Microb Ecol . 2005 Jan 11; {Epub ahead of print} Frequency and Diversity of Nitrate Reductase Genes among Nitrate-Dissimilating Pseudomonas in the Rhizosphere of Perennial Grasses Grown in Field Conditions; Roussel-Delif L et al.; A total of 1246 Pseudomonas strains were isolated from the rhizosphere of two perennial grasses (Lolium perenne and Molinia coerulea) with different nitrogen requirements . The plants were grown in their native soil under ambient and elevated atmospheric CO(2) content (pCO(2)) at the Swiss FACE (Free Air CO(2) Enrichment) facility . Root-, rhizosphere-, and non-rhizospheric soil-associated strains were characterized in terms of their ability to reduce nitrate during an in vitro assay and with respect to the genes encoding the membrane-bound (named NAR) and periplasmic (NAP) nitrate reductases so far described in the genus Pseudomonas . The diversity of corresponding genes was assessed by PCR-RFLP on narG and napA genes, which encode the catalytic subunit of nitrate reductases . The frequency of nitrate-dissimilating strains decreased with root proximity for both plants and was enhanced under elevated pCO(2) in the rhizosphere of L . perenne . NAR (54% of strains) as well as NAP (49%) forms were present in nitrate-reducing strains, 15.5% of the 439 strains tested harbouring both genes . The relative proportions of narG and napA detected in Pseudomonas strains were different according to root proximity and for both pCO(2) treatments: the NAR form was more abundant close to the root surface and for plants grown under elevated pCO(2) . Putative denitrifiers harbored mainly the membrane-bound (NAR) form of nitrate reductase . Finally, both narG and napA sequences displayed a high level of diversity . Anyway, this diversity was correlated neither with the root proximity nor with the pCO(2) treatment. Appl Microbiol Biotechnol . 2005 Jan 13; {Epub ahead of print} SspA, an outer membrane protein, is highly induced under salt-stressed conditions and is essential for growth under salt-stressed aerobic conditions in Rhodobacter sphaeroides f . sp . denitrificans; Tsuzuki M et al.; We have previously shown that an outer membrane protein, SspA, is prominently induced by salt stress in a photosynthetic bacterium, Rhodobacter sphaeroides f . sp . denitrificans IL106 (R . sphaeroides) . In this study, we investigated the physiological role of SspA under various stress conditions . Using recombinant SspA expressed in Escherichia coli as an antigen, the polyclonal antiserum of SspA was prepared . Western blot analysis demonstrated that SspA was highly induced by salt stress under both anaerobic and aerobic conditions . SspA was also induced, but to a lesser extent, by osmotic and acid stress . It is reduced under heat and cold compared to non-stressed conditions . While sspA-disrupted R . sphaeroides grew normally under anaerobic conditions in either the presence or absence of stress, it displayed significantly retarded growth under aerobic conditions in the dark, especially when osmotic or salt stress were imposed . In addition, the sspA disruptant, but not the wild type, formed cell aggregates when grown under both anaerobic and aerobic conditions, and this phenotype was significantly enhanced under salt-stressed aerobic conditions . Together, our findings suggest that SspA is critical under salt-stressed, aerobic growth conditions. Can J Microbiol, 2004 Sep, 50(9), 657 - 67 Experimental evidence for plasmid-borne nor-nir genes in Sinorhizobium meliloti JJ1c10; Chan YK et al.; In denitrification, nir and nor genes are respectively required for the sequential dissimilatory reduction of nitrite and nitric oxide to form nitrous oxide . Their location on the pSymA megaplasmid of Sinorhizobium meliloti was confirmed by Southern hybridization of its clones with specific structural gene probes for nirK and norCB . A 20-kb region of pSymA containing the nor-nir genes was delineated by nucleotide sequence analysis . These genes were linked to the nap genes encoding periplasmic proteins involved in nitrate reduction . The nor-nir-nap segment is situated within 30 kb downstream from the nos genes encoding nitrous oxide reduction, with a fix cluster intervening between nir and nos . Most of these predicted nor-nir and accessory gene products are highly homologous with those of related proteobacterial denitrifiers . Functional tests of Tn5 mutants confirmed the requirement of the nirV product and 1 unidentified protein for nitrite reduction as well as the norB-D products and another unidentified protein for nitric oxide reduction . Overall comparative analysis of the derived amino acid sequences of the S . meliloti gene products suggested a close relationship between this symbiotic N2 fixer and the free-living non-N2-fixing denitrifier Pseudomonas G-179, despite differences in their genetic organization . This relationship may be due to lateral gene transfer of denitrification genes from a common donor followed by rearrangement and recombination of these genes. Water Res, 2005 Jan-Feb, 39(2-3), 449 - 55 Epub 2004 Nov 05. Biodegradation and effect of formaldehyde and phenol on the denitrification process; Eiroa M et al.; Formaldehyde and phenol biodegradation during the denitrification process was studied at lab-scale, first in anoxic batch assays and then in a continuous anoxic reactor . The biodegradation of formaldehyde (260mgl(-1)) as single carbon source and at phenol concentrations ranging from 30 to 580mgl(-1) was investigated in batch assays, obtaining an initial biodegradation rate around 0.5g CH(2)OgVSS(-1)d(-1) . With regard to phenol, its complete biodegradation was only observed at initial concentrations of 30 and 180mgl(-1) . The denitrification process was inhibited at phenol concentrations higher than 360mgl(-1) . Studies were also done using a continuous anoxic upflow sludge blanket reactor in which formaldehyde removal efficiencies above 99.5% were obtained at all the applied formaldehyde loading rates, between 0.89 and 0.14g COD (CH(2)O)l(-1)d(-1) . The phenol loading rate was increased from 0.03 to 1.3g COD (C(6)H(6)O)l(-1)d(-1) . Phenol removal efficiencies above 90.6% were obtained at phenol concentrations in the influent between 27 and 755mgl(-1) . However, when the phenol concentration was increased to 1010mgl(-1), its removal efficiency decreased . Denitrification percentages around 98.4% were obtained with phenol concentrations in the influent up to 755mgl(-1) . After increasing phenol concentration to 1010mgl(-1), the denitrification percentage decreased because of the inhibition caused by phenol. Water Res, 2005 Jan-Feb, 39(2-3), 349 - 61 Epub 2004 Nov 24. Biodegradation of sediment-bound PAHs in field-contaminated sediment; Lei L et al.; The biodegradation of polycyclic aromatic hydrocarbons (PAHs) has been reported to occur under aerobic, sulfate reducing, and denitrifying conditions . PAHs present in contaminated sites, however, are known for their persistence . Most published studies were conducted in systems where PAHs were freshly spiked, and biodegradation was often tested using pure cultures or enrichments . This paper investigated the degradation potentials of PAHs that were present in aged contaminated sediment by indigenous bacteria, where the limited bioavailability of PAHs due to aging played an important role . The sediment and the overlaying water were collected from a contaminated site to prepare sediment-water slurries, and the sediment served as both the media containing PAH substrates and the habitat for the indigenous microorganisms . Reduced sulfur compounds present in the sediment caused rapid oxygen depletion due to extensive activities of sulfur-oxidizing bacteria and could result in a dramatic pH drop . Once oxygen depletion and acidification problems were avoided, substantial removals of two-, three-, four-, and five-ring PAHs were achieved aerobically, though the extent of degradations was smaller than what was reported for freshly spiked PAHs . The amendment of inorganic N and P, co-substrates, or surfactant Triton X-100 did not enhance the level of degradations appreciably . Under denitrifying conditions, no distinct PAH degradation was observed, while the complete denitrification of nitrate to nitrogen occurred stoichiometrically with a concomitant increase in sulfate concentration, indicating the dominance of autotrophic denitrifiers . The addition of ethanol or acetic acid did not stimulate PAH degradation . Substantial PAH degradation attributed to sulfate reduction was only observed for phenanthrene, the low-ring PAH existing in a highest initial concentration . Addition of ethanol or acetic acid did not change this finding . This is the first study to our knowledge that revealed the importance of indigenous bacteria involved in natural sulfur cycling in determining degradation behavior of PAHs. Res Microbiol, 2005 Jan-Feb, 156(1), 88 - 92 Electrochemical effect on denitrification in different microenvironments around anodes and cathodes; Zhang LH et al.; A bio-anode reactor and a bio-cathode reactor were developed to investigate the microenvironments around anodes and cathodes and their effects on denitrification . With an applied current of 40 mA, the oxidation-reduction potentials (ORPs) in the bio-cathode and bio-anode reactors were 100-200 mV lower and 50 mV higher, respectively, than that in the control reactor (a normal bio-reactor) . The cathode reaction enhanced denitrification and the anode reaction inhibited denitrification . At 40 mA, the denitrification rate in the bio-cathode reactor was 55.1% higher than that in the control reactor . At 75 mA, the denitrification rate in the bio-anode reactor was just 33.5% of that in control reactor . Electric current of less than 20 mA had no effect on the most probable number (MPN) of denitrifiers, but at 75 mA, the MPN of denitrifiers decreased by 90% in the bio-anode reactor . In the bio-cathode reactor, the MPN of denitrifiers increased more than 100% for the lower ORP environment produced by a cathode reaction at 75 mA. J Biol Chem . 