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Hydroxylamine, nitrous acid, and nitric oxide are obligate intermediates or side metabolites in different nitrogen-converting microorganisms. These compounds are unstable and susceptible to the formation of highly reactive nitrogen species, including nitrogen dioxide, dinitrogen trioxide, nitroxyl, and peroxynitrite. Due to the high reactivity and cytotoxicity, the buildup of reactive nitrogen can affect the interplay of microorganisms/microbial processes, stimulate the reactions with organic compounds like organic micropollutants (OMP) and act as the precursors of nitrous oxide (N2O). However, there is little understanding of the occurrence and significance of reactive nitrogen during biological nitrogen conversions in engineered water systems. In this review, we evaluate the formation and fate of reactive nitrogen produced by microorganisms involved in biological nitrogen removal (BNR) processes, i.e., nitritation/nitrification, denitratation/denitrification, anammox, and the combined processes. While the formation of reactive nitrogen intermediates is entirely controlled by microbial activities, the consumption can be either biological or purely chemical. Changes in environmental conditions, such as redox transition, pH, and substrate availability, can imbalance the production and consumption of these reactive intermediates, thus leading to the transient accumulation of species. Based on previous experimental evidence, environmental relevance of reactive nitrogen in BNR systems, particularly related to abiotic N2O production and OMP transformation, is demonstrated.

Urban wastewater systems (UWSs) are a main receptacle of excreted antibiotic resistance genes (ARGs) and their host microorganisms. However, we lack integrated and quantitative observations of the occurrence of ARGs in the UWS to characterize the sources and identify processes that contribute to their fate. We sampled the UWSs from three medium-size cities in Denmark, Spain, and the United Kingdom and quantified 70 clinically important extended-spectrum β-lactamase and carbapenemase genes along with the mobile genetic elements and microbial communities. Results from all three countries showed that sewage—especially from hospitals—carried substantial loads of ARGs (106–107copies per person equivalent), but these loads progressively declined along sewers and through sewage treatment plants, resulting in minimal emissions (101–104copies per person equivalent). Removal was primarily during sewage conveyance (65 ± 36%) rather than within sewage treatment (34 ± 23%). The extended-spectrum β-lactamase and carbapenemase genes were clustered in groups based on their persistence in the UWS compartments. The less-persistent groups were associated to putative host taxa (especially Enterobacteriaceae and Moraxellaceae), while the more persistent groups appeared horizontally transferred and correlated significantly with total cell numbers and mobile genetic elements. This documentation of a substantial ARG reduction during sewage conveyance provides opportunities for antibiotic resistance management and a caution for sewage-based antibiotic resistance surveillance.

Heterotrophic denitrification consists of the four-step sequential reduction of nitrate to dinitrogen gas over nitrite, nitric oxide, and nitrous oxide. Oxidation processes, commonly of organic compounds, provide the electrons needed for the sequential reaction steps. The intracellular electron distribution is a competitive process among the four reduction steps. In this study, a model describing organic carbon oxidation and four-step denitrification through electron competition is proposed [Activated Sludge Model–Electron Competition (ASM–EC)]. The model describes denitrification rates as an analogy to how current intensity varies through a parallel set of resistors in electric circuits. The ASM–EC model was calibrated with data from batch experiments with heterotrophic denitrifying communities, where reduction of mixtures of nitrogen oxides was monitored, while different carbon sources were supplied in excess. The carbon sources included methanol, ethanol, acetate, and their ternary mixture. The electron distribution preference and electron uptake rates varied between the carbon sources and were captured by the model structure for most of the experiments. The ASM–EC model uses fewer parameters compared to existing state-of-the-art denitrification models and performed equally well in the tested scenarios. We advocate the use of this model for denitrification in the activated sludge model, which can easily be integrated in existing model structures, because it provides a parsimonious description of electron competition during denitrification.

