Improving the electrochemical characteristics and performance of a neutral all-iron flow battery by using the iron reduction bacteria.

At present, the all-iron redox flow batteries (RFBs) have greater application potential due to high accessibility of electrolytes compared to traditional RFBs. Meanwhile, although electroactive bacteria can accelerate the electrons transfer, their potential to improve the performance of RFBs has been overlooked. Previously, we had confirmed that ferrous-oxidizing bacteria (FeOB) could enhance the performance of an all-iron RFB, therefore we conducted several batch experiments and chronopotentiometry experiments by using the ferric-reducing bacteria (FeRB) or mixed culture (FeOB and FeRB) to demonstrate whether they have the same or stronger effects on Fe3+-DTPA/Na4[Fe(CN)6] RFB. The results showed that the experimental reactors could achieve higher charging current density and initial cathodic potential during constant voltage charging process. The electrochemical impedance spectroscopy data and cyclic voltammetry curves demonstrated that the polarization impedance increased slower and reduction peak potential of experimental groups also emerged a positive shift compared to CK. According to chronopotentiometry experiments results, the microbes could function at maximum 0.3 M, 12 mA/cm2, and also improved the charging specific capacity. Combined the SEM pictures and microbial composition analysis, the main functional electroactive FeRB were Alcaligenes, Corynebacterium and Bacillus, which indicated to have important potential in improving the performance of RFBs.

Direct ammonia oxidation (Dirammox) is favored over cell growth in Alcaligenes ammonioxydans HO-1 to deal with the toxicity of ammonium.

Bacteria capable of direct ammonia oxidation (Dirammox) play important roles in global nitrogen cycling and nutrient removal from wastewater. Dirammox process, NH3 → NH2 OH → N2 , first defined in Alcaligenes ammonioxydans HO-1 and encoded by dnf gene cluster, has been found to widely exist in aquatic environments. However, because of multidrug resistance in Alcaligenes species, the key genes involved in the Dirammox pathway and the interaction between Dirammox process and the physiological state of Alcaligenes species remain unclear. In this work, ammonia removal via the redistribution of nitrogen between Dirammox and microbial growth in A. ammonioxydans HO-1, a model organism of Alcaligenes species, was investigated. The dnfA, dnfB, dnfC, and dnfR genes were found to play important roles in the Dirammox process in A. ammonioxydans HO-1, while dnfH, dnfG, and dnfD were not essential genes. Furthermore, an unexpected redistribution phenomenon for nitrogen between Dirammox and cell growth for ammonia removal in HO-1 was revealed. After the disruption of the Dirammox in HO-1, more consumed NH4 + was recovered as biomass-N via rapid metabolic response and upregulated expression of genes associated with ammonia transport and assimilation, tricarboxylic acid cycle, sulfur metabolism, ribosome synthesis, and other molecular functions. These findings deepen our understanding of the molecular mechanisms for Dirammox process in the genus Alcaligenes and provide useful information about the application of Alcaligenes species for ammonia-rich wastewater treatment.

Alcaligenes lipid A functions as a superior mucosal adjuvant to monophosphoryl lipid A via the recruitment and activation of CD11b+ dendritic cells in nasal tissue.

We previously demonstrated that Alcaligenes-derived lipid A (ALA), which is produced from an intestinal lymphoid tissue-resident commensal bacterium, is an effective adjuvant for inducing antigen-specific immune responses. To understand the immunologic characteristics of ALA as a vaccine adjuvant, we here compared the adjuvant activity of ALA with that of a licensed adjuvant (monophosphoryl lipid A, MPLA) in mice. Although the adjuvant activity of ALA was only slightly greater than that of MPLA for subcutaneous immunization, ALA induced significantly greater IgA antibody production than did MPLA during nasal immunization. Regarding the underlying mechanism, ALA increased and activated CD11b+ CD103- CD11c+ dendritic cells in the nasal tissue by stimulating chemokine responses. These findings revealed the superiority of ALA as a mucosal adjuvant due to the unique immunologic functions of ALA in nasal tissue.

Biodegradation of sulfametoxydiazine by Alcaligenes aquatillis FA: Performance, degradation pathways, and mechanisms.

