Identification and Analysis of the Biosynthetic Gene Cluster for the Hydrazide-Containing Aryl Polyene Spinamycin.

Natural products containing nitrogen-nitrogen (N-N) bonds have attracted much attention because of their bioactivities and chemical features. Several recent studies have revealed the nitrous acid-dependent N-N bond-forming machinery. However, the catalytic mechanisms of hydrazide synthesis using nitrous acid remain unknown. Herein, we focused on spinamycin, a hydrazide-containing aryl polyene produced by Streptomyces albospinus JCM3399. In the S. albospinus genome, we discovered a putative spinamycin biosynthetic gene (spi) cluster containing genes that encode a type II polyketide synthase and genes for the secondary metabolism-specific nitrous acid biosynthesis pathway. A gene inactivation experiment showed that this cluster was responsible for spinamycin biosynthesis. A feeding experiment using stable isotope-labeled sodium nitrite and analysis of nitrous acid-synthesizing enzymes in vitro strongly indicated that one of the nitrogen atoms of the hydrazide group was derived from nitrous acid. In vitro substrate specificity analysis of SpiA3, which is responsible for loading a starter substrate onto polyketide synthase, indicated that N-N bond formation occurs after starter substrate loading. In vitro analysis showed that the AMP-dependent ligase SpiA7 catalyzes the diazotization of an amino group on a benzene ring without a hydroxy group, resulting in a highly reactive diazo intermediate, which may be the key step in hydrazide group formation. Therefore, we propose the overall biosynthetic pathway of spinamycin. This study expands our knowledge of N-N bond formation in microbial secondary metabolism.

Bacterial Avenalumic Acid Biosynthesis Includes Substitution of an Aromatic Amino Group for Hydride by Nitrous Acid Dependent Diazotization.

The diazo group is an important functional group that can confer biological activity to natural products owing to its high reactivity. Recent studies have revealed that diazo groups are synthesized from amino groups using nitrous acid in secondary metabolites of actinomycetes. However, genome database analysis indicated that there are still many diazo group-biosynthesizing enzymes for unknown biosynthetic pathways. Here, we discovered an avenalumic acid biosynthesis gene cluster in Streptomyces sp. RI-77 by genome mining of enzymes involved in diazo group formation. Through heterologous expression, the gene cluster was revealed to direct avenalumic acid (AVA) biosynthesis via 3-aminoavenalumic acid (3-AAA). In vitro enzyme assays showed that AvaA6 and AvaA7 catalyzed the diazotization of 3-AAA using nitrous acid and substitution of the diazo group for hydride to synthesize AVA, respectively. This study revealed an unprecedented pathway for amino group removal via diazotization.

Further slowing down of hydrolysis of amylose heated with black soybean extract by treating with nitrite under gastric conditions.

Black soybean (BSB), which contains cyanidin-3-O-glucoside (C3G) and procyanidins, is cooked with rice in Japan. The color of the cooked rice is purplish red due to the binding of C3G and reddish oxidation products of procyanidins. These components can slowdown pancreatin-induced hydrolysis of amylose more significantly than the hydrolysis of amylopectin, and can react with nitrous acid in the stomach. This manuscript deals with the effects of nitrous acid on pancreatin-induced hydrolysis of amylose heated with BSB extract. The hydrolysis of amylose heated with BSB extract was slow, and the slowdown was due to the binding of C3G/its degradation products and degradation products of procyanidins. The amylose hydrolysis was slowed down further by treating with nitrite under gastric conditions. The further slowdown was discussed to be due to the binding of the products, which were formed by the reaction of procyanidins with nitrous acid, to amylose. In the products, dinitroprocyanidins were included. In this way, the digestibility of amylose heated with BSB extract can be slowed down further by reacting with nitrous acid in the stomach.

Water-driven microbial nitrogen transformations in biological soil crusts causing atmospheric nitrous acid and nitric oxide emissions.

