Hydroxylamine production by Alcaligenes faecalis challenges the paradigm of heterotrophic nitrification.

Heterotrophic nitrifiers continue to be a hiatus in our understanding of the nitrogen cycle. Despite their discovery over 50 years ago, the physiology and environmental role of this enigmatic group remain elusive. The current theory is that heterotrophic nitrifiers are capable of converting ammonia to hydroxylamine, nitrite, nitric oxide, nitrous oxide, and dinitrogen gas via the subsequent actions of nitrification and denitrification. In addition, it was recently suggested that dinitrogen gas may be formed directly from ammonium. Here, we combine complementary high-resolution gas profiles, 15N isotope labeling studies, and transcriptomics data to show that hydroxylamine is the major product of nitrification in Alcaligenes faecalis. We demonstrated that denitrification and direct ammonium oxidation to dinitrogen gas did not occur under the conditions tested. Our results indicate that A. faecalis is capable of hydroxylamine production from an organic intermediate. These results fundamentally change our understanding of heterotrophic nitrification and have important implications for its biotechnological application.

Dirammox-dominated microbial community for biological nitrogen removal from wastewater.

Direct ammonia oxidation (Dirammox) might be of great significance to advance the innovation of biological nitrogen removal process in wastewater treatment systems. However, it remains unknown whether Dirammox bacteria can be selectively enriched in activated sludge. In this study, a lab-scale bioreactor was established and operated for 2 months to treat synthetic wastewater with hydroxylamine as a selection pressure. Three Dirammox strains (Alcaligenes aquatilis SDU_AA1, Alcaligenes aquatilis SDU_AA2, and Alcaligenes sp. SDU_A2) were isolated from the activated sludge, and their capability to perform Dirammox process was confirmed. Although these three Dirammox bacteria were undetectable in the seed sludge (0%), their relative abundances rapidly increased after a month of operation, reaching 12.65%, 0.69%, and 0.69% for SDU_A2, SDU_AA1, and SDU_AA2, respectively. Among them, the most dominant Dirammox (SDU_A2) exhibited higher nitrogen removal rate (32.35%) than the other two strains (13.57% of SDU_AA1 and 14.52% of SDU_AA2). Comparative genomic analysis demonstrated that the most dominant Dirammox bacterium (SDU_A2) possesses fewer complete metabolic modules compared to the other two less abundant Alcaligenes strains. Our findings expanded the understanding of the application of Dirammox bacteria as key functional microorganisms in a novel biological nitrogen and carbon removal process if they could be well stabilized. KEY POINTS: • Dirammox-dominated microbial community was enriched in activated sludge bioreactor. • The addition of hydroxylamine played a role in Dirammox enrichment. • Three Dirammox bacterial strains, including one novel species, were isolated.

Hydroxylamine supplementation accelerated the rates of cell growth, aerobic denitrification and nitrous oxide emission of Pseudomonas taiwanensis EN-F2.

Hydroxylamine can disrupt the protein translation process of most reported nitrogen-converting bacteria, and thus hinder the reproduction of bacteria and nitrogen conversion capacity. However, the effect of hydroxylamine on the denitrification ability of strain EN-F2 is unclear. In this study, the cell growth, aerobic denitrification ability, and nitrous oxide (N2O) emission by Pseudomonas taiwanensis were carefully investigated by addition of hydroxylamine at different concentrations. The results demonstrated that the rates of nitrate and nitrite reduction were enhanced by 2.51 and 2.78 mg/L/h after the addition of 8.0 and 12.0 mg/L hydroxylamine, respectively. The N2O production from nitrate and nitrite reaction systems were strongly promoted by 4.39 and 8.62 mg/L, respectively, through the simultaneous acceleration of cell growth and both of nitrite and nitrate reduction. Additionally, the enzymatic activities of nitrate reductase and nitrite reductase climbed from 0.13 and 0.01 to 0.22 and 0.04 U/mg protein when hydroxylamine concentration increased from 0 to 6.0 and 12.0 mg/L. This may be the main mechanism for controlling the observed higher denitrification rate and N2O release. Overall, hydroxylamine supplementation supported the EN-F2 strain cell growth, denitrification and N2O emission rates.

