Quantification of SARS-CoV-2 in wastewater samples from hospitals treating COVID-19 patients during the first wave of the pandemic in Brazil.

The COVID-19 pandemic has caused a global health crisis, and wastewater-based epidemiology (WBE) has emerged as an important tool to assist public health decision-making. Recent studies have shown that the SARS-CoV-2 RNA concentration in wastewater samples is a reliable indicator of the severity of the pandemic for large populations. However, few studies have established a strong correlation between the number of infected people and the viral concentration in wastewater due to variations in viral shedding over time, viral decay, infiltration, and inflow. Herein we present the relationship between the number of COVID-19-positive patients and the viral concentration in wastewater samples from three different hospitals (A, B, and C) in the city of Belo Horizonte, Minas Gerais, Brazil. A positive and strong correlation between wastewater SARS-CoV-2 concentration and the number of confirmed cases was observed for Hospital B for both regions of the N gene (R = 0.89 and 0.77 for N1 and N2, respectively), while samples from Hospitals A and C showed low and moderate correlations, respectively. Even though the effects of viral decay and infiltration were minimized in our study, the variability of viral shedding throughout the infection period and feces dilution due to water usage for different activities in the hospitals could have affected the viral concentrations. These effects were prominent in Hospital A, which had the smallest sewershed population size, and where no correlation between the number of defecations from COVID-19 patients and viral concentration in wastewater was observed. Although we could not determine trends in the number of infected patients through SARS-CoV-2 concentrations in hospitals' wastewater samples, our results suggest that wastewater monitoring can be efficient for the detection of infected individuals at a local level, complementing clinical data.

Quinone Moieties Link the Microbial Respiration of Natural Organic Matter to the Chemical Reduction of Diverse Nitroaromatic Compounds.

Insensitive munitions compounds (IMCs) are emerging nitroaromatic contaminants developed by the military as safer-to-handle alternatives to conventional explosives. Biotransformation of nitroaromatics via microbial respiration has only been reported for a limited number of substrates. Important soil microorganisms can respire natural organic matter (NOM) by reducing its quinone moieties to hydroquinones. Thus, we investigated the NOM respiration combined with the abiotic reduction of nitroaromatics by the hydroquinones formed. First, we established nitroaromatic concentration ranges that were nontoxic to the quinone respiration. Then, an enrichment culture dominated by Geobacter anodireducens could indirectly reduce a broad array of nitroaromatics by first respiring NOM components or the NOM surrogate anthraquinone-2,6-disulfonate (AQDS). Without quinones, no nitroaromatic tested was reduced except for the IMC 3-nitro-1,2,4-triazol-5-one (NTO). Thus, the quinone respiration expanded the spectrum of nitroaromatics susceptible to transformation. The system functioned with very low quinone concentrations because NOM was recycled by the nitroaromatic reduction. A metatranscriptomic analysis demonstrated that the microorganisms obtained energy from quinone or NTO reduction since respiratory genes were upregulated when AQDS or NTO was the electron acceptor. The results indicated microbial NOM respiration sustained by the nitroaromatic-dependent cycling of quinones. This process can be applied as a nitroaromatic remediation strategy, provided that a quinone pool is available for microorganisms.

Bacteria Make a Living Breathing the Nitroheterocyclic Insensitive Munitions Compound 3-Nitro-1,2,4-triazol-5-one (NTO).

The nitroheterocyclic 3-nitro-1,2,4-triazol-5-one (NTO) is an ingredient of insensitive explosives increasingly used by the military, becoming an emergent environmental pollutant. Cometabolic biotransformation of NTO occurs in mixed microbial cultures in soils and sludges with excess electron-donating substrates. Herein, we present the unusual energy-yielding metabolic process of NTO respiration, in which the NTO reduction to 3-amino-1,2,4-triazol-5-one (ATO) is linked to the anoxic acetate oxidation to CO2 by a culture enriched from municipal anaerobic digester sludge. Cell growth was observed simultaneously with NTO reduction, whereas the culture was unable to grow in the presence of acetate only. Extremely low concentrations (0.06 mg L-1) of the uncoupler carbonyl cyanide m-chlorophenyl hydrazone inhibited NTO reduction, indicating that the process was linked to respiration. The ultimate evidence of NTO respiration was adenosine triphosphate production due to simultaneous exposure to NTO and acetate. Metagenome sequencing revealed that the main microorganisms (and relative abundances) were Geobacter anodireducens (89.3%) and Thauera sp. (5.5%). This study is the first description of a nitroheterocyclic compound being reduced by anaerobic respiration, shedding light on creative microbial processes that enable bacteria to make a living reducing NTO.

Microbial Enrichment Culture Responsible for the Complete Oxidative Biodegradation of 3-Amino-1,2,4-triazol-5-one (ATO), the Reduced Daughter Product of the Insensitive Munitions Compound 3-Nitro-1,2,4-triazol-5-one (NTO).

