Subsurface characterization of groundwater contaminated by landfill leachate using microbial community profile data and a nonparametric decision-making process

Microbial biodiversity in groundwater and soil presents a unique opportunity for improving characterization and monitoring at sites with multiple contaminants, yet few computational methods use or incorporate these data because of their high dimensionality and variability. We present a systematic, nonparametric decision-making methodology to help characterize a water quality gradient in leachate-contaminated groundwater using only microbiological data for input. The data-driven methodology is based on clustering a set of molecular genetic-based microbial community profiles. Microbes were sampled from groundwater monitoring wells located within and around an aquifer contaminated with landfill leachate. We modified a self-organizing map (SOM) to weight the input variables by their relative importance and provide statistical guidance for classifying sample similarities. The methodology includes the following steps: (1) preprocessing the microbial data into a smaller number of independent variables using principal component analysis, (2) clustering the resulting principal component (PC) scores using a modified SOM capable of weighting the input PC scores by the percent variance explained by each score, and (3) using a nonparametric statistic to guide selection of appropriate groupings for management purposes. In this landfill leachate application, the weighted SOM assembles the microbial community data from monitoring wells into groupings believed to represent a gradient of site contamination that could aid in characterization and long-term monitoring decisions. Groupings based solely on microbial classifications are consistent with classifications of water quality from hydrochemical information. These microbial community profile data and improved decision-making strategy compliment traditional chemical groundwater analyses for delineating spatial zones of groundwater contamination. © 2011 by the American Geophysical Union

Temporal dynamics of halogenated organic compounds in Marcellus Shale flowback

The chemistry of hydraulic fracturing fluids and wastewaters is complex and is known to vary by operator, geologic formation, and fluid age. A time series of hydraulic fracturing fluids, flowback fluids, and produced waters was collected from two adjacent Marcellus Shale gas wells for organic chemical composition analyses using ultrahigh resolution mass spectrometry. Hierarchical clustering was used to compare and extract ions related to different fluid ages and many halogenated organic molecular ions were identified in flowback fluids and early produced waters based on exact mass. Iodinated organic compounds were the dominant halogen class in these clusters and were nearly undetectable in hydraulic fracturing fluid prior to injection. The iodinated ions increased in flowback and remained elevated after ten months of well production. We suggest that these trends are mainly driven by dissolved organic matter reacting with reactive halogen species formed abiotically through oxidizing chemical additives applied to the well and biotically via iodide-oxidizing bacteria. Understanding the implications of these identified halogenated organic compounds will require future investigation in to their structures and environmental fate

Discrete organic phosphorus signatures are evident in pollutant sources within a Lake Erie tributary

Author Posting. © The Author(s), 2018. This is the author's version of the work. It is posted here under a nonexclusive, irrevocable, paid-up, worldwide license granted to WHOI. It is made available for personal use, not for redistribution. The definitive version was published in Environmental Science and Technology 52 (2018): 6771-6779, doi:10.1021/acs.est.7b05703.Phosphorus loads are strongly associated with the severity of harmful algal blooms in Lake Erie, a Great Lake situated between the United States and Canada. Inorganic and total phosphorus measurements have historically been used to estimate nonpoint and point source contributions, from contributing watersheds with organic phosphorus often neglected. Here, we used ultrahigh resolution mass spectrometry to characterize the dissolved organic matter and specifically dissolved organic phosphorus composition of several nutrient pollutant source materials and aqueous samples in a Lake Erie tributary. We detected between 23-313 organic phosphorus formulae across our samples, with manure samples having greater abundance of phosphorus- and nitrogen containing compounds compared to other samples. Manures also were enriched in lipids and protein-like compounds. The greatest similarities were observed between the Sandusky River and wastewater treatment plant effluent (WWTP), or the Sandusky River and agricultural edge of field samples. These sample pairs shared 84% of organic compounds and 59 to 73% of P-containing organic compounds, respectively. This similarity suggests that agricultural and/or WWTP sources dominate the supply of organic phosphorus compounds to the river. We identify formulae shared between the river and pollutant sources that could serve as possible markers of source contamination in the tributary.This research was supported by an Ohio State University Field to Faucet Institute award to P.J.M and by a Harmful Algal Bloom Research Initiative grant from the Ohio Department of Higher Education

