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Abstract The acidification of water in mining areas is a global environmental issue primarily catalyzed by sulfur-oxidizing bacteria (SOB). Little is known about microbial sulfur cycling in circumneutral pH mine tailing impoundment waters. Here we investigate biological sulfur oxidation over four years in a mine tailings impoundment water cap, integrating aqueous sulfur geochemistry, genome-resolved metagenomics and metatranscriptomics. The microbial community is consistently dominated by neutrophilic, chemolithoautotrophic SOB (relative abundances of ~76% in 2015, ~55% in 2016/2017 and ~60% in 2018). Results reveal two SOB strategies alternately dominate across the four years, influencing acid generation and sulfur speciation. Under oxic conditions, novel Halothiobacillus drive lower pH conditions (as low as 4.3) and lower [S2O3 2−] via the complete Sox pathway coupled to O2. Under anoxic conditions, Thiobacillus spp. dominate in activity, via the incomplete Sox and rDSR pathways coupled to NO3 −, resulting in higher [S2O3 2−] and no net significant acidity generation. This study provides genomic evidence explaining acidity generation and thiosulfate accumulation patterns in a circumneutral mine tailing impoundment and has significant environmental applications in preventing the discharge of sulfur compounds that can impact downstream environments. These insights illuminate opportunities for in situ biotreatment of reduced sulfur compounds and prediction of acidification events using gene-based monitoring and in situ RNA detection.

Drinking water stagnation can lead to degradation of chlorine residual, bacterial growth (including of opportunistic pathogens and nitrifiers), and metals release from plumbing materials; however, few studies have characterized building water quality and bacterial communities during the extended stagnation periods that occurred during COVID-19 pandemic-related building closures. Additionally, despite a lack of evidence-based guidance, flushing fixtures has been recommended to restore building water quality. We aimed to evaluate the impacts of reduced building occupancy (>2 months) and weekly restorative flushing on drinking water quality, bacterial communities, and the occurrence of undesirable microorganisms in three university buildings. Reduced occupancy led to diminished chloramine and elevated intact cell counts, but values remained stable after additional weeks of limited water use. Flushing temporarily improved water quality, with chlorine and cell counts remaining stable for at least 1 day but returning to levels measured prior to flushing within 1 week. Alpha diversity was lower under more stagnant conditions, and fixture identity, not flushing, was the most influential factor on bacterial community composition, suggesting a strong influence from local biofilm. Although Mycobacterium, Legionella, Pseudomonas, Nitrosomonas, and Nitrospira were detected in samples via amplicon sequencing, concentrations measured via qPCR of M. avium complex, L. pneumophila, P. aeruginosa, and ammonia-oxidizing bacteria were very low or were undetected, supporting that stagnation alone did not lead to high occurrence of undesirable microorganisms. Findings from this study contribute to our understanding of the effects of stagnation on building water microbiomes and the efficacy of flushing to improve water quality. Under the conditions of this case study, repeated flushing on a weekly timescale during low occupancy periods was not sufficient to maintain chlorine residual and prevent bacterial growth in fixtures. Building managers need to weigh the temporary water quality benefits of flushing against the labor and water resources required considering local context.

Wastewater surveillance for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA can be integrated with COVID-19 case data to inform timely pandemic response. However, more research is needed to apply and develop systematic methods to interpret the true SARS-CoV-2 signal from noise introduced in wastewater samples (e.g., from sewer conditions, sampling and extraction methods, etc.). In this study, raw wastewater was collected weekly from five sewersheds and one residential facility. The concentrations of SARS-CoV-2 in wastewater samples were compared to geocoded COVID-19 clinical testing data. SARS-CoV-2 was reliably detected (95% positivity) in frozen wastewater samples when reported daily new COVID-19 cases were 2.4 or more per 100,000 people. To adjust for variation in sample fecal content, four normalization biomarkers were evaluated: crAssphage, pepper mild mottle virus, Bacteroides ribosomal RNA (rRNA), and human 18S rRNA. Of these, crAssphage displayed the least spatial and temporal variability. Both unnormalized SARS-CoV-2 RNA signal and signal normalized to crAssphage had positive and significant correlation with clinical testing data (Kendall's Tau-b (τ)=0.43 and 0.38, respectively), but no normalization biomarker strengthened the correlation with clinical testing data. Locational dependencies and the date associated with testing data impacted the lead time of wastewater for clinical trends, and no lead time was observed when the sample collection date (versus the result date) was used for both wastewater and clinical testing data. This study supports that trends in wastewater surveillance data reflect trends in COVID-19 disease occurrence and presents tools that could be applied to make wastewater signal more interpretable and comparable across studies.

Clinical and surveillance testing for the SARS-CoV-2 virus relies overwhelmingly on RT-qPCR-based diagnostics, yet several popular assays require 2–3 separate reactions or rely on detection of a single viral target, which adds significant time, cost, and risk of false-negative results. Furthermore, multiplexed RT-qPCR tests that detect at least two SARS-CoV-2 genes in a single reaction are typically not affordable for large scale clinical surveillance or adaptable to multiple PCR machines and plate layouts. We developed a RT-qPCR assay using the Luna Probe Universal One-Step RT-qPCR master mix with publicly available primers and probes to detect SARS-CoV-2 N gene, E gene, and human RNase P (LuNER) to address these shortcomings and meet the testing demands of a university campus and the local community. This cost-effective test is compatible with BioRad or Applied Biosystems qPCR machines, in 96 and 384-well formats, with or without sample pooling, and has a detection sensitivity suitable for both clinical reporting and wastewater surveillance efforts.