This study reports the isolation and characterization of a novel bacterial strain Alcaligenes aquatillis FA with the ability to degrade sulfametoxydiazine (SMD), a commonly used sulfonamide antibiotic (SA) in livestock and poultry production. The biodegradation kinetics, pathways, and genomic background of SMD by FA were investigated. The results showed that strain FA had high specificity to degrade SMD, and was unable to effectively degrade its isomer, sulfamonomethoxine. The SMD biodegradation followed a first-order kinetic model with a rate constant of 27.39 mg·L-1·day-1 and a half-life of 5.98 days. The biodegradation pathways and detoxification processes of SMD were proposed based on the identification of its biodegradation byproducts and the biotoxicity assessment using both the ecological structure-activity relationship (ECOSAR) model and biological indicator. The involvement of novel degrading enzymes, such as dimethyllsulfone monooxygenase, 4-carboxymuconolactone decarboxylase, and 1,4-benzoquinone reductase, was inferred in the SMD biodegradation process. The presence of sul2 and dfrA genes in strain FA, which were constitutively expressed in its cells, suggests that multiple mechanisms were employed by the strain to resist SMD. This study provides new insights into the biodegradation of sulfonamide antibiotics (SAs) as it is the first to describe an SMD-degrading bacterium and its genetic information.

Engineered Alcaligenes sp. by chemical mutagen produces thermostable and acido-alkalophilic endo-1,4-ß-mannanases for improved industrial biocatalyst.

This study reported physicochemical properties of purified endo-1,4-ß-mannanase from the wild type, Alcaligenes sp. and its most promising chemical mutant. The crude enzymes from fermentation of wild and mutant bacteria were purified by ammonium sulfate precipitation, ion exchange and gel-filtration chromatography followed by an investigation of the physicochemical properties of purified wild and mutant enzymes. ß-mannanase from wild and mutant Alcaligenes sp. exhibited 1.75 and 1.6 purification-folds with percentage recoveries of 2.6 and 2.5% and molecular weights of 61.6 and 80 kDa respectively. The wild and mutant ß-mannanase were most active at 40 and 50 °C with optimum pH 6.0 for both and were thermostable with very high percentage activity but the wild-type ß-mannanase showed better stability over a broad pH activity. The ß-mannanase activity from the parent strain was stimulated in the presence of Mn2+, Co2+, Zn2+, Mg2+ and Na+. Vmax and Km for the wild type and its mutant were found to be 0.747 U//mL/min and 5.2 × 10-4 mg/mL, and 0.247 U/mL/min and 2.47 × 10-4 mg/mL, respectively. Changes that occurred in the nucleotide sequences of the most improved mutant may be attributed to its thermo-stability, thermo-tolerant and high substrate affinity- desired properties for improved bioprocesses.

TLR4 agonist activity of Alcaligenes lipid a utilizes MyD88 and TRIF signaling pathways for efficient antigen presentation and T cell differentiation by dendritic cells.

Alcaligenes faecalis was previously identified as an intestinal lymphoid tissue-resident commensal bacteria, and our subsequent studies showed that lipopolysaccharide and its core active element (i.e., lipid A) have a potent adjuvant activity to promote preferentially antigen-specific Th17 response and antibody production. Here, we compared A. faecalis lipid A (ALA) with monophosphoryl lipid A, a licensed lipid A-based adjuvant, to elucidate the immunological mechanism underlying the adjuvant properties of ALA. Compared with monophosphoryl lipid A, ALA induced higher levels of MHC class II molecules and costimulatory CD40, CD80, and CD86 on dendritic cells (DCs), which in turn resulted in strong T cell activation. Moreover, ALA more effectively promoted the production of IL-6 and IL-23 from DCs than did monophosphoryl lipid A, thus leading to preferential induction of Th17 and Th1 cells. As underlying mechanisms, we found that the ALA-TLR4 axis stimulated both MyD88- and TRIF-mediated signaling pathways, whereas monophosphoryl lipid A was biased toward TRIF signaling. These findings revealed the effects of ALA on DCs and T cells and its induction pattern on signaling pathways.

Long-term stability of reactor microbiome through bioaugmentation with Alcaligenes aquatilis AS1 promotes nitrogen removal of piggery wastewater.