Modified Lipid Extraction Methods for Deep Subsurface Shale

Growing interest in the utilization of black shales for hydrocarbon development and environmental applications has spurred investigations of microbial functional diversity in the deep subsurface shale ecosystem. Lipid biomarker analyses including phospholipid fatty acids (PLFAs) and diglyceride fatty acids (DGFAs) represent sensitive tools for estimating biomass and characterizing the diversity of microbial communities. However, complex shale matrix properties create immense challenges for microbial lipid extraction procedures. Here, we test three different lipid extraction methods: modified Bligh and Dyer (mBD), Folch (FOL), and microwave assisted extraction (MAE), to examine their ability in the recovery and reproducibility of lipid biomarkers in deeply buried shales. The lipid biomarkers were analyzed as fatty acid methyl esters (FAMEs) with the GC-MS, and the average PL-FAME yield ranged from 67 to 400 pmol/g, while the average DG-FAME yield ranged from 600 to 3,000 pmol/g. The biomarker yields in the intact phospholipid Bligh and Dyer treatment (mBD + Phos + POPC), the Folch, the Bligh and Dyer citrate buffer (mBD-Cit), and the MAE treatments were all relatively higher and statistically similar compared to the other extraction treatments for both PLFAs and DGFAs. The biomarker yields were however highly variable within replicates for most extraction treatments, although the mBD + Phos + POPC treatment had relatively better reproducibility in the consistent fatty acid profiles. This variability across treatments which is associated with the highly complex nature of deeply buried shale matrix, further necessitates customized methodological developments for the improvement of lipid biomarker recovery

The fate of SARS-CoV-2 viral RNA in coastal New England wastewater treatment plants

Municipal sewage carries SARS-CoV-2 viruses shed in the human stool by infected individuals to wastewater treatment plants (WWTPs). It is well-established that increasing prevalence of COVID-19 in a community increases the viral load in its WWTPs. Despite the fact that wastewater treatment facilities serve a critical role in protecting downstream human and environmental health through removal or inactivation of the virus, little is known about the fate of the virus along the treatment train. To assess the efficacy of differing WWTP size and treatment processes in viral RNA removal we quantified two SARS-CoV-2 nucleocapsid (N) biomarkers (N1 and N2) in both liquid and solids phases for multiple treatment train locations from seven coastal New England WWTPs. SARS-CoV-2 biomarkers were commonly detected in the influent, primary treated, and sludge samples (returned activated sludge, waste activated sludge, and digested sludge), and not detected after secondary clarification processes or disinfection. Solid fractions had 470 to 3,700-fold higher concentrations of viral biomarkers than liquid fractions, suggesting considerably higher affinity of the virus for the solid phase. Our findings indicate that a variety of wastewater treatment designs are efficient at achieving high removal of SARS CoV-2 from effluent; however, quantifiable viral RNA was commonly detected in wastewater solids at various points in the facility. This study supports the important role municipal wastewater treatment facilities serve in reducing the discharge of SARS-CoV-2 viral fragments to the environment and highlights the need to better understand the fate of this virus in wastewater solids

Enhanced detection of groundwater contamination from a leaking waste disposal site by microbial community profiles

Groundwater biogeochemistry is adversely impacted when municipal solid waste leachate, rich in nutrients and anthropogenic compounds, percolates into the subsurface from leaking landfills. Detecting leachate contamination using statistical techniques is challenging because well strategies or analytical techniques may be insufficient for detecting low levels of groundwater contamination. We sampled profiles of the microbial community from monitoring wells surrounding a leaking landfill using terminal restriction fragment length polymorphism (T-RFLP) targeting the 16S rRNA gene. Results show in situ monitoring of bacteria, archaea, and the family Geobacteraceae improves characterization of groundwater quality. Bacterial T-RFLP profiles showed shifts correlated to known gradients of leachate and effectively detected changes along plume fringes that were not detected using hydrochemical data. Experimental sediment microcosms exposed to leachate-contaminated groundwater revealed a shift from a -Proteobacteria and Actinobacteria dominated community to one dominated by Firmicutes and δ-Proteobacteria. This shift is consistent with the transition from oxic conditions to an anoxic, iron-reducing environment as a result of landfill leachate-derived contaminants and associated redox conditions. We suggest microbial communities are more sensitive than hydrochemistry data for characterizing low levels of groundwater contamination and thus provide a novel source of information for optimizing detection and long-term monitoring strategies at landfill sites. Copyright 2010 by the American Geophysical Union

Members of Marinobacter and Arcobacter Influence System Biogeochemistry During Early Production of Hydraulically Fractured Natural Gas Wells in the Appalachian Basin

Hydraulic fracturing is the prevailing method for enhancing recovery of hydrocarbon resources from unconventional shale formations, yet little is understood regarding the microbial impact on biogeochemical cycling in natural-gas wells. Although the metabolisms of certain fermentative bacteria and methanogenic archaea that dominate in later produced fluids have been well studied, few details have been reported on microorganisms prevelant during the early flowback period, when oxygen and other surface-derived oxyanions and nutrients become depleted. Here, we report the isolation, genomic and phenotypic characterization of Marinobacter and Arcobacter bacterial species from natural-gas wells in the Utica-Point Pleasant and Marcellus Formations coupled to supporting geochemical and metagenomic analyses of produced fluid samples. These unconventional hydrocarbon system-derived Marinobacter sp. are capable of utilizing a diversity of organic carbon sources including aliphatic and aromatic hydrocarbons, amino acids, and carboxylic acids. Marinobacter and Arcobacter can metabolize organic nitrogen sources and have the capacity for denitrification and dissimilatory nitrate reduction to ammonia (DNRA) respectively; with DNRA and ammonification processes partially explaining high concentrations of ammonia measured in produced fluids. Arcobacter is capable of chemosynthetic sulfur oxidation, which could fuel metabolic processes for other heterotrophic, fermentative, or sulfate-reducing community members. Our analysis revealed mechanisms for growth of these taxa across a broad range of salinities (up to 15% salt), which explains their enrichment during early natural-gas production. These results demonstrate the prevalence of Marinobacter and Arcobacter during a key maturation phase of hydraulically fractured natural-gas wells, and highlight the significant role these genera play in biogeochemical cycling for this economically important energy system

