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BinaRena: a dedicated interactive platform for human-guided exploration and binning of metagenomes

Background: Exploring metagenomic contigs and “binning” them into metagenome-assembled genomes (MAGs) are essential for the delineation of functional and evolutionary guilds within microbial communities. Despite the advances in automated binning algorithms, their capabilities in recovering MAGs with accuracy and biological relevance are so far limited. Researchers often find that human involvement is necessary to achieve representative binning results. This manual process however is expertise demanding and labor intensive, and it deserves to be supported by software infrastructure. Results: We present BinaRena, a comprehensive and versatile graphic interface dedicated to aiding human operators to explore metagenome assemblies via customizable visualization and to associate contigs with bins. Contigs are rendered as an interactive scatter plot based on various data types, including sequence metrics, coverage profiles, taxonomic assignments, and functional annotations. Various contig-level operations are permitted, such as selection, masking, highlighting, focusing, and searching. Binning plans can be conveniently edited, inspected, and compared visually or using metrics including silhouette coefficient and adjusted Rand index. Completeness and contamination of user-selected contigs can be calculated in real time. In demonstration of BinaRena’s usability, we show that it facilitated biological pattern discovery, hypothesis generation, and bin refinement in a complex tropical peatland metagenome. It enabled isolation of pathogenic genomes within closely related populations from the gut microbiota of diarrheal human subjects. It significantly improved overall binning quality after curating results of automated binners using a simulated marine dataset. Conclusions: BinaRena is an installation-free, dependency-free, client-end web application that operates directly in any modern web browser, facilitating ease of deployment and accessibility for researchers of all skill levels. The program is hosted at https://github.com/qiyunlab/binarena, together with documentation, tutorials, example data, and a live demo. It effectively supports human researchers in intuitive interpretation and fine tuning of metagenomic data.

59 BASIC BIOLOGICAL SCIENCES↗

Genomic Characterization and Wetland Occurrence of a Novel Campylobacter Isolate from Canada Geese

Populations of resident, non-migratory Canada geese are rapidly increasing. Canada geese are known to transmit viral and bacterial diseases, posing a possible threat to human health. The most prevalent pathogens vectored by geese are Campylobacter species, yet the current understanding of the identity and virulence of these pathogens is limited. In our previous study, we observed a high prevalence of Campylobacter spp. in the Banklick Creek wetland—a constructed treatment wetland (CTW) located in northern KY (USA) used to understand sources of fecal contamination originating from humans and waterfowl frequenting the area. To identify the types of Campylobacter spp. found contaminating the CTW, we performed genetic analyses of Campylobacter 16s ribosomal RNA amplified from CTW water samples and collected fecal material from birds frequenting those areas. Our results showed a high occurrence of a Campylobacter canadensis-like clade from the sampling sites. Whole-genome sequence analyses of an isolate from Canada goose fecal material, called MG1, were used to confirm the identity of the CTW isolates. Further, we examined the phylogenomic position, virulence gene content, and antimicrobial resistance gene profile of MG1. Lastly, we developed an MG1-specific real-time PCR assay and confirmed the presence of MG1 in Canada goose fecal samples surrounding the CTW. Our findings reveal that the Canada goose-vectored Campylobacter sp. MG1 is a novel isolate compared to C. canadensis that possesses possible zoonotic potential, which may be of human health concern.

59 BASIC BIOLOGICAL SCIENCES↗

Geobacter sp. Strain IAE Dihaloeliminates 1,1,2-Trichloroethane and 1,2-Dichloroethane