Bioaugmentation is considered as an attractive method for nitrogen removal in water treatment, but its effectiveness in actual high-strength piggery wastewater has not been adequately verified and the mechanism of bioaugmentation in actual wastewater treatment system is not very clear especially from the perspectives of microbial communities and functional genes. This study investigated the mechanisms of a heterotrophic nitrifying-aerobic denitrifying strain Alcaligenes aquatilis AS1 in the bioaugmentation of continuous biological nitrogen removal of actual piggery wastewater at laboratory scale. The addition of strain AS1 significantly improved the nitrogen removal efficiency (more than 95% of NH4+-N and 75% of TN were removed) and raised the activated sludge resistance to shock loading. AS1 addition also significantly shifted the microbiota structure and interactions among microbial networks were enhanced to obtain the stable bacterial communities. Moreover, strain AS1 achieved effective proliferation and long-term colonization in activated sludge with a relative abundance of genus Alcaligenes more than 70% during the whole operation process and played a dominant role in biological nitrogen removal, while different genera were respectively enriched and involved in pollutants removal at different stages in the control group. In addition, the abundances of most functional genes involved in carbon (C) degradation, carbon fixation and nitrogen (N), phosphorus (P), sulfur (S) cycling in activated sludge were significantly increased in reactor AS1, indicating that strain AS1 not only relied on its unique C and N metabolic activities, but also recruited microorganisms with diverse functions to jointly remove pollutants in wastewater, which could be a common bioaugmentation mechanism in open reactors. This study proves the promising application prospect of strain AS1 in the treatment of high-strength piggery wastewater and shows great importance for guiding bioaugmentation application of functional strains in practical wastewater treatment systems.

Exploring variation in the fecal microbial communities of Kasaragod Dwarf and Holstein crossbred cattle.

The gut microbiota and its impact on health and nutrition in animals, including cattle has been of intense interest in recent times. Cattle, in particular indigenous varieties like Kasaragod Dwarf cow, have not received the due consideration given to other non-native cattle breeds, and the composition of their fecal microbiome is yet to be established. This study applied 16S rRNA high-throughput sequencing of fecal samples and compared the Kasaragod Dwarf with the highly prevalent Holstein crossbred cattle. Variation in their microbial composition was confirmed by marker gene-based taxonomic analysis. Principle Coordinate Analysis (PCoA) showed the distinct microbial architecture of the two cattle types. While the two cattle types possess unique signature taxa, in Kasaragod Dwarf cattle, many of the identified genera, including Anaerovibrio, Succinivibrio, Roseburia, Coprococcus, Paludibacter, Sutterella, Coprobacillus, and Ruminobacter, have previously been shown to be present in higher abundance in animals with higher feed efficiency. This is the first report of Kasaragod Dwarf cattle fecal microbiome profiling. Our findings highlight the predominance of specific taxa potentially associated with different fermentation products and feed efficiency phenotypes in Kasaragod Dwarf cattle compared to Holstein crossbred cattle.

Identification and characterization of a novel hydroxylamine oxidase, DnfA, that catalyzes the oxidation of hydroxylamine to N2.

Nitrogen (N2) gas in the atmosphere is partially replenished by microbial denitrification of ammonia. Recent study has shown that Alcaligenes ammonioxydans oxidizes ammonia to dinitrogen via a process featuring the intermediate hydroxylamine, termed "Dirammox" (direct ammonia oxidation). However, the unique biochemistry of this process remains unknown. Here, we report an enzyme involved in Dirammox that catalyzes the conversion of hydroxylamine to N2. We tested previously annotated proteins involved in redox reactions, DnfA, DnfB, and DnfC, to determine their ability to catalyze the oxidation of ammonia or hydroxylamine. Our results showed that none of these proteins bound to ammonia or catalyzed its oxidation; however, we did find DnfA bound to hydroxylamine. Further experiments demonstrated that, in the presence of NADH and FAD, DnfA catalyzed the conversion of 15N-labeled hydroxylamine to 15N2. This conversion did not happen under oxygen (O2)-free conditions. Thus, we concluded that DnfA encodes a hydroxylamine oxidase. We demonstrate that DnfA is not homologous to any known hydroxylamine oxidoreductases and contains a diiron center, which was shown to be involved in catalysis via electron paramagnetic resonance experiments. Furthermore, enzyme kinetics of DnfA were assayed, revealing a Km of 92.9 ± 3.0 µM for hydroxylamine and a kcat of 0.028 ± 0.001 s-1. Finally, we show that DnfA was localized in the cytoplasm and periplasm as well as in tubular membrane invaginations in HO-1 cells. To the best of our knowledge, we conclude that DnfA is the first enzyme discovered that catalyzes oxidation of hydroxylamine to N2.

Unveiling steps of the TDP degradation pathway in Variovorax paradoxus TBEA6.