2005 Jan 5; {Epub ahead of print} BzdR, a repressor that controls the anaerobic catabolism of benzoate in Azoarcus sp . CIB, is the first member of a new subfamily of transcriptional regulators; Barragan MJ et al.; In this work we have studied the transcriptional regulation of the bzd operon involved in the anaerobic catabolism of benzoate in the denitrifying Azoarcus sp . strain CIB . The transcription start site of the PN promoter running the expression of the bzd catabolic genes was identified . Gel retardation assays and PN::lacZ translational fusion experiments performed both in Azoarcus sp . CIB and Escherichia coli cells have shown that bzdR encodes a specific repressor that controls the inducible expression of the adjacent bzd catabolic operon, being the first intermediate of the catabolic pathway, i.e., benzoyl-CoA, the actual inducer molecule . This is the first report of a transcriptional repressor and a CoA-derived aromatic inducer controlling gene expression in the anaerobic catabolism of aromatic compounds . DNase I footprinting experiments revealed that BzdR protected three regions (operators) at the PN promoter . The three operators contain direct repetitions of a TGCA sequence that forms part of longer palindromic structures . In agreement with the repressor role of BzdR, operator region I spans the transcription initiation site as well as the -10 sequence for recognition of the RNA polymerase . Primary sequence analyses of BzdR showed an unusual modular organization with an N-terminal region homologous to members of the HTH-XRE family of transcriptional regulators and a C-terminal region similar to shikimate kinases . A three-dimensional model of the N-terminal and C-terminal regions of BzdR, generated by comparison with the crystal structures of the SinR regulator from Bacillus subtilis and the shikimate kinase I protein from E . coli, strongly suggests that they contain the helix-turn-helix DNA-binding motif and the benzoyl-CoA binding groove, respectively . The BzdR protein constitutes, therefore, the prototype of a new subfamily of transcriptional regulators. Bioresour Technol, 2005 May, 96(8), 921 - 7 Epub 2004 Nov 11. Methane-dependent denitrification by a semi-partitioned reactor supplied separately with methane and oxygen; Waki M et al.; Methane (CH(4)) can be used as an alternative carbon source for denitrification with added oxygen (O(2)) . However, the off-gas of denitrification reactors using a CH(4)-O(2) mixture contains unused CH(4) and O(2) in proportions that make it unusable for fuel, carry explosion risks, and, if released into the atmosphere, contribute to the greenhouse effect . This study tested a novel reactor with a partition dividing the headspace completely and extending partly into the liquid layer . When CH(4) and O(2) were supplied separately to the liquid layer on opposite sides of the partition, the methane-dependent denitrification (MDD) activity was similar to that when the two gases were supplied as a mixture . In reactors with separate gas supplies, the off-gas from the CH(4) supply side was high in CH(4) and low in O(2), and was usable for fuel, and that from the O(2) supply side was very low in CH(4), and might be released into the atmosphere . MDD activity increased with the O(2) supply rate, and separate discharge of CH(4) and O(2) was maintained . The concentration of dissolved methane in the effluent was decreased by lowering the CH(4)/O(2) supply ratio to 1.0 and drawing the effluent from the O(2) supply side . This novel reactor enhances the safety of MDD, allows reuse of methane as fuel, and reduces methane leakage to the atmosphere. Bioresour Technol, 2005 May, 96(8), 879 - 88 High nitrate removal from synthetic wastewater with the mixed bacterial culture; Foglar L et al.; The applicability of the mixed bacterial culture, originated from two-stage anaerobic-aerobic industrial yeasts production wastewater treatment plant for high rate denitrification processes was investigated . After acclimation to nitrate, the dominant strains were Pseudomonas and Paracoccus sp . Complete denitrification with low accumulation of nitrite-N (0.1mg/l) was found in synthetic wastewater, obeying a zero-order reaction with respect to nitrate and a first-order reaction with respect to biomass concentration . Denitrification was then monitored in the continuous-flow stirred reactor at different hydraulic retention time, HRT (62-28h) in order to achieve the optimal HRT . Nitrate was completely removed during following 45days, at 25 degrees C with HRT, which we reduced from 62 to 28h . Yet still, at 28h HRT, high average specific denitrification rate of 142mg NO(3)(-)-N/g VSS h was obtained. Ying Yong Sheng Tai Xue Bao, 2004 Oct, 15(10), 1887 - 90 {Accumulation of soil inorganic nitrogen in mono-harvesting farmlands in northeast China in late autumn}; Chen X et al.; The study showed that in northeast China, the farmlands with a mono-harvesting agricultural system had a low accumulation of soil inorganic nitrogen after harvest . In late autumn, there was an interval of about 20-30 days when the temperature and humidity were appropriate to soil N mineralization, but the mineralized nitrogen could not be used by crops in this non-growing season . The accumulation of inorganic nitrogen in upland soil reached a high level before freezing . Its inorganic nitrogen content in 1 m depth soil was 99.9 kg x hm(-2) in the treatment of commercial fertilizers (N 150 kg x hm(-2); P 16.4 kg x hm(-2); K 62.3 kg x hm(-2))and 145.4 kg x hm(-2) in the treatment of commercial fertilizers plus manure (pig manure by a reutilization of 80% product) . While in rice field, its inorganic nitrogen content was relatively low before freezing . The reason might be that the soil permeability was not as good as that in upland for its long-term water-logging, and its insufficient aeration would be favorable to denitrification which induces the inorganic nitrogen loss via N2O emission. Syst Appl Microbiol, 2004 Nov, 27(6), 716 - 27 Filamentous Alphaproteobacteria associated with bulking in industrial wastewater treatment plants; Levantesi C et al.; The phylogeny and distribution of filamentous Alphaproteobacteria, morphologically similar to "Nostocoida limicola" and Eikelboom Type 021N that cause the solids separation problem of bulking in industrial activated sludge plants is described here . A combination of culture-dependent and culture-independent molecular methods has characterized 5 novel species . 16S rRNA targeted oligonucleotide probes were designed for their in situ identification by fluorescence in situ hybridisation (FISH) and used to monitor their presence in 86 WWTPs treating different industrial effluents in four European countries . The involvement of these bacteria in bulking in these plants was confirmed . Filaments hybridising with the ALF-968 probe for the Alphaproteobacteria were present in 65% of the WWTPs examined . They were dominant and therefore probably responsible for bulking in 25.5% of them . The heterogeneous filamentous alphaproteobacterial populations in these communities could be completely identified after application of the oligonucleotide probes used in this study in 91% of the plants containing them . The only filamentous Alphaproteobacteria retrieved in pure culture was isolated from three different industrial WWTPs plants . None of these isolates could grow anaerobically on glucose or denitrify, but all grew aerobically and heterotrophically on a range of carbon sources . Although morphologically similar to the Eikelboom Type 021N morphotype, they were not involved in sulphur metabolism . These bacteria accumulated lipidic storage granules that were associated with their presence under the unbalanced growth conditions existing in these plants. Wei Sheng Yan Jiu, 2004 Sep, 33(5), 527 - 30 {Factors affecting denitrification by denitrifican coated electrode}; Tan Y et al.; OBJECTIVE: To optimize the efficiency of coated electrode denitrifying reactor . METHODS: Synthetic groundwater was treated by coated electrode denitrifying reactor submitted to different operational parameters, such as electric current intensity (CI), oxidation reduction potential (ORP), hydraulic retention time (HRT) and temperature . RESULTS: Denitrification efficiencies of the reactor was found to be related with the applied electric current . The denitrification efficiency was 57.3% with a HRT of 12 hours and the optimum applied electric current intensity was 15 mA . Nitrate removal rate of the reactor was calculated to be 34.4 g NO3(-)-N/m3 x d . Denitrification efficiencies were also found to be related to HRT . The average denitrification velocity was 0.183 mg NO3(-)-N/h within 12 hours . The denitrification rates increased when the temperature of synthetic water raised from 5-35 degrees C . After the electrodes had been connected to the power supply for 1 hour, the concentration of dissolved oxygen (DO) and ORP decreased sharply to 1.08 mg/L and -40 mV, respectively . CONCLUSION: An adaptable reduction environment could be set up in the reactor for autotrophic denitrification shortly after the bio-electrochemical reaction began . The optimum electrode potential and current density were 2.5 V and 0.083 mA/cm2 . As the bio-electrochemical reaction went on, the pH volume decreased sharply, and nitrite accumulation was found correspondently, which leads to the inhibition of denitrification . HRT should be controlled within 12 hours . Anode oxidation reaction could offer inorganic carbon sources for autotrophic denitrifying bacteria, but might lead to pH increase, should be paid attention in the practical operation. Biochemistry, 2004 Dec 28, 43(51), 16311 - 9 Insights into redox partner interactions and substrate binding in nitrite reductase from Alcaligenes xylosoxidans: crystal structures of the Trp138His and His313Gln mutants; Barrett ML et al.; Dissimilatory nitrite reductase catalyses the reduction of nitrite to nitric oxide within the key biological process of denitrification . We present biochemical and structural results on two key mutants, one postulated to be important for the interaction with the partner protein and the other for substrate entry . Trp138, adjacent to one of the type-1 Cu ligands, is one of the residues surrounding a small depression speculated to be important in complex formation with the physiological redox partners, azurin I and II . Our data reveal that the Trp138His mutant is fully active using methyl viologen as an artificial electron donor, but there is a large decrease in activity using azurin I . These observations together with its crystal structure at a high resolution of 1.6 A confirm the importance of Trp138 in electron transfer and thus in productive interaction with azurin . A "hydrophobic pocket" on the protein surface has been identified as the channel through which nitrite may be guided to the catalytic type-2 Cu site . Glu133 and His313 at the opening of the pocket are conserved among most blue and green copper nitrite reductases (CuNiRs) . The failure to soak the substrate into our high-resolution crystal form of native and mutant CuNiRs has been linked to the observation of an extraneous poly(ethylene glycol) (PEG) molecule interacting with His313 . We present the crystal structure of His313Gln and the substrate-bound