We report that chlorinated ethanes, including 1,2-dichloroethane (1,2-DCA) and 1,1,2-trichloroethane (1,1,2-TCA), are widespread groundwater contaminants. Enrichment cultures XR DCA and XR TCA derived from river sediment dihaloeliminated 1,2-DCA to ethene and 1,1,2-TCA to vinyl chloride (VC), respectively. The XR TCA culture subsequently converted VC to ethene via hydrogenolysis. Microbial community profiling demonstrated the enrichment of Geobacter 16S rRNA gene sequences in both the XR DCA and XR TCA cultures, and Dehalococcoides mccartyi (Dhc) sequences were only detected in the ethene-producing XR TCA culture. The presence of a novel Geobacter population, designated as Geobacter sp. strain IAE, was identified by the 16S rRNA gene-targeted polymerase chain reaction and Sanger sequencing. Time-resolved population dynamics attributed the dihaloelimination activity to strain IAE, which attained the growth yields of 0.93 ± 0.06 x 10 7 and 1.18 ± 0.14 x 10 7 cells per μmol Cl -1 released with 1,2-DCA and 1,1,2-TCA as electron acceptors, respectively. In contrast, Dhc growth only occurred during VC-to-ethene hydrogenolysis. Our findings discover a Geobacter sp. strain capable of respiring multiple chlorinated ethanes and demonstrate the involvement of a broader diversity of organohalide-respiring bacteria in the detoxification of 1,2-DCA and 1,1,2-TCA.

1,1,2-trichloroethane↗

Description and Genome Characterization of Three Novel Fungal Strains Isolated from Mars 2020 Mission-Associated Spacecraft Assembly Facility Surfaces—Recommendations for Two New Genera and One Species

National Aeronautics and Space Administration’s (NASA) spacecraft assembly facilities are monitored for the presence of any bacteria or fungi that might conceivably survive a transfer to an extraterrestrial environment. Fungi present a broad and diverse range of phenotypic and functional traits to adapt to extreme conditions, hence the detection of fungi and subsequent eradication of them are needed to prevent forward contamination for future NASA missions. During the construction and assembly for the Mars 2020 mission, three fungal strains with unique morphological and phylogenetic properties were isolated from spacecraft assembly facilities. The reconstruction of phylogenetic trees based on several gene loci (ITS, LSU, SSU, RPB, TUB, TEF1) using multi-locus sequence typing (MLST) and whole genome sequencing (WGS) analyses supported the hypothesis that these were novel species. Here we report the genus or species-level classification of these three novel strains via a polyphasic approach using phylogenetic analysis, colony and cell morphology, and comparative analysis of WGS. The strain FJI-L9-BK-P1 isolated from the Jet Propulsion Laboratory Spacecraft Assembly Facility (JPL-SAF) exhibited a putative phylogenetic relationship with the strain Aaosphaeria arxii CBS175.79 but showed distinct morphology and microscopic features. Another JPL-SAF strain, FJII-L3-CM-DR1, was phylogenetically distinct from members of the family Trichomeriaceae and exhibited morphologically different features from the genera Lithohypha and Strelitziana. The strain FKI-L1-BK-DR1 isolated from the Kennedy Space Center facility was identified as a member of Dothideomycetes incertae sedis and is closely related to the family Kirschsteiniotheliaceae according to a phylogenetic analysis. The polyphasic taxonomic approach supported the recommendation for establishing two novel genera and one novel species. The names Aaosphaeria pasadenensis (FJI-L9-BK-P1 = NRRL 64424 = DSM 114621), Pasadenomyces melaninifex (FJII-L3-CM-DR1 = NRRL 64433 = DSM 114623), and Floridaphiala radiotolerans (FKI-L1-BK-DR1 = NRRL 64434 = DSM 114624) are proposed as type species. Furthermore, resistance to ultraviolet-C and presence of specific biosynthetic gene cluster(s) coding for metabolically active compounds are unique to these strains.

59 BASIC BIOLOGICAL SCIENCES↗

Coming-of-Age Characterization of Soil Viruses: A User’s Guide to Virus Isolation, Detection within Metagenomes, and Viromics

The study of soil viruses, though not new, has languished relative to the study of marine viruses. This is particularly due to challenges associated with separating virions from harboring soils. Generally, three approaches to analyzing soil viruses have been employed: (1) Isolation, to characterize virus genotypes and phenotypes, the primary method used prior to the start of the 21st century. (2) Metagenomics, which has revealed a vast diversity of viruses while also allowing insights into viral community ecology, although with limitations due to DNA from cellular organisms obscuring viral DNA. (3) Viromics (targeted metagenomics of virus-like-particles), which has provided a more focused development of ‘virus-sequence-to-ecology’ pipelines, a result of separation of presumptive virions from cellular organisms prior to DNA extraction. This separation permits greater sequencing emphasis on virus DNA and thereby more targeted molecular and ecological characterization of viruses. Employing viromics to characterize soil systems presents new challenges, however. Ones that only recently are being addressed. Here we provide a guide to implementing these three approaches to studying environmental viruses, highlighting benefits, difficulties, and potential contamination, all toward fostering greater focus on viruses in the study of soil ecology.

