Characterization of microbial communities removing nitrogen oxides from flue gas: the BioDeNOx process

BioDeNOx is an integrated physicochemical and biological process for the removal of nitrogen oxides (NOx) from flue gases. In this process, the flue gas is purged through a scrubber containing a solution of Fe(II)EDTA2-, which binds the NOx to form an Fe(II)EDTA.NO2- complex. Subsequently, this complex is reduced in the bioreactor to dinitrogen by microbial denitrification. Fe(II)EDTA2-, which is oxidized to Fe(III)EDTA- by oxygen in the flue gas, is regenerated by microbial iron reduction. In this study, the microbial communities of both lab- and pilot-scale reactors were studied using culture-dependent and -independent approaches. A pure bacterial strain, KT-1, closely affiliated by 16S rRNA analysis to the gram-positive denitrifying bacterium Bacillus azotoformans, was obtained. DNA-DNA homology of the isolate with the type strain was 89%, indicating that strain KT-1 belongs to the species B. azotoformans. Strain KT-1 reduces Fe(II)EDTA.NO2- complex to N2 using ethanol, acetate, and Fe(II)EDTA2- as electron donors. It does not reduce Fe(III)EDTA-. Denaturing gradient gel electrophoresis analysis of PCR-amplified 16S rRNA gene fragments showed the presence of bacteria closely affiliated with members of the phylum Deferribacteres, an Fe(III)-reducing group of bacteria. Fluorescent in situ hybridization with oligonucleotide probes designed for strain KT-1 and members of the phylum Deferribacteres showed that the latter were more dominant in both reactors.     

Oxidation of Fe(II)‐EDTA by nitrite and by two nitrate‐reducing Fe(II)‐oxidizing Acidovorax strains

The enzymatic oxidation of Fe(II) by nitrate‐reducing bacteria was first suggested about two decades ago. It has since been found that most strains are mixotrophic and need an additional organic co‐substrate for complete and prolonged Fe(II) oxidation. Research during the last few years has tried to determine to what extent the observed Fe(II) oxidation is driven enzymatically, or abiotically by nitrite produced during heterotrophic denitrification. A recent study reported that nitrite was not able to oxidize Fe(II)‐EDTA abiotically, but the addition of the mixotrophic nitrate‐reducing Fe(II)‐oxidizer, Acidovorax sp. strain 2AN, led to Fe(II) oxidation (Chakraborty & Picardal, 2013). This, along with other results of that study, was used to argue that Fe(II) oxidation in strain 2AN was enzymatically catalyzed. However, the absence of abiotic Fe(II)‐EDTA oxidation by nitrite reported in that study contrasts with previously published data. We have repeated the abiotic and biotic experiments and observed rapid abiotic oxidation of Fe(II)‐EDTA by nitrite, resulting in the formation of Fe(III)‐EDTA and the green Fe(II)‐EDTA‐NO complex. Additionally, we found that cultivating the Acidovorax strains BoFeN1 and 2AN with 10 mm nitrate, 5 mm acetate, and approximately 10 mm Fe(II)‐EDTA resulted only in incomplete Fe(II)‐EDTA oxidation of 47–71%. Cultures of strain BoFeN1 turned green (due to the presence of Fe(II)‐EDTA‐NO) and the green color persisted over the course of the experiments, whereas strain 2AN was able to further oxidize the Fe(II)‐EDTA‐NO complex. Our work shows that the two used Acidovorax strains behave very differently in their ability to deal with toxic effects of Fe‐EDTA species and the further reduction of the Fe(II)‐EDTA‐NO nitrosyl complex. Although the enzymatic oxidation of Fe(II) cannot be ruled out, this study underlines the importance of nitrite in nitrate‐reducing Fe(II)‐ and Fe(II)‐EDTA‐oxidizing cultures and demonstrates that Fe(II)‐EDTA cannot be used to demonstrate unequivocally the enzymatic oxidation of Fe(II) by mixotrophic Fe(II)‐oxidizers.     