Biological soil crusts (biocrusts) release the reactive nitrogen gases (Nr) nitrous acid (HONO) and nitric oxide (NO) into the atmosphere, but the underlying microbial process controls have not yet been resolved. In this study, we analyzed the activity of microbial consortia relevant in Nr emissions during desiccation using transcriptome and proteome profiling and fluorescence in situ hybridization. We observed that < 30 min after wetting, genes encoding for all relevant nitrogen (N) cycling processes were expressed. The most abundant transcriptionally active N-transforming microorganisms in the investigated biocrusts were affiliated with Rhodobacteraceae, Enterobacteriaceae, and Pseudomonadaceae within the Alpha- and Gammaproteobacteria. Upon desiccation, the nitrite (NO2-) content of the biocrusts increased significantly, which was not the case when microbial activity was inhibited. Our results confirm that NO2- is the key precursor for biocrust emissions of HONO and NO. This NO2- accumulation likely involves two processes related to the transition from oxygen-limited to oxic conditions in the course of desiccation: (i) a differential regulation of the expression of denitrification genes; and (ii) a physiological response of ammonia-oxidizing organisms to changing oxygen conditions. Thus, our findings suggest that the activity of N-cycling microorganisms determines the process rates and overall quantity of Nr emissions.

Influence of the heme distal pocket on nitrite binding orientation and reactivity in Sperm Whale myoglobin.

Nitrite binding to recombinant wild-type Sperm Whale myoglobin (SWMb) was studied using a combination of spectroscopic methods including room-temperature magnetic circular dichroism. These revealed that the reactive species is free nitrous acid and the product of the reaction contains a nitrite ion bound to the ferric heme iron in the nitrito- (O-bound) orientation. This exists in a thermal equilibrium with a low-spin ground state and a high-spin excited state and is spectroscopically distinct from the purely low-spin nitro- (N-bound) species observed in the H64V SWMb variant. Substitution of the proximal heme ligand, histidine-93, with lysine yields a novel form of myoglobin (H93K) with enhanced reactivity towards nitrite. The nitrito-mode of binding to the ferric heme iron is retained in the H93K variant again as a thermal equilibrium of spin-states. This proximal substitution influences the heme distal pocket causing the pKa of the alkaline transition to be lowered relative to wild-type SWMb. This change in the environment of the distal pocket coupled with nitrito-binding is the most likely explanation for the 8-fold increase in the rate of nitrite reduction by H93K relative to WT SWMb.

Novel insights into integrated fermentation and nitrogen removal by free nitrous acid (FNA) serving as treatment method.

Free nitrous acid (FNA) has only been studied as the pretreatment of waste activated sludge (WAS). Integrated fermentation and nitrogen removal using FNA as a primary means of treatment are seldom investigated. WAS fermentation was characterized under various FNA concentration. The production of COD, protein, and carbohydrate increased with FNA concentration (in the range of 0.197-1.97 mg/L) before the denitrification process. Volatile fatty acids (VFA) were only produced after complete denitrification. Potential FNA impact on fermentation step found FNA facilitated both solubilization and hydrolysis but inhibited acidification, acetogenesis, and methanogenesis processes. The types of fermentation were determined using threedimensional excitation-emission matrix (EEM) fluorescence spectroscopy. Protein-like substances and Tyrosine/Tryptophan were the most dominant dissolved organic matters (DOMs). The cell decay rate increased from 0.044 to 0.102/d based on the nonlinear fitting for the FNA concentration of 0.197-1.97 mg/L. The microbial biomass mortality reached 92.7% when the FNA in tight extracellular polymeric substances (T-EPS) exceeded 0.04 mg/L. In addition, the microbial diversity and microbial structure were substantially reduced by FNA during long-term operation, while the bacterial abundance associated with hydrolysis and acidification increased significantly.

Mining novel biosynthetic machineries of secondary metabolites from actinobacteria.

Secondary metabolites produced by actinobacteria have diverse structures and important biological activities, making them a useful source of drug development. Diversity of the secondary metabolites indicates that the actinobacteria exploit various chemical reactions to construct a structural diversity. Thus, studying the biosynthetic machinery of these metabolites should result in discovery of various enzymes catalyzing interesting and useful reactions. This review summarizes our recent studies on the biosynthesis of secondary metabolites from actinobacteria, including the biosynthesis of nonproteinogenic amino acids used as building blocks of nonribosomal peptides, the type II polyketide synthase catalyzing polyene scaffold, the nitrous acid biosynthetic pathway involved in secondary metabolite biosynthesis and unique cytochrome P450 catalyzing nitrene transfer. These findings expand the knowledge of secondary metabolite biosynthesis machinery and provide useful tools for future bioengineering.

Free nitrous acid-based nitrifying sludge treatment in a two-sludge system obtains high polyhydroxyalkanoates accumulation and satisfied biological nutrients removal.