mutant at high resolutions of 1.65 and 1.72 A, respectively . The observation of the substrate-bound structure for the His313Gln mutant and inhibitory studies with PEG establishes the role of the hydrophobic pocket as the port of substrate entry. Prikl Biokhim Mikrobiol, 2004 Nov-Dec, 40(6), 649 - 53 {Purification of soil from oil pollutants with the use of denitrifying hydrocarbon-oxidizing microorganisms}; Nitrate-dependent salicylate degradation by Pseudomonas butanovora under anaerobic conditions; Bay Zoltan Foundation for Applied Research, Institute for Biotechnology, Derkovits fasor 2., Szeged H-6726, HungaryNitrate-dependent salicylate degradation by the denitrifying Pseudomonas butanovora was investigated and the molar ratio of the cometabolism under anaerobic circumstances was determined . The bacterium was able to utilize salicylate as an electron donor for the reduction of nitrate . Salicylate was eliminated via catechol, which is degraded by means of catechol 2,3-oxygenases (meta-cleavage), forming 2-hydroxymuconic semialdehyde . The molar ratios of NO(3)(-)-N:salicylate existing during the experiment accorded well with the assumed 1:1 molar ratio . The tolerances of the growth, the salicylate degradation and the denitrification of P . butanovora to various heavy metal ions were also studied . Although the strain was tolerant to Pb(2+) and Cu(2+) up to 1mM in complete medium, salicylate utilization took place only up to a concentration of 0.1mM for both heavy metal ions . Of the heavy metal ions investigated, Cd(2+) (at a concentration of 0.05mM) displayed the highest inhibitory effect on salicylate degradation by P . butanovora. Aviakosm Ekolog Med, 2004 Sep-Oct, 38(5), 42 - 6 {Biotransformation of organic substances by an immobilized associative bacterial culture}; Nitric oxide reductases of prokaryotes with emphasis on the respiratory et al.; Lehrstuhl fur Mikrobiologie, Universitat Karlsruhe, Kaiserstrasse 12, D-76128 Karlsruhe, GermanyThe investigation of respiratory N-oxide reduction as part of a biogeochemical process sustained by prokaryotes has roots over a century ago and has laid the groundwork for microbial nitric oxide (NO) biology and recognizing that NO is of bioenergetic importance as an electron acceptor in anaerobic environments . NO is an obligatory respiratory substrate of nitrate- and nitrite-denitrifying prokaryotes that release nitrous oxide or dinitrogen as products . We witness currently a broadening of the scope of NO functionality and an increase in awareness that other heme-based NO-metabolizing systems contribute to the overall capability of the prokaryotic cell to cope with NO both in anaerobic and aerobic environments, including the pathogen-host interface . NO reduction of newly recognized physiological importance is catalyzed by the pentaheme nitrite reductase, cytochrome c', flavohemoglobin and flavorubredoxin . Respiratory NO reductases are heme-nonheme Fe proteins that can be classified either in a short-chain group, which are complexes with cytochrome c, or a long-chain group, which have a fused quinol oxidase domain . Even though NORs are not proton pumps, both reductase groups are structural homologues of heme-copper oxidases . As a unique case, the short-chain NOR of Roseobacter denitrificans acts on oxygen, based on a heme b(3)-Cu(B) center . In turn, certain heme-copper oxidases have significant turnover rates with NO . NOR mechanisms have been proposed from oxidase active site chemistry . Besides being a respiratory substrate, NO is also a signaling molecule that triggers gene expression of the principal components of NO respiration by members of the Crp-Fnr superfamily of transcription regulators. Appl Biochem Biotechnol, 2004 Dec, 119(3), 195 - 208 Denitrification in presence of benzene, toluene, and m-xylene: kinetics, mass balance, and yields; Pena-Calva A et al.; Denitrification of the electron donors toluene-C (15-100 mg/L), m-xylene-C (15-70 mg/L), benzene-C (5-25 mg/L), and acetate-C as experimental reference (50-140 mg/L) was carried out in batch culture . An initial concentration of 1.1 +/- 0.15 g of volatile suspended solids/L of denitrifying sludge without previous exposure to aromatic compounds was used as inoculum . The results showed toluene and nitrate consumption efficiency (ET and EN, respectively) of 100% . Toluene was completely mineralized (oxidized) to CO2 . In all cases, the N2 (YN2) and HCO3-yields (YHCO3) were 0.97 +/- 0.01 and 0.8 +/- 0.05, respectively . The consumption efficiency (EX) of m-xylene (53 +/- 5.7%) was partial . The YN2 and YHCO3 were 0.96 +/- 0.01 and 0.86 +/- 0.02, respectively . Benzene was not consumed under denitrifying conditions . The specific consumption rates of toluene (qT) and m-xylene (qX) were lower than that of acetate (qA) . The differences in specific consumption rates were probably owing to the negative effect of benzene, toluene, and isomers of xylene on the cell membrane. Sci Total Environ, 2005 Jan 5, 336(1-3), 255 - 68 Microbial reduction of (99)Tc in organic matter-rich soils; Abdelouas A et al.; For safety assessment purposes, it is necessary to study the mobility of long-lived radionuclides in the geosphere and the biosphere . Within this framework, we studied the behaviour of (99)Tc in biologically active organic matter-rich soils . To simulate the redox conditions in soils, we stimulated the growth of aerobic and facultative denitrifying and anaerobic sulphate-reducing bacteria (SRB) . In the presence of either a pure culture of denitrifiers (Pseudomonas aeruginosa) or a consortium of soil denitrifiers, the solubility of TcO(4)(-) was not affected . The nonsorption of TcO(4)(-) onto bacteria was confirmed in biosorption experiments with washed cells of P . aeruginosa regardless of the pH . At the end of denitrification with indigenous denitrifiers in soil/water batch experiments, the redox potential (E(H)) dropped and this was accompanied by an increase of Fe concentration in solution as a result of reduction of less soluble Fe(III) to Fe(II) from the soil particles . It is suggested that this is due to the growth of a consortium of anaerobic bacteria (e.g., Fe-reducing bacteria) . The drop in E(H) was accompanied by a strong decrease in Tc concentration as a result of Tc(VII) reduction to Tc(IV) . Thermodynamic calculations suggested the precipitation of TcO(2) . The stimulation of the growth of indigenous sulphate-reducing bacteria in soil/water systems led to even lower E(H) with final Tc concentration of 10(-8) M . Experiments with glass columns filled with soil reproduced the results obtained with batch cultures . Sequential chemical extraction of precipitated Tc in soils showed that this radionuclide is strongly immobilised within soil particles under anaerobic conditions . More than 90% of Tc is released together with organic matter (60-66%) and Fe-oxyhydroxides (23-31%) . The present work shows that ubiquitous indigenous anaerobic bacteria in soils play a major role in Tc immobilisation . In addition, organic matter plays a key role in the stability of the reduced Tc. Acta Crystallogr D Biol Crystallogr, 2004 Dec, 60(Pt 12 Pt 2), 2383 - 6 Epub 2004 Dec. Crystallization and preliminary X-ray crystallographic studies of dissimilatory nitrite reductase isolated from Hyphomicrobium denitrificans A3151; Xie Y et al.; Dissimilatory nitrite reductase isolated from Hyphomicrobium denitrificans A3151 (HdNIR) is a novel copper-containing nitrite reductase (CuNIR) composed of six identical subunits . One plastocyanin-like domain and one green CuNIR-like domain are connected to each other, suggesting that the HdNIR subunit structure resembles a complex of green CuNIR and pseudoazurin (or azurin) . Recombinant HdNIR protein was crystallized using the hanging-drop vapour-diffusion method with PEG 4000 as the precipitant at pH 8.9 . X-ray diffraction data were collected to 2.35 A resolution . The HdNIR crystal belonged to the tetragonal space group P4(1) (or P4(3)), with unit-cell parameters a = b = 221.9, c = 165.2 A, giving 12 molecules (two hexamers) per asymmetric unit and a solvent content of 64% . A mutant form of HdNIR, C260A, which lacks the type I copper ion in the CuNIR-like domain, was prepared and crystallized under wild-type HdNIR conditions . The C260A mutant crystal belonged to the cubic space group P4(3)32 (or P4(1)32), with unit-cell parameters a = b = c = 153.7 A, giving one molecule per asymmetric unit and a solvent content of 59% . X-ray diffraction data were collected to 3.5 A resolution . To solve the crystal structure of HdNIR, the multiwavelength anomalous dispersion (MAD) method and the molecular-replacement method are currently being used. Microbiology, 2004 Dec, 150(Pt 12), 4065 - 74 Evidence that the Rhizobium regulatory protein RirA binds to cis-acting iron-responsive operators (IROs) at promoters of some Fe-regulated genes; Yeoman KH et al.; Mutations in rirA of Rhizobium have been shown to deregulate expression of several genes that are normally repressed by iron . A conserved sequence, the iron-responsive operator (IRO), was identified near promoters of vbsC (involved in the synthesis of the siderophore vicibactin), rpoI (specifies an ECF sigma factor needed for vicibactin synthesis) and the two fhuA genes (encoding vicibactin receptor) . Removal of these IRO sequences abolished Fe-responsive repression . Most of these genes were constitutively expressed in the heterologous host, Paracoccus denitrificans, but introduction of the cloned rirA gene repressed expression of these Rhizobium genes in this heterologous host if the corresponding IRO sequences were also intact . These observations are the first to examine the mechanisms of RirA, which has no sequence similarity to well-known iron-responsive regulators such as Fur or DtxR . They provide strong circumstantial evidence that RirA is a transcriptional regulator that binds to cis-acting regulatory sequences near the promoters of at least some of the genes whose expression it controls in response to Fe availability. Environ Sci Technol, 2004 Nov 1, 38(21), 5729 - 36 Pumping-induced ebullition: a unified and simplified method for measuring multiple dissolved gases; Browne BA; The incorporation of multiple dissolved gas measurements in biogeochemical studies remains a difficult and expensive challenge . Incompatibilities in collection, handling, and storage procedures generally force the application of multiple sampling procedures for multiple gases . This paper introduces the concept and application of pumping-induced ebullition (PIE), a unified approach for routine measurement of multiple dissolved gases in natural waters and establishes a new platform for