微生物介导硝酸盐还原耦合亚铁氧化过程的动力学及其影响因素

【目的】探究不同菌浓度和亚铁浓度条件下,Acidovorax sp. strain BoFeN1介导的厌氧亚铁氧化耦合硝酸盐还原过程的动力学和次生矿物。【方法】构建包含菌BoFeN1、硝酸盐、亚铁的厌氧培养体系,测试硝酸根、亚硝酸根、乙酸根、亚铁等浓度,并收集次生矿物,采用XRD、SEM进行矿物种类和形貌表征。【结果】在微生物介导硝酸盐还原耦合亚铁氧化的体系中,高菌浓度促进硝酸盐还原,对亚铁氧化也有一定促进作用;高浓度亚铁在低菌浓度下氧化反应速率和程度降低,但是在高菌浓度下无明显影响;亚铁浓度越高次生矿物结晶度越高,但对硝酸盐还原具有一定抑制作用。在微生物介导亚硝酸盐还原耦合亚铁氧化的体系中,高的菌浓度和亚铁浓度都会促进亚硝酸盐还原,但亚铁氧化的次生矿物会对亚硝酸盐的微生物还原产生较强的抑制作用,次生矿物的种类和结晶度主要受亚铁浓度影响。【结论】硝酸盐还原主要是生物反硝化作用,亚硝酸盐还原包含生物反硝化和化学反硝化两部分,在硝酸盐体系中亚铁氧化与次生矿物生成是受生物和化学反硝化作用的共同影响,但亚硝酸盐体系中亚铁氧化与次生矿物生成主要是受化学反硝化作用影响。该研究可为深入理解厌氧微生物介导铁氮耦合反应机制提供基础数据和理论支撑。     

Reduction of Fe(II)EDTA-NO by a newly isolated <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. strain DN-2 in NO<Subscript><Emphasis Type="Italic">x</Emphasis></Subscript> scrubber solution

Biological reduction of nitric oxide (NO) chelated by ferrous ethylenediaminetetraacetate (Fe(II)EDTA) to N₂ is one of the core processes in a chemical absorption–biological reduction integrated technique for nitrogen oxide (NO _x ) removal from flue gases. A new isolate, identified as Pseudomonas sp. DN-2 by 16S rRNA sequence analysis, was able to reduce Fe(II)EDTA-NO. The specific reduction capacity as measured by NO was up to 4.17 mmol g DCW⁻¹ h⁻¹. Strain DN-2 can simultaneously use glucose and Fe(II)EDTA as electron donors for Fe(II)EDTA-NO reduction. Fe(III)EDTA, the oxidation of Fe(II)EDTA by oxygen, can also serve as electron acceptor by strain DN-2. The interdependency between various chemical species, e.g., Fe(II)EDTA-NO, Fe(II)EDTA, or Fe (III)EDTA, was investigated. Though each complex, e.g., Fe(II)EDTA-NO or Fe(III)EDTA, can be reduced by its own dedicated bacterial strain, strain DN-2 capable of reducing Fe(III)EDTA can enhance the regeneration of Fe(II)EDTA, hence can enlarge NO elimination capacity. Additionally, the inhibition of Fe(II)EDTA-NO on the Fe(III)EDTA reduction has been explored previously. Strain DN-2 is probably one of the major contributors for the continual removal of NO _x due to the high Fe(II)EDTA-NO reduction rate and the ability of Fe(III)EDTA reduction.     

