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Chlorite dismutase is a heme enzyme that catalyzes the conversion of the toxic compound ClO2−(chlorite) to innocuous Cl−and O2. The reaction is a very rare case of enzymatic O–O bond formation, which has sparked the interest to elucidate the reaction mechanism using pre-steady-state kinetics. During stopped-flow experiments, spectroscopic and structural changes of the enzyme were observed in the absence of a substrate in the time range from milliseconds to minutes. These effects are a consequence of illumination with UV–visible light during the stopped-flow experiment. The changes in the UV–visible spectrum in the initial 200 s of the reaction indicate a possible involvement of a ferric superoxide/ferrous oxo or ferric hydroxide intermediate during the photochemical inactivation. Observed EPR spectral changes after 30 min reaction time indicate the loss of the heme and release of iron during the process. During prolonged illumination, the oligomeric state of the enzyme changes from homo-pentameric to monomeric with subsequent protein precipitation. Understanding the effects of UV–visible light illumination induced changes of chlorite dismutase will help us to understand the nature and mechanism of photosensitivity of heme enzymes in general. Furthermore, previously reported stopped-flow data of chlorite dismutase and potentially other heme enzymes will need to be re-evaluated in the context of the photosensitivity.Graphic abstractIllumination of recombinantly expressedAzospira oryzaeChlorite dismutase (AoCld) with a high-intensity light source, common in stopped-flow equipment, results in disruption of the bond between FeIIIand the axial histidine. This leads to the enzyme losing its heme cofactor and changing its oligomeric state as shown by spectroscopic changes and loss of activity.

An iron reducing enrichment was obtained from sulfate reducing sludge and was evaluated on the capability of reducing Fe3+ coupled to acetate oxidation in a microbial fuel cell (MFC). Three molar ratios for acetate/Fe3+ were evaluated (2/16, 3.4/27 and 6.9/55 mM). The percentages of Fe3+ reduction were in a range of 80–90, 60–70 and 40–50% for the MFCs at closed circuit for the molar ratios of 2/16, 3.4/27 and 6.9/55 mM, respectively. Acetate consumption was in a range of 80–90% in all cases. The results obtained at closed circuit for current density were: 11.37 mA/m2, 4.5 mA/m2 and 7.37 mA/m2 for the molar ratios of 2/16, 3.4/27 and 6.9/55 mM, respectively. Some microorganisms that were isolated and identified in the MFCs were Azospira oryzae, Cupriavidus metallidurans CH34, Enterobacter bugandensis 247BMC, Citrobacter freundii ATCC8090 and Citrobacter murliniae CDC2970-59, these bacteria have been reported as exoelectrogens in MFC and in MFC involving metals removal but not all of them have been reported to utilize acetate as preferred substrate. The results demonstrate that the isolates can utilize acetate as the sole source of carbon and suggest that Fe3+ reduction was carried out by a combination of different mechanisms (direct contact and redox mediators) utilized by the bacteria identified in the MFC. Storage of the energy generated from the 2/16 mM MFC system arranged in a series of three demonstrated that it is possible to utilize the energy to charge a battery.

To speed up the degradation of corn stover directly returned to soil at low temperature, the corn stover-degrading microbial consortium GF-20, acclimated to biological decomposition in the frigid region, was successfully constructed under a long-term limiting substrate. To evaluate its potential in accelerating the decomposition of un-pretreated corn stover, the decomposing property, fermentation dynamic and the microbial diversity were analyzed. GF-20 degraded corn stover by 32% after 15-day fermentation at 10°C. Peak activities of filter paperlyase (FPA), β-glucosidases (CB), endoglucanases (Cx), and cellobiohydrolases (C1) were 1.15, 1.67, 1.73, and 1.42 U mL−1, appearing at the 6th, 3rd, 11th, and 9th d, respectively. The pH averaged at 6.73–8.42, and the optical density (OD) value peaked at 1.87 at the 120 h of the degradation process. Cellulase, hemicellulase and lignin in corn stover were persistently degraded by 44.85, 43.85 and 25.29% at the end of incubation. Result of denaturing gradient gel electrophoresis (DGGE) profiles demonstrated that GF-20 had a stable component structure under switching the temperature and pH. The composition of the GF-20 was also analyzed by constructing bacterial 16S rDNA clone library and fungal 18SrDNA-PCR-DGGE. Twenty-two bacterial clones and four fungal bands were detected and identified dominant bacteria represented by Cellvibrio mixtus subsp., Azospira oryzae, Arcobacter defluyii, and Clostridium populeti and the fungi were mainly identified as related to Trichosporon sp.

