A positive contribution to nitrogen removal by a novel NOB in a full-scale duck wastewater treatment system.

Nitrite-oxidizing bacteria (NOB) are undesirable in the anaerobic ammonium oxidation (anammox)-driven nitrogen removal technologies in the modern wastewater treatment plants (WWTPs). Diverse strategies have been developed to suppress NOB based on their physiological properties that we have understood. But our knowledge of the diversity and mechanisms employed by NOB for survival in the modern WWTPs remains limited. Here, Three NOB species (NOB01-03) were recovered from the metagenomic datasets of a full-scale WWTP treating duck breeding wastewater. Among them, NOB01 and NOB02 were classified as newly identified lineage VII, tentatively named Candidatus (Ca.) Nitrospira NOB01 and Ca. Nitrospira NOB02. Analyses of genomes and in situ transcriptomes revealed that these two novel NOB were active and showed a high metabolic versatility. The transcriptional activity of Ca. Nitrospira could be detected in all tanks with quite different dissolved oxygen (DO) (0.01-5.01 mg/L), illustrating Ca. Nitrospira can survive in fluctuating DO conditions. The much lower Ca. Nitrospira abundance on the anammox bacteria-enriched sponge carrier likely originated from the intensification substrate (NO2 -) competition from anammox and denitrifying bacteria. In particular, a highlight is that Ca. Nitrospira encoded and treanscribed cyanate hydratase (CynS), amine oxidase, urease (UreC), and copper-containing nitrite reductase (NirK) related to ammonium and NO production, driving NOB to interact with the co-existed AOB and anammox bacteria. Ca. Nitrospira strains NOB01 and NOB02 showed quite different niche preference in the same aerobic tank, which dominanted the NOB communities in activated sludge and biofilm, respectively. In addition to the common rTCA cycle for CO2 fixation, a reductive glycine pathway (RGP) was encoded and transcribed by NOB02 likely for CO2 fixation purpose. Additionally, a 3b group hydrogenase and respiratory nitrate reductase were uniquely encoded and transcribed by NOB02, which likely confer a survival advantage to this strain in the fluctuant activated sludge niche. The discovery of this new genus significantly broadens our understanding of the ecophysiology of NOB. Furthermore, the impressive metabolic versatility of the novel NOB revealed in this study advances our understanding of the survival strategy of NOB and provides valuable insight for suppressing NOB in the anammox-based WWTP.

Distinct microbial nitrogen cycling processes in the deepest part of the ocean.

The Mariana Trench (MT) is the deepest part of the ocean on Earth. Previous studies have described the microbial community structures and functional potential in the seawater and surface sediment of MT. Still, the metabolic features and adaptation strategies of the microorganisms involved in nitrogen cycling processes are poorly understood. In this study, comparative metagenomic approaches were used to study microbial nitrogen cycling in three MT habitats, including hadal seawater [9,600-10,500 m below sea level (mbsl)], surface sediments [0-46 cm below seafloor (cmbsf) at a water depth between 7,143 and 8,638 mbsl], and deep sediments (200-306 cmbsf at a water depth of 8,300 mbsl). We identified five new nitrite-oxidizing bacteria (NOB) lineages that had adapted to the oligotrophic MT slope sediment, via their CO2 fixation capability through the reductive tricarboxylic acid (rTCA) or Calvin-Benson-Bassham (CBB) cycle; an anammox bacterium might perform aerobic respiration and utilize sedimentary carbohydrates for energy generation because it contains genes encoding type A cytochrome c oxidase and complete glycolysis pathway. In seawater, abundant alkane-oxidizing Ketobacter species can fix inert N2 released from other denitrifying and/or anammox bacteria. This study further expands our understanding of microbial life in the largely unexplored deepest part of the ocean. IMPORTANCE: The metabolic features and adaptation strategies of the nitrogen cycling microorganisms in the deepest part of the ocean are largely unknown. This study revealed that anammox bacteria might perform aerobic respiration in response to nutrient limitation or O2 fluctuations in the Mariana Trench sediments. Meanwhile, an abundant alkane-oxidizing Ketobacter species could fix N2 in hadal seawater. This study provides new insights into the roles of hadal microorganisms in global nitrogen biogeochemical cycles. It substantially expands our understanding of the microbial life in the largely unexplored deepest part of the ocean.