Copper is a cofactor of the ammonia monooxygenase, an essential enzyme for the activity of ammonia oxidizing prokaryotes (AOP). Copper dosing at less than 1 μg/L stimulated ammonium removal in the poorly nitrifying biological filters of three full-scale drinking water treatment plants. Upon copper dosing, the ammonium concentration in the effluent decreased from up to 0.18 to less than 0.01 mg NH4+-N/L. To investigate how copper dosing affected the filter microbial community, we applied amplicon sequencing and qPCR targeting key nitrifying groups, including complete ammonia oxidizing (comammox) Nitrospira. Copper dosing increased the abundance of different nitrifiers. Multiple Nitrosomonasvariants (betaproteobacterial ammonia oxidizers), which initially collectively represented 1% or less of the total community, increased almost 10-fold. Comammox Nitrospirawere abundant and increased too, but their relative abundance within the AOP decreased because of Nitrosomonasproliferation. No other consistent change in the filter communities was detected, as well as no adverse effect of copper on the filters functionality. Our results show that copper dosing in three independent treatment plants was associated with consistent growth of AOP and that efficient nitrification was achieved through the joint contribution of comammox Nitrospiraand an increasing fraction of betaproteobacterial ammonia oxidizers.

The recent discovery of nitrous oxide (N2O)-reducing bacteria suggests a potential biological sink for the potent greenhouse gas N2O. For an application toward N2O mitigation, characterization of more isolates will be required. Here, we describe the successful enrichment and isolation of high-affinity N2O-reducing bacteria using a N2O-fed reactor (N2OFR). Two N2OFRs, where N2O was continuously and directly supplied as the sole electron acceptor to a biofilm grown on a gas-permeable membrane, were operated with acetate or a mixture of peptone-based organic substrates as an electron donor. In parallel, a NO3–-fed reactor (NO3FR), filled with a nonwoven sheet substratum, was operated using the same inoculum. We hypothesized that supplying N2O vs NO3–would enhance the dominance of distinct N2O-reducing bacteria. Clade II type nosZbacteria became rapidly enriched over clade I type nosZbacteria in the N2OFRs, whereas the opposite held in the NO3FR. High-throughput sequencing of 16S rRNA gene amplicons revealed the dominance of Rhodocyclaceaein the N2OFRs. Strains of the Azospiraand Dechloromonasgenera, canonical denitrifiers harboring clade II type nosZ, were isolated with high frequency from the N2OFRs (132 out of 152 isolates). The isolates from the N2OFR demonstrated higher N2O uptake rates (Vmax: 4.23 × 10–3–1.80 × 10–2pmol/h/cell) and lower N2O half-saturation coefficients (Km,N2O: 1.55–2.10 μM) than a clade I type nosZisolate from the NO3FR. Furthermore, the clade II type nosZisolates had higher specific growth rates on N2O than nitrite as an electron acceptor. Hence, continuously and exclusively supplying N2O in an N2OFR allows the enrichment and isolation of high-affinity N2O-reducing strains, which may be used as N2O sinks in bioaugmentation efforts.

Global paddy soil is the primary source of methane, a potent greenhouse gas. It is therefore highly important to understand the carbon cycling in paddy soil. Microbial reduction of iron, which is widely found in paddy soil, is likely coupled with the oxidation of dissolved organic matter (DOM) and suppresses methanogenesis. However, little is known about the biotransformation of small molecular DOM accumulated under flooded conditions and the effect of iron reduction on the biotransformation pathway. Here, we carried out anaerobic incubation experiments using field-collected samples amended with ferrihydrite and different short-chain fatty acids. Our results showed that less than 20% of short-chain fatty acids were mineralized and released to the atmosphere. Using Fourier transform ion cyclotron resonance mass spectrometry, we further found that a large number of recalcitrant molecules were produced during microbial consumption of these short-chain fatty acids. Moreover, the biotransformation efficiency of short-chain fatty acids decreased with the increasing length of carbon chains. Ferrihydrite addition promoted microbial assimilation of short-chain fatty acids as well as enhanced the activation and biotransformation of indigenous stable carbon in the soil replenished with formate. This study demonstrates the significance of ferrihydrite in the biotransformation of labile DOM and promotes a more comprehensive understanding of the coupling of iron reduction and carbon cycling in paddy soils.