Nanopore Identification of N-Acetylation by Hydroxylamine Deacetylation (NINAHD).

N-Acetyl modification, a chemical modification commonly found on biomacromolecules, plays a crucial role in the regulation of cell activities and is related to a variety of diseases. However, due to the instability of N-acetyl modification, accurate and rapid identification of N-acetyl modification with a low measurement cost is still technically challenging. Here, based on hydroxylamine deacetylation and nanopore single molecule chemistry, a universal sensing strategy for N-acetyl modification has been developed. Acetohydroxamic acid (AHA), which is produced by the hydroxylamine deacetylation reaction and serves as a reporter for N-acetylation identification, is specifically sensed by a phenylboronic acid (PBA)-modified Mycobacterium smegmatis porin A (MspA). With this strategy, N-acetyl modifications on RNA, DNA, proteins, and glycans were identified, demonstrating its generality. Specifically, histones can be treated with hydroxylamine deacetylation, from which the generated AHA can represent the amount of N-acetyl modification detected by a nanopore sensor. The unique event features of AHA also demonstrate the robustness of sensing against other interfering analytes in the environment.

Discovery of a Hydroxylamine-Based Brain-Penetrant EGFR Inhibitor for Metastatic Non-Small-Cell Lung Cancer.

Metastases to the brain remain a significant problem in lung cancer, as treatment by most small-molecule targeted therapies is severely limited by efflux transporters at the blood-brain barrier (BBB). Here, we report the discovery of a selective, orally bioavailable, epidermal growth factor receptor (EGFR) inhibitor, 9, that exhibits high brain penetration and potent activity in osimertinib-resistant cell lines bearing L858R/C797S and exon19del/C797S EGFR resistance mutations. In vivo, 9 induced tumor regression in an intracranial patient-derived xenograft (PDX) murine model suggesting it as a potential lead for the treatment of localized and metastatic non-small-cell lung cancer (NSCLC) driven by activating mutant bearing EGFR. Overall, we demonstrate that an underrepresented functional group in medicinal chemistry, the trisubstituted hydroxylamine moiety, can be incorporated into a drug scaffold without the toxicity commonly surmised to accompany these units, all while maintaining potent biological activity and without the molecular weight creep common to drug optimization campaigns.

Role of Nitric Oxide in Hydroxylamine Oxidation by Ammonia-Oxidizing Bacteria.

An important role of nitric oxide (NO) as either a free intermediate in the NH3 oxidation pathway or a potential oxidant for NH3 or NH2OH has been proposed for ammonia-oxidizing bacteria (AOB) and archaea (AOA), respectively. However, tracing NO metabolism at low concentrations remains notoriously difficult. Here, we use electrochemical sensors and the mild NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO) to trace apparent NO concentration and determine production rates at low micromolar concentrations in the model AOB strain Nitrosomonas europaea. In agreement with previous studies, we found that PTIO does not affect NH3 oxidation instantaneously in both Nitrosospira briensis and Nitrosomonas europaea, unlike inhibitors for ammonia oxidation such as allylthiourea and acetylene, although it effectively scavenged NO from the cell suspensions. Quantitative analysis showed that NO production by N. europaea amounted to 3.15% to 6.23% of NO2- production, whereas N. europaea grown under O2 limitation produced NO equivalent to up to 40% of NO2- production at high substrate concentrations. In addition, we found that PTIO addition to N. europaea grown under O2 limitation abolished N2O production. These results indicate different turnover rates of NO during NH3 oxidation under O2-replete and O2-limited growth conditions in AOB. The results suggest that NO may not be a free intermediate or remain tightly bound to iron centers of enzymes during hydroxylamine oxidation and that only NH3 saturation and adaptation to O2 limitation may lead to significant dissociation of NO from hydroxylamine dehydrogenase. IMPORTANCE Ammonia oxidation by chemolithoautotrophic ammonia-oxidizing bacteria (AOB) is thought to contribute significantly to global nitrous oxide (N2O) emissions and leaching of oxidized nitrogen, particularly through their activity in nitrogen (N)-fertilized agricultural production systems. Although substantial efforts have been made to characterize the N metabolism in AOB, recent findings suggest that nitric oxide (NO) may play an important mechanistic role as a free intermediate of hydroxylamine oxidation in AOB, further implying that besides hydroxylamine dehydrogenase (HAO), additional enzymes may be required to complete the ammonia oxidation pathway. However, the NO spin trap PTIO was found to not inhibit ammonia oxidation in AOB. This study provides a combination of physiological and spectroscopic evidence that PTIO indeed scavenges only free NO in AOB and that significant amounts of free NO are produced only during incomplete hydroxylamine oxidation or nitrifier denitrification under O2-limited growth conditions.