Metabolic and phylogenetic diversity in the phylum Nitrospinota revealed by comparative genome analyses.

The most abundant known nitrite-oxidizing bacteria in the marine water column belong to the phylum Nitrospinota. Despite their importance in marine nitrogen cycling and primary production, there are only few cultured representatives that all belong to the class Nitrospinia. Moreover, although Nitrospinota were traditionally thought to be restricted to marine environments, metagenome-assembled genomes have also been recovered from groundwater. Over the recent years, metagenomic sequencing has led to the discovery of several novel classes of Nitrospinota (UBA9942, UBA7883, 2-12-FULL-45-22, JACRGO01, JADGAW01), which remain uncultivated and have not been analyzed in detail. Here, we analyzed a nonredundant set of 98 Nitrospinota genomes with focus on these understudied Nitrospinota classes and compared their metabolic profiles to get insights into their potential role in biogeochemical element cycling. Based on phylogenomic analysis and average amino acid identities, the highly diverse phylum Nitrospinota could be divided into at least 33 different genera, partly with quite distinct metabolic capacities. Our analysis shows that not all Nitrospinota are nitrite oxidizers and that members of this phylum have the genomic potential to use sulfide and hydrogen for energy conservation. This study expands our knowledge of the phylogeny and potential ecophysiology of the phylum Nitrospinota and offers new avenues for the isolation and cultivation of these elusive bacteria.

Facet impact of CeO2@C 2D core-shell structure on electrochemical reaction kinetic factor and efficient detection of nitrite.

Fine-tuning the surface structure of transition metal oxides at the atomic level is a promising way to improve the catalytic properties of materials. However, the influence of crystal surface structure on electrode reaction kinetics is still limited. In this study, we propose an in-situ synthesis strategy to obtain two-dimensional carbon/cerium oxide core-shell nanosheets by thermal decomposition of Ce-MOF nanosheets grown on the surface of carbon nanostructures, and fine-tuning the surface structure by introducing oxygen vacancies through defect engineering during the oxide nucleation process is conducted to obtain controllable exposed {111} and {110} surface CeO2@C composites. Both experiments and theoretical calculations show that the {110} -dominated nanocomplex (CeO2@C-350S) has better kinetic behavior and catalytic activity due to its abundant surface defects, which is manifested in higher active surface area, richer carrier concentration, and better promotion of diffusion and adsorption. In addition, CeO2@C-350S electrode has an extremely wide linear range and good stability in the electrochemical detection of nitrite. After 1000 times of the accelerated cycle experiments, CeO2@C-350S electrode still maintains 79.3 % of its initial current response, and recovers to 87.3 % after 10 min of stopping the test. The electrode stability is excellent, which is attributed to the clever carbon shell structure of the material. This synthesis strategy can be extended to other carbon-based oxide composite catalysts to improve the electrocatalytic performance and overall stability by adjusting the surface structure.

Nitrite Oxidation in Wastewater Treatment: Microbial Adaptation and Suppression Challenges.

Microbial nitrite oxidation is the primary pathway that generates nitrate in wastewater treatment systems and can be performed by a variety of microbes: namely, nitrite-oxidizing bacteria (NOB). Since NOB were first isolated 130 years ago, the understanding of the phylogenetical and physiological diversities of NOB has been gradually deepened. In recent endeavors of advanced biological nitrogen removal, NOB have been more considered as a troublesome disruptor, and strategies on NOB suppression often fail in practice after long-term operation due to the growth of specific NOB that are able to adapt to even harsh conditions. In line with a review of the history of currently known NOB genera, a phylogenetic tree is constructed to exhibit a wide range of NOB in different phyla. In addition, the growth behavior and metabolic performance of different NOB strains are summarized. These specific features of various NOB (e.g., high oxygen affinity of Nitrospira, tolerance to chemical inhibitors of Nitrobacter and Candidatus Nitrotoga, and preference to high temperature of Nitrolancea) highlight the differentiation of the NOB ecological niche in biological nitrogen processes and potentially support their adaptation to different suppression strategies (e.g., low dissolved oxygen, chemical treatment, and high temperature). This review implicates the acquired physiological characteristics of NOB to their emergence from a genomic and ecological perspective and emphasizes the importance of understanding physiological characterization and genomic information in future wastewater treatment studies.