development of in situ real-time dissolved gas monitoring tools . Ebullition (spontaneous formation of bubbles) is induced by pumping a water sample through a narrow-diametertube (a "restrictor") to decrease hydrostatic pressure (PH) below total dissolved gas pressure (PT) . Buoyancy is used to trap bubbles within a collection tower where gas accumulates rapidly (1 mL/min) to support multiple chemical analyses . Providing for field collection of an essentially unlimited and unified volume of gas sample, PIE afforded accurate and precise measurements of major (N2, 02, Ar), trace (CO2, N20, CH4) and ultratrace (CFC11, CFC12, CFC113, SF6) dissolved gases in Wisconsin groundwater, revealing interrelationships between denitrification, apparent recharge age-dates, and historical land use . Compared to conventional approaches, PIE eliminates multiple gas-specific sampling methods, reduces data computations, simplifies laboratory instrumentation, and avoids aqueous production and consumption of biogenic gases during sample storage . A lake depth profile for CO2 demonstrates PIE's flexibility as an in situ real-time platform for dissolved gas measurements . The apparent departures of some gases (SF6, H2, N2O, CO2) from solubility equilibrium behavior warrant further confirmation and theoretical investigation. Water Sci Technol, 2004, 50(8), 217 - 24 Characteristics of granular sludge in a single upflow sludge blanket reactor treating high levels of nitrate and simple organic compounds; Lee CY et al.; Simultaneous denitrification and methanogenesis were accomplished in a single upflow sludge blanket (USB) reactor . More than 99% and 95% of nitrate and chemical oxygen demand (COD) removal rates were obtained at a loading of 600 mg NO3-N/L x d and 3,300 mg COD/L x d, respectively . The specific denitrification rate (SDR) increased as COD/NO3-N ratios decreased . Maximum SDR with acetate could reach 1.05 g NO3-N/gVSS x d . Significant sludge flotation was observed at the top of the reactor due to the change of microbial composition and the formation of hollow granules . Granules became fluffy and buoyant due to the growth of denitrifiers . Microscopic examination showed that granules exhibited layered structure and they were mainly composed of Methanosarcina sp., Pseudomonas sp., and rod-shaped bacteria. Water Sci Technol, 2004, 50(8), 145 - 51 Nitrate removal with low N2O emission by application of sulfur denitrification in actual agricultural field; Hasegawa K et al.; Sulfur denitrification was applied to the agricultural field and the characteristics of the treatment were evaluated from the viewpoints of nitrate removal efficiency and nitrous oxide (N2O) emission . Two actual sites where sulfur denitrification was performed were surveyed . One is a valley bottom field, where groundwater contaminated with nitrate is coming up as spring water . The nitrate concentration in influent was about 45 mgN/L . The other was wastewater from a plastic greenhouse . The nitrate concentration in inflow water was about 200 mgN/L . Nitrate was almost removed by the containers packed with sulfur (S0)-CaCO3 blocks in both sites . Increase of sulfate indicated that nitrate was removed by sulfur denitrification . This was also estimated stoichiometrically from the relationships between the removed nitrate and produced sulfate . The N2O was supersaturated in water at most sampling points and the highest concentration of dissolved N2O reached 900 microgN/L in Saitama in March . It seemed that insufficient nitrate removal caused accumulation of intermediates during denitrification, such as nitrite and N2O, in this month . However, the emission ratio of N2O to the removed nitrate during these processes was kept low, ranging from 0.01 to 0.19%, at both two sites throughout all surveys. Water Sci Technol, 2004, 50(8), 111 - 8 Application of a bio-electrochemical reactor process to direct treatment of metal pickling wastewater containing heavy metals and high strength nitrate; Watanabe T et al.; The fundamental performance of a bio-electrochemical reactor for the direct treatment of metal pickling wastewater was investigated experimentally . In the reactor, carbon anode and cathode were installed . On the cathode, denitrifying microorganisms were immobilized . Continuous experiments were carried out by feeding a synthetic wastewater containing nitrate and binary heavy metal ions, copper and lead, under different operating conditions . Acetate as well as the electric current was supplied at the minimum amount for stoichiometry of the dissimilatory denitrification reaction . The results indicated that the dissolved copper and lead removal, denitrification and neutralization could be achieved simultaneously in a single bio-electrochemical reactor . The dissolved heavy metals were removed by electrochemical deposition on cathode and by the other phenomena such as the formation of insoluble suspensions and the sorption on suspended bacterial sludge . Denitrification proceeded effectively with the utilization of both added acetate and hydrogen gas generated by electrolysis of water . The pH value increased up to around neutral due to the occurrence of denitrification in the reactor, although the influent pH was less than 3 . The removal efficiencies of heavy metals and nitrate increased with increasing the current density . The applied electric current was indispensable for sustaining the stable treatment in the reactor. Water Sci Technol, 2004, 50(8), 33 - 40 Improvement of denitrification by denitrifying phosphorus removing bacteria using sequentially combined carbon; Cho ES et al.; The effects of sequentially combined carbon (SCC) using a symbiotic relationship of methanol and acetic acid on biological nutrient removal were investigated in both the continuous bench scale process consisting of an anoxic, an aerobic and a final settling tank and intensive batch tests . Compared to the use of respective sole carbon sources, methanol and acetic acid, the use of SCC showed superior removal efficiency of nitrogen (98.3%) and phosphorus (approximately 100%) . Furthermore, the use of SCC enhanced simultaneous denitrification and phosphorus uptake by denitrifying phosphorus removal bacteria (DPB), resulting in the highest specific denitrification rate (SDNR) of 0.252 g NO3-N/g VSS/d achieved from the first anoxic zone with methanol of 30 mg COD/I . From batch tests performed under carbon limited anoxic conditions, 1 g of nitrate was used by DPB for P-uptake of 1.19 g . According to this result, 0.205 g NO3-N/g VSS/d was accomplished by normal denitrifiers using methanol, and 0.047 g NO3-N/g VSS/d was achieved by DPB . This research also demonstrated that the increase of poly-beta-hydroxybutyrate (PHB) stored by phosphorus accumulating organisms (PAOs) could be of importance in improving aerobic denitrification . The use of SCC produced the highest P-release in the anoxic zone, indicating the amount of PHB would be higher compared to the use of other sole carbons . Therefore, the SCC could be a very effective carbon source for the enhancement of aerobic denitrification as well. Water Res, 2004 Dec, 38(20), 4524 - 34 Effects of oxygen on biodegradation of benzoate and 3-chlorobenzoate in a denitrifying chemostat; Deniz T et al.; A mixed microbial culture degraded a mixture of benzoate (863 mg/L), 3-chlorobenzoate (3-CB) (69.7 mg/L), and pyruvate (244 mg/L) under denitrifying conditions in a chemostat . Biodegradation under denitrifying conditions was stable, complete (effluent concentrations below detection limits), and proceeded without the production of toxic intermediates like chlorocatechols . The addition of oxygen at mass input rates of 6.2%, 15.5%, and 43.9% of the mass input rate of chemical oxygen demand (COD) (337 mg COD/h) did not induce the synthesis of aerobic biodegradation pathways and thus did not disrupt biodegradation . Rather, the oxygen was used as a terminal electron acceptor, displacing a stoichiometric amount of nitrate, leading to microaerobic conditions (dissolved oxygen concentration <0.050 mg/L) in which oxygen utilization and denitrification occurred simultaneously . The reduction of nitrate occurred fully to N(2) gas with no accumulation of nitrite, nitrous oxide, or nitric oxide, although the ability of the culture to transfer electrons to the nitrogen oxides decreased as the oxygen input was increased . The anoxic benzoate uptake capability was unaffected by the increase in oxygen addition, but the anoxic 3-CB uptake capability increased, as did the level of benzoyl-CoA reductase in the cells. Mikrobiol Z, 2004 Sep-Oct, 66(5), 68 - 75 {Microflora of damaged ferroconcrete structures under the conditions of inhibitory protection}; Commissioning and operational experiences for the 160ML/d Woodman Point Sequencing Batch Reactor--control of settleability and denitrification using bioselectors; Environmental Solutions International Ltd, Burswood, Western Australia, WA 6100, Australia . wayneb@environ.com.au Achieving and maintaining good biomass settling characteristics is a critical process design objective for any activated sludge wastewater treatment plant (WWTP), whether intermittent or continuous technology . One way of ensuring good sludge settleability in intermittent WWTPs is the incorporation of bioselectors in the process . A bioselector is essentially a small discrete reactor volume designed primarily for carbon absorption, in which activated sludge organisms are exposed to a high substrate concentration for a relatively short time . It is normally very much smaller than an anoxic zone and the activated sludge recycle is only a fraction of that typically adopted in continuous plants . With proper conditioning, recycled biomass rapidly absorbs and stores soluble organic wastewater components before transfer to the main treatment basin . This absorption and storage mechanism, and careful management of aeration throughout the intermittent treatment cycle, plays a crucial role in many subsequent growth and treatment processes, including sludge floc formation, denitrification and biological phosphorus removal . This paper examines some design considerations, and reviews the benefits of bioselectors by reference to the commissioning and initial operation of the new 160ML/d Woodman Point Sequencing Batch Reactor in Perth, Western Australia . The applicability of bioselectors in continuous plants is discussed. Water Sci Technol, 2004, 50(7), 105 - 12 Denitrification rate and carbon source consumption in full-scale wastewater filtration; Jonsson L; In response to new demands for increased removal of nitrogen and phosphorus, the Henriksdal and Bromma treatment plants, with hydraulic loads