Hydroxylamine (NH2OH) and nitrite (NO2–), intermediates during the nitritation process, can engage in chemical (abiotic) reactions that lead to nitrous oxide (N2O) generation. Here, we quantify the kinetics and stoichiometry of the relevant abiotic reactions in a series of batch tests under different and relevant conditions, including pH, absence/presence of oxygen, and reactant concentrations. The highest N2O production rates were measured from NH2OH reaction with HNO2, followed by HNO2reduction by Fe2+, NH2OH oxidation by Fe3+, and finally NH2OH disproportionation plus oxidation by O2. Compared to other examined factors, pH had the strongest effect on N2O formation rates. Acidic pH enhanced N2O production from the reaction of NH2OH with HNO2indicating that HNO2instead of NO2–was the reactant. In departure from previous studies, we estimate that abiotic N2O production contributes little (< 3% of total N2O production) to total N2O emissions in typical nitritation reactor systems between pH 6.5 and 8. Abiotic contributions would only become important at acidic pH (≤ 5). In consideration of pH effects on both abiotic and biotic N2O production pathways, circumneutral pH set-points are suggested to minimize overall N2O emissions from nitritation systems.

A myriad of nonantibiotic compounds is released into the environment, some of which may contribute to the dissemination of antimicrobial resistance by stimulating conjugation. Here, we analyzed a collection of studies to (i) identify patterns of transfer stimulation across groups and concentrations of chemicals, (ii) evaluate the strength of evidence for the proposed mechanisms behind conjugal stimulation, and (iii) examine the plausibility of alternative mechanisms. We show that stimulatory nonantibiotic compounds act at concentrations from 1/1000 to 1/10 of the minimal inhibitory concentration for the donor strain but that stimulation is always modest (less than 8-fold). The main proposed mechanisms for stimulation via the reactive oxygen species/SOS cascade and/or an increase in cell membrane permeability are not unequivocally supported by the literature. However, we identify the reactive oxygen species/SOS cascade as the most likely mechanism. This remains to be confirmed by firm molecular evidence. Such evidence and more standardized and high-throughput conjugation assays are needed to create technologies and solutions to limit the stimulation of conjugal gene transfer and contribute to mitigating global antibiotic resistance.

Surveillance of antibiotic resistance genes (ARGs) has been increasingly conducted in environmental sectors to complement the surveys in human and animal sectors under the “One-Health” framework. However, there are substantial challenges in comparing and synthesizing the results of multiple studies that employ different test methods and approaches in bioinformatic analysis. In this article, we consider the commonly used quantification units (ARG copy per cell, ARG copy per genome, ARG density, ARG copy per 16S rRNA gene, RPKM, coverage, PPM, etc.) for profiling ARGs and suggest a universal unit (ARG copy per cell) for reporting such biological measurements of samples and improving the comparability of different surveillance efforts.

Although nitrogen removal by partial nitritation and anammox is more cost-effective than conventional nitrification and denitrification, one downside is the production and accumulation of nitrous oxide (N2O). The potential exploitation of N2O-reducing bacteria, which are resident members of anammox microbial communities, for N2O mitigation would require more knowledge of their ecophysiology. This study investigated the phylogeny of resident N2O-reducing bacteria in an anammox microbial community and quantified individually the processes of N2O production and N2O consumption. An up-flow column-bed anammox reactor, fed with NH4+and NO2–and devoid of oxygen, emitted N2O at an average conversion ratio (produced N2O: influent nitrogen) of 0.284%. Transcriptionally active and highly abundant nosZgenes in the reactor biomass belonged to the Burkholderiaceae(clade I type) and Chloroflexusgenera (clade II type). Meanwhile, less abundant but actively transcribing nosZstrains were detected in the genera Rhodoferax, Azospirillum, Lautropia, and Bdellovibrioand likely act as an N2O sink. A novel 15N tracer method was adapted to individually quantify N2O production and N2O consumption rates. The estimated true N2O production rate and true N2O consumption rate were 3.98 ± 0.15 and 3.03 ± 0.18 mgN·gVSS–1·day–1, respectively. The N2O consumption rate could be increased by 51% (4.57 ± 0.51 mgN·gVSS–1·day–1) with elevated N2O concentrations but kept comparable irrespective of the presence or absence of NO2–. Collectively, the approach allowed the quantification of N2O-reducing activity and the identification of transcriptionally active N2O reducers that may constitute as an N2O sink in anammox-based processes.