59 BASIC BIOLOGICAL SCIENCES↗

The military gear microbiome: risk factors surrounding the warfighter

ABSTRACT Combat extremity wounds are highly susceptible to contamination from surrounding environmental material. This bioburden could be partially transferred from materials in immediate proximity to the wound, including fragments of the uniform and gear. However, the assessment of the microbial bioburden present on military gear during operational conditions of deployment or training is relatively unexplored. Opportunistic pathogens that can survive on gear represent risk factors for infection following injury, especially following combat blasts, where fibers and other materials are embedded in wounded tissue. We utilized 16S rRNA sequencing to assess the microbiome composition of different military gear types (boot, trouser, coat, and canteen) from two operational environments (training in Hawai’i and deployed in Indonesia) across time (days 0 and 14). We found that microbiome diversity, stability, and composition were dependent on gear type, training location, and sampling timepoint. At day 14, species diversity was significantly higher in Hawai’i samples compared to Indonesia samples for boot, coat, and trouser swabs. In addition, we observed the presence of potential microbial risk factors, as opportunistic pathogenic species, such as Acinetobacter , Pseudomonas, and Staphylococcus , were found to be present in all sample types and in both study sites. These study outcomes will be used to guide the design of antimicrobial materials and uniforms and for infection control efforts following combat blasts and other injuries, thereby improving treatment guidance during military training and deployment. IMPORTANCE Combat extremity wounds are vulnerable to contamination from environments of proximity to the warfighter, leading to potential detrimental outcomes such as infection and delayed wound healing. Therefore, microbial surveillance of such environments is necessary to aid the advancement of military safety and preparedness through clinical diagnostics, treatment protocols, and uniform material design.

59 BASIC BIOLOGICAL SCIENCES↗

Classifying the full SDSS-IV MaNGA Survey using optical diagnostic diagrams: Presentation of AGN catalogs in flexible apertures

Accurate active galactic nucleus (AGN) identifications in large galaxy samples are crucial for the assessment of the role of AGN and AGN feedback in the co-evolution of galaxies and their central supermassive black holes. Emission-line flux-ratio diagnostics are commonly used to identify AGN in optical spectra. New large samples of integral field unit observations allow exploration of the role of aperture size in the classification process. In this paper, we present galaxy classifications for all 10010 galaxies observed within the Mapping Nearby Galaxies at Apache Point Observatory (MaNGA) survey. We use Baldwin-Philips-Terlevich line flux-ratio diagnostics combined with an Hα equivalent threshold in 60 apertures of varying size for the classification, and provide the corresponding catalogs. MaNGA-selected AGN primarily lie below the main sequence of star-forming galaxies, and reside in massive galaxies with stellar masses of ~10 11 M ⊙ and a median Hα-derived star formation rate of ~1.44M ⊙ yr -1 . We find that the number of “fake” AGN increases significantly beyond selection apertures of >1.0 R eff because of increased contamination from diffuse ionized gas (DIG). A comparison with previous works shows that the treatment of the underlying stellar continuum and flux measurements can significantly impact galaxy classification. Our work provides the community with AGN catalogs and galaxy classifications for the full MaNGA survey.

79 ASTRONOMY AND ASTROPHYSICS↗

Bioremediation of copper in sediments from a constructed wetland ex situ with the novel bacterium Cupriavidus basilensis SRS

