Revealing the degrading-possibility of methyl red by two azoreductases of Anoxybacillus sp. PDR2 based on molecular docking.

Azo dyes, as the most widely used synthetic dyes, are considered to be one of the culprits of water resources and environmental pollution. Anoxybacillus sp. PDR2 is a thermophilic bacterium with the ability to degrade azo dyes, whose genome contains two genes encoding azoreductases (named AzoPDR2-1 and AzoPDR2-2). In this study, through response surface methodology (RSM), when the initial pH, inoculation volume and Mg2+ addition amount were 7.18, 10.72% and 0.1 g/L respectively, the decolorization rate of methyl red (MR) (200 mg/L) could reach its maximum (98.8%). The metabolites after biodegradation were detected by UV-Vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), and liquid chromatography mass spectrometry (LC-MS/MS), indicating that MR was successfully decomposed into 4-aminobenzoic acid and other small substrates. In homologous modeling, it was found that both azoreductases were flavin-dependent azoreductases, and belonged to the α/ß structure, using the Rossmann fold. In their docking results with the cofactor flavin mononucleotide (FMN), FMN bound to the surface of the protein dimer. Nicotinamide adenine dinucleotide (NADH) was superimposed on the plane of the pyrazine ring between FMN and the activity pocket of protein. Besides, both azoreductase complexes (azoreductase-FMN-NADH) exhibited a substrate preference for MR. Asn104 and Tyr74 played an important role in the combination of the azoreductase AzoPDR2-1 complex and the azoreductase AzoPDR2-2 complex with MR, respectively. This provided assistance for studying the mechanism of azoreductase biodegradation of azo dyes in thermophilic bacteria.

Molecular response of Anoxybacillus sp. PDR2 under azo dye stress: An integrated analysis of proteomics and metabolomics.

Treating azo dye wastewater using thermophilic bacteria is considered a more efficient bioremediation strategy. In this study, a thermophilic bacterial strain, Anoxybacillus sp. PDR2, was regarded as the research target. This strain was characterized at different stages of azo dye degradation by using TMT quantitative proteomic and non-targeted metabolome technology. A total of 165 differentially expressed proteins (DEPs) and 439 differentially metabolites (DMs) were detected in comparisons between bacteria with and without azo dye. It was found that Anoxybacillus sp. PDR2 can degrade azo dye Direct Black G (DBG) through extracellular electron transfer with glucose serving as electron donors. Most proteins related to carbohydrate metabolism, including acetoacetate synthase, and malate synthase G, were overexpressed to provide energy. The bacterium can also self-synthesize riboflavin as a redox mediator of in vitro electron transport. These results lay a theoretical basis for industrial bioremediation of azo dye wastewater.

Genome-based reclassification of Anoxybacillus karvacharensis Panosyan et al. as a later heterotypic synonym of Anoxybacillus kestanbolensis Dulger et al. 2004; A. flavithermus subsp. yunnanensis CCTCC AB2010187T Dai et al. 2011 as a later heterotypic synonym of A. tengchongensis DSM 23211T Zhang et al. 2011.

In the present study, we attempted to clarify the taxonomic positions of Anoxybacillus karvacharensis K1T, Anoxybacillus kestanbolensis NCIMB 13971T, Anoxybacillus flavithermus subsp. yunnanensis CCTCC AB2010187T, and Anoxybacillus tengchongensis DSM 23211T using whole-genome phylogenetic analysis. The genome sequence of A. kestanbolensis NCIMB13971T was not available in any database, so it was sequenced in this study. The 16S rRNA gene sequence obtained from the genome of A. kestanbolensis NCIMB13971T had 99.93% similarity with A. karvacharensis K1T. The average nucleotide identity (ANI), average amino acid identity (AAI), and digital DNA-DNA hybridization (DDH) values between A. karvacharensis K1T and A. kestanbolensis NCIMB13971T and between A. flavithermus subsp. yunnanensis CCTCCAB 2010187T and A. tengchongensis DSM 23211T were greater than the threshold values for species demarcation. The present results indicate that A. karvacharensis K1T is a later heterotypic synonym of A. kestanbolensis NCIMB13971T; A. flavithermus subsp. yunnanensis CCTCCAB 2010187T is a later heterotypic synonym of A. tengchongensis DSM 23211T.

Cloning, biochemical characterization and molecular docking of novel thermostable ß-glucosidase BglA9 from Anoxybacillus ayderensis A9 and its application in de-glycosylation of Polydatin.

