The description of comammox Nitrospira spp., performing complete ammonia-to-nitrate oxidation, and their co-occurrence with canonical β-proteobacterial ammonia oxidizing bacteria (β-AOB) in the environment, calls into question the metabolic potential of comammox Nitrospira and the evolutionary history of their ammonia oxidation pathway. We report four new comammox Nitrospira genomes, constituting two novel species, and the first comparative genomic analysis on comammox Nitrospira. Unlike canonical Nitrospira, comammox Nitrospira genomes lack genes for assimilatory nitrite reduction, suggesting that they have lost the potential to use external nitrite nitrogen sources. By contrast, compared to canonical Nitrospira, comammox Nitrospira harbor a higher diversity of urea transporters and copper homeostasis genes and lack cyanate hydratase genes. Additionally, the two comammox clades differ in their ammonium uptake systems. Contrary to β-AOB, comammox Nitrospira genomes have single copies of the two central ammonia oxidation pathway operons. Similar to ammonia oxidizing archaea and some oligotrophic AOB strains, they lack genes involved in nitric oxide reduction. Furthermore, comammox Nitrospira genomes encode genes that might allow efficient growth at low oxygen concentrations. Regarding the evolutionary history of comammox Nitrospira, our analyses indicate that several genes belonging to the ammonia oxidation pathway could have been laterally transferred from β-AOB to comammox Nitrospira. We postulate that the absence of comammox genes in other sublineage II Nitrospira genomes is the result of subsequent loss.

The recent discovery of completely nitrifying Nitrospira demands a re-examination of nitrifying environments to evaluate their contribution to nitrogen cycling. To approach this challenge, tools are needed to detect and quantify comammox Nitrospira. We present primers for the simultaneous quantification and diversity assessement of both comammox Nitrospira clades. The primers cover a wide range of comammox diversity, spanning all available high quality sequences. We applied these primers to 12 groundwater-fed rapid sand filters, and found comammox Nitrospira to be abundant in all filters. Clade B comammox comprise the majority (∼75%) of comammox abundance in all filters. Nitrosomonadaceae were present in all filters, although at low abundance (mean = 1.8%). Ordination suggests that temperature impacts the structure of nitrifying communities, and in particular that increasing temperature favours Nitrospira. The nitrogen content of the filter material, sulfate concentration and surface ammonium loading rates shape the structure of the comammox guild in the filters. This work provides an assay for simultaneous detection and diversity assessment of clades A and B comammox Nitrospira, expands our current knowledge of comammox Nitrospira diversity and demonstrates a key role for comammox Nitrospira in nitrification in groundwater-fed biofilters.

In this study, a lab-scale rotating biological contactor (RBC) treating a synthetic NH 4 ؉ wastewater devoid of organic carbon and showing high N losses was examined for several important physiological and microbial characteristics. The RBC biofilm removed 89% ؎ 5% of the influent N at the highest surface load of approximately 8.3 g of N m؊2 day ؊1 , with N 2 as the main end product. In batch tests, the RBC biomass showed good aerobic and anoxic ammonium oxidation (147.8 ؎ 7.6 and 76.5 ؎ 6.4 mg of NH 4 ؉ -N g of volatile suspended solids [VSS] ؊1 day ؊1 , respectively) and almost no nitrite oxidation (< 1 mg of N g of VSS ؊1 day ؊1 ). The diversity of aerobic ammonia-oxidizing bacteria (AAOB) and planctomycetes in the biofilm was characterized by cloning and sequencing of PCR-amplified partial 16S rRNA genes. Phylogenetic analysis of the clones revealed that the AAOB community was fairly homogeneous and was dominated by Nitrosomonas-like species. Close relatives of the known anaerobic ammonia-oxidizing bacterium (AnAOB) Kuenenia stuttgartiensis dominated the planctomycete community and were most probably responsible for anoxic ammonium oxidation in the RBC. Use of a less specific planctomycete primer set, not amplifying the AnAOB, showed a high diversity among other planctomycetes, with representatives of all known groups present in the biofilm. The spatial organization of the biofilm was characterized using fluorescence in situ hybridization (FISH) with confocal scanning laser microscopy (CSLM). The latter showed that AAOB occurred side by side with putative AnAOB (cells hybridizing with probe PLA46 and AMX820/KST1275) throughout the biofilm, while other planctomycetes hybridizing with probe PLA886 (not detecting the known AnAOB) were present as very conspicuous spherical structures. This study reveals that long-term operation of a lab-scale RBC on a synthetic NH 4 ؉ wastewater devoid of organic carbon yields a stable biofilm in which two bacterial groups, thought to be jointly responsible for the high autotrophic N removal, occur side by side throughout the biofilm.Sustainable wastewater treatment systems are being developed that minimize energy consumption, CO 2 emission, and sludge production. However, these systems typically yield effluents rich in ammonium-nitrogen (NH 4 ϩ -N) and poor in biodegradable organic carbon, thereby making them less suitable for biological N removal through the conventional nitrification-denitrification sequence.Different N removal processes that could be successfully integrated in a sustainable wastewater treatment system are being studied. The Sharon process (single-reactor high-activity ammonium removal over nitrite) (15) uses the principle that at higher temperatures (30 to 35°C), pH 7 to 8, and a cell residence time of 1 day, aerobic ammonia-oxidizing bacteria (AAOB) are able to maintain themselves in the system while nitrite-oxidizing bacteria (NOB) are washed out. Given the reaction stoichiometry of the two groups of nitrifying bacteria (equations 1 and 2), this proces...