The H-02 constructed wetland was designed to remove metals (primarily copper and zinc) to treat building process water and storm water runoff from multiple sources associated with the Tritium Facility at the DOE-Savannah River Site, Aiken, SC. The concentration of Cu and Zn in the sediments has increased over the lifetime of the wetland and is a concern. A bioremediation option was investigated at the laboratory scale utilizing a newly isolated bacterium of the copper metabolizing genus Cupriavidus isolated from Tim’s Branch Creek, a second-order stream that eventually serves as a tributary to the Savannah River, contaminated with uranium and other metals including copper, nickel, and mercury. Cupriavidus basilensis SRS is a rod-shaped, gram-negative bacterium which has been shown to have predatory tendencies. The isolate displayed resistance to the antibiotics ofloxacin, tetracycline, ciprofloxacin, select fungi, as well as Cu 2+ and Zn 2+ . Subsequent ribosomal sequencing demonstrated a 100% confidence for placement in the genus Cupriavidus and a 99.014% match to the C. basilensis type strain. When H-02 wetland samples were inoculated with Cupriavidus basilensis SRS samples showed significant (p < 0.05) decrease in Cu 2+ concentrations and variability in Zn 2+ concentrations. Over the 72-h incubation there were no significant changes in the inoculate densities (10 6 –10 8 cells/ML) indicating Cupriavidus basilensis SRS resiliency in this environment. This research expands our understanding of the Cupriavidus genus and demonstrates the potential for Cupriavidus basilensis SRS to bioremediate sites impacted with heavy metals, most notably copper.

54 ENVIRONMENTAL SCIENCES↗

Genomic and environmental controls on Castellaniella biogeography in an anthropogenically disturbed subsurface

Castellaniella species have been isolated from a variety of mixed-waste environments including the nitrate and multiple metal-contaminated subsurface at the Oak Ridge Reservation (ORR). Previous studies examining microbial community composition and nitrate removal at ORR during biostimulation efforts reported increased abundances of members of the Castellaniella genus concurrent with increased denitrification rates. Thus, we asked how genomic and abiotic factors control the Castellaniella biogeography at the site to understand how these factors may influence nitrate transformation in an anthropogenically impacted setting. We report the isolation and characterization of several Castellaniella strains from the ORR subsurface. Five of these isolates match at 100% identity (at the 16S rRNA gene V4 region) to two Castellaniella amplicon sequence variants (ASVs), ASV1 and ASV2, that have persisted in the ORR subsurface for at least 2 decades. However, ASV2 has consistently higher relative abundance in samples taken from the site and was also the dominant blooming denitrifier population during a prior biostimulation effort. We found that the ASV2 representative strain has greater resistance to mixed metal stress than the ASV1 representative strains. We attribute this resistance, in part, to the large number of unique heavy metal resistance genes identified on a genomic island in the ASV2 representative genome. Additionally, we suggest that the relatively lower fitness of ASV1 may be connected to the loss of the nitrous oxide reductase (nos) operon (and associated nitrous oxide reductase activity) due to the insertion at this genomic locus of a mobile genetic element carrying copper resistance genes. This study demonstrates the value of integrating genomic, environmental, and phenotypic data to characterize the biogeography of key microorganisms in contaminated sites.

59 BASIC BIOLOGICAL SCIENCES↗

Concentration-response gene expression analysis in zebrafish reveals phenotypically-anchored transcriptional responses to retene

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants and are associated with human disease. Canonically, many PAHs induce toxicity via activation of the aryl hydrocarbon receptor (AHR) pathway. While the interaction between PAHs and the AHR is well-established, understanding which AHR-regulated transcriptional effects directly result in observable phenotypes and which are adaptive or benign is important to better understand PAH toxicity. Retene is a frequently detected PAH in environmental sampling and has been associated with AHR2-dependent developmental toxicity in zebrafish, though its mechanism of toxicity has not been fully elucidated. To interrogate transcriptional changes causally associated with retene toxicity, we conducted whole-animal RNA sequencing at 48 hours post-fertilization after exposure to eight retene concentrations. The concentrations were selected to produce effects ranging from no phenotype to mortality and malformations in 100% of animals at 5 days post-fertilization. We identified a concentration-response relationship between retene teratogenicity and differential gene expression in both number of DEGs and magnitude of expression change. Elevated expression of cyp1a at retene concentrations below the threshold for teratogenicity suggested that while cyp1a expression is a sensitive biomarker of AHR activation, it may be too sensitive to serve as a biomarker of AHR-dependent teratogenicity. Genes differentially expressed at only non-teratogenic concentrations were enriched for transforming growth factor-ß (TGF-ß) signaling pathway disruption while DEGs identified at only teratogenic concentrations were significantly enriched for response to xenobiotic stimulus and reduction-oxidation reaction activity. DEGs which spanned both non-teratogenic and teratogenic concentrations showed similar disrupted biological processes to those unique to teratogenic concentrations, indicating these processes were disrupted at low exposure concentrations. Gene co-expression network analysis identified several gene modules, including those associated with PAHs and AHR2 activation. One, Module 7, was strongly enriched for AHR2-associated genes and contained the strongest responses to retene. Benchmark concentration (BMC) of Module 7 genes identified a median BMC of 7.5 µM, nearly the highest retene concentration with no associated teratogenicity, supporting the hypothesis that Module 7 genes are largely responsible for retene toxicity.