The global rise and spread of antibiotic resistance greatly challenge the treatment of bacterial infections. Wastewater treatment plants (WWTPs) harbor and discharge antibiotic resistance genes (ARGs) as environmental contaminants. However, the knowledge gap on the host identity, activity, and functionality of ARGs limits transmission and health risk assessment of the WWTP resistome. Hereby, a genome-centric quantitative metatranscriptomic approach was exploited to realize high-resolution qualitative and quantitative analyses of bacterial hosts of ARGs (i.e., multiresistance, pathogenicity, activity, and niches) in the 12 urban WWTPs. We found that ∼45% of 248 recovered genomes expressed ARGs against multiple classes of antibiotics, among which bacitracin and aminoglycoside resistance genes in Proteobacteria were the most prevalent scenario. Both potential pathogens and indigenous denitrifying bacteria were transcriptionally active hosts of ARGs. The almost unchanged relative expression levels of ARGs in the most resistant populations (66.9%) and the surviving ARG hosts including globally emerging pathogens (e.g., Aliarcobacter cryaerophilus) in treated WWTP effluent prioritize future examination on the health risks related to resistance propagation and human exposure in the receiving environment.

Organic micropollutants (OMPs) are a threat to aquatic environments, and wastewater treatment plants may act as a source or a barrier of OMPs entering the environment. Understanding the fate of OMPs in wastewater treatment processes is needed to establish efficient OMP removal strategies. Enhanced OMP biotransformation has been documented during biological nitrogen removal and has been attributed to the cometabolic activity of ammonia-oxidizing bacteria (AOB) and, specifically, to the ammonia monooxygenase (AMO) enzyme. Yet, the exact mechanisms of OMP biotransformation are often unknown. This critical review aims to fundamentally and quantitatively evaluate the role of ammonia oxidation in OMP biotransformation during wastewater treatment processes. OMPs can be transformed by AOB via direct and indirect enzymatic reactions: AMO directly transforms OMPs primarily via hydroxylation, while biologically produced reactive nitrogen species (hydroxylamine (NH2OH), nitrite (NO2–), and nitric oxide (NO)) can chemically transform OMPs through nitration, hydroxylation, and deamination and can contribute significantly to the observed OMP transformations. OMPs containing alkyl, aliphatic hydroxyl, ether, and sulfide functional groups as well as substituted aromatic rings and aromatic primary amines can be biotransformed by AMO, while OMPs containing alkyl groups, phenols, secondary amines, and aromatic primary amines can undergo abiotic transformations mediated by reactive nitrogen species. Higher OMP biotransformation efficiencies and rates are obtained in AOB-dominant microbial communities, especially in autotrophic reactors performing nitrification or nitritation, than in non-AOB-dominant microbial communities. The biotransformations of OMPs in wastewater treatment systems can often be linked to ammonium (NH4+) removal following two central lines of evidence: (i) Similar transformation products (i.e., hydroxylated, nitrated, and desaminated TPs) are detected in wastewater treatment systems as in AOB pure cultures. (ii) Consistency in OMP biotransformation (rbio, μmol/g VSS/d) to NH4+removal (rNH4+, mol/g VSS/d) rate ratios (rbio/rNH4+) is observed for individual OMPs across different systems with similar rNH4+and AOB abundances. In this review, we conclude that AOB are the main drivers of OMP biotransformation during wastewater treatment processes. The importance of biologically driven abiotic OMP transformation is quantitatively assessed, and functional groups susceptible to transformations by AMO and reactive nitrogen species are systematically classified. This critical review will improve the prediction of OMP transformation and facilitate the design of efficient OMP removal strategies during wastewater treatment.