Dual-edged effects and mechanisms of hydroxylamine in partial denitrification-anaerobic ammonium oxidation system.

The combination of partial denitrification (PD) and anaerobic ammonium oxidation (anammox) is a novel and promising nitrogen removal process. Regulating the synergistic reaction between denitrifiers and anammox bacteria (AnAOB) is the key to achieving stable and efficient PD-anammox performance. In this study, 10 mg/L of hydroxylamine (NH2OH) was considered to efficiently promote the bacterial activity, microbial energy flow, and the synergy of functional microflora. As a result, the nitrogen removal rate (NRR) significantly increased from 0.05 to 0.30 g N/L/d in parallel with an increase in the nitrogen loading rate (NLR) from 0.10 to 0.40 g N/L/d. However, the dual-edged effect of NH2OH was also confirmed. The long-term presence of NH2OH caused overgrowth of complete-denitrifying bacteria and decreased the NRR to 0.11 g N/L/d. Additionally, NH2OH enhanced nitrous oxide (N2O) emissions via chemical pathways as well as enhanced denitrification Fortunately, the inhibition caused by NH2OH was reversible by stopping the dosing, the reactor restored to stable operation with an NRR of 0.27 g N/L/d. Analysis of metabolic intensity and pathways revealed the effecting process and mechanism of NH2OH on the PD-anammox system. This study verified the dual-edged effects and mechanisms of NH2OH, therefore proving a theoretical basis and technical reference for the application of PD-anammox.

Proteomics reveals the enhancing mechanism for eliminating toxic hydroxylamine from water by nanocompartments containing hydroxylamine oxidase.

Hydroxylamine (NH2OH) is a potentially toxic pollutant when it is present in water, as it can damage both bacteria and the human body. It is still difficult to eliminate the toxic NH2OH in water. Here, we showed that the model bacterium (Escherichia coli) with nanocompartments encapsulated with hydroxylamine oxidase (HAO) can remove NH2OH from water. In addition, the removal efficiency of NH2OH by genetically modified bacteria (with HAO-nanocompartments) was 3.87 mg N L-1 h-1, and that of wild-type bacteria (without HAO-nanocompartments) was only 1.86 mg N L-1 h-1. Label-free quantitative proteomics indicated that the nanocompartments containing HAO enhanced bacterial activity by inducing the up-regulation of proteins involved in stress and stimulus responses, and decreased their intracellular NH2OH concentration. Moreover, the synthesis of proteins involved in energy metabolism, gene expression, and other processes in bacterial was enhanced under hydroxylamine stress, and these changes increased the resistance of bacterial to NH2OH. This work can aid our understanding of the toxic effects of NH2OH on bacteria as well as the development of new approaches to eliminate NH2OH in water.

Mechanisms of NO and N2O production by enriched nitrifying sludge in a sequencing batch reactor: Effects of hydroxylamine.