Toxin, Zebrafish, Retene, Transcriptomics, network↗

Metagenome-assembled genomes from soil samples in control and warming plots in Blodgett Forest, CA (2014-2021)

The pathways of carbon transport and loss through and from soils—soil organic matter (SOM) depolymerization to dissolved organic carbon and mineralization to carbon dioxide (CO2)—are fundamentally driven by microbial activity, which is strongly regulated by environmental conditions. As part of LBNL (Lawrence Berkeley National Laboratory) TES (Terrestrial Ecosystem Science) Belowground Biogeochemistry Science Focus Area (SFA), we have established a novel whole-soil long-term warming experiment at the University of California (UC) Blodgett Forest Research Station (Sierra Nevada) in 2014, where we study the role of biogeochemical, microbial and geochemical process interactions in SOM decomposition and stabilization.Here, we present metagenome-assembled genomes (MAGs) for the bacterial and archaeal community from soil depth profiles collected from 2014 to 2021 from three paired control and warming plots. We collected soil samples across a range of depth profiles (spanning surface to 90 cm deep) from three paired control and warming plots from a temperate mixed forest in Northern California. Each paired plot had been subjected to experimental warming since June 2014 to simulate a predicted climate change scenario for northern California. 101 soil metagenomes were sequenced at JGI (Joint Genome Institute) and UCSF (University of California San Francisco) Center for Advanced Technology and can be found under the JGI (Joint Genome Institute) GOLD (Genomes Online Database) Sequencing project Gs0151586 and NCBI (National Center for Biotechnology Information) Projects PRJNA1225762 and PRJEB39497. Metagenomes were assembled using JGI (Joint Genome Institute) Metagenome Workflow (10.1128/mSystems.00804-20). For each metagenome, the assembled contigs were binned into genomes using 3 binning algorithms (cocacola, metabat, and maxbin) and the resulting bins were consolidated using dastool. The consolidated bins from all metagenomes were pooled, filtered by completeness (>50%) and contamination (<25%), and dereplicated at 99% ANI (average nucleotide identity) using dRep (https://github.com/MrOlm/drep).The dataset includes a zip file of 2321 MAG (Metagenome Assembled Genome) fasta files, the accession numbers for the underlying metagenomes, and a csv file with MAG (Metagenome Assembled Genome) quality metrics and taxonomic classification (GTDB -Genome Taxonomy Database-RS220). This dataset also includes a file-level metadata (flmd.csv) file that lists each file contained in the dataset with associated metadata and a data dictionary (dd.csv) file that contains column/row headers used throughout the files along with a definition, units, and data type. A sample metadata file (samples.csv) that contains site information has also been included.

54 ENVIRONMENTAL SCIENCES↗

Diverse PFAS produce unique transcriptomic changes linked to developmental toxicity in zebrafish

Per- and polyfluoroalkyl substances (PFAS) are a widespread and persistent class of contaminants posing significant environmental and human health concerns. Comprehensive understanding of the modes of action underlying toxicity among structurally diverse PFAS is mostly lacking. To address this need, we recently reported on our application of developing zebrafish to evaluate a large library of PFAS for developmental toxicity. In the present study, we prioritized 15 bioactive PFAS that induced significant morphological effects and performed RNA-sequencing to characterize early transcriptional responses at a single timepoint (48 h post fertilization) after early developmental exposures (8 h post fertilization). Internal concentrations of 5 of the 15 PFAS were measured from pooled whole fish samples across multiple timepoints between 24–120 h post fertilization, and additional temporal transcriptomics at several timepoints (48–96 h post fertilization) were conducted for Nafion byproduct 2. A broad range of differentially expressed gene counts were identified across the PFAS exposures. Most PFAS that elicited robust transcriptomic changes affected biological processes of the brain and nervous system development. While PFAS disrupted unique processes, we also found that similarities in some functional head groups of PFAS were associated with the disruption in expression of similar gene sets. Body burdens after early developmental exposures to select sulfonic acid PFAS, including Nafion byproduct 2, increased from the 24–96 h post fertilization sampling timepoints and were greater than those of sulfonamide PFAS of similar chain lengths. In parallel, the Nafion byproduct 2-induced transcriptional responses increased between 48 and 96 h post fertilization. PFAS characteristics based on toxicity, transcriptomic effects, and modes of action will contribute to further prioritization of PFAS structures for testing and informed hazard assessment.