A novel Nitrospira lineage isolated from activated sludge using elevated temperatures.

The genus Nitrospira represents the dominant nitrite-oxidizing clade in most wastewater treatment plants (WWTPs) globally, and several Nitrospira strains have been isolated from activated sludge. Using a pre-enrichment strategy with alternating nitrifying and denitrifying conditions, followed by incubation at elevated temperatures, we isolated a novel Nitrospira species, named Nitrospira tepida. This moderately thermophilic species with optimal growth between 37 and 45°C is only distantly related to other Nitrospira and forms a novel lineage VII within the genus, together with few environmental 16S rRNA gene sequences predominantly detected in thermal wastewater or oxygen-limited systems. Genomic and physiological analyses revealed remarkable differences between N. tepida and two other isolates previously obtained from the same WWTP, suggesting niche differentiation between these nitrite oxidizers. N. tepida grows in aggregates, and tolerates nitrite and nitrate concentrations of up to 20 mM and 40 mM, respectively. The Km value for nitrite of N. tepida is 77 ± 26 µM. In summary, this novel Nitrospira lineage seems to be well-adapted for wastewater treatment processes at elevated temperatures and limited aeration, conditions that potentially reduce operational costs of such systems.

Design strategy and mechanism of nitrite oxidation suppression of elevated loading rate partial nitritation system.

There is a current need for a low operational intensity, effective and small footprint system to achieve stable partial nitritation for subsequent anammox treatment at mainstream municipal wastewaters. This research identifies a unique design strategy using an elevated total ammonia nitrogen (TAN) surface area loading rate (SALR) of 5 g TAN/m2.d to achieve cost-effective, stable, and elevated rates of partial nitritation in a moving bed biofilm reactor (MBBR) system under mainstream conditions. The elevated loaded partial nitritation MBBR system achieves a TAN surface area removal rate (SARR) of 2.01 ± 0.07 g TAN/m2.d and NO2 --N: NH4 +-N stoichiometric ratio of 1.15:1, which is appropriate for downstream anammox treatment. The elevated TAN SALR design strategy promotes nitrite-oxidizing bacteria (NOB) activity suppression rather than a reduction in NOB population as the reason for the suppression of nitrite oxidation in the mainstream elevated loaded partial nitritation MBBR system. NOB activity is limited at an elevated TAN SALR likely due to thick biofilm embedding the NOB population and competition for dissolved oxygen (DO) with ammonia-oxidizing bacteria for TAN oxidation to nitrite within the biofilm structure, which ultimately limits the uptake of DO by NOB in the system. Therefore, this design strategy offers a cost-effective and efficient alternative for mainstream partial nitritation MBBR systems at water resource recovery facilities.

Nitrification kinetics, N2O emission, and energy use in intermittently aerated hybrid reactor under different organic loading rates.

This study investigated the impact of intermittent aeration strategies and reduction in the reactor's organic and nitrogen loading rates on the course of particular stages of the nitrification process, as well as energy consumption and N2O emissions in a hybrid reactor with nitrification/denitrification. Each of the analysed series revealed the greatest ammonia oxidation activity in activated sludge flocs. The highest activity of nitrite nitrogen oxidation was demonstrated in the case of biofilm. A reduction in the reactor's organic and nitrogen loading rate value had a greater effect on changes in the activity of ammonia-oxidizing bacteria than nitrite-oxidizing bacteria. In a system where the operation of air pumps was controlled through switching them and off according to the adopted ratio between non-aerated and aerated sub-phase times and the assumed oxygen concentration, a reduction in the duration of aerated sub-phases caused no decrease in energy use for aeration. Lower N2O emission was recorded when the reactor operated with a longer duration of aerated sub-phases. Supplementary Information: The online version contains supplementary material available at 10.1007/s13762-022-04715-6.