of 283,000 and 148,000 m3/d, respectively, built filtration steps as a final process step in the plants . The denitrification rates in a full-scale and in a pilot plant filter are calculated to 13.1 and 21.3 g (NO3+NO2)-N/(m3 x h), respectively, in the total filter bed after 2.5-24.2 and 16.0-28.0 h of operational time, and 6.4 and 18.7 g (NO3+NO2)-N/(m3 x h), respectively, after 1.0 and 0.1-0.9 h of operational time . In composite samples, the denitrification rate in the total filter bed is 10-20 g (NO3+NO2)-N/(m3 x h) in the full-scale filter . The average values for k = deltaCODf/deltaC(T) are 1.6 and around 3 in the total filter bed in steady state and in the beginning of the experiments, respectively, both in the full-scale and in the pilot plant study . The carbon source costs for reducing the concentration of nitrate nitrogen in the Bromma plant from 12 to 8 mg/l in the effluent are 117,400 EUR and 147,400 EUR with methanol and ethanol, respectively, as a carbon source. Water Sci Technol, 2004, 50(7), 97 - 103 Six years of operation of a fluidised bed reactor for denitrification; Harri A et al.; At Himmerfjarden wastewater treatment plant, a fluidised bed reactor for nitrogen removal has been operated since 1997 . Despite its small footprint, the system enables a far-reaching nitrogen removal . The current nitrate reduction in the reactor is 95% . The reduction of total nitrogen at the wastewater treatment plant is 80-90% at normal operation . The concentration of nitrate in the effluent is easily controlled by changing the dose of carbon . As part of a series of full-scale experiments, the plant has, for the last two years, been operated without denitrification for a couple of months during spring/summer, in order to benefit a favourable N/P-ratio in the recipient and to counteract the growth of nitrogen fixing blue-green algae, When resuming the dosage of carbon, full denitrification was re-established in about two weeks . Important factors to take into consideration when operating the fluidised bed reactor are the abrasive characteristics of the carrier material (sand), the increased concentration of suspended solids in the effluent, and the importance of a suitable N/P-ratio in the influent, as lack of phosphorus might cause an uncontrolled microbiological growth. Water Sci Technol, 2004, 50(7), 27 - 34 The Polish perspective on adopting EU standards for nitrogen removal at large WWTPs--case studies; Makinia J et al.; The most challenging issue for existing large WWTPs (>100,000 PE) in Poland will be achievement of the new effluent standards for total nitrogen . Consequently, reliable and accurate information concerning the dimensioning of anoxic compartments is necessary . This study focused on validating to what extent the denitrification rates determined from batch tests were comparable with the rates calculated based on a mass balance over a full-scale activated sludge reactor . The experiments were conducted at two large WWTPs in northern Poland: "Wschod" in Gdansk and "Debogorze" in Gdynia . Two types of batch tests were used to determine the denitrification capability of activated sludge . Lower nitrate utilization rates observed during the full-scale experiments could potentially result from the local disturbances such as nitrate limitation ("Wschod" WWTP) or oxygen penetration to the anoxic zone ("Debogorze" WWTP) . These factors should be taken into consideration during the design phase of the anoxic compartments. Biol Chem, 2004 Oct, 385(10), 875 - 83 Structural basis of denitrification; Einsle O et al.; Denitrification represents an important part of the biogeochemical cycle of the essential element nitrogen . It constitutes the predominant pathway of the reductive dissimilation of nitrate in the environment . Via four enzymatic reactions, nitrate is transformed stepwise to nitrite (NO2-), nitric oxide (NO), and nitrous oxide (N2O), to finally yield dinitrogen gas (N2) . All steps within this metabolic pathway are catalyzed by complex multi-site metalloenzymes with unique spectroscopic and structural features . In recent years, high-resolution crystal structures have become available for these enzymes with the exception of the structure for NO reductase. Environ Technol, 2004 Oct, 25(10), 1201 - 10 Nitrate removal from saline water using autotrophic denitrification by the bacterium Thiobacillus denitrificans MP-1; Zhao Z et al.; Autotrophic denitrification of synthetic wastewater by Thiobacillus denitrificans MP-1 isolated from mangrove sediment was investigated in both up-flow packed-bed reactors and fermentor . More than 97.5% and 90% of the nitrate in inflow was removed after 8.8 and 161 hours at 250 and 195 mg l(-1) for the packed-bed reactor and fermentor system, respectively . The nitrate was quickly denitrified at very low flow rates (0.11 m h(-1)) for the packed-bed reactors, but as the flow rate was greater than 0.13 m h(-1), the nitrate removal rate increased as the flow rate increased . In the static fermentor system, the denitrification can be described by a secondary reaction, but at a flow rate between 1.31 to 1.49 m h(-1), the reactor performance can be described using the zero-order reaction in the packed-bed reactor . As the speed increases, the zero-order reaction translates into half-order reaction as the penetration efficiency of nitrate decreases . The mass ratios between the nitrate removed and the sulfate produced were determined to be 6.81 and 9.32 in the reactor column and fermentor, respectively . The results of this study suggest that efficient removal of high concentrations of nitrate in water or wastewater can be achieved effectively using autotrophic bacteria immobilized on surfaces of sulphur granules in packed-bed reactor. Arch Microbiol, 2005 Jan, 183(1), 27 - 36 Epub 2004 Nov 13. The genome sequence of an anaerobic aromatic-degrading denitrifying bacterium, strain EbN1; Rabus R et al.; Recent research on microbial degradation of aromatic and other refractory compounds in anoxic waters and soils has revealed that nitrate-reducing bacteria belonging to the Betaproteobacteria contribute substantially to this process . Here we present the first complete genome of a metabolically versatile representative, strain EbN1, which metabolizes various aromatic compounds, including hydrocarbons . A circular chromosome (4.3 Mb) and two plasmids (0.21 and 0.22 Mb) encode 4603 predicted proteins . Ten anaerobic and four aerobic aromatic degradation pathways were recognized, with the encoding genes mostly forming clusters . The presence of paralogous gene clusters (e.g., for anaerobic phenylacetate oxidation), high sequence similarities to orthologs from other strains (e.g., for anaerobic phenol metabolism) and frequent mobile genetic elements (e.g., more than 200 genes for transposases) suggest high genome plasticity and extensive lateral gene transfer during metabolic evolution of strain EbN1 . Metabolic versatility is also reflected by the presence of multiple respiratory complexes . A large number of regulators, including more than 30 two-component and several FNR-type regulators, indicate a finely tuned regulatory network able to respond to the fluctuating availability of organic substrates and electron acceptors in the environment . The absence of genes required for nitrogen fixation and specific interaction with plants separates strain EbN1 ecophysiologically from the closely related nitrogen-fixing plant symbionts of the Azoarcus cluster . Supplementary material on sequence and annotation are provided at the Web page http://www.micro-genomes.mpg.de/ebn1/. J Biol Chem . 2004 Nov 17; {Epub ahead of print} A dynamic Zn redox switch; Neculai AM et al.; The crystal structures of glutathione-dependent formaldehyde-activating enzyme (Gfa) from Paracoccus denitrificans, that catalyzes the formation of S-hydroxymethylglutathione from formaldehyde and glutathione, and its complex with glutathione (Gfa-GTT) have been determined . Gfa has a new fold with two Zn-Sulfur centers, one structural (Zn tetracoordinated), and one catalytic (Zn apparently tricoordinated) . In Gfa-GTT, the catalytic Zn is displaced due to disulfide bond formation of glutathione with one of the Zn coordinating cysteines . Soaking crystals of Gfa-GTT with formaldehyde restores the holoenzyme . Accordingly, the displaced Zn forms a complex by scavenging formaldehyde and glutathione . The activation of formaldehyde and of glutathione in this Zn complex favors the final nucleophilic addition, followed by relocation of Zn in the catalytic site . Therefore, the structures of Gfa and Gfa-GTT draw the critical association between a dynamic Zn redox switch and a nucleophilic addition as a new facet of the redox activity of Zn-Sulfur sites. Int J Syst Evol Microbiol, 2004 Nov, 54(Pt 6), 2307 - 12 Pseudovibrio denitrificans gen . nov., sp . nov., a marine, facultatively anaerobic, fermentative bacterium capable of denitrification; Shieh WY et al.; Two denitrifying strains of heterotrophic, facultatively anaerobic, marine bacteria, designated DN34(T) and DN33, were isolated from sea-water samples collected in Nanwan Bay, Kenting National Park, Taiwan . They were Gram-negative . Cells in late exponential to early stationary phase of growth were predominantly straight or curved rods, but Y- or V-shaped forms were also observed . They were motile by means of one to several lateral or subpolar flagella . Both strains required NaCl for growth and exhibited optimal growth at about 30 degrees C, pH 8 and 3 % NaCl . They were capable of anaerobic growth by carrying out denitrifying metabolism using nitrate, nitrite or nitrous oxide as terminal electron acceptors or, alternatively, by fermenting glucose, mannose, sucrose or trehalose as substrates . Anaerobic fermentative growth on glucose resulted in formation of various organic acids, including formate, lactate, acetate, pyruvate and fumarate . The major cellular fatty acids were 2-OH-14 : 0, 3-OH-14 : 0 and 16 : 0 . DN34(T) and DN33 had DNA G+C contents of 51.7 and 51.6 mol%, respectively . Physiological characterization, together with phylogenetic analysis based on 16S rRNA gene sequence analysis, revealed that the two denitrifying strains could be accommodated in a novel genus, for which the name Pseudovibrio gen . nov . is proposed . Pseudovibrio denitrificans sp . nov . is the type species, with DN34(T) (=BCRC 17323(T)=JCM 12308(T)) as the type strain. Int J Syst Evol Microbiol, 2004 Nov, 54(Pt 6), 2297 - 300 Sulfuricurvum kujiense gen . nov., sp . nov., a facultatively anaerobic, chemolithoautotrophic, sulfur-oxidizing bacterium isolated from an underground crude-oil storage cavity; Kodama Y et al.; A facultatively