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Water and sanitation represent a key battlefront in combatting the spread of antimicrobial resistance (AMR). Basic water sanitation infrastructure is an essential first step towards protecting public health, thereby limiting the spread of pathogens and the need for antibiotics. AMR presents unique human health risks, meriting new risk assessment frameworks specifically adapted to water and sanitation-borne AMR. There are numerous exposure routes to AMR originating from human waste, each of which must be quantified for its relative risk to human health. Wastewater treatment plants play a vital role in centralized collection and treatment of human sewage, but there are numerous unresolved issues in terms of the microbial ecological processes occurring within them and the extent to which they attenuate or amplify AMR. Research is needed to advance understanding of the fate of resistant bacteria and antibiotic resistance genes in various waste management systems, depending on the local constraints and intended reuse applications. World Health Organization and national AMR action plans would benefit from a more holistic 'One Water' understanding. In this article we provide a framework for research, policy, practice and public engagement aimed at limiting the spread of AMR from water and sanitation in low-, medium- and high-income countries.

The goal of this study was to explore the relationship between metal extracellular sorption, intracellular accumulation, and nitrification inhibition. Metal sorption on nitrifying biomass was rapid and could be described by linear partitioning with partition coefficients (Kp) of 20.3 +/- 0.1, 0.4 +/- 0.0, 0.1 +/- 0.0, and 0.2 +/- 0.0 L/g biomass chemical oxygen demand for Cu, Zn, Ni, and Cd, respectively. On the other hand, intracellular Zn, Ni, and Cd concentrations continued to increase with time beyond 12 h after metal addition, whereas intracellular Cu attained equilibrium after 4 h. Metal internalization kinetics could be described by an intraparticle diffusion model, with characteristic diffusion time constants (td) of 9.4, 64.6, 80.5, and 66.1 h for Cu, Zn, Ni, and Cd, respectively. Ultimate internalized percentages of the total cell-associated metal were 1.4 +/- 0.0, 4.3 +/- 0.5,7.6 +/- 1.0, and 2.7 +/- 0.2% for Cu, Zn, Ni, and Cd, respectively. Nitrification inhibition was not a function of the sorbed metal fraction but correlated well with intracellular Zn, Ni, or Cd fractions. An intraparticle diffusion model coupled with a saturation-type biological toxicity model fit the inhibition data for varying initial Cd concentrations and exposure periods. In contrast, no relationship between intracellular or sorbed Cu concentrations and nitrification inhibition was observed. In the presence of 1 mM Cu, less than 13.3 +/- 10.5% cells remained viable as compared to 72.8 +/- 7.5,104.8 +/- 1.7, and 84.7 +/- 7.0% (assumed 100% viable cells in metal-free control) in the presence of 1 mM Zn, Ni, and Cd, respectively. Hence, the observations that inhibition by metals such as Zn, Ni, and Cd is related to their intracellular fraction and the slow kinetics of metal internalization indicate that metal inhibition can easily be underpredicted from short-term batch assays. Furthermore, the inhibitory mechanism of Cu was very different from Zn, Ni, and Cd and may involve rapid loss of membrane integrity.

Heavy metals have been postulated to cause significant nitrification inhibition. Using biomass from a well-controlled continuously operated lab-scale nitrifying bioreactor, the effect of nickel and cadmium on ammonium and nitrite oxidation was quantified. The extent of inhibition was calculated from the kinetics of ammonium oxidation and nitrite oxidation, inferred from maximum specific oxygen uptake rates (SOUR) measured in batch respirometric assays. Nickel and cadmium inhibited ammonium oxidation but not nitrite oxidation up to total analytical concentrations of approximately 1.0 mM. Metal fractions (total and free ion) were correlated with the extent of ammonium oxidation inhibition in the presence of various metal complexing agents (EDTA, NTA, citrate, SO4(2-)). Inhibition was not a function of the total analytical metal concentration but strongly correlated with free cation concentration, [Ni2+] or [Cd2+]. This relationship could be described by either an empirical noncompetitive inhibition model for [Ni2+] or a linear model in the case of [Cd2+]. Furthermore, the free Ni2+ or Cd2+ concentrations could be modulated by the addition of a strong chelating agent (e.g., EDTA), resulting in reduced deleterious effects. However, at elevated doses, EDTA itself impaired nitrification. In sum, predictions and mandatory strategies of nitrification inhibition by heavy metals should be based on free cation concentrations and not on total metal concentrations.

25Wastewater treatment plants (WWTPs) have long been suggested as reservoirs and sources of 26 antibiotic resistance genes (ARGs) in the environment. In a WWTP ecosystem, human enteric and 27 environmental bacteria are mixed and exposed to pharmaceutical residues, potentially favoring 28 genetic exchange and thus ARG transmission. However, the contribution of microbial communities 29 in WWTP to ARG dissemination remains poorly understood. Here, we examined for the first time 30 plasmid permissiveness of an activated sludge microbial community, by utilizing an established 31 fluorescent bioreporter system. The activated sludge microbial community was challenged in 32 standardized filter matings with one of the three multi-drug resistance plasmids (pKJK5, pB10 and 33 RP4) harbored by Escherichia coli or Pseudomonas putida. Different donor-plasmid combinations 34 had distinct transfer frequencies, ranging from 3 to 50 conjugation events per 100,000 cells of the 35 WWTP microbial community. In addition, transfer was observed to a broad phylogenetic range of 36 13 bacterial phyla with several taxa containing potentially pathogenic species. Preferential transfer 37 to taxa belonging to the predicted evolutionary host range of the plasmids was not observed. 38Overall, the ARG dissemination potential uncovered in WWTP communities calls for a thorough 39 risk assessment of ARG transmission across the wastewater system, before identifying possible 40 mitigation strategies. 41 42 43 44 45 46 47 48 3 Introduction 49