Since the role of biobased plastics increases every year, the search for alternatives to petrol-based polymers is very important. Variovorax paradoxus TBEA6 is able to grow with 3,3'-thiodipropionic acid (TDP) as sole source for carbon and energy. TDP can be used as a precursor substrate for the synthesis of polythioesters (PTE). To increase the feasibility of PTE synthesis, a good understanding of the degradation pathway of TDP in V. paradoxus TBEA6 is essential. Therefore, two putative 3-hydroxyisobutyryl-CoA hydrolases (VPARA_03110 & VPARA_05510) and two putative 3-hydroxypropionate dehydrogenases (VPARA_41140 & VPARA_54550) were investigated in this study. The deletion mutant V. paradoxus ∆VPARA_05510 showed a TDP-negative phenotype during growth experiments. The ability to grow with TDP as sole carbon source was successfully restored by complementation. Supernatant analysis revealed that the deletion mutant did not metabolize TDP or 3MP anymore. A specific enzyme activity up to 0.032 U/mg for the purified 3-hydroxyisobutyryl-CoA hydrolase VPARA_05510 was determined. A shift in the proteins (VPARA_54550) melting temperature of 6 °C with 2000 µM 3HP in comparison to protein without ligand was observed during thermal shift assays with the putative 3-hydroxypropionate dehydrogenase.

Tetracycline biotransformation by a novel bacterial strain Alcaligenes sp. T17.

Comprehensive knowledge on the biotransformation of tetracycline (TC) is critical for the improvement of TC removal in the bioremediation process. This work isolated a novel TC-degrading bacterial strain Alcaligenes sp. T17 and explored its degradation ability under different conditions. Temperature and pH could affect the degradation efficiency, and higher temperature as well as neutral and weakly acidic conditions were conducive to the biotransformation. Response surface methodology predicted the maximum degradation rate of TC (94.35%) under the condition of 25.15 mg/L TC, pH 7.23, and inoculation dosage 1.17% at 40 °C. According to the result of disk diffusion tests, the biodegradation products had lower antimicrobial potency than the parent compound. Five potential biodegradation products were identified, and a possible degradation pathway (degrouping, oxidation and ring-opening) was proposed. The draft genome of strain T17 was also determined. Genomic analysis indicated that strain T17 harbored multiple genes that participated in the metabolism of aromatic compounds as well as genes encoding oxygenases. These functional genes may be relevant to TC biotransformation. This study could provide new insights towards the biotransformation of TC mediated by bacteria.

Characterization of Alcaligenes aquatilis as a novel member of heterotrophic nitrifier-aerobic denitrifier and its performance in treating piggery wastewater.

A novel strain AS1 with heterotrophic nitrifying-aerobic denitrifying capacity in the species of Alcaligenes aquatilis was isolated from the aerobic activated sludge. It showed a great capability of ammonia removal, and the aerobic metabolic pathways to yield gaseous-nitrogen by hydroxylamine oxidation and nitrite denitrification were proposed. AS1 could efficiently remove ammonia under a wide range of environmental conditions, including the ratio of chemical oxygen demand to total nitrogen: 15-30, pH: 6-10, NaCl: 0-60 g/L, shaking speed of 0-180 rpm, and succinate, acetate, or citrate as carbon source. In the treatment of actual piggery wastewater, 95.3%, 95.1% and 84.9% of NH4+-N was removed by AS1 when the initial ammonia concentration was 500, 1300, and 2000 mg/L, respectively, with the maximum NH4+-N removal rate of 30.5 mg/L/h and 569.7 mg/L/d. Furthermore, plate colony-counting showed that AS1 achieved an efficient proliferation. These results imply the application potential of AS1 in treating high-ammonia wastewater.

Sulphonated azo dye decolorization by Alcaligenes faecalis subsp. phenolicus MB207: Insights from laboratory and computational analysis.

Microbial decolorization of azo dyes, mediated by an enzymatic mechanism is an intricate cost-effective, and eco-friendly treatment method of genotoxic azo pollutants. Scientists are on the constant lookout for microbes, enzymes, and mechanisms that could aid remediation of the environment at a fast pace. Alcaligenes faecalis subsp. phenolicus MB207 is one such bacteria, consisting of azoreductase (AzoR) and laccase/multicopper oxidase enzyme responsible for sulphonated mono-azo dye (Methyl orange) and di-azo dye (Congo red) degradation. AzoR degrades dyes by a ping-pong setup while multicopper oxidase achieves this through a non-specified radical approach. We have coupled experimental analysis with bioinformatics for deciphering intricacies of this procedure in tiny scale enzymatic machines of this biotope. The degradation assays were followed by molecular docking of the enzyme-substrate complexes. Key anchoring bonds were detected and mapped H-bonding, electron exchange, ionic interactions, as well as hydrophobic interactions, provided insights into dye-enzyme and NADH-enzyme binding. This study establishes a foundation of the molecular basis of dye interaction with azoR and multicopper oxidase in A. facealis subsp. phenolicus MB207.