anaerobic, chemolithoautotrophic, sulfur-oxidizing bacterium, strain YK-1(T), was isolated from an underground crude-oil storage cavity at Kuji in Iwate, Japan . The cells were motile, curved rods and had a single polar flagellum . Optimum growth occurred in a low-strength salt medium at pH 7.0 and 25 degrees C . It utilized sulfide, elemental sulfur, thiosulfate and hydrogen as the electron donors and nitrate as the electron acceptor under anaerobic conditions, but it did not use nitrite . Oxygen also served as the electron acceptor under the microaerobic condition (O(2) in the head space 1 %) . It did not grow on sugars, organic acids or hydrocarbons as carbon and energy sources . The DNA G+C content of strain YK-1(T) was 45 mol% . Phylogenetic analysis, based on the 16S rRNA gene sequence, showed that its closest relative was Thiomicrospira denitrificans in the 'Epsilonproteobacteria', albeit with low homology (90 %) . On the basis of physiological and phylogenetic data, strain YK-1(T) should be classified into a novel genus and species, for which the name Sulfuricurvum kujiense gen . nov., sp . nov . is proposed . The type strain is YK-1(T) (=JCM 11577(T)=MBIC 06352(T)=ATCC BAA-921(T)). Int J Syst Evol Microbiol, 2004 Nov, 54(Pt 6), 2181 - 4 Jonesia quinghaiensis sp . nov., a new member of the suborder Micrococcineae; Schumann P et al.; A coryneform strain isolated from soda lake mud in China corresponded in chemotaxonomic characteristics such as peptidoglycan type A4alpha l-lys-l-ser-d-Glu and major menaquinone MK-9, as well as in its DNA base composition (57 mol% G+C), to its phylogenetic neighbour Jonesia denitrificans . Differences in phenotypic characteristics and the phylogenetic distance (96.6 % 16S rRNA gene sequence similarity) from J . denitrificans justify the proposal of a second species of the genus Jonesia, Jonesia quinghaiensis sp . nov., with the type strain QH3A7(T) (=DSM 15701(T)=CGMCC 1.3459(T)). J Environ Qual, 2004 Nov-Dec, 33(6), 2367 - 74 Denitrification in a coastal plain riparian zone contiguous to a heavily loaded Swine wastewater spray field; Hunt PG et al.; Riparian zones are recognized as landscape features that buffer streams from pollutants, particularly nitrogen . The objectives of this experiment were to (i) assess denitrification activity within a riparian zone and (ii) determine the influence of physical, chemical, and landscape features on denitrification . This experiment was conducted from 1994 to 1997 in North Carolina on a riparian zone contiguous to a spray field that was heavily loaded with swine lagoon wastewater . Denitrification enzyme activity (DEA) was measured on soils collected from (i) the soil surface, (ii) midway between the soil surface and water table, and (iii) above the water table . The DEA ranged from 3 to 1660 microg N(2)O-N kg(-1) soil h(-1) . The DEA was highest next to the stream and lowest next to the spray field . Nitrate was found to be the limiting factor for denitrification . The DEA generally decreased with soil depth; means for the surface, middle, and bottom depths were 147, 83, and 67 microg N(2)O-N kg(-1) soil h(-1), respectively . These DEA values are higher than those reported for riparian zones adjoining cropland of the southeastern United States, but are lower than those reported for a constructed wetland used for treatment of swine wastewater . Regression analysis indicated that soil total nitrogen was the highest single factor correlated to DEA (r(2) = 0.65) . The inclusion of water table depth, soil depth, and distance from the spray field improved the R(2) to 0.86 . This riparian zone possessed sufficient soil area with high denitrifying conditions to be a significant factor in the removal of excess nitrogen in the ground water. Water Sci Technol, 2004, 50(6), 261 - 8 The effect of anoxic selectors on sludge bulking; Tampus MV et al.; The effect of the substrate feeding pattern on sludge settleability in denitrifying systems was investigated . The feeding pattern was simulated in two sequencing batch reactors (SBR) . SBR1 simulates a system with a limiting substrate (acetate) and a non-limiting electron acceptor (nitrate), while SBR2 simulates the opposite condition . Sludge volume index (SVI) was the main observed parameter . The stoichiometry, substrate uptake rate, storage capacity as well as sludge morphology and mixed culture population dynamics were also determined . Results show that independent of the feeding pattern, the settleability of the sludge remained good (SVI approximately 50 mL/g) and stable . Neither the concentration of the acetate nor of the nitrate offered any limitations on sludge settleability, assuring that a good bio-P activity is present in nitrate limiting systems . Filamentous bacteria belonging to the TM group were usually present, although in low numbers . Despite the diffused, irregularly shaped and open floc structure, good settleability was observed . The results obtained were in line with the hypothesis on diffusion based selection. Water Sci Technol, 2004, 50(6), 251 - 60 Model-based evaluation of nitrogen removal in a tannery wastewater treatment plant; Moussa MS et al.; Computer modelling has been used in the last 15 years as a powerful tool for understanding the behaviour of activated sludge wastewater treatment systems . However, computer models are mainly applied for domestic wastewater treatment plants (WWTPs) . Application of these types of models to industrial wastewater treatment plants requires a different model structure and an accurate estimation of the kinetics and stoichiometry of the model parameters, which may be different from the ones used for domestic wastewater . Most of these parameters are strongly dependent on the wastewater composition . In this study a modified version of the activated sludge model No . 1 (ASM 1) was used to describe a tannery WWTP . Several biological tests and complementary physical-chemical analyses were performed to characterise the wastewater and sludge composition in the context of activated sludge modelling . The proposed model was calibrated under steady-state conditions and validated under dynamic flow conditions . The model was successfully used to obtain insight into the existing plant performance, possible extension and options for process optimisation . The model illustrated the potential capacity of the plant to achieve full denitrification and to handle a higher hydraulic load . Moreover, the use of a mathematical model as an effective tool in decision making was demonstrated. Water Sci Technol, 2004, 50(6), 217 - 25 Removal of nitrogen and phosphorus from industrial wastewaters by phytoremediation using water hyacinth (Eichhornia crassipes (Mart.) Solms); Jayaweera MW et al.; This paper elucidates the phytoremediation potential of water hyacinth (Eichhornia crassipes {Mart.} Solms) for TN and TP rich industrial wastewaters determined for 15 weeks under different set-ups of 2-fold (56 TN mg/l and 15.4 TP mg/l), 1-fold, 1/2-fold, 1/4-fold and 1/8-fold and a control with no nutrients in duplicate . A mass balance was conducted to evaluate the phytoremediation efficiencies and to identify the key mechanisms of nutrient removal from the wastewaters . Our results manifested that water hyacinth is a promising candidate for a batch removal of TN and TP from wastewaters . 100% removal of both TN and TP was observed at the end of the 9th week in all the set-ups mainly due to assimilation and the period between 6-9weeks became the optimum period after which complete harvesting is recommended . Plants having an age of 6 weeks are ideal to commence the free-floating wetland and 21 days hydraulic retention time (HRT) is recommended for optimum removal of TN and TP . Assimilation and denitrification were the key mechanisms of TN removal while assimilation and sorption became the prominent mechanisms in the removal of TP from wastewaters. Water Sci Technol, 2004, 50(6), 173 - 8 Control of nutrients after discharge to lakes through wastewater; Zhang XH et al.; Control of nutrients as nitrogen and phosphorus after discharge into lakes is necessary since it is difficult and costly to control within wastewater plants currently in China . This paper studied the cycling of phosphorus and nitrogen with water and sediments from two lakes in China . It is found that oxygen plays a critical role in regulating phosphorus and nitrogen cycling within water and sediments . Three different oxygenation methods including aeration, calcium peroxide and hydrogen peroxide were studied to control phosphorus and nitrogen in overlying water . In anoxic conditions, the P concentration in water increased from an average 14 microg/L to 115.2 microg/L for Xili Lake, and from an average 24 microg/L to 1,000 microg/L for Jinchun Lake . The concentration of ammonia increased under anoxic conditions, while the concentration of nitrate increased under oxic conditions . In anoxic conditions, the nitrate concentration decreased probably through denitrification . Both N and P accumulation processes can be controlled under the three treatments . The phosphorus removal efficiency from the water body was in the order of CaO2 addition > aeration > H2O2 addition, while controlling effectiveness for ammonia was in the order of aeration > CaO2 addition > H2O2 addition. Water Sci Technol, 2004, 50(6), 141 - 8 Treatment of landfill leachate by a pilot-scale modified Ludzack-Ettinger and sulfur-utilizing denitrification process; Bae JH et al.; Nitrogen removal efficiency of a pilot-scale system consisted of Modified Ludzack-Ettinger (MLE) followed by sulfur-utilizing denitrification (SUDNR) process was evaluated with a landfill leachate . For SUDNR, a down-flow mode sulfur packed bed reactor (SPBR) filled with sulfur and limestone particles was used . Although total nitrogen removal efficiency of the MLE process was about 80% at the recycle ratio of 4, effluent contained 350-450 mg/L NO(3-)-N . Up to a loading rate of 1.2 kg NO(3-)-N/m3-day, the SPBR could achieve complete removal of nitrate, and nitrate removal rate was kept to that level even at higher loading rate . When a COD/N ratio of MLE process was maintained at 2 instead of 4, more organics with molecular weight less than 500 were utilized for heterotrophic denitrification although denitrification was not complete with the lack of electron donors . Clogging in the SPBR, mainly by the accumulation of nitrogen gas in the pores, could easily be removed by introducing the effluent in an upward direction for 1 min at 1 hr intervals . The proposed treatment system could achieve nitrate free effluent with a slight increase in chemical cost . Furthermore, depending on further COD removal