A novel strategy to achieve substantial polyhydroxyalkanoates (PHA) accumulation in waste activated sludge (WAS) was developed, which was conducted in a two-sludge system consisted of an anaerobic/anoxic/oxic reactor (AAO-SBR) and a nitrifying reactor (N-SBR), where the nitrifying-sludge was treated by free nitrous acid (FNA). Initially, 0.98 ±â€¯0.09 and 1.46 ±â€¯0.10 mmol-c/g VSS of PHA were respectively determined in the control-SBR and AAO-SBR. When 1/16 of nitrifying sludge was daily treated with 1.49 mg N/L FNA for 24 h, ∼46.5% of nitrite was accumulated in the N-SBR, ∼2.43 ±â€¯0.12 mmol-c/g VSS of PHA was accumulated in WAS in AAO-SBR without deteriorating nutrient removal. However, nutrient removal of control-SBR was completely collapsed after implementing the same FNA treatment. Further investigations revealed that the activity and abundance of nitrite oxidizing bacteria (NOB) was decreased significantly after FNA treatment. Finally, sludge with high PHA level to generate more methane was confirmed.

Soil HONO emissions at high moisture content are driven by microbial nitrate reduction to nitrite: tackling the HONO puzzle.

Nitrous acid (HONO) is a precursor of the hydroxyl radical (OH), a key oxidant in the degradation of most air pollutants. Field measurements indicate a large unknown source of HONO during the day time. Release of nitrous acid (HONO) from soil has been suggested as a major source of atmospheric HONO. We hypothesize that nitrite produced by biological nitrate reduction in oxygen-limited microzones in wet soils is a source of such HONO. Indeed, we found that various contrasting soil samples emitted HONO at high water-holding capacity (75-140%), demonstrating this to be a widespread phenomenon. Supplemental nitrate stimulated HONO emissions, whereas ethanol (70% v/v) treatment to minimize microbial activities reduced HONO emissions by 80%, suggesting that nitrate-dependent biotic processes are the sources of HONO. High-throughput Illumina sequencing of 16S rRNA as well as functional gene transcripts associated with nitrate and nitrite reduction indicated that HONO emissions from soil samples were associated with nitrate reduction activities of diverse Proteobacteria. Incubation of pure cultures of bacterial nitrate reducers and gene-expression analyses, as well as the analyses of mutant strains deficient in nitrite reductases, showed positive correlations of HONO emissions with the capability of microbes to reduce nitrate to nitrite. Thus, we suggest biological nitrate reduction in oxygen-limited microzones as a hitherto unknown source of atmospheric HONO, affecting biogeochemical nitrogen cycling, atmospheric chemistry, and global modeling.

Beyond esterase-like activity of serum albumin. Histidine-(nitro)phenol radical formation in conversion cascade of p-nitrophenyl acetate and the role of infrared light.

Serum albumin, recognized mainly for its capacity to act as a carrier protein for many compounds, can also actively transform some organic molecules. As a starting point in this study, we consider esterase-like activity of bovine serum albumin (BSA) toward p-nitrophenyl acetate (p-NPA). Our results reveal that the reaction goes beyond ester hydrolysis step. In fact, the transformation product, p-nitrophenol (p-NP), becomes a substrate for further reaction with BSA in which its nitro group in subtracted and released in the form of HNO2 . Spectral data indicate that this cascade of events proceeds through formation of phenoxyl radical via proton-coupled electron transport (PCET) between OH group of p-NP and imidazole ring of histidine from the protein. Furthermore, the effect of application of electromagnetic radiation in the infrared range suggests that this remote physical trigger can support interactions based on PCET mechanism by acting on polarization and mutual alignment of water dipoles serving as effective water wires.

Dual nitrogen species involved in foliar uptake of nitrogen dioxide in Arabidopsis thaliana.

Foliar uptake of nitrogen dioxide (NO2) is governed by its reactive absorption mechanism, by which NO2 molecules diffuse through cell wall layers and simultaneously react with apoplastic ascorbate to form nitrous acid, which freely diffuses across plasmalemma. However, whether free diffusion of nitrous acid is the sole mechanism of foliar uptake of NO2 remains unknown. The involvement of ammonia-inhibitable nitrite transporters in the foliar uptake of NO2, as reported in nitrite transport in Arabidopsis roots, is also unknown. In this study, we treated Arabidopsis thaliana leaves with methionine sulfoximine (MSX) to inhibit incorporation of ammonia into glutamate and exposed them to 4 ppm 15N-labeled NO2 for 4 h in light followed by quantification of total nitrogen, reduced nitrogen, and ammonia nitrogen derived from NO2 using mass spectrometry and capillary electrophoresis. The total nitrogen derived from NO2 in leaves without MSX treatment was 587.0 nmol NO2/g fresh weight, of which more than 65% was recovered as reduced nitrogen. In comparison, MSX treatment decreased the total nitrogen and reduced nitrogen derived from NO2 by half. Thus, half of the foliar uptake of NO2 is not attributable to passive diffusion of nitrous acid but to ammonia-inhibitable nitrite transport. Foliar uptake of NO2 is mediated by a dual mechanism in A. thaliana: nitrous acid-free diffusion and nitrite transporter-mediated transport.