3-Nitro-1,2,4-triazol-5-one (NTO) is one of the main ingredients of many insensitive munitions, which are being used as replacements for conventional explosives. As its use becomes widespread, more research is needed to assess its environmental fate. Previous studies have shown that NTO is biologically reduced to 3-amino-1,2,4-triazol-5-one (ATO). However, the final degradation products of ATO are still unknown. We have studied the aerobic degradation of ATO by enrichment cultures derived from the soil. After multiple transfers, ATO degradation was monitored in closed bottles through measurements of inorganic carbon and nitrogen species. The results indicate that the members of the enrichment culture utilize ATO as the sole source of carbon and nitrogen. As ATO was mineralized to CO2, N2, and NH4+, microbial growth was observed in the culture. Co-substrates addition did not increase the ATO degradation rate. Quantitative polymerase chain reaction analysis revealed that the organisms that enriched using ATO as carbon and nitrogen source were Terrimonas spp., Ramlibacter-related spp., Mesorhizobium spp., Hydrogenophaga spp., Ralstonia spp., Pseudomonas spp., Ectothiorhodospiraceae, and Sphingopyxis. This is the first study to report the complete mineralization of ATO by soil microorganisms, expanding our understanding of natural attenuation and bioremediation of the explosive NTO.

Ecotoxicity of the insensitive munitions compound 3-nitro-1,2,4-triazol-5-one (NTO) and its reduced metabolite 3-amino-1,2,4-triazol-5-one (ATO).

The insensitive munitions compound 3-nitro-1,2,4-triazol-5-one (NTO) was recently approved by the U.S. Army to replace cyclotrimethylene trinitramine (RDX) in conventional explosives. As its use becomes widespread, concern about the potential toxicity of NTO increases. NTO can undergo microbial reduction to 3-amino-1,2,4-triazol-5-one (ATO), which is recalcitrant in waterlogged soils. In this study, the acute toxicity of NTO and ATO towards various organisms, including microorganisms (i.e., methanogenic archaea, aerobic heterotrophs, and Aliivibrio fischeri (Microtox assay)), the microcrustacean Daphnia magna (ATO only), and zebrafish embryos (Danio rerio), was assessed. NTO was notably more inhibitory to methanogens than ATO (IC50=1.2mM,>62.8mM, respectively). NTO and ATO did not cause noteworthy inhibition on aerobic heterotrophs even at the highest concentrations tested (32.0mM). High concentrations of both NTO and ATO were required to inhibit A. fischeri (IC20=19.2, 22.4mM, respectively). D. magna was sensitive to ATO (LC50=0.27mM). Exposure of zebrafish embryos to NTO or ATO (750µM) did not cause lethal or developmental effects (22 endpoints tested). However, both compounds led to swimming behavior abnormalities at low concentrations (7.5µM). The results indicate that the reductive biotransformation of NTO could enhance or lower its toxicity according to the target organism.

Environmental Fate of 14C Radiolabeled 2,4-Dinitroanisole in Soil Microcosms.

2,4-Dinitrosanisole (DNAN) is an insensitive munitions component replacing conventional explosives. While DNAN is known to biotransform in soils to aromatic amines and azo-dimers, it is seldom mineralized by indigenous soil bacteria. Incorporation of DNAN biotransformation products into soil as humus-bound material could serve as a plausible remediation strategy. The present work studied biotransformation of DNAN in soil and sludge microcosms supplemented with uniformly ring-labeled 14C-DNAN to quantify the distribution of label in soil, aqueous, and gaseous phases. Electron donor amendments, different redox conditions (anaerobic, aerobic, sequential anaerobic-aerobic), and the extracellular oxidoreductase enzyme horseradish peroxidase (HRP) were evaluated to maximize incorporation of DNAN biotransformation products into the nonextractable soil humus fraction, humin. Irreversible humin incorporation of 14C-DNAN occurred at higher rates in anaerobic conditions, with a moderate increase when pyruvate was added. Additionally, a single dose of HRP resulted in an instantaneous increased incorporation of 14C-DNAN into the humin fraction. 14C-DNAN incorporation to the humin fraction was strongly correlated (R2 = 0.93) by the soil organic carbon (OC) amount present (either intrinsic or amended). Globally, our results suggest that DNAN biotransformation products can be irreversibly bound to humin in soils as a remediation strategy, which can be enhanced by adding soil OC.

Sequential anaerobic-aerobic biodegradation of emerging insensitive munitions compound 3-nitro-1,2,4-triazol-5-one (NTO).

Insensitive munitions, such as 3-nitro-1,2,4-triazol-5-one (NTO), are being considered by the U.S. Army as replacements for conventional explosives. Environmental emissions of NTO are expected to increase as its use becomes widespread; but only a few studies have considered the remediation of NTO-contaminated sites. In this study, sequential anaerobic-aerobic biodegradation of NTO was investigated in bioreactors using soil as inoculum. Batch bioassays confirmed microbial reduction of NTO under anaerobic conditions to 3-amino-1,2,4-triazol-5-one (ATO) using pyruvate as electron-donating cosubstrate. However, ATO biodegradation was only observed after the redox condition was switched to aerobic. This study also demonstrated that the high-rate removal of NTO in contaminated water can be attained in a continuous-flow aerated bioreactor. The reactor was first fed ATO as sole energy and nitrogen source prior to NTO addition. After few days, ATO was removed in a sustained fashion by 100%. When NTO was introduced together with electron-donor (pyruvate), NTO degradation increased progressively, reaching a removal efficiency of 93.5%. Mineralization of NTO was evidenced by the partial release of inorganic nitrogen species in the effluent, and lack of ATO accumulation. A plausible hypothesis for these findings is that NTO reduction occurred in anaerobic zones of the biofilm whereas ATO was mineralized in the bulk aerobic zones of the reactor.