59 BASIC BIOLOGICAL SCIENCES↗

Genome collection processing for “Conserved upper thermal limits and small safety margins in soil copiotrophic bacteria”

We extracted the genomic DNA of 400 randomly selected isolates using a Quick-DNA Microprep Kit (Zymo Research D3020) according to the manufacturer’s protocol. We then submitted the extracted gDNA samples for short-read Illumina sequencing (200 Mbp) at SeqCoast Genomics (Portsmouth, NH, USA). After preprocessing the sequences using Trimmommatic (Bolger et al. 2014), we assembled the genomes using SPADES (Bankevich et al. 2012) and checked the quality of each assembly using QUAST (Gurevich et al. 2013). We processed the genome assemblies using a KBase (v1.4.0) pipeline (Allen et al. 2017; Arkin et al. 2018). Briefly, we used DRAM (v0.1.2) with default settings to annotate the genome assemblies. We then evaluated genome quality and possible contamination levels using CheckM (v1.0.18) (Parks et al. 2015) and retained genomes with completeness above 98% and contamination below 5% (n = 354), following the authors' guidelines. We then obtained taxonomic assignments for all remaining isolates using the Genome Taxonomy Database tool GTDB-Tk (v2.3.2, database version r214) (Chaumeil et al. 2019). We constructed a phylogenetic tree using the tool SpeciesTree (v2.2.0). We then trimmed the tree (using Trim SpeciesTree to GenomeSet- v1.4.0), retaining only tips within our collection with measured thermal performance.

59 BASIC BIOLOGICAL SCIENCES↗

Spirochaete genome identified in red abalone sample represents a novel genus Candidatus Haliotispira gen. nov. within the order Spirochaetales

A fully assembled spirochaete genome was identified as a contaminating scaffold in our red abalone ( Haliotis rufescens ) genome assembly. In this paper, we describe the analysis of this bacterial genome. The assembled spirochaete genome is 3.25 Mb in size with 48.5 mol% G+C content. The proteomes of 38 species were compared with the spirochaete genome and it was discovered to form an independent branch within the family Spirochaetaceae on the phylogenetic tree. The comparison of 16S rRNA sequences and average nucleotide identity scores between the spirochaete genome with known species of different families in Spirochaetia indicate that it is an unknown species. Further, the percentage of conserved proteins compared to neighbouring taxa confirm that it does not belong to a known genus within Spirochaetaceae . We propose the name Candidatus Haliotispira prima gen. nov., sp. nov. based on its taxonomic placement and origin. We also tested for the presence of this species in different species of abalone and found that it is also present in white abalone ( Haliotis sorenseni ). In addition, we highlight the need for better classification of taxa within the class Spirochaetia .

Microbiology↗

Comparative physiological and genomic characterization of a novel Nitrobacter vulgaris strain from a nitrate-contaminated subsurface