Achieving nitritation by treating sludge with free nitrous acid: The effect of starvation.

Side-stream sludge treatment using free nitrous acid (FNA) is a novel strategy to achieve nitritation in mainstream of wastewater treatment plants (WWTPs). To optimize nitritation, the effect of starvation on this strategy was investigated in this study. The results showed that pre-starvation, which is the starvation before FNA treatment, enhanced the resistance of sludge to FNA. This led to a decrease in the nitrite accumulation rate (NAR), which dropped from 70% to 27% after aerobic pre-starvation. This was further confirmed in the FNA treatment using the sludge collected from the secondary settling tank (anoxic pre-starvation) and the aerobic zone (without starvation) of an Anaerobic-Anoxic-Oxic system. The post-starvation, which was the starvation after FNA treatment, decreased NAR from 63% to 14%. To obtain a higher NAR, the sludge used for FNA treatment should be collected from aerobic zone, and be returned to aerobic zone after treatment to avoid pre-starvation and post-starvation.

Novel desferrioxamine derivatives synthesized using the secondary metabolism-specific nitrous acid biosynthetic pathway in Streptomyces davawensis.

Recently, a novel nitrous acid biosynthetic pathway composed of two enzymes was discovered to be involved in the biosynthesis of cremeomycin for the formation of its diazo group. In this pathway, CreE oxidizes L-aspartic acid to nitrosuccinic acid and CreD liberates nitrous acid from nitrosuccinic acid. Bioinformatic analysis showed that various actinobacteria have putative secondary metabolite biosynthesis gene clusters containing creE and creD homologs, suggesting that this pathway is widely used for the biosynthesis of various natural products. Here, we focused on creE and creD homologs (BN159_4422 and BN159_4421) in Streptomyces davawensis. In vitro analysis of recombinant BN159_4422 and BN159_4421 proteins showed that these enzymes synthesized nitrous acid from L-aspartic acid. Secondary metabolites produced by this gene cluster were investigated by comparing the metabolic profiles of the wild-type and ΔBN159_4422 strains. When these strains were co-cultured with Tsukamurella pulmonis TP-B0596, three compounds were specifically produced by the wild-type strain. These compounds were identified as novel desferrioxamine derivatives containing either of two unique five-membered heterocyclic ring structures and shown to have iron-binding properties. A putative desferrioxamine biosynthetic gene cluster was found in the S. davawensis genome, and inactivation of a desD homolog (BN159_5485) also abolished the production of these compounds. We propose that these compounds should be synthesized by the modification of desferrioxamine B and a shorter chain analog using nitrous acid produced by the CreE and CreD homologs. This study provides an important insight into the diverse usage of the secondary metabolism-specific nitrous acid biosynthetic pathway in actinomycetes.

Preparation of low molecular weight heparins from bovine and ovine heparins using nitrous acid degradation.

Low molecular weight heparins (LMWHs) are important anticoagulant drugs. Nitrous acid degradation is a major approach to produce LMWHs, such as dalteparin. Due to the foreseeable shortage of porcine intestinal mucosa heparin and other potential risks, expansion of other animal tissues for heparin preparation is necessary. Heparins from different tissues differ in structure and bioactivity potency, and these variations may be carried over to the LMWH products. Sophisticated analytical techniques have been applied to compare various versions of dalteparins produced from porcine intestinal, bovine lung and ovine intestinal heparins to elucidate the effects of different animal tissues starting materials and processing conditions on the properties of final dalteparin products. With adjusted depolymerization conditions, versions of dalteparins that qualify under the European Pharmacopeia (EP) specifications were manufactured using non-porcine heparins. Dissimilarities among the three interspecies animal tissue heparin-derived dalteparins regarding fine structures are also disclosed, and their origins are discussed.

Crystal structure of the nitrosuccinate lyase CreD in complex with fumarate provides insights into the catalytic mechanism for nitrous acid elimination.