Simultaneous biodegradation of dimethyl sulfide and 1-propanethiol by Pseudomonas putida S-1 and Alcaligenes sp. SY1: "Lag" cause, reduction, and kinetics exploration.

Simultaneous biodegradation of malodorous 1-propanethiol (PT) and dimethyl sulfide (DMS) by Pseudomonas putida S-1 and Alcaligenes sp. SY1 was investigated and the interactions implicated were explored. Results showed that PT was completely degraded in 33 h, while a lag of 10 h was observed for DMS degradation alone, and the lag was even extended to 81 h in the binary mixture. Mechanism analysis found that the lag was mainly attributed to the exposure of DMS degrader (Alcaligenes sp. SY1), rather than PT metabolites and PT degrader. The exposure time and PT concentration also influenced the lag duration much. Citric acid could effectively reduce the lag. Pseudo-first-order model was proved suitable for the description of PT degradation, revealing that PT degradation could be enhanced in presence of DMS with a concentration of < 50 mg L-1. A modified Gompertz model, incorporated the lag phase, was developed for the description of DMS degradation in the mixture, revealing that DMS degradation depended on the initial PT concentration, and when the lag was not considered, PT with low-concentration could promote DMS biodegradation, while a higher concentration (> 20 mg L-1) cast negative effect.

Microbiologically influenced corrosion of Cu by marine ammonifying Alcaligenes aquatilis bacterium.

In this work, we studied the microbiologically influenced corrosion mechanism of Cu by marine ammonifying bacterium Alcaligenes aquatilis. Through immersion experiments, we found that A. aquatilis could accelerate the corrosion rate of copper, resulting in the development of pits. In the presence of A. aquatilis, the morphology and composition of the corrosion products differed from the abiotic samples, and we found that Cu2O was the main corrosion product. By analyzing the biotic medium and experimental NH3 addition, we verified that NH3 was the main component that intensified copper corrosion. Furthermore, we found that NH3 played a catalytic role in the corrosion of Cu in the presence of A. aquatilis.

Weilan gum oligosaccharide ameliorates dextran sulfate sodium­induced experimental ulcerative colitis.

Ulcerative colitis (UC) is a global disease, characterized by periods of relapse that seriously affects the quality of life of patients. Oligosaccharides are considered to be a prospective strategy to alleviate the symptoms of UC. The present study aimed to evaluate the effect of weilan gum oligosaccharide (WLGO) on a mouse UC model induced by dextran sulfate sodium (DSS). WLGO structural physical properties were characterized by electrospray mass spectrometry and fourier tansform infrared spectroscopy. MTT assays were performed to evaluate the non­toxic concentration of WLGO. RT­qPCR and ELISAs were conducted to determine the levels of inflammatory factors. The clinical symptoms and mucosal integrity of the DSS­induced UC model were assessed by DAI and histological assessment. LPS­induced Caco­2 cells and DSS­induced UC mice were used to explore the effects of WLGO on UC. Treatment of the mice with 4.48 g/kg/day WLGO via gavage for 7 days significantly relieved the symptoms of DSS­induced UC model mice, whereas significant effects were not observed for all symptoms of DSS­induced UC in the WLGO­low group. The disease activity index score was decreased and the loss of body weight was reduced in DSS­induced UC model mice treated with WLGO. Moreover, colonic damage and abnormally short colon length shortenings were relieved following WLGO treatment. WLGO treatment also reduced the concentration and mRNA expression levels of proinflammatory cytokines, including interleukin­1ß, interleukin­6 and tumor necrosis factor α, in DSS­induced UC model mice and lipopolysaccharide­treated Caco­2 cells. These results indicated that WLGO may be an effective strategy for UC treatment.

Removal of harmful algae by Shigella sp. H3 and Alcaligenes sp. H5: algicidal pathways and characteristics.