Comammox Nitrospira Clade B is the most abundant complete ammonia oxidizer in a dairy pasture soil and inhibited by dicyandiamide and high ammonium concentrations.

The recent discovery of comammox Nitrospira, a complete ammonia oxidizer, capable of completing the nitrification on their own has presented tremendous challenges to our understanding of the nitrification process. There are two divergent clades of comammox Nitrospira, Clade A and B. However, their population abundance, community structure and role in ammonia and nitrite oxidation are poorly understood. We conducted a 94-day microcosm study using a grazed dairy pasture soil amended with urea fertilizers, synthetic cow urine, and the nitrification inhibitor, dicyandiamide (DCD), to investigate the growth and community structure of comammox Nitrospira spp. We discovered that comammox Nitrospira Clade B was two orders of magnitude more abundant than Clade A in this fertile dairy pasture soil and the most abundant subcluster was a distinctive phylogenetic uncultured subcluster Clade B2. We found that comammox Nitrospira Clade B might not play a major role in nitrite oxidation compared to the role of canonical Nitrospira nitrite-oxidizers, however, comammox Nitrospira Clade B is active in nitrification and the growth of comammox Nitrospira Clade B was inhibited by a high ammonium concentration (700 kg synthetic urine-N ha-1) and the nitrification inhibitor DCD. We concluded that comammox Nitrospira Clade B: (1) was the most abundant comammox in the dairy pasture soil; (2) had a low tolerance to ammonium and can be inhibited by DCD; and (3) was not the dominant nitrite-oxidizer in the soil. This is the first study discovering a new subcluster of comammox Nitrospira Clade B2 from an agricultural soil.

Ammonia oxidation by novel "Candidatus Nitrosacidococcus urinae" is sensitive to process disturbances at low pH and to iron limitation at neutral pH.

Acid-tolerant ammonia-oxidizing bacteria (AOB) can open the door to new applications, such as partial nitritation at low pH. However, they can also be problematic because chemical nitrite oxidation occurs at low pH, leading to the release of harmful nitrogen oxide gases. In this publication, the role of acid-tolerant AOB in urine treatment was explored. On the one hand, the technical feasibility of ammonia oxidation under acidic conditions for source-separated urine with total nitrogen concentrations up to 3.5 g-N L-1 was investigated. On the other hand, the abundance and growth of acid-tolerant AOB at more neutral pH was explored. Under acidic conditions (pH of 5), ammonia oxidation rates of 500 mg-N L-1 d-1 and 10 g-N g-VSS-1 d-1 were observed, despite high concentrations of 15 mg-N L-1 of the AOB-inhibiting compound nitrous acid and low concentration of 0.04 mg-N L-1 of the substrate ammonia. However, ammonia oxidation under acidic conditions was very sensitive to process disturbances. Even short periods of less than 12 h without oxygen or without influent resulted in a complete cessation of ammonia oxidation with a recovery time of up to two months, which is a problem for low maintenance applications such as decentralized treatment. Furthermore, undesirable nitrogen losses of about 10% were observed. Under acidic conditions, a novel AOB strain was enriched with a relative abundance of up to 80%, for which the name "Candidatus (Ca.) Nitrosacidococcus urinae" is proposed. While Nitrosacidococcus members were present only to a small extent (0.004%) in urine nitrification reactors operated at pH values between 5.8 and 7, acid-tolerant AOB were always enriched during long periods without influent, resulting in an uncontrolled drop in pH to as low as 2.5. Long-term experiments at different pH values showed that the activity of "Ca. Nitrosacidococcus urinae" decreased strongly at a pH of 7, where they were also outcompeted by the acid-sensitive AOB Nitrosomonas halophila. The experiment results showed that the decreased activity of "Ca. Nitrosacidococcus urinae" correlated with the limited availability of dissolved iron at neutral pH.

The Stochastic Assembly of Nitrobacter winogradskyi-Selected Microbiomes with Heterotrophs from Sewage Sludge or Grassland Soil.