NO and N2O as important greenhouse gases andtheir production mechanisms during nitrification are not completely understood. This study aimed to analyze the effect of hydroxylamine (NH2OH) on NO and N2O produced by nitrifying bacteria from activated sludge in a sequencing batch reactor (SBR). Experimental results showed that when nitrite (NO2-) accumulated during aerobic ammonia (NH4+) oxidation, N2O was the main product. The total amount of NO and N2O produced by NH2OH oxidation was positively correlated with dissolved oxygen (DO) levels. The imbalance of NH4+ oxidation caused by NH2OH addition was more conducive to the generation of NO and N2O under high DO conditions. When NH2OH was added into the reactor with NO2- as the substrate, the production of NO and N2O under high DO levels was mainly related to NH2OH oxidation. Under low DO conditions, NO and N2O from the biotic/abiotic hybrid pathways were more significant in the reactor of the coexistence of NO2- and NH2OH, which could be mainly caused by the pathways of nitrifier denitrification and abiotic reaction. Besides, limited amount of NO and N2O was generated by heterotrophic denitrification pathway during autotrophic nitrification. The implications for the above results are important for understanding the production of NO and N2O under NH2OH stress in nitrifying sludge reactor.

Non-Hydroxamate Zinc-Binding Groups as Warheads for Histone Deacetylases.

Histone deacetylases (HDACs) remove acetyl groups from acetylated lysine residues and have a large variety of substrates and interaction partners. Therefore, it is not surprising that HDACs are involved in many diseases. Most inhibitors of zinc-dependent HDACs (HDACis) including approved drugs contain a hydroxamate as a zinc-binding group (ZBG), which is by far the biggest contributor to affinity, while chemical variation of the residual molecule is exploited to create more or less selectivity against HDAC isozymes or other metalloproteins. Hydroxamates have a propensity for nonspecificity and have recently come under considerable suspicion because of potential mutagenicity. Therefore, there are significant concerns when applying hydroxamate-containing compounds as therapeutics in chronic diseases beyond oncology due to unwanted toxic side effects. In the last years, several alternative ZBGs have been developed, which can replace the critical hydroxamate group in HDACis, while preserving high potency. Moreover, these compounds can be developed into highly selective inhibitors. This review aims at providing an overview of the progress in the field of non-hydroxamic HDACis in the time period from 2015 to present. Formally, ZBGs are clustered according to their binding mode and structural similarity to provide qualitative assessments and predictions based on available structural information.

Heme P460: A (Cross) Link to Nitric Oxide.