Physiology of phototrophic iron(II)-oxidizing bacteria: implications for modern and ancient environments

Phototrophic iron(II) [Fe(II)]-oxidizing bacteria are present in modern environments and evidence suggests that this metabolism was present already on early earth. We determined Fe(II) oxidation rates depending on pH, temperature, light intensity, and Fe(II) concentration for three phylogenetically different phototrophic Fe(II)-oxidizing strains (purple nonsulfur bacterium Rhodobacter ferrooxidans sp. strain SW2, purple sulfur bacterium Thiodictyon sp. strain F4, and green sulfur bacterium Chlorobium ferrooxidans strain KoFox). While we found the overall highest Fe(II) oxidation rates with strain F4 (4.5 mmol L(-1) day(-1), 800 lux, 20 degrees C), the lowest light saturation values [at which maximum Fe(II) oxidation occurred] were determined for strain KoFox with light saturation already below 50 lux. The oxidation rate per cell was determined for R. ferrooxidans strain SW2 to be 32 pmol Fe(II) h(-1) per cell. No significant toxic effect of Fe(II) was observed at Fe(II) concentrations of up to 30 mM. All three strains are mesophiles with upper temperature limits of c. 30 degrees C. The main pigments were identified to be spheroidene, spheroidenone, OH-spheroidenone (SW2), rhodopinal (F4), and chlorobactene (KoFox). This study will improve our ecophysiological understanding of iron cycling in modern environments and will help to evaluate whether phototrophic iron oxidizers may have contributed to the formation of Fe(III) on early earth.     

Steady-state nitric oxide concentrations during denitrification

Three species of denitrifying bacteria, Paracoccus denitrificans, Pseudomonas stutzeri strain JM300, and Achromobacter cycloclastes, were allowed to reduce nitrate or nitrite in anaerobic, closed vials while the equilibration of gases between aqueous and gas phases was facilitated by vigorous stirring. The gas phase was sampled and analyzed for NO with use of a chemiluminescence detector calibrated against bottled NO standards or against NO produced by the nitrite-iodide reaction. [NOaq] was inferred from [NOg] and the solubility of NO. NO was detected only during denitrification in amounts that, once established, did not change with time, were independent of the initial concentration of nitrate or nitrite, and were largely independent of cell concentration, at least when nitrate was the oxidant. The usual level of NO was promptly re-established following the addition of exogenous NO or following the loss of NO by sparging. The aforementioned properties are expected for a steady-state intermediate in denitrification. Steady-state [NOaq] ranged between 1 and 65 nM depending on species and conditions. Similar results were also obtained in a related experiment in which P. stutzeri strain ZoBell respired nitrite under growth conditions. The very low steady-state [NOaq] observed during denitrification imply that the maximum activity of nitric oxide reductase in vivo, if it could be realized, would be large relative to that for nitrite reductase. This circumstance allows NO to be an intermediate without reaching toxic steady-state levels.     

Potential Role of Nitrite for Abiotic Fe(II) Oxidation and Cell Encrustation during Nitrate Reduction by Denitrifying Bacteria

Microorganisms have been observed to oxidize Fe(II) at neutral pH under anoxic and microoxic conditions. While most of the mixotrophic nitrate-reducing Fe(II)-oxidizing bacteria become encrusted with Fe(III)-rich minerals, photoautotrophic and microaerophilic Fe(II) oxidizers avoid cell encrustation. The Fe(II) oxidation mechanisms and the reasons for encrustation remain largely unresolved. Here we used cultivation-based methods and electron microscopy to compare two previously described nitrate-reducing Fe(II) oxidizers ( Acidovorax sp. strain BoFeN1 and Pseudogulbenkiania sp. strain 2002) and two heterotrophic nitrate reducers (Paracoccus denitrificans ATCC 19367 and P. denitrificans Pd 1222). All four strains oxidized ∼8 mM Fe(II) within 5 days in the presence of 5 mM acetate and accumulated nitrite (maximum concentrations of 0.8 to 1.0 mM) in the culture media. Iron(III) minerals, mainly goethite, formed and precipitated extracellularly in close proximity to the cell surface. Interestingly, mineral formation was also observed within the periplasm and cytoplasm; intracellular mineralization is expected to be physiologically disadvantageous, yet acetate consumption continued to be observed even at an advanced stage of Fe(II) oxidation. Extracellular polymeric substances (EPS) were detected by lectin staining with fluorescence microscopy, particularly in the presence of Fe(II), suggesting that EPS production is a response to Fe(II) toxicity or a strategy to decrease encrustation. Based on the data presented here, we propose a nitrite-driven, indirect mechanism of cell encrustation whereby nitrite forms during heterotrophic denitrification and abiotically oxidizes Fe(II). This work adds to the known assemblage of Fe(II)-oxidizing bacteria in nature and complicates our ability to delineate microbial Fe(II) oxidation in ancient microbes preserved as fossils in the geological record.     