Chemolitho-autotrophic microorganisms like the nitrite-oxidizing Nitrobacter winogradskyi create an environment for heterotrophic microorganisms that profit from the production of organic compounds. It was hypothesized that the assembly of a community of heterotrophic microorganisms around N. winogradskyi depends on the ecosystem from which the heterotrophs are picked. To test this hypothesis, pure cultures of N. winogradskyi were grown in continuously nitrite-fed bioreactors in a mineral medium free of added organic carbon that had been inoculated with diluted sewage sludge or with a suspension from a grassland soil. Samples for chemical and 16S rRNA gene amplicon analyses were taken after each volume change in the bioreactor. At the end of the enrichment runs, samples for shotgun metagenomics were also collected. Already after two volume changes, the transformations in community structure became less dynamic. The enrichment of heterotrophs from both sewage and soil was highly stochastic and yielded different dominant genera in most of the enrichment runs that were independent of the origin of the inoculum. Hence, the hypothesis had to be refuted. Notwithstanding the large variation in taxonomic community structure among the enrichments, the functional compositions of the communities were statistically not different between soil- and sludge-based enrichments. IMPORTANCE In the process of aerobic nitrification, nitrite-oxidizing bacteria together with ammonia-oxidizing microorganisms convert mineral nitrogen from its most reduced appearance, i.e., ammonium, into its most oxidized form, i.e., nitrate. Because the form of mineral nitrogen has large environmental implications, nitrite-oxidizing bacteria such as Nitrobacter winogradskyi play a central role in the global biogeochemical nitrogen cycle. In addition to this central role, the autotrophic nitrite-oxidizing bacteria also play a fundamental role in the global carbon cycle. They form the basis of heterotrophic food webs, in which the assimilated carbon is recycled. Little is known about the heterotrophic microorganisms that participate in these food webs, let alone their assembly in different ecosystems. This study showed that the assembly of microbial food webs by N. winogradskyi was a highly stochastic process and independent of the origin of the heterotrophic microorganisms, but the functional characteristics of the different food webs were similar.

Binary organic-inorganic nanocomposite of polyaniline-MnO2 for non-enzymatic electrochemical detection of environmental pollutant nitrite.

Due to wide usage as nitrogen fertilizer in agriculture and food additive in industry, nitrite, as one of inorganic environmental pollutants, could cause detrimental effects to the ecological environment. Therefore, accurate, sensitive and rapid detection of nitrite is necessary. In this work, binary hybrid polyaniline-MnO2 organic-inorganic nanocomposite is prepared chemically and characterized via X-ray diffraction spectroscopy, Fourier-transform infrared spectroscopy, scanning electron microscopy and energy-dispersive X-ray spectroscopy. Polyaniline-MnO2 organic-inorganic nanocomposite serves as excellent electrode modifier for electrochemical sensing of nitrite by two modes of cyclic voltammetry and chronoamperometry, achieving broad linear ranges and low limits of detection for both methods. Moreover, the organic-inorganic nanocomposite displays satisfactory sensing performance in real water sample analysis. Amine and imino groups of polyaniline contribute to the better adsorption behavior of nitrite onto the nanocomposite, which improves the nanocomposite's sensing performance. In summary, the synergistic effects between polyaniline and MnO2 is taken advantaged in the nanocomposite for effective electrochemical sensor development.

Nitrite oxidation in oxygen-deficient conditions during landfill leachate treatment.

Until recently, all known nitrite oxidation occurred in oxygen-rich conditions but now the oxidation of nitrite into nitrate within a low oxygen or anoxic environment has been observed in the ocean. However, this phenomenon is rarely reported in wastewater treatments and its mechanism is unknown. In this study, the partial nitrification and nitrite oxidation were conducted in no enough oxygen in order to remove nitrogen from landfill leachate, save energy, and save money. The results show that the NH4+-N removal efficiency was 99.4%. During phase I of the anaerobic sequential batch reactor (ASBR), no change in Chemical Oxygen Demand (COD) and ammonium were detected. The nitrite concentration decreased from 107 ± 3 mg/L to 0.16 mg/L during 96 h of oxygen- deficiency, while NO3--N increased from 152.5 ± 3 mg/L to 253.65 ± 3 mg/L. The main microorganisms involved in this reaction in the ASBR were Nitrite-Oxidizing Bacteria (NOB), including Nitrospira and Nitrolancea, their relative abundances were 3.56% and 0.13%, respectively. The major NOB (Nitrospira) were confirmed by the further metagenomic binning analysis. This finding shows that nitrite oxidation can occur in oxygen-deficient conditions with specific NOB.

