vadinBB60, Ruminococcaceae, Marinilabiaceae, Methanobacterium, and Thermoplasmatales Incertae Sedis) that were selected for in microbial communities in stressed reactors under perturbed conditions (feedstock changes). Collectively, results from these studies suggested that weeks of acclimation are required to build up sufficient quantities of desired hydrolyzing microbes; thus, hydrolysis processes operated in batch mode should be inoculated with each new batch, and desired microorganisms should be maintained in the system via properly developed inoculation strategies. To identify improved methods of maintaining such communities in multi-stage reactor systems, reactor performance under elevated ammonia and salinity was compared for leach bed reactors (LBRs) seeded with unacclimated inoculum and different ratios of acclimated inoculum (0-60% by mass) at start-up. Additionally, the effect of seeding methods was examined by identifying the optimal ratio of fresh waste to previously digested waste in multi-stage systems incorporating leachate recycle during long-term operation. Results demonstrated that high quantities of inoculum (~60%) increase waste hydrolysis and are beneficial at start-up or when inhibitors are increasing. After start-up (~112 days) with high inoculum quantities, leachate recirculation leads to accumulation of inhibitor-tolerant hydrolyzing bacteria in leachate. During long-term operation, low inoculum quantities (~10%) effectively increase waste hydrolysis relative to without solids-derived inoculum. Additionally, molecular analyses indicated that combining digested solids with leachate-based inoculum doubles quantities of Bacteria contacting waste over a batch and supplies additional desirable phylotypes Bacteriodes and Clostridia. To provide detailed insight into microbial community activity during degradation, metatranscriptomic analyses were conducted on reactors fed food waste and manure under low ammonia, and several common active (e.g., Methanomicrobia, Methanosaeta concilii, and Clostridia) and unique active (e.g., Enterobacteriaceae, Clostridium thermocellum, and Clostridium celluloyticum) phylotypes between the reactors were identified. Functional classification of the active microbial communities generally revealed several similarities between the reactors despite the differences in feedstock. However, similarities or differences in transcript abundance for specific gene categories (e.g. one-carbon metabolism or fermentation) might indicate some potentially useful biomarkers for monitoring process health. Additionally, data from this experiment expanded the gene sequence database for assay development, which is particularly key for improving current functional gene-targeted assays to more accurately characterize microbial communities. Overall, results from this study have provided operational guidance for establishing and maintaining desired microbial communities as inocula to enhance waste hydrolysis for a variety of feedstocks.Item Open Access The innovative application of random packing material to enhance the hydraulic disinfection efficiency of small scale water systems(Colorado State University. Libraries, 2021) Baker, Jessica L., author; Venayagamoorthy, Subhas Karan, advisor; De Long, Susan K., advisor; Niemann, Jeffrey D., committee member; Leisz, Stephen J., committee memberIn a world where the quality of our water supplies is declining and our infrastructure is deteriorating, let alone the lack of available water in arid regions, the treatment of drinking water is becoming ever more challenging – especially for small scale systems that lack technical and financial support. The innovative application of random packing material (RPM) has been proposed as a possible tool to aid small water treatment systems (SWTSs) improve their disinfection contact systems in order to meet the Safe Drinking Water Act (SDWA) standards and provide the communities they serve with safe drinking water. While it has been demonstrated at the laboratory–scale that RPM can significantly improve the hydraulic disinfection efficiency of a contact basin in terms of baffling factor (BF) there was a lack of fundamental understanding of why RPM is so effective. Conceptually, the RPM slows and spreads the jet flow from a sharp inlet. Yet the mechanics of a jet flow through a highly porous material such as RPM is not well understood. Insight into the dynamics of such a flow is important in order to be able to use RPM in a manner that maximizes the benefits and minimizes the (unintended) drawbacks. The main aim of this dissertation is to use laboratory-scale experiments to study the mechanics of a turbulent jet flow from a long pipe through RPM and the impact on the hydraulic disinfection efficiency and final water quality for a disinfection contactor. There are three main objectives in this work: (1) To gain fundamental insights regarding turbulent jet flow through a highly porous media (such as RPM); (2) To address practical concerns for the application of the use of RPM in disinfection contactors; and (3) To provide guidance in terms of best practice for the innovative use of RPM to enhance hydraulic disinfection efficiency in SWTSs. The first part of this dissertation focuses on the resulting flow fields of a turbulent jet flow (5-20 gpm) through a wall of RPM of various thicknesses (L). An experiment was conducted in a flume using a Particle Image Velocimetry (PIV) system to map the flow fields downstream of the jet up to x⁄dj ≈ 30 (where dj is the diameter of the jet, i.e. inlet pipe). Once the PIV data were verified using a Laser-Doppler Anemometry (LDA) system and validated for a jet into an ambient (provided as a baseline), the velocity fields of the jet flow downstream of the walls of RPM were analyzed. A second order relationship was observed between the thickness of RPM and the spread of the flow. It was also observed that the jet velocities decay exponentially through RPM. With respect to flow rate, the spreading rate increased slightly, but there was a slight decrease in the decay of the jet as the flow rate increased. While the maximum velocities were reduced by over 90% after L ≈ 5dj, it was only after L ≈ 15dj that the flow downstream of the RPM was nearly uniform. Furthermore, the coefficients of drag showed a non-monotonic relationship with respect to the particle Reynolds number (Redp) that followed the well-established trend of a uniform flow around an infinitely long