This study reports a novel BglA9 gene of 1345 bp encoding ß-glucosidase from Anoxybacillus ayderensis A9, which was amplified and expressed in E. coli BL21 (DE3): pLysS cells, purified with Ni-NTA column having molecular weight of 52.6 kDa and was used in the bioconversion of polydatin to resveratrol. The kinetic parameters values using pNPG as substrate were Km (0.28 mM), Vmax (43.8 µmol/min/mg), kcat (38.43 s-1) and kcat/Km (135.5 s-1 mM-1). The BglA9 was active in a broad pH range and had an activity half-life around 24 h at 50 °C. The de-glycosylation efficiency of BglA9 for polydatin was determined by estimating the amount of glucose released after enzymatic reaction by a dinitrosalicylic acid (DNS) assay. The kinetic parameters of BglA9 for polydatin were 5.5 mM, 20.84 µmol/min/mg, 18.28 s-1and 3.27 s-1 mM-1 for Km, Vmax, kcat, and kcat/Km values, respectively. The Ki value for glucose was determined to be 1.7 M. The residues Gln19, His120, Glu355, Glu409, Glu178, Asn222 may play a crucial role in the deglycosylation as revealed by the 3D structure of enzyme docked with polydatin.

Detoxification of azo dye Direct Black G by thermophilic Anoxybacillus sp. PDR2 and its application potential in bioremediation.

Direct Black G (DBG) is a highly toxic synthetic azo dye which is difficult to degrade. Biological treatment seems to be a promising option for the treatment of azo dye containing effluent. A thermophilic bacterial strain (Anoxybacillus sp. PDR2) previously isolated from the soil can effectively remove DBG. However, the molecular underpinnings of DBG degradation and the microbial detoxification ability remains unknown. In the present study, the genetic background of PDR2 for the efficient degradation of DBG and its adaptation to azo dye-contaminated environments was revealed by bioinformatics. Moreover, the possible biodegradation pathways were speculated based on the UV-vis spectral analysis, FTIR, and intermediates identified by LC-MS. Additionally, phytotoxicity and the comet experiment studies clearly indicated that PDR2 converts toxic azo dye (DBG) into low toxicity metabolites. The combination of biodegradation pathways and detoxification analysis were utilized to explore the molecular degradation mechanism and bioremediation of azo dye for future applications. These findings will provide a valuable theoretical basis for the practical treatment of azo dye wastewater.

Genome and transcriptome analysis of a newly isolated azo dye degrading thermophilic strain Anoxybacillus sp.

Understanding azo dye degrading enzymes and the encoding of their functional genes is crucial for the elucidation of their molecular mechanisms. In this study, a thermophilic strain capable of degrading azo dye was isolated from the soil near a textile dye manufacturing factory. Based on its morphological, physiological and biochemical properties, as well as 16S rRNA gene sequence analysis, the strain was identified as Anoxybacillus sp. PDR2. The decolorization ratios of 100-600 mg/L Direct Black G (DBG) by strain PDR2 reached 82.12-98.39% within 48 h of dyes. Genome analysis revealed that strain PDR2 contains a circular chromosome of 3791144 bp with a G + C content of 42.48%. The genetic basis of azo dye degradation by strain PDR2 and its capacity to adapt to harsh environments, were further elucidated through bioinformatics analysis. RNA-Seq and qRT-PCR technology confirmed that NAD(P)H-flavin reductase, 2Fe-2S ferredoxin and NAD(P)-dependent ethanol dehydrogenase genes expressed by strain PDR2, were the key genes involved in DBG degradation. The combination of genome and transcriptome analysis was utilized to explore the key genes of strain PDR2 involved in azo dye biodegradation, with these findings providing a valuable theoretical basis for the practical treatment of azo dye wastewater.

Structure and mechanism of the Mrp complex, an ancient cation/proton antiporter.

Multiple resistance and pH adaptation (Mrp) antiporters are multi-subunit Na+ (or K+)/H+ exchangers representing an ancestor of many essential redox-driven proton pumps, such as respiratory complex I. The mechanism of coupling between ion or electron transfer and proton translocation in this large protein family is unknown. Here, we present the structure of the Mrp complex from Anoxybacillus flavithermus solved by cryo-EM at 3.0 Å resolution. It is a dimer of seven-subunit protomers with 50 trans-membrane helices each. Surface charge distribution within each monomer is remarkably asymmetric, revealing probable proton and sodium translocation pathways. On the basis of the structure we propose a mechanism where the coupling between sodium and proton translocation is facilitated by a series of electrostatic interactions between a cation and key charged residues. This mechanism is likely to be applicable to the entire family of redox proton pumps, where electron transfer to substrates replaces cation movements.