Effect of peracetic acid solution on a nitrifying culture: Kinetics, inhibition, cellular and transcriptional responses.

Peracetic acid (PAA) has been widely used as a disinfectant in many industries. However, information related to the potential inhibitory effect of PAA solutions (PAA and H2O2) on biological wastewater treatment processes is very limited. The work reported here assessed the effect of PAA and H2O2 solutions on nitrification kinetics and inhibition, cellular level responses and gene expression of a suspended-growth nitrifying culture. The initial ammonia removal and nitrate production rates significantly decreased at 1/0.14 to 3/0.42 mg/L PAA/H2O2. H2O2 up to 3 mg/L did not impact nitrification, cell viability or related respiratory activities; thus, the impact of the PAA solution is attributed to PAA alone or in some combination with H2O2. Nitrification inhibition by PAA was predominantly related to enzyme inhibition, rather than to loss of cell viability and/or cell lysis. PAA and H2O2 negatively affected Nitrosomonas but resulted in Nitrosospira enrichment. Most nitrogen metabolism-related genes (e.g., hydroxylamine oxidoreductase and nitrite oxidoreductase genes) as well as oxidase genes (e.g., cytochrome c oxidase, catalase-peroxidase, and peroxidase genes) were upregulated in PAA- and H2O2-amended cultures. Major ATPase genes were downregulated while ATP synthase genes upregulated under the effect of PAA and/or H2O2. Upregulation of ATP-dependent protease genes indicates protein damage predominantly caused by PAA rather than H2O2. The transcriptional level of genes related to cell division and DNA repair did not show a particular pattern; thus, cell division functionality and DNA integrity were not significantly affected by PAA or H2O2. The results of this study have significant implications in the design and operation of effective biological nitrogen removal systems for the treatment of PAA-bearing wastewater.

Response of substrate kinetics and biological mechanisms to various pH constrains for cultured Nitrobacter and Nitrospira in nitrifying bioreactor.

Nitrite (NO2-) oxidation is an essential step of biological nitrogen cycling in natural ecosystems, and is performed by chemolithoautotrophic nitrite-oxidizing bacteria (NOB). Although Nitrobacter and Nitrospira are regarded as representative NOB in nitrification systems, little attention has focused on kinetic characterisation of the coexistence of Nitrobacter and Nitrospira at various pH values. Here, we evaluate the substrate kinetics, biological mechanism and microbial community dynamics of an enrichment culture including Nitrobacter (17.5 ± 0.9%) and Nitrospira (7.2 ± 0.6%) in response to various pH constrains. Evaluation of the Monod equation at pH 6.0, 6.5, 7.0, 7.5, 8.0 and 8.5 showed that the enrichment had maximum rate (rmax) and maximum substrate affinity (KS) for NO2- oxidation at pH 7.0, which was also supported by the largest absolute abundance of Nitrobacter nxrA (5.26 × 107 copies per g wet sludge) and Nitrospira nxrB (1.975 × 109 copies per g wet sludge) genes. Moreover, the predominant species for the Nitrobacter-like nxrA were N. vulgaris and N. winogradskyi, while for the Nitrospira-like nxrB, the predominant species were N. japonica, N. calida and Ca. N. bockiana. Furthermore, the rmax was strongly and positively correlated with the abundance of the Nitrobacter nxrA or Nitrospira nxrB genes, or N. winogradsk, whereas KS was positively correlated with the abundance of Nitrobacter nxrA or Nitrospira nxrB genes or Ca. N. bockiana. Overall, this study could improve basis kinetic parameters and biological mechanism of NO2- oxidation in WWTPs.

