It has recently been suggested that oxygenic dismutation of NO into N 2 and O 2 may occur in the anaerobic methanotrophic "Candidatus Methylomirabilis oxyfera" and the alkane-oxidizing gammaproteobacterium HdN1. It may represent a new pathway in microbial nitrogen cycling catalyzed by a putative NO dismutase (Nod). The formed O 2 enables microbes to employ aerobic catabolic pathways in anoxic habitats, suggesting an ecophysiological niche space of substantial appeal for bioremediation and water treatment. However, it is still unknown whether this physiology is limited to "Ca. Methylomirabilis oxyfera" and HdN1 and whether it can be coupled to the oxidation of electron donors other than alkanes. Here, we report insights into an unexpected diversity and remarkable abundance of nod genes in natural and engineered water systems. Phylogenetically diverse nod genes were recovered from a range of contaminated aquifers and N-removing wastewater treatment systems. Together with nod genes from "Ca. Methylomirabilis oxyfera" and HdN1, the novel environmental nod sequences formed no fewer than 6 well-supported phylogenetic clusters, clearly distinct from canonical NO reductase (quinoldependent NO reductase [qNor] and cytochrome c-dependent NO reductase [cNor]) genes. The abundance of nod genes in the investigated samples ranged from 1.6 ϫ 10 7 to 5.2 ϫ 10 10 copies · g Ϫ1 (wet weight) of sediment or sludge biomass, accounting for up to 10% of total bacterial 16S rRNA gene counts. In essence, NO dismutation could be a much more widespread physiology than currently perceived. Understanding the controls of this emergent microbial capacity could offer new routes for nitrogen elimination or pollutant remediation in natural and engineered water systems.IMPORTANCE NO dismutation into N 2 and O 2 is a novel process catalyzed by putative NO dismutase (Nod). To date, only two bacteria, the anaerobic methaneoxidizing bacterium "Ca. Methylomirabilis oxyfera" and the alkane-oxidizing gammaproteobacterium HdN1, are known to harbor nod genes. In this study, we report efficient molecular tools that can detect and quantify a wide diversity of nod genes in environmental samples. A surprisingly high diversity and abundance of nod genes were found in contaminated aquifers as well as wastewater treatment systems. This evidence indicates that NO dismutation may be a much more widespread physiology in natural and man-made environments than currently perceived. The molecular tools presented here will facilitate further studies on these enigmatic microbes in the future.KEYWORDS nitric oxide (NO) dismutation, NO dismutase, oxygenic denitrification,

Deammonification's performance and associated nitrous oxide emissions (NO) depend on operational conditions. While studies have investigated factors for high performances and low emissions separately, this study investigated optimizing deammonification performance while simultaneously reducing NO emissions. Using a design of experiment (DoE) method, two models were developed for the prediction of the nitrogen removal rate and NO emissions during single-stage deammonification considering three operational factors (i.e., pH value, feeding and aeration strategy). The emission factor varied between 0.7±0.5% and 4.1±1.2% at different DoE-conditions. The nitrogen removal rate was predicted to be maximized at settings of pH 7.46, intermittent feeding and aeration. Conversely, emissions were predicted to be minimized at the design edges at pH 7.80, single feeding, and continuous aeration. Results suggested a weak positive correlation between the nitrogen removal rate and NO emissions, thus, a single optimizing operational set-point for maximized performance and minimized emissions did not exist.

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Abstract:A two-stage deammonification pilot plant with two different second-stage reactors, namely a sequencing batch reactor (SBR) with suspended sludge and a moving bed biofilm reactor (MBBR) with biofilm carriers, was investigated over a 1.5-year period to compare reactor performances. Additionally, dissolved nitrous oxide (N 2 O) was measured to determine the reactors' N 2 O formation potential. Although the nitritation performance was moderate (NO 2 -N/NH 4 -N effluent ratio of 0.32 ± 0.15 in combination with SBR and 0.25 ± 0.14 with MBBR), nitrogen turnover and degradation rates exceeding 500 g N/(m 3 ·day) and 80%, respectively, were achieved in both second stages, yet requiring additional aeration. The SBR's average nitrogen removal was 19% higher than the MBBR's; however, the SBR's nitrite influent concentration was comparably elevated. Concerning N 2 O formation, the nitritation reactor exhibited the lowest N 2 O concentrations, while the buffer tank, interconnecting the first and second stages, exhibited the highest N 2 O concentrations of all reactors. Given these high concentrations, a transfer of N 2 O into the second stage was observed, where anoxic phases enabled N 2 O reduction. Frequent biomass removal and a decreased hydraulic retention time in the buffer tank would likely minimize N 2 O formation. For the second stage, enabling anoxic periods in the intermittent aeration cycles right after feeding to support N 2 O reduction and thus minimize the stripping effects or the implementation of a complete anoxic ammonium oxidation will mitigate N 2 O emissions.

Different configurations of photoacoustic (PA) setups for the online-measurement of gaseous NO, employing semiconductor lasers at 2.9 and 4.5 μm, were developed and tested. Their performance was assessed with respect to the analysis of NO emissions from wastewater treatment plants. For this purpose, the local NO emissions of a wastewater treatment bioreactor was sampled by a dedicated mobile sampling device, and the total NO emissions were analyzed in the gastight headspace of the bioreactor. We found that the use of a quantum-cascade laser emitting at about 4.53 μm, operated in a wavelength modulation mode, in combination with a conventional longitudinal PA cell yielded the highest sensitivity (<100 ppbv). However, we also observed a strong cross-sensitivity to humidity, which can be explained by increased V-T relaxation. This observation in combination with the limited dynamic range (max conc. ∼ 3000 ppmv) led us to the use of the less-sensitive but spectroscopically more robust 2.9 μm laser. A detection limit below 1 ppmv, a dynamic range of more than 4 orders of magnitude, no influence of humidity or any other substance relevant to the off-gas analysis, as well as a comparable low price of the laser source made it the ideal tool for NO analyses of the off-gas of a wastewater treatment plant. Such a system was implemented successfully in a full-scale wastewater treatment plant. The results regarding the comparison of different PA setups can be transferred to other systems, and the optimum performance can be selected according to the specific demands.