Characterization of Microbial Communities Removing Nitrogen Oxides from Flue Gas: the BioDeNOx Process

BioDeNOx is an integrated physicochemical and biological process for the removal of nitrogen oxides (NOx) from flue gases. In this process, the flue gas is purged through a scrubber containing a solution of Fe(II)EDTA²⁻, which binds the NOx to form an Fe(II)EDTA·NO²⁻ complex. Subsequently, this complex is reduced in the bioreactor to dinitrogen by microbial denitrification. Fe(II)EDTA²⁻, which is oxidized to Fe(III)EDTA⁻ by oxygen in the flue gas, is regenerated by microbial iron reduction. In this study, the microbial communities of both lab- and pilot-scale reactors were studied using culture-dependent and -independent approaches. A pure bacterial strain, KT-1, closely affiliated by 16S rRNA analysis to the gram-positive denitrifying bacterium Bacillus azotoformans, was obtained. DNA-DNA homology of the isolate with the type strain was 89%, indicating that strain KT-1 belongs to the species B. azotoformans. Strain KT-1 reduces Fe(II)EDTA·NO²⁻ complex to N₂ using ethanol, acetate, and Fe(II)EDTA²⁻ as electron donors. It does not reduce Fe(III)EDTA⁻. Denaturing gradient gel electrophoresis analysis of PCR-amplified 16S rRNA gene fragments showed the presence of bacteria closely affiliated with members of the phylum Deferribacteres, an Fe(III)-reducing group of bacteria. Fluorescent in situ hybridization with oligonucleotide probes designed for strain KT-1 and members of the phylum Deferribacteres showed that the latter were more dominant in both reactors.     

Nitric oxide reduction in BioDeNOx reactors: kinetics and mechanism

Biological reduction of nitric oxide (NO) to di-nitrogen (N(2)) gas in aqueous Fe(II)EDTA(2-) solutions is a key reaction in BioDeNOx, a novel process for NOx removal from flue gases. The mechanism and kinetics of the first step of NO reduction, that is, the conversion of NO to N(2)O, was determined in batch experiments using various types of inocula. Experiments were performed in Fe(II)EDTA(2-) medium (5-25 mM) under BioDeNOx reactor conditions (55 degrees C, pH 7.2 +/- 0.2) with ethanol as external electron donor. BioDeNOx reactor mixed liquor gave the highest NO reduction rates (+/-0.34 nmol s(-1) mg(prot)(-1)) with an estimated K(m) value for NO lower than 10 nM. The specific NO (to N(2)O) reduction rate depended on the NO (aq) and Fe(II)EDTA(2-) concentration as well as the temperature. The experimental results, complemented with kinetic and thermodynamic considerations, show that Fe(II)EDTA(2-), and not ethanol, is the primary electron donor for NO reduction, that is, the BioDeNOx reactor medium (the redox system Fe(II)EDTA(2-)/Fe(III)EDTA(-)) interferes with the NO reduction electron transfer chain and thus enhances the NO denitrification rate.     