Chlorite dismutase (Cld) is a key enzyme of perchlorate and chlorate respiration. This heme-based protein reduces the toxic compound chlorite into the innocuous chloride anion in a very efficient way while producing molecular oxygen. A sequence comparison between Cld homologues shows a highly conserved family. The crystal structure of Azospira oryzae strain GR-1 Cld is reported to 2.1 A resolution. The structure reveals a hexameric organization of the Cld, while each monomer exhibits a ferredoxin-like fold. The six subunits are organized in a ring structure with a maximal diameter of 9 nm and an inner diameter of 2 nm. The heme active-site pocket is solvent accessible both from the inside and the outside of the ring. Moreover, a second anion binding site that could accommodate the assumed reaction intermediate ClO(-) for further transformation has been identified near the active site. The environment of the heme cofactor was investigated with electron paramagnetic resonance spectroscopy. Apart from the high-spin ferric signal of the five-coordinate resting-state enzyme, two low-spin signals were found corresponding to six-coordinate species. The current crystal structure confirms and complements a recently proposed catalytic mechanism that proceeds via a ferryl species and a ClO(-) anion. Our structural data exclude cooperativity between the iron centers.

A bacterium that reduces the soluble selenium oxyanions, selenate and selenite, to insoluble elemental red selenium (Se(0)) was isolated from a laboratory reactor developed to remove selenate from groundwater. Gene sequence alignment of the 16S rRNA allowed identification of the isolate as Azospira oryzae. Biochemical and morphologic characterization confirm the identification. The isolate reduces selenate and selenite to Se(0) under microaerophilic and denitrifying conditions but not under aerobic conditions. It does not use selenate or selenite as terminal e(-) donors. Se oxyanion reduction causes the formation of Se nanospheres that are 0.25 +/- 0.04 microm in diameter. Nanospheres may be associated with the cells or free in the medium. The enzymatic activity associated with the reduction of selenate has a molecular mass of approximately 500 kD, and the enzymatic activity associated with the reduction of selenite has a mass of approximately 55 kD. Selenite reduction was inhibited by tungsten. The molecular masses of these activities were different from those associated with the reduction of dimethylsulfoxide, sulfate, and nitrite. This bacterium, or perhaps its enzymes or DNA, might be useful for the remediation of waters contaminated with Se oxyanions.

Recent studies on the occurrence of (per)chlorate-reducing bacteria have resulted in the characterization of strains capable of dissimilatory (per)chlorate reduction. Phylogenetic analysis has shown that these bacteria are members of the Proteobacteria. Strains have been isolated from polluted and pristine sites, but only strains from polluted sites have been characterized in detail and deposited in culture collections. Herein we describe the isolation and characterization of perchlorate-reducing bacterium strain MA-1T and chlorate-reducing bacterium strain ASK-1, respectively isolated from a pristine and a chlorate-polluted site. Both isolates are members of the Proteobacteria. The 16S rRNA gene sequence similarity of MA-1T to Dechloromonas agitata DSM 13637T is 97·6 %, but the relatedness in DNA–DNA reassociation is only 37 %. Therefore, we propose to classify strain MA-1T (=DSM 15637T=ATCC BAA-776T) as the type strain of a novel species, Dechloromonas hortensis sp. nov. Strain ASK-1 and a previously described strain GR-1 show 100 and 99 % 16S rRNA gene sequence similarity to Pseudomonas chloritidismutans DSM 13592T and Dechlorosoma suillum DSM 13638T, respectively. DNA–DNA hybridization studies indicated that strains ASK-1 and GR-1 are related at the species level to P. chloritidismutans DSM 13592T (79 %) and Dechlorosoma suillum DSM 13638T (85 %), respectively. As suggested previously, Dechlorosoma suillum appears to be a later heterotypic synonym of Azospira oryzae. Although strain ASK-1 is identified as P. chloritidismutans, its morphology and growth requirements are different from those of the type strain.