Enzymes belonging to the aspartase/fumarase superfamily catalyze elimination of various functional groups from succinate derivatives and play an important role in primary metabolism and aromatic compound degradation. Recently, an aspartase/fumarase superfamily enzyme, CreD, was discovered in cremeomycin biosynthesis. This enzyme catalyzes the elimination of nitrous acid from nitrosuccinate synthesized from aspartate by CreE, a flavin-dependent monooxygenase. Nitrous acid generated by this pathway is an important precursor of the diazo group of cremeomycin. CreD is the first aspartase/fumarase superfamily enzyme that was reported to catalyze the elimination of nitrous acid, and therefore we aimed to analyze its reaction mechanism. The crystal structure of CreD was determined by the molecular replacement native-single anomalous diffraction method at 2.18 Å resolution. Subsequently, the CreD-fumarate complex structure was determined at 2.30 Å resolution by the soaking method. Similar to other aspartase/fumarase superfamily enzymes, the crystal structure of CreD was composed of three domains and formed a tetramer. Two molecules of fumarate were observed in one subunit of the CreD-fumarate complex. One of them was located in the active site pocket formed by three different subunits. Intriguingly, no histidine residue, which usually functions as a catalytic acid in aspartase/fumarase superfamily enzymes, was found around the fumarate molecule in the active site. Based on the mutational analysis, we propose a catalytic mechanism of CreD, in which Arg325 acts as a catalytic acid. DATABASES: The crystal structures of CreD and the CreD-fumarate complex were deposited to PDB under the accession numbers 5XNY and 5XNZ, respectively. ENZYMES: Nitrosuccinate lyase CreD, EC4.3.

Hydroxylamine released by nitrifying microorganisms is a precursor for HONO emission from drying soils.

Nitrous acid (HONO) is an important precursor of the hydroxyl radical (OH), the atmosphere´s primary oxidant. An unknown strong daytime source of HONO is required to explain measurements in ambient air. Emissions from soils are one of the potential sources. Ammonia-oxidizing bacteria (AOB) have been identified as possible producers of these HONO soil emissions. However, the mechanisms for production and release of HONO in soils are not fully understood. In this study, we used a dynamic soil-chamber system to provide direct evidence that gaseous emissions from nitrifying pure cultures contain hydroxylamine (NH2OH), which is subsequently converted to HONO in a heterogeneous reaction with water vapor on glass bead surfaces. In addition to different AOB species, we found release of HONO also in ammonia-oxidizing archaea (AOA), suggesting that these globally abundant microbes may also contribute to the formation of atmospheric HONO and consequently OH. Since biogenic NH2OH is formed by diverse organisms, such as AOB, AOA, methane-oxidizing bacteria, heterotrophic nitrifiers, and fungi, we argue that HONO emission from soil is not restricted to the nitrifying bacteria, but is also promoted by nitrifying members of the domains Archaea and Eukarya.

Effect of free ammonium and free nitrous acid on the activity, aggregate morphology and extracellular polymeric substance distribution of ammonium oxidizing bacteria in partial nitrification.

Successful partial nitrification not only guarantees the inhibition of nitrite oxidation, but also does not excessively retard the ammonia oxidation rate. Therefore, the performance of ammonium oxidizing bacteria (AOB) during partial nitrification is fundamental to this process. In this study, two lab-scale partial nitrification bioreactors containing different inhibition conditions-one with free ammonium (FA) inhibition, the other with free nitrous acid (FNA) inhibition-were used to compare the differences between activity, quantity, aggregation morphology and extracellular polymeric substance (EPS) distribution of AOB. The results showed that although stable, long-term, partial nitrification was achieved in both reactors, there were differences in AOB activity, microbial spatial distribution and EPS characteristic. In the FA bioreactor, FA concentration was conducted at more than 40 mg/L, which had a strong impact on the metabolism of AOB. The activity and quantity decreased by 50%. Higher EPS (42.44 ± 2.31 mg g-1 mixed liquor volatile suspended solids [MLVSS]) and protein were introduced into the EPS matrix. However, in the FNA bioreactor, the FNA concentration was about 0.23 mg/L. It did not reach a level to affect AOB metabolism. The AOB activity and quantity were maintained at high levels and the total EPS content was 28.29 ± 2.04 mg g-1 MLVSS. Additionally, the microscopic results showed that in the FA bioreactor, AOB cells aggregated in microcolonies, while they appeared to be self-flocculating with no specific conformation in the other reactor. ß-polysaccharides located inside sludge flocs in the FA bioreactor but only accumulated around the outer layer of activated sludge flocs in the FNA condition.