15N-tracer and NMR studies on the pathway of denitrification. Evidence against trioxodinitrate but for nitroxyl as an intermediate

The effects of four species of denitrifying bacteria on the conversion of [15N]nitrite to trioxodinitrate (HN2O3-) and N2O and of trioxodinitrate to N2O were studied. For all species, the N2O produced in the presence of [15N]nitrite and trioxodinitrate was isotopically randomized throughout the period of incubation and was not composed at the outset predominantly of 14N2O or 14N2O plus 15N2O. The N2O produced was also heavily enriched in 15N at times when the trioxodinitrate pool was only weakly enriched in 15N. By 15N NMR, the N(2) position, but not the N(1) position, of trioxodinitrate was found to become progressively labeled with 15N during incubation with [15N]nitrite. These results argue that (a) the N-N bond of trioxodinitrate is not preserved in its conversion to N2O, (b) trioxodinitrate can be neither a free nor enzyme-bound intermediate in denitrifying bacteria, and (c) the pathways from nitrite and trioxodinitrate involve a common mononitrogen intermediate. The conclusion that this intermediate is probably nitroxyl (HNO), at least with Paracoccus denitrificans and Pseudomonas stutzeri, provides indirect evidence that N-N bond formation in denitrification can occur through the dimerization of nitroxyl.     

Time-resolved infrared spectroscopy reveals a stable ferric heme-NO intermediate in the reaction of Paracoccus pantotrophus cytochrome cd1 nitrite reductase with nitrite

Cytochrome cd(1) is a respiratory enzyme that catalyzes the physiological one-electron reduction of nitrite to nitric oxide. The enzyme is a dimer, each monomer containing one c-type cytochrome center and one active site d(1) heme. We present stopped-flow Fourier transform infrared data showing the formation of a stable ferric heme d(1)-NO complex (formally d(1)Fe(II)-NO(+)) as a product of the reaction between fully reduced Paracoccus pantotrophus cytochrome cd(1) and nitrite, in the absence of excess reductant. The Fe-(14)NO nu(NO) stretching mode is observed at 1913 cm(-1) with the corresponding Fe-(15)NO band at 1876 cm(-1). This d(1) heme-NO complex is still readily observed after 15 min. EPR and visible absorption spectroscopic data show that within 4 ms of the initiation of the reaction, nitrite is reduced at the d(1) heme, and a cFe(III) d(1)Fe(II)-NO complex is formed. Over the next 100 ms there is an electron redistribution within the enzyme to give a mixed species, 55% cFe(III) d(1)Fe(II)-NO and 45% cFe(II) d(1)Fe(II)-NO(+). No kinetically competent release of NO could be detected, indicating that at least one additional factor is required for product release by the enzyme. Implications for the mechanism of P. pantotrophus cytochrome cd(1) are discussed.     

Effect of copper dosing on sulfide inhibited reduction of nitric and nitrous oxide

The stimulating effect of copper addition on the reduction rate of nitrous oxide (N(2)O) to dinitrogen (N(2)) in the presence of sulfide was investigated in batch experiments (pH 7.0; 55 degrees C). N(2)O was dosed either directly as a gas to the headspace of the bottles or formed as intermediate during the denitrification of nitrite in Fe(II)EDTA(2-)-containing medium and nitrate in Fe(II)EDTA(2-)-free medium. Sulfide was either dosed externally or generated from endogenous sulfur sources during anaerobic incubation of the sludge. In the presence of sulfide (from 15 microM to 1mM), heterotrophic denitrification using ethanol as electron donor was incomplete, i.e., N(2)O accumulated instead of N(2) or was transiently formed. Copper addition (60 microM) rapidly stimulated the reduction of N(2)O to N(2). Zinc addition (60 microM) did not have a similar strong stimulating effect as observed for copper and the N(2)O reduction rate was not stimulated at all upon supply of FeCl(3) (2 mM). Thus, a copper deficiency for N(2)O reduction is most likely developed in the presence of sulfide. It is suggested that sulfide induces this deficiency as it readily precipitates as copper sulfide and thus scavenges copper in the medium or that sulfide inactivates the N(2)OR reductase as it sequesters the copper of this metalloenzyme.     