The extremophile Anoxybacillus sp. PC2 isolated from Brazilian semiarid region (Caatinga) produces a thermostable keratinase.

The aim of this study was to select and identify thermophilic bacteria from Caatinga biome (Brazil) able to produce thermoactive keratinases and characterize the keratinase produced by the selected isolate. After enrichment in keratin culture media, an Anoxybacillus caldiproteolyticus PC2 was isolated. This thermotolerant isolate presents a remarkable feature producing a thermostable keratinase at 60°C. The partially purified keratinase, identified as a thermolysin-like peptidase, was active at a pH range of 5.0-10.0 with maximal activity at a temperature range of 50-80°C. The optimal activity was observed at pH 7.0 and 50-60°C. These characteristics are potentially useful for biotechnological purposes such as processing and bioconversion of keratin.

Resistance, removal, and bioaccumulation of Ni (II) and Co (II) and their impacts on antioxidant enzymes of Anoxybacillus mongoliensis.

In this study, it was hypothesis that A. mongoliensis could be used as bioindicator for Ni (II) and Co (II). Thus, Ni (II) and Co (II) resistance, removal, bioaccumulation, and the impacts of them on antioxidant enzyme systems of thermophilic Anoxybacillus mongoliensis were investigated in details. The bioaccumulation of Ni (II) and Co (II) on the cell membrane of thermophilic A. mongoliensis, variations on surface macrostructure and functionality by FT-IR and SEM, and determination of antioxidant enzyme activities were also tested. The highest bioaccumulation values of Co (II) and Ni (II) were detected as 102.0 mg metal/g of dry bacteria at 10 mg/L for the 12th h and 90.4 mg metal/g of dry bacteria for the 24th h, respectively, and the highest Ni (II) and Co (II) cell membrane bioaccumulation capacities of A. mongoliensis were determined as 268.5 and 274.9 mg metal/g wet membrane, respectively at the 24th h. In addition, increasing on SOD and CAT activities were observed on depend of concentration of Ni (II) and Co (II) with respect to control. The antioxidant enzyme activity results also indicated that A. mongoliensis might be used as a bioindicator for Ni (II) and Co (II) pollution in environmental water specimens.

An evolution-based designing and characterization of mutants of cyclomaltodextrinase: Molecular modeling and spectroscopic studies.

Cyclomaltodextrinase (CDase) is a member of the alpha-amylase family GH13, the subfamily GH13_20. In addition to CDase and neopullulanase, this subfamily also contains maltogenic amylase. They have common structural features, but different substrate specificity. In current work, a combination of bioinformatics and experimental tools were used for designing and constructions of single and double mutants of a new variant of CDase from Anoxybacillus flavithermus. Considering the evolutionary variable positions 123 and 127 at the dimer interface of subunits in the alpha-amylase family, these positions in CDase were modified and three mutants, including A123V, C127Q and A123V/C127Q were constructed. The tertiary structure of WT and mutants were made with the MODELLER program, and the phylogenetic tree of homologous protein sequences was built with selected programs in Phylip package. Enzyme kinetic studies revealed that the catalytic efficiency of mutants, especially double one, is lower than the WT enzyme. Heat-induced denaturation experiments were monitored by measuring the UV/Vis signal at 280 nm, and it was found that WT protein is structurally more stable at 25 °C. However, it is more susceptible to changes in temperature compared to the double mutant. It was concluded that the positions 123 and 127 at the dimeric interface of CDase, not only could affect the conformational stability; but also; the catalytic properties of the enzyme by setting up the active site configuration in the dimeric state.

Association of Anoxybacillus sp. with acid off-flavor development in a spoiled, boiled, rice dish.