cylinder. This relationship provides valuable insight into the different regimes of the highly complex flow within and/or downstream of a highly porous material. Next, the potential improvement in the hydraulic disinfection efficiency and the possible energy loss as a result of the presence of random packing material in a laboratory-scale chlorine contactor were investigated. Tracer tests were conducted on a 55-gal drum tank filled with RPM in varying amounts in different configurations to measure the efficiency of each setup in terms of baffling factor. The bulk pressure drop was measured to determine the energy loss for each configuration. The results of this study show that securing RPM near the inlet, in any amount, improves the BF by 300% to more than 900%. The amount of RPM begins to have an impact at or above an inlet jet Reynolds number of 27,700. Also, changes in head loss due to the presence of RPM (in any amount, configuration, and/or flow rate) were generally considered to be negligible. Finally, a concern surrounding the potential for excessive biofilm growth is addressed through a long-term study. The inflow, outflow, and RPM were monitored for heterotrophic bacteria (via heterotrophic plate counts) and Pseudomonas aeruginosa as indicators of bacteriological water quality and the presence of biofilm. The results of this study show that there was no substantial biofilm growth in a lab-scale chlorine contactor and no substantial increase in bacterial counts for the bulk outflow over a 10-week period. Thus, the potential for excessive biofilm growth should not be considered a barrier concerning the use of RPM to improve the hydraulic disinfection efficiency of chlorine contactors in small drinking water treatment systems. Overall, this dissertation work aims to contribute a foundational understanding of turbulent jet flow through a highly porous material such as RPM as well as address some practical concerns for the innovative application of RPM to improve the hydraulic disinfection efficiency. From the results of the studies conducted, best practice guidelines have been developed to maximize the potential benefit of using RPM in disinfection contactors. Ultimately, the hope of this work is to promote the use of RPM to help SWTSs that are struggling to meet SDWA standards and to provide the communities they serve with safe drinking water.Item Open Access Thermally enhanced bioremediation of LNAPL(Colorado State University. Libraries, 2013) Zeman, Natalie Rae, author; De Long, Susan K., advisor; Sale, Thomas, advisor; Stromberger, Mary, committee memberInadvertent releases of petroleum liquids into the environment have led to widespread soil and groundwater contamination. Petroleum liquids, referred to as Light Non-Aqueous Phase Liquid (LNAPL), pose a threat to the environment and human health. The purpose of the research described herein was to evaluate thermally enhanced bioremediation as a sustainable remediation technology for rapid cleanup of LNAPL zones. Thermally enhanced LNAPL attenuation was investigated via a thermal microcosm study that considered six different temperatures: 4¡ÆC, 9¡ÆC, 22¡ÆC, 30¡ÆC, 35¡ÆC, and 40¡ÆC. Microcosms were run for a period of 188 days using soil, water and LNAPL from a decommissioned refinery in Evansville, WY. The soil microcosms simulated anaerobic subsurface conditions where sulfate reduction and methanogenesis were the pathways for biodegradation. To determine the optimal temperature range for thermal stimulation and provide guidance for design of field-scale application, both contaminant degradation and soil microbiology were monitored. CH4 and CO2 generation occurred in microcosms at 22¡ÆC, 30¡ÆC, 35¡ÆC and 40¡ÆC but was not observed at 4¡ÆC and 9¡ÆC. The total volume of biogas generated after 188 days of incubation was 19 times higher in microcosms at 22¡ÆC and 30¡ÆC compared to the microcosms at 35¡ÆC. When compared to microcosms at 40¡ÆC, the total biogas generated was 3 times higher at both 22¡ÆC and 30¡ÆC. The onset of CH4 and CO2 production occurred first within the microcosms held at 30¡ÆC beginning after 28 days of incubation, and second within microcosms at 22¡ÆC beginning after 58 days of incubation. A delay in CH4 and CO2 production was observed within microcosms held at 35¡ÆC and 40¡ÆC (beginning after 173 and 138 days after incubation) contributing to lower cumulative biogas generation at these temperatures relative to 22¨¬C and 30¨¬C. Microcosms incubated at 4¨¬C, 22¡ÆC, 30¨¬C, 35 ¨¬C and 40 ¨¬C showed statistically significant biological removal of gasoline range organics (GRO) at the ¥á=0.05 level over the course of the 188-day incubation period. Biotic removal of GRO was significantly higher at 22¨¬C and 30¨¬C when compared to 4¨¬C and 9¨¬C. The observed removals at 22 ¨¬C, 30 ¨¬C, 35 ¨¬C and 40¨¬C were not statistically different from each other. The biological removal of DRO compounds was found to be statistically significant at 22¨¬C, 30 ¨¬C, 35 ¨¬C and 40 ¨¬C and was significantly higher at 22¨¬C when compared to 4¨¬C and 9¨¬C. The percent biological removal of DRO compounds at these temperatures was not statistically different from each other and ranged from 18-22%. Statistically significant biological degradation of all the BTEX compounds only occurred in microcosms at 22¡ÆC and 30¡ÆC. Benzene, toluene, and xylene biodegradation was observed to be statistically significant in microcosms at 35¡ÆC. Phylogenetic analysis of the microbial communities present at 22¡ÆC via a 16S rRNA gene archaeal clone library and pyrosequencing revealed the presence of both hydrogenotrophic and acetoclastic methanogens. Pyrosequencing data from microcosms at 4¡ÆC, 22¡ÆC and 35¡ÆC revealed that temperature impacted both archaeal and bacterial community structure. A potential requirement for changes in microbial community structure at the higher temperatures (35¡ÆC and 40¡ÆC) could explain the observed delay in biogas production. The results of the thermal microcosm study led to the development of a pilot study to investigate the efficacy of Sustainable Thermally Enhanced LNAPL Attenuation STELA at the site in Evansville, WY. The thermal pilot study approach and preliminary data is included in this thesis. The outcome and results of the pilot study will be presented in the masters theses of other students.Item Embargo 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 memberAlthough 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.