Evaluation of complexed NO reduction mechanism in a chemical absorption–biological reduction integrated NO<Subscript><Emphasis Type="BoldItalic">x</Emphasis></Subscript> removal system

Biological reduction of nitric oxide (NO) from Fe(II) ethylenediaminetetraacetic acid (EDTA)-NO to dinitrogen (N(2)) is a core process for the continual nitrogen oxides (NO(x)) removal in the chemical absorption-biological reduction integrated approach. To explore the biological reduction of Fe(II)EDTA-NO, the stoichiometry and mechanism of Fe(II)EDTA-NO reduction with glucose or Fe(II)EDTA as electron donor were investigated. The experimental results indicate that the main product of complexed NO reduction is N(2), as there was no accumulation of nitrous oxide, ammonia, nitrite, or nitrate after the complete depletion of Fe(II)EDTA-NO. A transient accumulation of nitrous oxide (N(2)O) suggests reduction of complexed NO proceeds with N(2)O as an intermediate. Some quantitative data on the stoichiometry of the reaction are experimental support that reduction of complexed NO to N(2) actually works. In addition, glucose is the preferred and primary electron donor for complexed NO reduction. Fe(II)EDTA served as electron donor for the reduction of Fe(II)EDTA-NO even in the glucose excessive condition. A maximum reduction capacity as measured by NO (0.818 mM h(-1)) is obtained at 4 mM of Fe(II)EDTA-NO using 5.6 mM of glucose as primary electron donor. These findings impact on the understanding of the mechanism of bacterial anaerobic Fe(II)EDTA-NO reduction and have implication for improving treatment methods of this integrated approach.     

一株嗜酸化能异养菌Acidiphilium sp.的分离鉴定及其对Fe(Ⅲ)代谢的研究

从云南省腾冲热泉酸性泥土样品中分离得到一株好氧嗜酸异养细菌Teng-A。菌株Teng-A细胞大小0.6～0.8μm×1.0～1.5μm,单生或成链状排列,革兰氏染色反应为阴性,有周生鞭毛,不产芽孢,该菌适宜的生长温度为29～33℃、pH为3.0～4.0,可以利用许多有机物生长,但不能利用Fe(Ⅱ)、S、Na2S2O3、K2S4O6等为能源生长。菌株Teng-A基因组DNA的(G C)mol%为69.6mol%,其16S rRNA基因与Acidiphilium属菌种的16S rRNA基因的最高相似性大于99%。根据形态学、生理生化特点及系统发育分析表明,菌株Teng-A是Acidiphilium属的一个新成员,崭定名为Acidiphilium sp.strainTeng-A。厌氧条件下,菌株Teng-A可以葡萄糖或H2为电子供体,将Fe(Ⅲ)还原为Fe(Ⅱ),还原速率分别为11.56mg/L.day与15.34mg/L.day。菌株Teng-A与Acidithiobacillus ferrooxidansLJ-1和Leptospirilum ferriphilum LJ-2共同培养,前3dFe2 氧化速度分别为0.44g/L.day和0.41g/L.day,比LJ-1(0.64g/L.day)和LJ-2(0.60g/L.day)单独培养时氧化Fe(Ⅱ)的速率稍慢,但当培养时间超过5d时,Fe(Ⅱ)最终被全部氧化,并且发现在共培养时,Fe(Ⅱ)氧化生成的沉淀物的形态不同于At.ferrooxidans和L.ferriphilum单独培养时产生的沉淀物的形态。最后讨论了Acidiphilium对生物浸矿和生物成矿作用的影响。     

Structure of microbial communities performing the simultaneous reduction of Fe(II)EDTA.NO2−and Fe(III)EDTA−