Application of algicidal bacteria is a promising technology to control harmful algal blooms (HABs). In this study, algicidal bacteria strains Shigella sp. H3 and Alcaligenes sp. H5 were obtained via two different isolation methods from the same lake water sample, with optimal algicidal efficiencies 96% and 74% against algae mixture. The Shigella sp. H3 and Alcaligenes sp. H5 lysed algae cells through cells-to-cells direct contact and secretion of algicidal metabolites, respectively. The stronger algicidal capability of Shigella sp. H3 was also attributable to its higher efficiency for triggering reactive oxygen species, which led to broken down of the antioxidant system and more severe damage to the bacterial cells. The antioxidant enzyme activities in Alcaligenes sp. H5 group were still expressed because of its relatively weaker algicidal capability and some intact algal cells were remained. The liquid carbohydrates from algal lysis in both groups increased significantly, whereas the quantities of liquid protein decreased, which might be assimilated by algicidal bacteria. Nonetheless, the whole algicidal process resulted in the increase of total released organic matters content. This study revealed the algicidal pathways of diverse bacterial strains, and the possible secondary environmental problem caused by the algal released organic matters should be considered when applying bacteria to control HABs.

Characterization of biofilm formation and reduction of hexavalent chromium by bacteria isolated from tannery sludge.

Biofilm formation ability of bacteria makes them potential in the field of tannery effluent treatment. However, the hazardous nature of effluent and environmental conditions may disturb the biofilm formation ability of bacteria which ultimately affects their effluent treatment efficiency. Accordingly, we isolated and characterized biofilm-forming bacteria Bacillus vallismortis (MT027009), Bacillus haynesii (MT027008), and Alcaligenes aquatilis (MT027005) from tannery sludge and examined them for biofilm formation under variable environmental conditions. Biofilm formation in tryptic soy broth (TSB) at different incubation times (24-120 h) revealed that the biofilm formation activity of the strain B. haynesii was not affected by incubation time, whereas the increase in biofilm formation was observed in the case of B. vallismortis (28 %) and A. aquatilis (52 %) after 48 h. The medium pH (pH 5.0-9.0) had a limited effect on biofilm formation except in the case of A. aquatilis at pH 5.0 (94 %) and pH 9.0 (80 %). Furthermore, compared to the controls (only TSB), the strains B. vallismortis, B. haynesii, and A. aquatilis showed enhanced biofilm formation in undiluted tannery effluent (28, 33, and 21 %) and 25 mg L-1 Cr(VI) (23 %, 48 % 32 %). The biofilm structure was influenced by Cr(VI) as revealed by scanning electron microscopy (SEM) analysis. The results of Cr(VI) bioreduction studies suggest that bacterial biofilm (60-99 %) has a greater potential to remove Cr(VI) than planktonic cells (43-94 %). The results of the study provide important data on biofilm formation by indigenous bacteria in effluent environment conditions, making them potential isolates for tannery effluent treatment.

Novel Alcaligenes ammonioxydans sp. nov. from wastewater treatment sludge oxidizes ammonia to N2 with a previously unknown pathway.

Heterotrophic nitrifiers are able to oxidize and remove ammonia from nitrogen-rich wastewaters but the genetic elements of heterotrophic ammonia oxidation are poorly understood. Here, we isolated and identified a novel heterotrophic nitrifier, Alcaligenes ammonioxydans sp. nov. strain HO-1, oxidizing ammonia to hydroxylamine and ending in the production of N2 gas. Genome analysis revealed that strain HO-1 encoded a complete denitrification pathway but lacks any genes coding for homologous to known ammonia monooxygenases or hydroxylamine oxidoreductases. Our results demonstrated strain HO-1 denitrified nitrite (not nitrate) to N2 and N2 O at anaerobic and aerobic conditions respectively. Further experiments demonstrated that inhibition of aerobic denitrification did not stop ammonia oxidation and N2 production. A gene cluster (dnfT1RT2ABCD) was cloned from strain HO-1 and enabled E. coli accumulated hydroxylamine. Sub-cloning showed that genetic cluster dnfAB or dnfABC already enabled E. coli cells to produce hydroxylamine and further to 15 N2 from (15 NH4 )2 SO4 . Transcriptome analysis revealed these three genes dnfA, dnfB and dnfC were significantly upregulated in response to ammonia stimulation. Taken together, we concluded that strain HO-1 has a novel dnf genetic cluster for ammonia oxidation and this dnf genetic cluster encoded a previously unknown pathway of direct ammonia oxidation (Dirammox) to N2 .