Free nitrous acid and pH determine the predominant ammonia-oxidizing bacteria and amount of N2O in a partial nitrifying reactor.

We investigated the effects of free ammonia (FA) and free nitrous acid (FNA) concentrations on the predominant ammonia-oxidizing bacteria (AOB) and the emission of nitrous oxide (N2O) in a lab-scale sequencing batch reactor for partial nitrification. The reactor was operated with stepwise increases in the NH4+ loading rate, which resulted in a maximum FA concentration of 29.3 mg-N/L at pH 8.3. Afterwards, FNA was increased by a gradual decrease of pH, reaching its maximum concentration of 4.1 mg-N/L at pH 6.3. Fluorescence in situ hybridization indicated that AOB remained predominant during the operation, achieving specific nitrification rates of 1.04 and 0.99 g-N/g-VSS/day at the highest accumulations of FA and FNA, respectively. These rates were in conjunction with partial nitrification efficiencies of >84%. The N2O emission factor of oxidized NH4+ was 0.90% at pH 7.0, which was higher than those at pH 8.3 (0.11%) and 6.3 (0.12%), the pHs with the maximum FA and FNA concentrations, respectively. High-throughput sequencing of 16S ribosomal RNA genes showed that increases in FNA drastically changed the predominant AOB species, although increased FA produced no significant changes. This study demonstrates that the FNA concentration and pH are the main drivers that determine the predominant AOB species and N2O-emission in a partial nitrifying bioreactor.

Resilience of sulfate-reducing granular sludge against temperature, pH, oxygen, nitrite, and free nitrous acid.

Sulfate-reducing granular sludge has recently been developed and characterized in detail as part of the development of the sulfate reduction, autotrophic denitrification, nitrification integrated (SANI) process. However, information regarding temperature of granules to environmental fluctuation is lacking, an aspect that is important in dealing with real wastewater. A comprehensive assessment of sulfate-reducing granular sludge performance under various environmental conditions was thus conducted in this study, including temperature, pH, oxygen, nitrite, and free nitrous acid (FNA) as possible encountering conditions in the removal of organics and/or nitrate. Specific chemical oxygen demand removal rate of the granules was determined to be reduced by 65 % when the temperature varied between 10-15 °C, reduced by 70 % when dissolved oxygen (DO) was 0.5 mg/L or greater, and at least, reduced by 75 % when nitrite was 30 mg N/L or above. Nevertheless, the sludge activity recovered by 82, 100, and 86 % from exposure to high oxygen and nitrite and low temperature levels, respectively. Combined inhibition of nitrite and FNA on the sludge is strong and complex, while FNA alone reduced cell viability from 60 to 40 % when its concentration increased to 2.3 mg N/L. The present study demonstrates that sulfate-reducing bacteria (SRB) granules possess high resilience against varying environmental conditions, showing the high application potential of sulfate-reducing granular sludge in dealing with brackish and saline industrial or domestic wastewaters.

Achieving Stable Nitritation for Mainstream Deammonification by Combining Free Nitrous Acid-Based Sludge Treatment and Oxygen Limitation.

Stable nitritation is a critical bottleneck for achieving autotrophic nitrogen removal using the energy-saving mainstream deammonification process. Herein we report a new strategy to wash out both the Nitrospira sp. and Nitrobacter sp. from the treatment of domestic-strength wastewater. The strategy combines sludge treatment using free nitrous acid (FNA) with dissolved oxygen (DO) control in the nitritation reactor. Initially, the nitrifying reactor achieved full conversion of NH4(+) to NO3(-). Then, nitrite accumulation at ~60% was achieved in the reactor when 1/4 of the sludge was treated daily with FNA at 1.82 mg N/L in a side-stream unit for 24 h. Fluorescence in-situ hybridization (FISH) revealed FNA treatment substantially reduced the abundance of nitrite oxidizing bacteria (NOB) (from 23.0 ± 4.3 to 5.3 ± 1.9%), especially that of Nitrospira sp. (from 15.7 ± 3.9 to 0.4 ± 0.1%). Nitrite accumulation increased to ~80% when the DO concentration in the mainstream reactor was reduced from 2.5-3.0 to 0.3-0.8 mg/L. FISH revealed the DO limitation further reduced the abundance of NOB (to 2.1 ± 1.0%), especially that of Nitrobacter sp. (from 4.9 ± 1.2 to 1.8 ± 0.8%). The strategy developed removes a major barrier for deammonification in low-strength domestic wastewater.