Off-flavor is one of the most common food complaints. In this study, we demonstrated that acetic acid produced by Anoxybacillus sp. contamination of takikomi-gohan (boiled rice with sweet potato mixed in advance) was considered the causative agent of acid off-flavor development. First, we conducted whole genome sequencing of the bacterial strain (S1674) isolated from the remains of the contaminated takikomi-gohan, and phylogenetic analysis of k-mer diversity demonstrated that S1674 belongs to the Anoxybacillus genus. Gene expression analysis of S1674 RNA sequencing (RNA-seq) and quantitative reverse transcription polymerase chain reaction (qRT-PCR) indicated that the genes encoding enzymes responsible for acetic acid formation, namely ackA1, eutD, pflA, pflB, and pykA, were upregulated in high-temperature cultures in Thermus medium supplemented with soluble starch. Additionally, we succeeded in reproducing the acid off-flavor by adding S1674 to boiled rice stored at 37 °C, 45 °C, and 60 °C. The most strongly detected organic acid was acetic acid, at the odor threshold value or more in both the air and condensation samples. Our findings suggest that some Anoxybacillus sp. produce acetic acid as a byproduct of carbohydrate metabolism, potentially causing the complaint of acid off-flavor even under high-temperature conditions in which other bacteria cannot survive.

Anoxybacillus sediminis sp. nov., a novel moderately thermophilic bacterium isolated from a hot spring.

A Gram-stain positive, moderately thermophilic, aerobic, spore-forming and rod-shaped bacterium, designated YIM 73012T, was isolated from a sediment sample collected from a hot spring located in Tibet, China, and was characterized by using a polyphasic taxonomy approach. The strain is oxidase positive and catalase negative. Growth occurred at 37-65 °C (optimum, 45-50 °C), at pH 6.0-8.5 (optimum, pH 7.0-7.5) and with 0.5-3.5% NaCl (optimum, 0.5-1.0%, w/v). The major fatty acids were iso-C15:0, iso-C16:0 and C16:0. The major polar lipids comprised of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine and phosphatidylglycerol. The cell wall peptidoglycan contained meso-diaminopimelic acid. The respiratory quinone was MK-7. The G+C content of genomic DNA was 43.6 mol%. Phylogenetic analyses based on 16S rRNA gene sequences showed that the strain YIM 73012T forms a distinct lineage with respect to the genus Anoxybacillus in the family Bacillaceae. Based on 16S rRNA gene sequence identities the closely related phylogenetic neighbours are Anoxybacillus caldiproteolyticus DSM 15730T (96.7%) and Saccharococcus thermophilus DSM 4749T (96.6%). Strain YIM 73012T was distinguishable from the closely related reference strains by the differences in phenotypic, chemotaxonomic and genotypic characteristics, and represents a novel species of the genus Anoxybacillus, for which the name Anoxybacillus sp. nov. is proposed. The type species is Anoxybacillus sediminis sp. nov., with the type strain YIM 73012T (= KCTC 33884T = DSM 103835T).

The N-Terminal Domain of the Pullulanase from Anoxybacillus sp. WB42 Modulates Enzyme Specificity and Thermostability.

Anoxybacillus sp. WB42 pullulanase (PulWB42) is a novel thermophilic amylopullulanase that was assigned to the glycoside hydrolase family 13 subfamily 14 (GH13_14) type I pullulanases in the carbohydrate-active enzymes database. Its N-terminal domain (Met1-Phe101) was identified as the carbohydrate-binding module 68 (CBM68) by homology modeling. The N-domain-deleted PulWB42 exhibited an equivalent Michaelis constant (Km ) for pullulan and significant decreases in pullulytic activity, amylose selectivity, and thermostability relative to PulWB42 having a high α-amylase-to-pullulanase activity ratio. Furthermore, the replacement of Ala90 or Arg93 significantly changed the substrate specificity and catalytic efficiency of PulWB42, whereas Q87A, L173D, and H5A/R6A/T7A showed improvements in thermostability and changes in catalytic kinetics. Therefore, the N domain of PulWB42 is not essential for catalysis, but it does modulate enzyme catalysis, especially with respect to substrate specificity. The modulation was achieved mainly by the Leu86-Arg93 segment adjacent to the CBM48 domain and the catalytic A domain in the modeled structure of PulWB42.

Short communication: Growth of dairy isolates of Geobacillus thermoglucosidans in skim milk depends on lactose degradation products supplied by Anoxybacillus flavithermus as secondary species.