Ammonia-oxidizing bacteria (AOB) convert ammonia (NH3) to nitrite (NO2-) as their primary metabolism and thus provide a blueprint for the use of NH3 as a chemical fuel. The first energy-producing step involves the homotrimeric enzyme hydroxylamine oxidoreductase (HAO), which was originally reported to oxidize hydroxylamine (NH2OH) to NO2-. HAO uses the heme P460 cofactor as the site of catalysis. This heme is supported by seven other c hemes in each monomer that mediate electron transfer. Heme P460 cofactors are c-heme-based cofactors that have atypical protein cross-links between the peptide backbone and the porphyrin macrocycle. This cofactor has been observed in both the HAO and cytochrome (cyt) P460 protein families. However, there are differences; specifically, HAO uses a single tyrosine residue to form two covalent attachments to the macrocycle whereas cyt P460 uses a lysine residue to form one. In Nitrosomonas europaea, which expresses both HAO and cyt P460, these enzymes achieve the oxidation of NH2OH and were both originally reported to produce NO2-. Each can inspire means to effect controlled release of chemical energy.Spectroscopically studying the P460 cofactors of HAO is complicated by the 21 non-P460 heme cofactors, which obscure the active site. However, monoheme cyt P460 is more approachable biochemically and spectroscopically. Thus, we have used cyt P460 to study biological NH2OH oxidation. Under aerobic conditions substoichiometric production of NO2- was observed along with production of nitrous oxide (N2O). Under anaerobic conditions, however, N2O was the exclusive product of NH2OH oxidation. We have advanced our understanding of the mechanism of this enzyme and have showed that a key intermediate is a ferric nitrosyl that can dissociate the bound nitric oxide (NO) molecule and react with O2, thus producing NO2- abiotically. Because N2O was the true product of one P460 cofactor-containing enzyme, this prompted us to reinvestigate whether NO2- is enzymatically generated from HAO catalysis. Like cyt P460, we showed that HAO does not produce NO2- enzymatically, but unlike cyt P460, its final product is NO, establishing it as an intermediate of nitrification. More broadly, NO can be recognized as a molecule common to the primary metabolisms of all organisms involved in nitrogen "defixation".Delving deeper into cyt P460 yielded insights broadly applicable to controlled biochemical redox processes. Studies of an inactive cyt P460 from Nitrosomonas sp. AL212 showed that this enzyme was unable to oxidize NH2OH because it lacked a glutamate residue in its secondary coordination sphere that was present in the active N. europaea cyt P460 variant. Restoring the Glu residue imbued activity, revealing that a second-sphere base is Nature's key to controlled oxidation of NH2OH. A key lesson of bioinorganic chemistry is reinforced: the polypeptide matrix is an essential part of dictating function. Our work also exposed some key functional contributions of noncanonical heme-protein cross-links. The heme-Lys cross-link of cyt P460 enforces the relative position of the cofactor and second-sphere residues. Moreover, the cross-link prevents the dissociation of the axial histidine residue, which stops catalysis, emphasizing the importance of this unique post-translational modification.

An Untargeted Approach for Revealing Electrophilic Metabolites.

Reactive electrophilic intermediates such as coenzyme A esters play central roles in metabolism but are difficult to detect with conventional strategies. Here, we introduce hydroxylamine-based stable isotope labeling to convert reactive electrophilic intermediates into stable derivatives that are easily detectable via LC-MS. In the model system Caenorhabditis elegans, parallel treatment with 14NH2OH and 15NH2OH revealed >1000 labeled metabolites, e.g., derived from peptide, fatty acid, and ascaroside pheromone biosyntheses. Results from NH2OH treatment of a pheromone biosynthesis mutant, acox-1.1, suggested upregulation of thioesterase activity, which was confirmed by gene expression analysis. The upregulated thioesterase contributes to the biosynthesis of a specific subset of ascarosides, determining the balance of dispersal and attractive signals. These results demonstrate the utility of NH2OH labeling for investigating complex biosynthetic networks. Initial results with Aspergillus and human cell lines indicate applicability toward uncovering reactive metabolomes in diverse living systems.

Characterization of N-(2,6-dimethylphenyl)hydroxylamine adducts of 2'-deoxyguanosine under weakly basic conditions.

Aromatic amines are a class of chemical carcinogens that are activated by cytochrome P450 enzymes to form arylhydroxylamines that are conjugated to form N-acetoxyarylamines or N-sulfonyloxyarylamines. These conjugates undergo N-O bond cleavage to become reactive nitrenium ions that may form DNA adducts. Numerous studies in the past using N-acetoxyarylamines to investigate DNA adduct formation were conducted, however, less is known in regard to DNA adduct formation directly from arylhydroxylamines - especially under conditions that mimic the physiological conditions of cells such as weakly basic conditions. In this study, 2'-deoxyguanosine (dG) was exposed to N-(2,6-dimethylphenyl)hydroxylamine (2,6-DMPHA) and N-phenylhydroxylamine (PHA) at pH 7.4 without enzymes and analyzed by liquid chromatography high resolution mass spectrometry (LC-HRMS). 2,6-DMPHA exposure resulted in the production of relatively low amounts of adducts however the identities of at least six different adducts that were formed through reactions with carbon, nitrogen and oxygen of 2'-deoxyguanosine were proposed based upon different analytical approaches including HRMS CID fragmentation and NMR analyses. Contrastively, PHA exposure under identical conditions resulted in one adduct at the C8 position. It was concluded from these results and results of theoretical calculations that nitrenium ions produced from 2,6-DMPHA were relatively more stable resulting in longer nitrenium ion lifetimes which ultimately led to greater potential for 2,6-DMPHA nitrenium ions to react with multiple sites on dG.