In order to clarify the taxonomic position of Dechlorosoma suillum, which shares 99.9% 16S rDNA sequence identity (1433 of 1435 bp) with Azospira oryzae, we compared the two species in a polyphasic taxonomic approach. Results of 122 physiological and biochemical tests for D. suillum DSM 13638T and Azospira oryzae 6a3T were identical, except for the lack of growth of Azospira oryzae 6a3T with perchlorate as the terminal electron acceptor. Presence of a nifH gene and nitrogenase activity, a key feature of Azospira, were also detected in D. suillum by Southern hybridization and by the acetylene reduction assay, respectively. Whole-cell SDS-PAGE profiles of SDS-soluble proteins of strains DSM 13638T and 6a3T were almost identical. DNA-DNA hybridization studies showed more than 90% binding between D. suillum and two strains of Azospira oryzae. These data provide evidence that the two bacteria belong to the same species and that D. suillum is a later subjective synonym of Azospira oryzae.

Chlorite dismutase, a homotetrameric haem-based protein, is one of the key enzymes of (per)chlorate-reducing bacteria. It is highly active (2 kU mg(-1)) in reducing the toxic compound chlorite to the innocuous chloride anion and molecular oxygen. Chlorite itself is produced as the intermediate product of (per)chlorate reduction. The chlorite dismutase gene in Azospira oryzae strain GR-1 employing degenerate primers has been identified and the active enzyme was subsequently overexpressed in Escherichia coli. Chlorite dismutase was purified, proven to be active and crystallized using sitting drops with PEG 2000 MME, KSCN and ammonium sulfate as precipitants. The crystals belonged to space group P2(1)2(1)2 and were most likely to contain six subunits in the asymmetric unit. The refined unit-cell parameters were a = 164.46, b = 169.34, c = 60.79 A. The crystals diffracted X-rays to 2.1 A resolution on a synchrotron-radiation source and a three-wavelength MAD data set has been collected. Determination of the chlorite dismutase structure will provide insights into the active site of the enzyme, for which no structures are currently available.

Enhanced biohydrogen production via cascade acid stress on microbial communities, structure patterns of the microbial communities revealed by PLFAs, and the succession of biohydrogen related species against cascade acid stress were all investigated. It was found that hydrogen production could be improved from 48.7 to 79.4 mL/gVS after cascade acid stress. In addition, the Gram negative (G−) bacteria were found to be more tolerant to organic acids than those of the Gram positive (G+) bacteria, regardless of the dominance of G+ bacteria within the microbial communities. Moreover, Clostridium butyricum, Clostridium aciditolerans and Azospira oryzae, were proved to be enriched, and then might play indispensable roles for the enhanced biohydrogen production after cascade acid stress, as which were responsible for the biohydrogen accumulation, acid tolerance and nitrogen removal, respectively.

Existing methods for perchlorate remediation are hampered by the common co-occurrence of nitrate, which is structurally similar and a preferred electron acceptor. In this work, the potential for perchlorate removal using cell-free bacterial enzymes as biocatalysts was investigated using crude cell lysates and soluble protein fractions of Azospira oryzae PS, as well as soluble protein fractions encapsulated in lipid and polymer vesicles. The crude lysates showed activities between 41 700 to 54 400 U L(-1) (2.49 to 3.06 U mg(-1) total protein). Soluble protein fractions had activities of 15 400 to 29 900 U L(-1) (1.70 to 1.97 U mg(-1)) and still retained an average of 58.2% of their original activity after 23 days of storage at 4 °C under aerobic conditions. Perchlorate was removed by the soluble protein fraction at higher rates than nitrate. Importantly, perchlorate reduction occurred even in the presence of 500-fold excess nitrate. The soluble protein fraction retained its function after encapsulation in lipid or polymer vesicles, with activities of 13.8 to 70.7 U L(-1), in agreement with theoretical calculations accounting for the volume limitation of the vesicles. Further, encapsulation mitigated enzyme inactivation by proteinase K. Enzyme-based technologies could prove effective at perchlorate removal from water cocontaminated with nitrate or sulfate.