Thermophilic bacilli such as Anoxybacillus and Geobacillus are important contaminants in dairy powder products. Remarkably, one of the common contaminants, Geobacillus thermoglucosidans, showed poor growth in skim milk, whereas significant growth of G. thermoglucosidans was observed in the presence of an Anoxybacillus flavithermus dairy isolate. In the present study, we investigated the underlying reason for this growth dependence of G. thermoglucosidans. Whole-genome sequences of 4 A. flavithermus strains and 4 G. thermoglucosidans strains were acquired, with special attention given to carbohydrate utilization clusters and proteolytic enzymes. Focusing on traits relevant for dairy environments, comparative genomic analysis revealed that all G. thermoglucosidans strains lacked the genes necessary for lactose transport and metabolism, showed poor growth in skim milk, and produced white colonies on X-gal plates, indicating the lack of ß-galactosidase activity. The A. flavithermus isolates scored positive in these tests, consistent with the presence of a putative lactose utilization gene cluster. All tested isolates from both species showed proteolytic activity on milk plate count agar plates. Adding glucose or galactose to liquid skim milk supported growth of G. thermoglucosidans isolates, in line with the presence of the respective monosaccharide utilization gene clusters in the genomes. Analysis by HPLC of A. flavithermus TNO-09.006 culture filtrate indicated that the previously described growth dependence of G. thermoglucosidans in skim milk was based on the supply of glucose and galactose by A. flavithermus TNO-09.006.

Changes in the membrane fatty acid composition in Anoxybacillus flavithermus subsp. yunnanensis E13T as response to solvent stress.

Anoxybacillus flavithermus subsp. yunnanensis is currently the first species of strictly thermophilic bacteria that is able to tolerate a broad range of solvents. Unlike most of solvent-tolerant mesophilic bacteria, the bacterium does not synthesize unsaturated fatty acids. Our results revealed that in growing cells of A. flavithermus subsp. yunnanensis E13T, ethanol and toluene resulted in an increase in straight-chain fatty acids, mainly C16:0, leading to a more rigid membrane. Moreover, the increase in straight-chain fatty acids caused by ethanol was much higher than that of toluene. High temperature had little effect on the fatty acid composition by itself, whereas the combined conditions of high temperature and ethanol caused the dramatic increase in straight-chain fatty acids (mainly C16:0), that was balanced by decreasing branched fatty acids. The increase was also temperature dependent. The proportion of C16:0 further increased above 60 °C. No similar evidence was found in four other species of Anoxybacillus. The results suggested that A. flavithermus subsp. yunnanesis seems to develop a different response to solvents compared to its mesophilic counterparts, which consist of an increase in the saturated straight/branched ratio.

High-throughput pyrosequencing used for the discovery of a novel cellulase from a thermophilic cellulose-degrading microbial consortium.

OBJECTIVES: To analyze the microbial diversity and gene content of a thermophilic cellulose-degrading consortium from hot springs in Xiamen, China using 454 pyrosequencing for discovering cellulolytic enzyme resources. RESULTS: A thermophilic cellulose-degrading consortium, XM70 that was isolated from a hot spring, used sugarcane bagasse as sole carbon and energy source. DNA sequencing of the XM70 sample resulted in 349,978 reads with an average read length of 380 bases, accounting for 133,896,867 bases of sequence information. The characterization of sequencing reads and assembled contigs revealed that most microbes were derived from four phyla: Geobacillus (Firmicutes), Thermus, Bacillus, and Anoxybacillus. Twenty-eight homologous genes belonging to 15 glycoside hydrolase families were detected, including several cellulase genes. A novel hot spring metagenome-derived thermophilic cellulase was expressed and characterized. CONCLUSIONS: The application value of thermostable sugarcane bagasse-degrading enzymes is shown for production of cellulosic biofuel. The practical power of using a short-read-based metagenomic approach for harvesting novel microbial genes is also demonstrated.

Purification and characterization of an extracellular cellulase from Anoxybacillus gonensis O9 isolated from geothermal area in Turkey.

In the present study, cellulase was purified and characterized from Anoxybacillus gonensis (Gen bank Number: KM596794) which was isolated and characterized from Agri Diyadin Hot spring. It was found to synthesize cellulase which had a wide range of industrial applications. Twenty four-hour-cultured bacteria induced cellulase production and specific activities during the purification steps were 1.47, 81.06 and 109.4 EU mg(-1) protein at crude extract, ammonium sulphate precipitated and DEAE-Sephadex purification steps. The highest enzyme activity was observed at 50°C and the optimum range of pH was 3-10. Molecular weight of enzyme was determined approximately 40kDa. The kinetic parameters of cellulase against carboxymethylcellulose (CMC) were 153.4 pmol min(-1) mg for Vmax and 0.46mM for Km. Among effectors of the enzyme, Zn2+, Ca2+, Co2+ and EDTA decreased enzyme activity.