BioDeNOx is a combined physicochemical and biological process for the removal of nitrogen oxides (NOx) from flue gas. In the present study, two anaerobic bioreactors performing BioDeNOx were run consecutively (RUN-1 and RUN-2) at a dilution rate of 0.01 h⁻¹ with Fe(II)EDTA.NO²⁻ and Fe(III)EDTA⁻ as electron acceptors and ethanol as electron donor. The measured protein concentration of the reactor biomass of both runs was 120 mg/l. Different molecular methods were used to determine the identity and abundance of the bacterial populations in both bioreactors. Bacillus azotoformans strain KT-1 was recognized as a key player in Fe(II)EDTA.NO²⁻ reduction. PCR-denaturing gradient gel electrophoresis analysis of the reactor biomass showed a greater diversity in RUN-2 than in RUN-1. Enrichments of Fe(II)EDTA.NO²⁻ and Fe(III)EDTA⁻ reducers and activity assays were conducted using the biomass from RUN-2 as an inoculum. The results on substrate turnover, overall microbial diversity, and enrichments and finally activity assays confirmed that ethanol was used as electron donor for Fe(II)EDTA.NO²⁻ reduction. In addition, the Fe(III)EDTA⁻ reduction rate of the microbial community proved to be feasible enough to run the bioreactors, ruling out the chemical reduction of Fe(III)EDTA⁻ with sulfide as was proposed by other researchers.     

Quantum mechanical interpretation of nitrite reduction by cytochrome cd1 nitrite reductase from Paracoccus pantotrophus

The reduction of nitrite to nitric oxide in respiratory denitrification is catalyzed by a cytochrome cd(1) nitrite reductase in Paracoccus pantotrophus (formerly known as Thiosphaera pantotropha LMD 92.63). High-resolution structures are available for the fully oxidized [Fül?p, V., Moir, J. W., Ferguson, S. J., and Hajdu, J. (1995) Cell 81, 369-377; Baker, S. C., Saunders, N. F., Willis, A. C., Ferguson, S. J., Hajdu, J., and Fül?p, V. (1997) J. Mol. Biol. 269, 440-455] and fully reduced forms of this enzyme, as well as for various intermediates in its catalytic cycle [Williams, P. A., Fül?p, V., Garman, E. F., Saunders, N. F., Ferguson, S. J., and Hajdu, J. (1997) Nature 389, 406-412]. On the basis of these structures, quantum mechanical techniques (QM), including density functional methods (DFT), were combined with simulated annealing (SA) and molecular mechanics techniques (MM) to calculate the electronic distribution of molecular orbitals in the active site during catalysis. The results show likely trajectories for electrons, protons, substrates, and products in the process of nitrite reduction, and offer an interpretation of the reaction mechanism. The calculations indicate that the redox state of the d(1) heme and charges on two histidines in the active site orchestrate catalysis locally. Binding of nitrite to the reduced iron is followed by proton transfer from His345 and His388 to one of the oxygens of nitrite, creating a water molecule and an [Fe(II)-NO(+)] complex. Valence isomerization within this complex gives [Fe(III)-NO]. The release of NO from the ferric iron is influenced by the protonation state of His345 and His388, and by the orientation of NO on the d(1) heme. Return of Tyr25 to a hydrogen-bonding position between His345 and His388 facilitates product release, but a rebinding of Tyr25 to the oxidized iron may be bypassed in steady-state catalysis.     

A new approach for Fe(III)EDTA reduction in NOx scrubber solution using bio-electro reactor

A new process for the removal of NO_x by a combined Fe(II)EDTA absorption and microbial reduction has been demonstrated, in which part of the Fe(II)EDTA will be oxidized by oxygen in the flue gas to form Fe(III)EDTA. In former studies, strain FR-2 has been found to reduce Fe(III)EDTA efficiently. Otherwise, it has been reported that bio-electro reactor could efficiently provide a chance for simultaneous denitrification and metal ion removal. Therefore, a use of bio-electro reactor is suggested to promote the reduction of Fe(III)EDTA by strain FR-2 in this paper. The results showed that the concentration of Fe(III)EDTA decreased rapidly when electric current was applied, and that as the current density rose, the Fe(III)EDTA reduction rate increased while followed by a decrease afterward. The formation of the biofilm on the electrode was observed by ESEM (Environmental Scan Electro-Microscope). In addition, the Fe(III)EDTA reduction rate obviously decreased with the existence of NaNO₂.     