Genome Sequence of the Deltaproteobacterial Strain NaphS2 and Analysis of Differential Gene Expression during Anaerobic Growth on Naphthalene

Anaerobic polycyclic hydrocarbon (PAH) degradation coupled to sulfate reduction may be an important mechanism for in situ remediation of contaminated sediments. Steps involved in the anaerobic degradation of 2-methylnaphthalene have been described in the sulfate reducing strains NaphS3, NaphS6 and N47. Evidence from N47 suggests that naphthalene degradation involves 2-methylnaphthalene as an intermediate, whereas evidence in NaphS2, NaphS3 and NaphS6 suggests a mechanism for naphthalene degradation that does not involve 2-methylnaphthalene. To further characterize pathways involved in naphthalene degradation in NaphS2, the draft genome was sequenced, and gene and protein expression examined.Draft genome sequencing, gene expression analysis, and proteomic analysis revealed that NaphS2 degrades naphthoyl-CoA in a manner analogous to benzoyl-CoA degradation. Genes including the previously characterized NmsA, thought to encode an enzyme necessary for 2-methylnaphthalene metabolism, were not upregulated during growth of NaphS2 on naphthalene, nor were the corresponding protein products. NaphS2 may possess a non-classical dearomatizing enzyme for benzoate degradation, similar to one previously characterized in Geobacter metallireducens. Identification of genes involved in toluene degradation in NaphS2 led us to determine that NaphS2 degrades toluene, a previously unreported capacity. The genome sequence also suggests that NaphS2 may degrade other monoaromatic compounds.This study demonstrates that steps leading to the degradation of 2-naphthoyl-CoA are conserved between NaphS2 and N47, however while NaphS2 possesses the capacity to degrade 2-methylnaphthalene, naphthalene degradation likely does not proceed via 2-methylnaphthalene. Instead, carboxylation or another form of activation may serve as the first step in naphthalene degradation. Degradation of toluene and 2-methylnaphthalene, and the presence of at least one bss-like and bbs-like gene cluster in this organism, suggests that NaphS2 degrades both compounds via parallel mechanisms. Elucidation of the key genes necessary for anaerobic naphthalene degradation may provide the ability to track naphthalene degradation through in situ transcript monitoring

Sulfide Generation by Dominant Halanaerobium Microorganisms in Hydraulically Fractured Shales

Hydraulic fracturing of black shale formations has greatly increased United States oil and natural gas recovery. However, the accumulation of biomass in subsurface reservoirs and pipelines is detrimental because of possible well souring, microbially induced corrosion, and pore clogging. Temporal sampling of produced fluids from a well in the Utica Shale revealed the dominance of Halanaerobium strains within the in situ microbial community and the potential for these microorganisms to catalyze thiosulfate-dependent sulfidogenesis. From these field data, we investigated biogenic sulfide production catalyzed by a Halanaerobium strain isolated from the produced fluids using proteogenomics and laboratory growth experiments. Analysis of Halanaerobium isolate genomes and reconstructed genomes from metagenomic data sets revealed the conserved presence of rhodanese-like proteins and anaerobic sulfite reductase complexes capable of converting thiosulfate to sulfide. Shotgun proteomics measurements using a Halanaerobium isolate verified that these proteins were more abundant when thiosulfate was present in the growth medium, and culture-based assays identified thiosulfate-dependent sulfide production by the same isolate. Increased production of sulfide and organic acids during the stationary growth phase suggests that fermentative Halanaerobium uses thiosulfate to remove excess reductant. These findings emphasize the potential detrimental effects that could arise from thiosulfate-reducing microorganisms in hydraulically fractured shales, which are undetected by current industry-wide corrosion diagnostics. IMPORTANCE Although thousands of wells in deep shale formations across the United States have been hydraulically fractured for oil and gas recovery, the impact of microbial metabolism within these environments is poorly understood. Our research demonstrates that dominant microbial populations in these subsurface ecosystems contain the conserved capacity for the reduction of thiosulfate to sulfide and that this process is likely occurring in the environment. Sulfide generation (also known as “souring”) is considered deleterious in the oil and gas industry because of both toxicity issues and impacts on corrosion of the subsurface infrastructure. Critically, the capacity for sulfide generation via reduction of sulfate was not detected in our data sets. Given that current industry wellhead tests for sulfidogenesis target canonical sulfate-reducing microorganisms, these data suggest that new approaches to the detection of sulfide-producing microorganisms may be necessary