Nitrite-oxidizing bacteria (NOB) represent a crucial node in the global nitrogen cycle. By catalyzing the second step of nitrification—the oxidation of nitrite to nitrate to generate energy for growth—NOB activity controls the fate of nitrite (NO 2 - ) in aerobic environments. Despite thriving in diverse environments, including soils, freshwater, marine ecosystems, subsurface habitats, and water treatment systems, organisms capable of nitrite oxidation are confined to Nitrobacter, Nitrospira, Nitrospina, Nitrotoga, and a few other specific lineages. The genus Nitrobacter, recognized for its facultative heterotrophic metabolism, is often associated with high-nitrogen environments. Here, we report the physiological characterization of a novel strain, Nitrobacter vulgaris strain MLSD-S22, isolated from a nitrate- and heavy-metal-contaminated subsurface. Growth inhibition experiments revealed that strain MLSD-S22 and the N. vulgaris type strain Z exhibited similar sensitivities to nitrite and nitrate, with nitrite being the most inhibitory. Microrespirometry demonstrated that the two N. vulgaris strains and Nitrobacter winogradskyi Nb-255 possessed higher affinities for nitrite and oxygen than previously reported for Nitrobacter, suggesting potential to compete in low-substrate environments. Long-read DNA sequencing provided a complete genome for strain MLSD-S22, revealing two plasmids and an intact nitrous oxide (N 2 O) reduction operon—an unexpected feature for Nitrobacter. While N 2 O reduction activity was not observed under the tested conditions, this discovery raises questions about the contribution of Nitrobacter NOB to the N 2 O sink. These findings broaden the physiological and genomic diversity of Nitrobacter, offering new insights into their adaptation strategies and providing a framework for future evaluation of their potential roles in nitrogen loss.

Nitrobacter↗

Semi-Analytical Leakage Solutions for Aquifers (SALSA) v1

SALSA is a semi-analytical modeling tool that can provide assessments of pressure perturbations and brine leakage because of fluid injection and extraction activities in sedimentary basins. Sedimentary basins typically contain vertical sequences of near-horizontal aquifers separated by less-permeable aquitards. In the context of geologic carbon sequestration (GCS), while aquitards are relied upon to contain injected buoyant free-phase CO2 and limit inter-aquifer brine flow, the large number of leaky wells (e.g., unused/unsuccessful exploration wells, or improperly plugged water or oil/gas wells) that are present in sedimentary basins creates concerns for potential leakage and groundwater contamination. The mathematical theory and solution method included in SALSA is presented in our recent manuscript under review. SALSA is the first to account for the coupled leakage through aquitards and leaky wells, with geologic pressurization sources. SALSA based on semi-analytical solutions can be computationally very efficient for problems containing many injection and leaky wells in multilayered sedimentary systems, compared to any numerical simulation software requiring local mesh refinement around each well to obtain accurate results. Therefore, stakeholders (e.g., regulators, operators) for site screening, injection and post-injection pressure behavior and assessing leakage risks can use SALSA as a fast-predictive tool in GCS applications in multilayered aquifer systems.

Cihan, Abdullah↗

MR13A-3183: Microbial and Geochemical Characterization of Groundwater: Implications for Underground Hydrogen Storage Leakage

Underground hydrogen storage (UHS) in geological formations is a key element of the clean energy transition as it enables the decarbonization of the transportation and industrial sectors by decoupling hydrogen production and storage. UHS has many benefits, including low cost, much wider availability, large storage capacity, well-established infrastructure, and increased safety because of geological sealing capabilities. However, the impact of hydrogen (H2) biogeochemical interactions in the presence of subsurface microorganisms is largely neglected from UHS perspectives. These interactions might affect the effectiveness of storage and can even cause H2 to leak into the shallow aquifers. Leakage of H2 into groundwater can change the geochemistry and induce several microbial-driven processes. Microorganisms, such as sulfate-reducers, are naturally abundant in groundwater and consume H2 to produce hydrogen sulfide (H2S), which can contaminate the freshwater drinking groundwater and cause damage to infrastructure. Hydrogen leakage can also trigger microbial reactions responsible for metal mobility, which can impact the water quality. However, the kinetics of these reactions and the temporal impact of hydrogen leakage in groundwater are still unknown. Therefore, a time series hydrogen-groundwater interaction experiment was conducted, and the changes in fluid chemistry and headspace gas composition will be analyzed along with DNA sequencing results to understand the extent and kinetics of biogeochemical reactions that occur if hydrogen leaks into groundwater. In the experiments, Ultra High Purity (UHP) hydrogen gas will be injected into glass vials with groundwater samples, for a designated time period. For each glass-sealed vial, 16S rRNA gene sequencing, IC, ICP-MS, and GC-TCD will be performed. The experiments provide insights into plausible impacts of hydrogen leakage into shallow drinking water aquifers.