Biosynthesis of hydrazine from ammonium and hydroxylamine using an anaerobic ammonium oxidizing bacterium.

OBJECTIVES: To synthesize hydrazine (N2H4) from ammonium and hydroxylamine (NH2OH) using an anaerobic ammonium oxidation (anammox) bacterium, Candidatus Kuenenia stuttgartiensis. RESULTS: K. stuttgartiensis cells were anoxically cultivated with the addition of ammonium (2 mM) and NH2OH (1-100 mM) at pH 6-10.5, and 4-65 °C to examine the favorable cultivation conditions for N2H4 production. The influence of NH2OH concentration was more prominent than that of pH and temperature, and NH2OH concentration higher than 1 mM deteriorated N2H4 yields significantly. The following conditions were found to be favorable for N2H4 production using K. stuttgartiensis cells: pH 9, 38 °C, and < 1 mM NH2OH. In a continuous-feed system operated at these conditions, K. stuttgartiensis cells produced N2H4 with a maximum concentration of 0.65 mM, which is the highest N2H4 concentration previously reported in biological processes. CONCLUSIONS: Optimal cultivation conditions for K. stuttgartiensis for N2H4 production were successfully determined, and the present study is the first to document potential biological N2H4 production using anammox bacteria.

Synthesis and breakdown of universal metabolic precursors promoted by iron.

Life builds its molecules from carbon dioxide (CO2) and breaks them back down again through the intermediacy of just five metabolites, which are the universal hubs of biochemistry1. However, it is unclear how core biological metabolism began and why it uses the intermediates, reactions and pathways that it does. Here we describe a purely chemical reaction network promoted by ferrous iron, in which aqueous pyruvate and glyoxylate-two products of abiotic CO2 reduction2-4-build up 9 of the 11 intermediates of the biological Krebs (or tricarboxylic acid) cycle, including all 5 universal metabolic precursors. The intermediates simultaneously break down to CO2 in a life-like regime that resembles biological anabolism and catabolism5. Adding hydroxylamine6-8 and metallic iron into the system produces four biological amino acids in a manner that parallels biosynthesis. The observed network overlaps substantially with the Krebs and glyoxylate cycles9,10, and may represent a prebiotic precursor to these core metabolic pathways.

Photobacterium sp. NNA4, an efficient hydroxylamine-transforming heterotrophic nitrifier/aerobic denitrifier.

An efficient heterotrophic nitrifying/aerobic denitrifying strain, Photobacterium sp. NNA4 was isolated from a recirculating aquaculture system (RAS). NNA4 was capable of utilizing ammonia, nitrate or nitrite as sole N-source with maximal removal rates of 12.5 mg/L/h for NH4+N, 16.4 mg/L/h for NO3--N, and 4.5 mg/L/h for NO2--N, respectively. Optimal nitrification conditions were: sodium succinate as C-source, 30-37°C, NaCl 1-4%, pH 7.0-8.0, dissolved oxygen 5.89 mg/L, C/N > 10. Gas chromatography/mass spectrometry and gas chromatography/isotope ratio mass spectrometry analyses showed that N2 and N2O were aerobic denitrification products of nitrite and nitrate. NNA4 could tolerate high concentration of hydroxylamine and displayed efficient hydroxylamine-transforming capability. Hydroxylamine oxidoreductase activity using potassium ferricyanide as electron acceptor was 0.042 U. Results revealed that strain NNA4 could oxidize NH2OH directly to N2O at aerobic conditions. In view of its high removal ability of inorganic nitrogen pollutants and broad salinity tolerance range, NNA4 has great potential in denitrification treatment of types of wastewater with either low salinity (e.g., municipal facilities) or high salinity (e.g., aquaculture, seafood processing).