[Adaptation of Anoxybacillus flavithermus ssp. yunnanesis E13(T) to toluene at the level of fatty acid composition of membrane].

OBJECTIVE: Anoxybacillus flavithermus subsp. yunnanensis is now the only species of thermophilic bacteria able to tolerate toxic solvents at high temperature. The adaptive responses of A. flavithermus subsp. yunnanensis E13(T) to toluene on the level of fatty acid composition of membrane were studied in detail. METHODS: The extraction of fatty acids was performed according to the method described in the Sherlock Microbial Identification System manual. The fatty acid compositions were analyzed by gas chromatography mass spectrometry (GC-MS). RESULTS: In presence of 0.3% (V/V) toluene, key moment to adapt the saturated straight-chain fatty acids was that when cells grew from the lag phase to the initial growth phase in liquid. The saturated straight-chain fatty acids were continuously decreased as the strain E13(T) to grow. In survival of the cells in 100% toluene, the saturated straight-chain fatty acids increased significantly. CONCLUSION: A. flavithermus ssp. yunnanesis E13(T) alters its membrane fluidity via fatty acid composition to become more rigid when it is exposed to solvent, which is consistent that commonly found in mesophilic organic solvent-tolerant bacteria. However, it adapted its membrane by increasing straight-chain saturated fatty acids, rather than unsaturated fatty acids, which was demonstrated in mesophilic organic solvent-tolerant bacteria.

Characteristics of a novel thermophilic heterotrophic bacterium, Anoxybacillus contaminans HA, for nitrification-aerobic denitrification.

A strain bacterium that is thermophilic, heterotrophic nitrifying, and aerobic denitrifying was isolated and identified as Anoxybacillus contaminans HA for the first time. The identification was based on morphological and physiological characterizations, together with phylogenetic analysis of 16S rDNA sequence. The strain possessed excellent tolerance to high temperatures, with 55 °C as its optimum and 60 °C as viable. Moreover, NH4 (+)-N and NO3 (-)-N could be efficiently removed under thermophilic and solely aerobic conditions, with little intermediate accumulation. Average removal efficiencies of NH4 (+)-N and NO3 (-)-N at 55 °C reached 71.0 and 74.7 %, respectively, with removal rates of 5.83 and 32.08 mg l(-1) h(-1), respectively. Single-factor experiments suggested that the optimal conditions for both heterotrophic nitrification and aerobic denitrification were glucose as carbon source, NH4 (+)-N range of 50-200 mg l(-1), and wide NO3 (-)-N range of 200-1000 mg l(-1). These results indicated that strain HA had heterotrophic nitrification and aerobic denitrification abilities, as well as the notable ability to remove ammonium under thermophilic condition. Thus, this strain has potential application in waste-gas treatment.

Fe(II)EDTA-NO reduction by a newly isolated thermophilic Anoxybacillus sp. HA from a rotating drum biofilter for NOx removal.

The reduction of Fe(II)EDTA-NO is one of the core processes in BioDeNOx, an integrated physicochemical and biological technique for NOx removal from industrial flue gases. A newly isolated thermophilic Anoxybacillus sp. HA, identified by 16S rRNA sequence analysis, could simultaneously reduce Fe(II)EDTA-NO and Fe(III)EDTA. A maximum NO removal efficiency of 98.7% was achieved when 3mM Fe(II)EDTA-NO was used in the nutrient solution at 55°C. Results of this study strongly indicated that the biological oxidation of Fe(II)EDTA played an important role in the formation of Fe(III)EDTA in the anaerobic system. Fe(II)EDTA-NO was more competitive than Fe(III)EDTA as an electron acceptor, and the presence of Fe(III)EDTA slightly affected the reduction rate of Fe(II)EDTA-NO. At 55°C, the maximum microbial specific growth rate µmax reached the peak value of 0.022h(-1). The maximum NO removal efficiency was also measured (95.4%) under this temperature. Anoxybacillus sp. HA, which grew well at 50°C-60°C, is a potential microbial resource for Fe(II)EDTA-NO reduction at thermophilic temperatures.