The diversity elucidation by amplified ribosomal DNA restriction analysis and 16S rDNA sequencing of 96 associative diazotrophs, isolated from the feeder roots of tea on enriched nitrogen-free semisolid media, revealed the predominance of Gram-positive over Gram-negative bacteria within the Kangra valley in Himachal Pradesh, India. The Gram-positive bacteria observed belong to two taxonomic groupings; Firmicutes, including the genera Bacillus and Paenibacillus; and Actinobacteria, represented by the genus Microbacterium. The Gram-negative bacteria included alpha-Proteobacteria genera Brevundimonas, Rhizobium, and Mesorhizobium; gamma-Proteobacteria genera Pseudomonas and Stenotrophomonas; and beta-Proteobacteria genera Azospira, Burkholderia, Delftia, Herbaspirillum and Ralstonia. The low level of similarity of two isolates, with the type strains Paenibacillus xinjiangensis and Mesorhizobium albiziae, suggests the possibility of raising species novum. The bacterial strains of different phylogenetic groups exhibited distinct carbon-source utilization patterns and fatty acid methyl ester profiles. The strains differed in their nitrogenase activities with relatively high activity seen in the Gramnegative strains exhibiting the highest similarity to Azospira oryzae, Delftia lacustris and Herbaspirillum huttiense.

A novel, Gram-negative bacterial strain, SUA2T, isolated from groundwater, was characterized using a polyphasic approach. Cells are Gram-negative, non-spore-forming, straight to curved rods with a single polar flagellum. Strain SUA2T is oxidase- and catalase-positive and is able to fix nitrogen. Poly-β-hydroxybutyrate storage granules are produced. Dominant fatty acids when grown in R2A and VM ethanol media for 72 h at 37 °C are C16 : 0, C16 : 1 ω7c, C17 : 0 cyclo, C10 : 0 3-OH, C18 : 1 ω7c, C12 : 0 and C15 : 0. DNA G+C content is 67.9 mol%. Phenotypic and phylogenetic data indicate that strain SUA2T is related to, but clearly differentiated from Azospira oryzae. Strain SUA2T is thus proposed as a novel species of the genus Azospira with the name Azospira restricta sp. nov. The description of the genus Azospira is emended to include the characteristics of this novel species. The type strain of Azospira restricta is SUA2T (=NRRL B-41660T=DSM 18626T=LMG 23819T).

The taxonomic structure of members of the genus Azoarcus sensu lato was reassessed in a polyphasic approach. Two species, Azoarcus communis and Azoarcus indigens, three unnamed species containing diazotrophs associated with Kallar grass roots (groups C, D) and a group of strains (E) isolated from fungi were analysed. They were compared by PAGE analyses of cellular proteins, genomic fingerprints, morphological and nutritional features to new isolates from rice roots. All strains within groups C, D and E containing 5-12 isolates showed group-specific cell and colony morphology and carbon source utilization patterns, with exception of the obligately microaerobic strain BS20-3, a member of group C. All strains, with this exception, also had almost indistinguishable electrophoretic protein patterns and genomic fingerprints generated with tDNA-directed primers, suggesting they belong to the same species. Phylogenetic analyses of almost complete 16S rDNA sequences carried out with three different algorithms (neighbour-joining, maximum-likelihood, parsimony) revealed that Azoarcus sensu lato is not monophyletic. Groups C, D and E formed three distinct lineages located between the Azoarcus/Thauera and the Rhodocyclus clusters. Phylogenetic distances between groups C, D and E were as large as between other genera (93-94% sequence similarity). This suggested they have the rank of three different genera. Since it was possible to differentiate them from each other and other related bacteria by phenotypic features, three new genera with one type species each are proposed: Azovibrio restrictus gen. nov., sp. nov., Azospira oryzae gen. nov., sp. nov. and Azonexus fungiphilus gen. nov., sp. nov.