Ammonium transformed into nitrous oxide via nitric oxide by Pseudomonas putida Y-9 under aerobic conditions without hydroxylamine as intermediate.

Previous studies have reported that hydroxylamine (NH2OH) is an inevitable intermediate of the ammonium (NH4+) oxidation pathway under aerobic conditions. In this study, Pseudomonas putida Y-9 was found to oxidize ammonium into N2O via NO without the accumulation of NH2OH and NO2- under aerobic conditions. NH2OH was nearly completely transformed into NO2- whether NH4+ was present in the medium, and NH4+ could accelerate the transformation of NH2OH to NO2- by promoting Y-9 growth. NH4+ was oxidized rapidly by Y-9 with or without the presence of NH2OH in the medium, and the decrease of total nitrogen reached 30.65 mg/L and 39.38 mg/L, respectively, which indicates that NH2OH inhibits the transformation efficiency of NH4+ to N2O. Gene amplification and enzyme assays demonstrated that ammonia monooxygenase doesn't exist in Y-9. All results show that NH4+ can be transformed into N2O via NO by Y-9 under aerobic conditions without NH2OH as intermediate.

Nitric Oxide Production from Nitrite Reduction and Hydroxylamine Oxidation by Copper-containing Dissimilatory Nitrite Reductase (NirK) from the Aerobic Ammonia-oxidizing Archaeon, Nitrososphaera viennensis.

Aerobic ammonia-oxidizing archaea (AOA) play a crucial role in the global nitrogen cycle by oxidizing ammonia to nitrite, and nitric oxide (NO) is a key intermediate in AOA for sustaining aerobic ammonia oxidation activity. We herein heterologously expressed the NO-forming, copper-containing, dissimilatory nitrite reductase (NirK) from Nitrososphaera viennensis and investigated its enzymatic properties. The recombinant protein catalyzed the reduction of 15NO2- to 15NO, the oxidation of hydroxylamine (15NH2OH) to 15NO, and the production of 14-15N2O from 15NH2OH and 14NO2-. To the best of our knowledge, the present study is the first to document the enzymatic properties of AOA NirK.

Nitrite accumulation inside sludge flocs significantly influencing nitrous oxide production by ammonium-oxidizing bacteria.

This work aims to clarify the role of potential nitrite (NO2-) accumulation inside sludge flocs in N2O production by ammonium-oxidizing bacteria (AOB) at different dissolved oxygen (DO) levels with focus on the conditions of no significant bulk NO2- accumulation (<0.2 mg N/L). To this end, an augmented nitrifying sludge with much higher abundance of nitrite-oxidizing bacteria (NOB) than AOB was enriched and then used for systematically designed batch tests, which targeted a range of DO levels from 0 to 3.0 mg O2/L at a fixed ammonium concentration of 10 mg N/L. A two-pathway N2O model was applied to facilitate the interpretation of batch experimental data, thus shedding light on the relationships between N2O production pathways and key process parameters (i.e., DO and NO2- accumulation inside sludge flocs). The results demonstrated (i) the biomass specific N2O production rate firstly increased and then decreased with DO, with the maximum value of 3.03 ±â€¯0.05 mg N/h/g VSS obtained at DO level of 0.75 mg O2/L, (ii) the AOB denitrification pathway for N2O production was dominant (98.0%) at all DO levels tested even without significant bulk NO2- accumulation (<0.2 mg N/L) observed in the system, but its contribution decreased with DO, (iii) DO had a positive impact on the hydroxylamine pathway for N2O production which therefore increased with DO, and (iv) the nitrite accumulation existed inside the sludge flocs and induced significant N2O production from the AOB denitrification pathway.

Biochemistry of N-mustard tumour control compounds reconsidered.

The biochemicals and reactions involved in the present mechanism of degradation of tumour cells during chemotherapy are reconsidered and limitations noted. Alternative mechanisms and treatment methods are detailed.