Comparison of dentrification by Paracoccus denitrificans, Pseudomonas stutzeri and Pseudomonas aeruginosa.

The course of denitrification of nitrate, nitrite and both compounds together by static cultures of Paracoccus denitrificans, Pseudomonas stutzeri and Pseudomonas aeruginosa was studied. These strains represent three different types of denitrification: 1. reduction of nitrate to gaseous nitrogen without accumulation of nitrite (P. denitrificans); 2. partial accumulation of nitrite in growing cultures during reduction of nitrate to gaseous nitrogen (P. aeruginosa) and 3. two-phase denitrification that includes reduction of nitrates at the very beginning of the process, and then, after depletion of the former, the reduction of nitrates to gaseous nitrogen (P. stutzeri). These observations differ from the results reported in the literature and possible reasons are discussed.     

Ferrovibrio denitrificans gen. nov., sp. nov., a novel neutrophilic facultative anaerobic Fe(II)-oxidizing bacterium

A neutrophilic Fe(II)-oxidizing bacterium was isolated from the redox zone of a low-salinity spring in Krasnodar krai (Russia), at the FeS-Fe(OH)(3) interface deposited at the sediment surface. The cells of strain Sp-1 were short, thin motile vibrioids with one polar flagellum dividing by binary fission. The optimal values and ranges for pH and temperature were pH 6.2 (5.5-8) and 35?°C (5-45?°C), respectively. The organism was a facultative anaerobe. Strain Sp-1 was capable of organotrophic, lithoheterotrophic and mixotrophic growth with Fe(II) as an electron donor. The denitrification chain was 'disrupted'. Oxidation of Fe(II) was coupled to reduction of NO3 - to NO2 - or of N(2) O to N(2) , as well as under microaerobic conditions, with O(2) as an electron acceptor. The DNA G+C content was 64.2?mol%. According to the results of phylogenetic analysis, the strain was 10.6-12% remote from the closest relatives, members of the genera Sneathiella, Inquilinus, Oceanibaculum and Phaeospirillum within the Alphaproteobacteria. Based on its morphological, physiological and taxonomic characteristics, together with the results of phylogenetic analysis, strain Sp-1 is described as a member of a new genus Ferrovibrio gen. nov., with the type species Ferrovibrio denitrificans sp. nov. and the type strain Sp-1(T) (=?LMG 25817(T) =?VKM B-2673(T) ).     

日本三宅岛火山土壤中硫代硫酸盐氧化菌的分离、生物学特性及鉴定

【目的】利用培养法从日本三宅岛火山土壤(堆积年限131年)中分离到一株能氧化分解硫代硫酸盐的细菌MU2A-22T。【方法】用培养法对该菌株MU2A-22T进行了生理生化性质以及分类学位置上的确定。【结果】菌株MU2A-22T为革兰氏阴性,短杆状或球状。理化性质表明该菌株能利用葡萄糖、L-阿拉伯糖、葡萄糖酸盐、己二酸酯、dL-苹果酸钠、硫代硫酸钠(最适浓度为2.5 mmol/L)为唯一碳源进行自养生长。最适生长温度为25°C 30°C,最适pH为6.0 8.0。菌株MU2A-22T的16S rRNA序列与菌株Paracoccus solventivorans 6637T亲缘关系最近,序列相似性为97%,编码核酮糖-1,5-二磷酸羧化酶/加氧酶(Rubisco)的基因也被确定。对Paracoccus属内几种近缘菌的脂肪酸分析,证明菌株MU2A-22T中含有Paracoccus属的特征氨基酸,其中含量大于10%的分别为C18:1(74.7%)和C18:0(12.1%)。DNA-DNA杂交实验表明,菌株MU2A-22T与Paracoccus solventivorans 6637TDNA的相似度为49.3%。MU2A-22T菌株G+C含量为66.5%66.7%。【结论】菌株MU2A-22T为Paracoccus属内的一新种菌(登录号GQ452286),命名为Paracoccus scorialis sp.nov.。