Facile Controlled Synthesis of Pd-ZnO Nanostructures for Nitrite Detection.

The electrocatalytic characteristics of nanostructures are significantly affected by surface structure. The strict regulation of structural characteristics is highly beneficial for the creation of novel nanocatalysts with enhanced electrocatalytic performance. This work reports a nitrite electrochemical sensor based on novel flower-like Pd-ZnO nanostructures. The Pd-ZnO nanocatalysts were synthesized through a simple hydrothermal method, and their morphology and structure were characterized via field-emission scanning electron microscopy (FE-SEM), X-ray photoelectron spectroscopy (XPS), and X-ray diffraction (XRD). Their electrocatalytical performance in the nitrite oxidation reaction was studied via cyclic voltammetry (CV) and the amperometric technique. Compared to pure ZnO and Pd nanoparticles, the Pd-ZnO nanostructures exhibited enhanced electrochemical performance in the nitrite oxidation reaction. In order to investigate the relationships between the structures of Pd-ZnO nanocatalysts and the corresponding electrocatalytic performances, different surface morphologies of Pd-ZnO nanocatalysts were fabricated by altering the solution pH. It was found that the flower-like Pd-ZnO nanostructures possessed larger effective surface areas and faster electron transfer rates, resulting in the highest electrocatalytic performance in the nitrite oxidation reaction. The designed nitrite sensor based on flower-like Pd-ZnO displayed a wide concentration linear range of 1 µM-2350 µM, a low detection limit of 0.2 µM (S/N of 3), and high sensitivity of 151.9 µA mM-1 cm-2. Furthermore, the proposed sensor exhibited perfect selectivity, excellent reproducibility, and long-time stability, as well as good performance in real sample detection.

Activity-Based Cell Sorting Reveals Resistance of Functionally Degenerate Nitrospira during a Press Disturbance in Nitrifying Activated Sludge.

Managing and engineering activated sludge wastewater treatment microbiomes for low-energy nitrogen removal requires process control strategies to stop the oxidation of ammonium at nitrite. Our ability to out-select nitrite-oxidizing bacteria (NOB) from activated sludge is challenged by their metabolic and physiological diversity, warranting measurements of their in situ physiology and activity under selective growth pressures. Here, we examined the stability of nitrite oxidation in activated sludge during a press disturbance induced by treating a portion of return activated sludge with a sidestream flow containing free ammonia (FA) at 200 mg NH3-N/liter. The nitrite accumulation ratio peaked at 42% by day 40 in the experimental bioreactor with the press disturbance, while it did not increase in the control bioreactor. A subsequent decrease in nitrite accumulation within the experimental bioreactor coincided with shifts in dominant Nitrospira 16S rRNA amplicon sequence variants (ASVs). We applied bioorthogonal noncanonical amino acid tagging (BONCAT) coupled with fluorescence-activated cell sorting (FACS) to investigate changes in the translational activity of NOB populations throughout batch exposure to FA. BONCAT-FACS confirmed that the single Nitrospira ASV washed out of the experimental bioreactor had reduced translational activity following exposure to FA, whereas the two Nitrospira ASVs that emerged after process acclimation were not impacted by FA. Thus, the coexistence of functionally degenerate and physiologically resistant Nitrospira populations provided resilience to the nitrite-oxidizing function during the press disturbance. These results highlight how BONCAT-FACS can resolve ecological niche differentiation within activated sludge and inform strategies to engineer and control microbiome function. IMPORTANCE Nitrogen removal from activated sludge wastewater treatment systems is an energy-intensive process due to the large aeration requirement for nitrification. This energy footprint could be minimized with engineering control strategies that wash out nitrite-oxidizing bacteria (NOB) to limit oxygen demands. However, NOB populations can have a high degree of physiological diversity, and it is currently difficult to decipher the behavior of individual taxa during applied selective pressures. Here, we utilized a new substrate analog probing approach to measure the activity of NOB at the cellular translational level in the face of a press disturbance applied to the activated sludge process. Substrate analog probing corroborated the time series reactor sampling, showing that coexisting and functionally degenerate Nitrospira populations provided resilience to the nitrite oxidation process. Taken together, these results highlight how substrate analog approaches can illuminate in situ ecophysiologies within shared niches, and can inform strategies to improve microbiome engineering and management.