Clark, Allison [West Virginia University (WVU)]↗

Opportunities to Implement Solutions to Achieve Remedial Action Objectives in Consideration of Stakeholder Interests - 20365

A case study in the implementation of a soils excavation and removal project conducted at a Formerly Utilized Sites Remedial Action Program (FUSRAP) Maywood Superfund Site (FMSS) vicinity property; an active commercial business, in a densely populated area, with significant operational, technical and, logistical constraints that were expected to limit areas that could be remediated. The United States Army Corps of Engineers-led (USACE) team successfully navigated complex overlapping stakeholder interests to achieve a more efficient and complete removal of contaminated soils and debris while minimizing unnecessary excess costs to the Government. This paper centers on FUSRAP activities at the FMSS vicinity property located at 149-151 Maywood Avenue, Maywood, Bergen County, New Jersey. For most of its FUSRAP history, this ∼109,000 square meter (27-acre) property housed a now-demolished ∼26,000 square meter (6.5-acre) warehouse operated by Sears Logistics Services, a unit of the Sears Roebuck Company (Figure 2). Sears ended its property lease and vacated the warehouse in December 2016. Given that long history and for the purposes of this paper, the property will be referred to as the 'Sears property' or simply 'the property.' The Sears property is approximately 109,000 square meters (27-acres) and is currently zoned for commercial use. The property is bound to the north and northwest by 100 West Hunter Avenue (the Stepan Company), to the northeast by 205 Maywood Avenue, to the east by Maywood Avenue, to the south by 23 West Howcroft Road, and to the west by businesses on NJ Route 17 and the NJ Route 17 roadway. Until December 2016, the property housed a warehouse and distribution center operated by Sears Logistical Services. On-site structures were demolished by the property owner in 2017. The warehouse covered the north section of the property along the Stepan Company property line. A railroad spur from the adjacent property now known as the Maywood Interim Storage Site (MISS) ended at the northeast corner of the warehouse. Wetlands were located east of the warehouse; the rest of the property was covered with paved parking lots and grassed areas. During remediation at the property, opportunities frequently arose where the project team was able to coordinate effectively with stakeholders to sequence remediation activities to facilitate removal of otherwise inaccessible soils and identify opportunities for materials reuse when supported by residual radiological and chemical levels. Some specific opportunities include: - remediating individual truck loading dock bays to maintain tenant operations; - working along a busy highway and in a utility corridor containing a 30-inch high pressure gas main; - remediating Lodi Brook and associated wetlands requiring bypass pumping and other diversionary structures to maintain local community stormwater drainage; - protecting during remediation and supporting access to facilitate tenant/owner maintenance of critical fire protection and water supply system service lines buried in contaminated soils and necessary for safe warehouse operations; and - coordinating with stakeholders to ensure non-radiological contaminants of concern for the site (unaffiliated with FUSRAP) were addressed by the responsible party in a manner that maximized benefit to all parties while supporting an efficient overall site remediation program. Once the property was vacant and the warehouse structure and radiologically non-impacted above-grade structures were demolished, the project identified an opportunity to significantly reduce the volume of waste associated with the foundation of the former warehouse, a foundation suspected of being partially constructed in radiologically contaminated soils. The project developed and implemented supplemental radiological verification survey and sampling strategies based on radiological cross-contamination risk potential with process flow-charts and screening-level based decision points; a program that classified saw-cut sections of concrete based on observed residual surface radioactivity conditions using a combination of gamma sensitive sodium-iodide scans and beta sensitive Geiger-Muller direct measurements. The proposed approach was reviewed with stakeholders with feedback, including consideration of applicable State of New Jersey Site Remediation Program criteria and guidance before implementation. Once classified, additional sampling was performed at frequencies driven by screening. The sampling methods, stockpile sampling frequencies, criteria and, approach to data evaluation were developed in consideration of existing site cleanup standards, regional background ranges, State of New Jersey radiological remediation program guidance, and specific stakeholder input With the property remediation and survey efforts nearly completed, it is relevant to examine retrospectively the objectives, assumptions and limitations in the remedial design (i.e., what was planned) versus what was able to be accomplished through effective teaming. Benefits to other environmental remediation site programs include better understanding of how to work effectively with stakeholders to achieve win-win outcomes, and approaches to site remediation, waste minimization and materials reuse that were successful in their overall outcomes. (authors)

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