Novel insights into overcoming nitrite oxidation bacteria acclimatization problem in treatment of high-ammonia wastewater through partial nitrification.

A partial nitrification sequencing batch reactor was operated to reveal mechanisms behind nitrite oxidation bacteria (NOB) acclimatization in high-ammonia wastewater treatment. The influent NH4+-N increased stepwise from 499.7 ± 4.2 mg/L to 6994.5 ± 7.5 mg/L with initial free ammonia (FA) concentration rising from 37.9 ± 3.2 mg NH3-N/L to 715.3 ± 47.3 mg NH3-N/L, respectively. NOB acclimatized this FA range with NO3--N production increasing from 29.2 ± 2.6 mg/L to 144.1 ± 31.0 mg/L in a cycle, which was caused by the shift of dominant NOB genus from Nitrospira to Nitrolencea. Nitrosomonas as ammonia oxidation bacteria, could sustain its activity of 62.1 ± 0.1 mg NH4+-N/(gVSS∙L∙h) under the same condition. Hydroxylamine addition could be implemented as an emergency measure to alleviate NOB acclimatization in short-term operation. The findings expanded knowledge about NOB acclimatization types and provided novel insights for addressing this problem in a targeted way.

Hydroxylamine and the nitrogen cycle: A review.

Aerobic ammonium oxidizing bacteria were first isolated more than 100 years ago and hydroxylamine is known to be an intermediate. The enzymatic steps involving hydroxylamine conversion to nitrite are still under discussion. For a long time it was assumed that hydroxylamine was directly converted to nitrite by a hydroxylamine oxidoreductase. Recent enzymatic evidences suggest that the actual product of hydroxylamine conversion is NO and a third, yet unknown, enzyme further converts NO to nitrite. More recently, ammonium oxidizing archaea and complete ammonium oxidizing bacteria were isolated and identified. Still the central nitrogen metabolism of these microorganisms presents to researchers the same puzzle: how hydroxylamine is transformed to nitrite. Nitrogen losses in the form of NO and N2O have been identified in all three types of aerobic ammonium oxidizing microorganisms and hydroxylamine is known to play a significant role in the formation. Yet, the pathways and the factors promoting the greenhouse gas emissions are to be fully characterized. Hydroxylamine also plays a yet poorly understood role on anaerobic ammonium oxidizing bacteria and is known to inhibit nitrite oxidizing bacteria. In this review, the role of this elusive intermediate in the metabolism of different key players of the nitrogen cycle is discussed, as well as the putative importance of hydroxylamine as a key nitrogen metabolite for microbial interactions within microbial communities and engineered systems. Overall, for the first time putting together the acquired knowledge about hydroxylamine and the nitrogen cycle over the years in a review, setting potential hypothesis and highlighting possible next steps for research.

Worm-like gold nanowires assembled carbon nanofibers-CVD graphene hybrid as sensitive and selective sensor for nitrite detection.

The development of a rapid, selective, and sensitive sensor to precisely monitor nitrite oxidation is of growing importance, given the strong interest in the protection of drinking water quality, treatment of wastewater, food production, and control of remediation processes. In this research, we successfully fabricated a hybrid originated from worm-like gold nanowires (Au WNWs) assembled on a high-quality carbon nanofibers-graphene (CNFs-Gr) hybrid network through a facile synthesis method. The hybrid as a binder-free sensor exhibited excellent activity towards nitrite detection in phosphate buffer solution (pH of 7.4) with a wide linear detection range of (1.98 µM - 3.77 mM), excellent sensitivity of 836 µA cm-2 mM-1, low detection limit of 1.24 µM, and long-term durability. The results were attributed to a special synergistic effect originating from unique hybridization of Au WNWs with large-area CNFs-Gr network to produce more electroactive sites and excellent conductivity, favorably boosting catalytic performance of the sensor. The successful fabrication of Au WNWs/CNFs-Gr suggested an interesting candidate for practically determining low-level nitrite in analytical applications.