requirement after biological treatment, the proposed treatment system can be an economical solution. Biochemistry, 2004 Nov 16, 43(45), 14370 - 8 ATR-FTIR spectroscopy and isotope labeling of the PM intermediate of Paracoccus denitrificans cytochrome c oxidase; Iwaki M et al.; The structure of the P(M) intermediate of Paracoccus denitrificans cytochrome c oxidase was investigated by perfusion-induced attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy . Transitions from the oxidized to P(M) state were initiated by perfusion with CO/oxygen buffer, and the extent of conversion was quantitated by simultaneously monitoring visible absorption changes . In prior work, tentative assignments of bands were proposed for heme a(3), a change in the environment of the protonated state of a carboxylic acid, and a covalently linked histidine-tyrosine ligand to Cu(B) that has been found in the catalytic site . In this work, reduced minus oxidized difference spectra at pH 6.5 and 9.0 and P(M) minus oxidized difference spectra at pH 9.0 were compared in unlabeled, universally (15)N-labeled, and tyrosine-ring-d(4)-labeled proteins to improve these assignments . In the reduced minus oxidized difference spectrum, (15)N labeling resulted in large changes in the amide II region and a 9 cm(-1) downshift in a 1105 cm(-1) trough that is attributed to histidine . In contrast, changes induced by tyrosine-ring-d(4) labeling were barely detectable where the isotope-sensitive bands are expected . Both isotope substitutions had large effects on P(M) minus oxidized difference spectra . A prominent trough at 1542 cm(-1) was shifted to 1527 cm(-1) with (15)N labeling, and its magnitude was diminished with the appearance of a 1438 cm(-1) trough with tyrosine-ring-d(4) labeling . Both isotope substitutions also had large effects on a 1314 cm(-1) trough in the same spectra . These shifts indicate that the bands are linked to both a nitrogenous compound and a tyrosine, the most obvious candidate being the covalent histidine-tyrosine ligand of Cu(B) . Comparison with model material data suggests that the tyrosine hydroxyl group is protonated when the binuclear center is oxidized but deprotonated in the P(M) intermediate . Positive bands at 1519 and 1570 cm(-1) were replaced with bands at 1504 and 1556 cm(-1), respectively, with tyrosine-ring-d(4) labeling, are characteristic of upsilon(7a)(C-O) and upsilon(C-C) bands of neutral phenolic radicals, and most likely reflect the formation of the neutral radical state of the histidine-tyrosine ligand in P(M). Microbiology, 2004 Nov, 150(Pt 11), 3527 - 46 Nitrate reduction and the nitrogen cycle in archaea; Cabello P et al.; The nitrogen cycle (N-cycle) in the biosphere, mainly driven by prokaryotes, involves different reductive or oxidative reactions used either for assimilatory purposes or in respiratory processes for energy conservation . As the N-cycle has important agricultural and environmental implications, bacterial nitrogen metabolism has become a major research topic in recent years . Archaea are able to perform different reductive pathways of the N-cycle, including both assimilatory processes, such as nitrate assimilation and N(2) fixation, and dissimilatory reactions, such as nitrate respiration and denitrification . However, nitrogen metabolism is much less known in archaea than in bacteria . The availability of the complete genome sequences of several members of the eury- and crenarchaeota has enabled new approaches to the understanding of archaeal physiology and biochemistry, including metabolic reactions involving nitrogen compounds . Comparative studies reveal that significant differences exist in the structure and regulation of some enzymes involved in nitrogen metabolism in archaea, giving rise to important conclusions and new perspectives regarding the evolution, function and physiological relevance of the different N-cycle processes . This review discusses the advances that have been made in understanding nitrate reduction and other aspects of the inorganic nitrogen metabolism in archaea. Water Environ Res, 2004 Sep-Oct, 76(5), 404 - 12 Carbon and nutrient removal from on-site wastewater using extended-aeration activated sludge and ion exchange; Safferman SI et al.; The need to improve on-site wastewater treatment processes is being realized as populations move into more environmentally sensitive regions and regulators adopt the total maximum daily load approach to watershed management . Under many conditions, septic systems do not provide adequate treatment; therefore, advanced systems are required . These systems must remove significant amounts of biochemical oxygen demand (BOD) and suspended solids, and substantially nitrify, denitrify, and remove phosphorus . Many existing advanced on-site wastewater systems effectively remove BOD, suspended solids, and ammonia, but few substantially denitrify and uptake phosphorus . The purpose of this research was to design and test modifications to an existing on-site wastewater treatment system to improve denitrification and phosphorus removal . The Nayadic (Consolidated Treatment Systems, Inc., Franklin, Ohio), an established, commercially available, extended-aeration, activated sludge process, was used to represent a typical existing system . Several modifications were considered based on a literature review, and the option with the best potential was tested . To improve denitrification, a supplemental treatment tank was installed before the Nayadic and a combination flow splitter, sump, and pump box with a recirculation system was installed after it . A recirculation pump returned a high proportion of the system effluent back to the supplemental treatment tank . Two supplemental treatment tank sizes, three flowrates, and three recirculation rates were tested . Actual wastewater was dosed as brief slugs to the system in accordance with a set schedule . Several ion-exchange resins housed in a contact column were tested on the effluent for their potential to remove phosphorus . Low effluent levels of five-day biochemical oxygen demand, suspended solids, and total nitrogen were achieved and substantial phosphorous removal was also achieved using a 3780-L supplemental treatment tank, a recirculation ratio of 5:1, and a fine-grain activated aluminum-oxide-exchange media . Good results were also obtained with an 1890-L supplemental treatment tank and a recirculation ratio of 3:1 . The most significant benefit of the supplemental treatment tank, in combination with the recirculation system, appears to be the low nitrogen concentration dosed to the Nayadic . By reducing the nitrogen concentration and spreading out its mass over time during no-flow periods, the Nayadic's inherent low-level denitrifying capacity was more closely matched and effective treatment was achieved. Water Environ Res, 2004 Sep-Oct, 76(5), 388 - 93 Denitrification of nitrate wastewater using packed-bed columns; Lee LY et al.; Columnar packed-bed (PB) reactors with a specific surface area of 127 m2/m3 were investigated in this study for treating nitrate wastewater . This study demonstrated that a single-stage packed bed was able to achieve total nitrogen (TN) and chemical oxygen demand (COD) removal efficiencies higher than 83 and 75%, respectively . The highest achievable TN and COD removal rates were 47.2 g N/m2 x d and 158.0 g COD/m2 x d, respectively . The substrate removal rate in the PB column was found to follow half-order reaction kinetics, with a reaction coefficient, kappa, of 53.62 (mg/L)1/2/d . A dual-stage PB system was capable of achieving TN and COD removal efficiencies greater than 99 and 98%, respectively . Effluent TN and COD concentrations less than 6.5 mg NO3(-)-N/L and 50.0 mg COD/L, respectively, were obtained when the dual PB system was used. J Biotechnol, 2004 Nov 9, 114(3), 289 - 97 Use of volatile fatty acids from an acid-phase digester for denitrification; Elefsiniotis P et al.; This study investigates the ability of naturally-produced volatile fatty acids (VFAs) to act as a carbon source for the removal of nitrate-Nitrogen concentrations in the range of 20 to 200 mg/L . The VFAs were generated from an anaerobic digester treating a 1:1 mixture of starch-rich industrial and municipal wastewater . The experiments were carried out at an ambient temperature of 22+/-2 degrees C using 600 mL batch reactors containing VFA-rich effluent from the digester and prepared primary sludge . Nitrates added in the form of a 1 M potassium nitrate solution were observed to completely disappear in conjunction with significant recovery of pH and alkalinity (i.e . biological denitrification) . In all cases, denitrification followed zero-order kinetics with an average rate constant of 2.67 mg/{Lh} and a mean specific denitrification rate of 0.0111 g NOx-N/g volatile suspended solids (VSS) per day . The denitrifier population had a preference for acetic acid which occurred in the largest quantity and was exhausted the most rapidly . The next two most abundant VFAs were butyric acid and propionic acid, which were consumed only after acetate concentrations began to decline . The third "choice" of the denitrifiers was valeric acid, thus it appears that denitrifying bacteria have a sequential preference for VFAs . In addition, they prefer VFAs over other more complex soluble organic carbon forms. J Mol Biol, 2004 Nov 19, 344(2), 419 - 33 Structure/function studies on a S-adenosyl-L-methionine-dependent uroporphyrinogen III C methyltransferase (SUMT), a key regulatory enzyme of tetrapyrrole biosynthesis; Vevodova J et al.; The crystallographic structure of the Pseudomonas denitrificans S-adenosyl-L-methionine-dependent uroporphyrinogen III methyltransferase (SUMT), which is encoded by the cobA gene, has been solved by molecular replacement to 2.7A resolution . SUMT is a branchpoint enzyme that plays a key role in the biosynthesis of modified tetrapyrroles by controlling flux to compounds such as vitamin B(12) and sirohaem, and catalysing the transformation of uroporphyrinogen III into precorrin-2 . The overall topology of the enzyme is similar to that of the SUMT module of sirohaem synthase (CysG) and the cobalt-precorrin-4 methyltransferase CbiF and, as with the latter structures, SUMT has the product S-adenosyl-L-homocysteine bound in the crystal . The roles of a number of residues within the SUMT structure are discussed with respect to their conservation either across the broader family of cobalamin biosynthetic methyltransferases or within the sub-group of SUMT members . The D47N, L49A, F106A, T130A, Y183A and M184A variants of SUMT were generated by mutagenesis of the cobA gene, and tested for SAM binding and enzymatic activity . Of these variants, only D47N and L49A bound the co-substrate S-adenosyl-L-methionine . Consequently, all the mutants were severely restricted in their capacity to synthesise precorrin-2, although both the D47N and L49A variants produced significant quantities of precorrin-1, the monomethylated derivative of uroporphyrinogen III . The activity of these variants is interpreted with respect to the structure of the enzyme. Biochemistry, 2004 Nov 9, 43(44), 14180 - 8 Characterization of two type 1 Cu sites of Hyphomicrobium denitrificans nitrite reductase: a new class of copper-containing nitrite reductases; Yamaguchi K et al.; We report (1) the amino acid sequence of Hyphomicrobium denitrificans nitrite reductase (HdNIR), containing two type 1 Cu sites and one type 2 Cu site; (2) the expression and preparation of wild-type HdNIR and two mutants replacing the Cys ligand of each type 1 Cu with Ala; and (3) their spectroscopic and functional characterization . The open-reading frame of 50-kDa HdNIR is composed of the 15-kDa N-terminal domain having a type 1 Cu-binding motif like cupredoxins and the 35-kDa C-terminal domain having type 1 Cu-binding and type 2 Cu-binding motifs such as common nitrite reductases (NIRs) . Moreover, the amino acid sequences of the N- and C-terminal domains are homologous to those of plastocyanins and NIRs, respectively . The point mutation of the Cys ligand of each type 1 Cu with Ala gives two mutants, C114A and C260A, possessing one type 1 Cu and one type 2 Cu . The spectroscopic data of C114A reveal that the C-terminal NIR-like domain has the green type 1 Cu (type 1 Cu(C)), showing two intense absorption peaks at 455 (epsilon = 2600 M(-1) cm(-1)) and 600 nm (epsilon = 2800 M(-1) cm(-1)) and a rhombic EPR signal like those of the green type 1 Cu of Achromobacter cycloclastes NIR (AcNlR) . The spectroscopic data of C260A elucidate that the N-terminal Pc-like domain in HdNIR contains the blue type 1 Cu (type 1 Cu(N)), exhibiting an intense absorption band at 605 nm (epsilon = 2900 M(-1) cm(-1)) and an axial EPR signal like those of the blue type 1 Cu of Alcaligenes xylosoxidans NIR (AxNIR) . The sum of the visible absorption or EPR spectra of C114A and C260A is almost equal to the corresponding spectrum of wild-type HdNIR . The spectroscopic characterization of the type 1 Cu indicates that the geometries of the type 1 Cu(N) and Cu(C) sites are slightly distorted tetrahedral (or axially elongated bipyramidal) and flattened tetrahedral, respectively . In the cyclic voltammograms, the midpoint potentials (E(1/2)), probably because of the type 1 Cu ions of C114A and C260A, are observed at +321 and +336 mV versus normal hydrogen electrode (NHE) at pH 7.0, respectively . These values, which are close to each other, are more positive than those ( approximately +0.24-0.28 V at pH 7.0) of the type 1 Cu sites of AcNIR and AxNIR . The electron-accepting capability of C114A from cytochrome c(550) is almost similar to that of wild-type HdNIR, whereas that of C260A is very low . This suggests that the type 1 Cu(C) in the C-terminal domain is essential for the enzyme functions of HdNIR. Biochemistry, 2004 Nov 9, 43(44), 14118 - 27 NO binding and dynamics in reduced heme-copper oxidases aa3 from Paracoccus denitrificans and ba3 from Thermus thermophilus; Pilet E et al.; Cytochrome c oxidase (CcO) has a high affinity for nitric oxide (NO), a property involved in the regulation of respiration . It has been shown that the recombination kinetics of photolyzed NO with reduced CcO from Paracoccus denitrificans on the picosecond time scale depend strongly on the NO/enzyme stoichiometry and inferred that more than one NO can be accommodated by the active site, already at mildly suprastoichiometric NO concentrations . We have largely extended these studies by monitoring rebinding dynamics from the picosecond to the microsecond time scale, by performing parallel steady-state low-temperature electron paramagnetic resonance (EPR) characterizations on samples prepared similarly as for the optical experiments and comparing them with molecular-modeling results . A comparative study was performed on CcO ba(3) from Thermus thermophilus, where two NO molecules cannot be copresent in the active site in the steady state because of its NO reductase activity . The kinetic results allow discrimination between different models of NO-dependent recombination and show that the overall NO escape probability out of the protein is high when only one NO is bound to CcO aa(3), whereas strong rebinding on the 15-ns time scale was observed for CcO ba(3) . The EPR characterizations show similar results for aa(3) at substoichiometric NO/enzyme ratios and for ba(3), indicating formation of a 6-coordinate heme-NO complex . The presence of a second NO molecule in the aa(3) active site strongly modifies the heme-NO EPR spectrum and can be rationalized by a rotation of the Fe-N-O plane with respect to the histidine that coordinates the heme iron . This proposal is supported by molecular-modeling studies that indicate a approximately 63 degrees rotation of heme-bound NO upon binding of a second NO to the close-lying copper center CuB . It is argued that the second NO binds to CuB. Huan Jing Ke Xue, 2004 Jul, 25(4), 54 - 8 {Effect of carbon source and nitrate concentration on denitrifying dephosphorus removal and variation of ORP}; Wang YY et al.; Effect of added carbon source and nitrate concentration on the denitrifying phosphorus removal by SBR process was systematicaly studied, at the same time the variation of oxidation reductiun potential (ORP) was investigated . The results showed the phosphate release rate and the denitrifying and dephosphorus uptake rate in anoxic phase increased with the high carbon source concentration under anaerobic condition (100-300mg/L) . However when the carbon source added in anaerobic phase was high to 300mg/L, the residual COD inhibited the succeed denitrifying dephosphorus uptake . High nitrate concentration (5, 15, 40mg/L) in anoxic phase increased the initial denitrifying dephosphorus rate . Once the nitrate depletes, phosphate uptake changed to phosphate release . Moreover, the time of the turning point occurred later with the higher nitrate addition . ORP can be used as a control parameter of phosphorus release, and it can also indicate the denitrificaiton react degree during the anoxic phosphorus removal but can't be used as control parameter of phosphorus uptake. Environ Technol, 2004 Sep, 25(9), 1041 - 9 Comparison of different carbon sources for ground water denitrification; Lorrain MJ et al.; This study presents a comparison of denitrification rates and denitrification stoichiometry when using different sources of carbon . Denitrification tests were carried out in test bottles containing water and soil samples acquired at a nitrate-contaminated site and supplemented with either sucrose, acetate or ethanol . The tests demonstrated nitrate removal in all carbon source supplemented bottles . The rate of denitrification and the required amount of a carbon source, however, depended on the choice of substrate . Ethanol and acetate were found to provide the highest denitrification rate, that of 1.4 - 1.6 mg-N l(-1) d(-1) . Sucrose-supplemented bottles demonstrated a significantly slower denitrification rate, that of 0.6 - 0.9 mg-N l(-1) d(-1) . In addition to slow denitrification rates, sucrose-supported denitrification required more carbon source. J Biol Chem, 2004 Dec 31, 279(53), 55247 - 54 Epub 2004 Oct 25. NO reduction by nitric-oxide reductase from denitrifying bacterium Pseudomonas aeruginosa: characterization of reaction intermediates that appear in the single turnover cycle; Kumita H et al.; Nitric-oxide reductase (NOR) of a denitrifying bacterium catalyzes NO reduction to N(2)O at the binuclear catalytic center consisting of high spin heme b(3) and non-heme Fe(B) . The structures of the reaction intermediates in the single turnover of the NO reduction by NOR from Pseudomonas aeruginosa were investigated using optical absorption and EPR spectroscopies combined with an originally designed freeze-quench device . In the EPR spectrum of the sample, in which the fully reduced NOR was mixed with an NO solution and quenched at 0.5 ms after the mixing, two characteristic signals for the ferrous Fe(B)-NO and the penta-coordinated ferrous heme b(3)-NO species were observed . The CO inhibition of its formation indicated that two NO molecules were simultaneously distributed into the two irons of the same binuclear center of the enzyme in this state . The time- and temperature-dependent EPR spectral changes indicated that the species that appeared at 0.5 ms is a transient reaction intermediate prior to the N(2)O formation, in good agreement with the so-called "trans" mechanism . It was also found that the final state of the enzyme in the single turnover cycle is the fully oxidized state, in which the mu-oxo-bridged ligand is absent between the two irons of its binuclear center, unlike the resting form of NOR as isolated . On the basis of these present findings, we propose a newly developed mechanism for the NO reduction reaction conducted by NOR. Arch Microbiol, 2004 Dec, 182(6), 514 - 9 Epub 2004 Dec. Degradation pathway of 2-chloroethanol in Pseudomonas stutzeri strain JJ under denitrifying conditions; Dijk JA et al.; The pathway of 2-chloroethanol degradation in the denitrifying Pseudomonas stutzeri strain JJ was investigated . In cell-free extracts, activities of a phenazine methosulfate (PMS)-dependent chloroethanol dehydrogenase, an NAD-dependent chloroacetaldehyde dehydrogenase, and a chloroacetate dehalogenase were detected . This suggested that the 2-chloroethanol degradation pathway in this denitrifying strain is the same as found in aerobic bacteria that degrade chloroethanol . Activity towards primary alcohols, secondary alcohols, diols, and other chlorinated alcohols could be measured in cell-free extracts with chloroethanol dehydrogenase (CE-DH) activity . PMS and phenazine ethosulfate (PES) were used as primary electron acceptors, but not NAD, NADP or ferricyanide . Cells of strain JJ cultured in a continuous culture under nitrate limitation exhibited chloroethanol dehydrogenase activity that was a 12 times higher than in cells grown in batch culture . However, under chloroethanol-limiting conditions, CE-DH activity was in the same range as in batch culture . Cells grown on ethanol did not exhibit CE-DH activity . Instead, NAD-dependent ethanol dehydrogenase (E-DH) activity and PMS-dependent E-DH activity were detected. Environ Geochem Health, 2004 Jun-Sep, 26(2-3), 245 - 51 Nitrogen and phosphorus cycling in shrimp ponds and the measures for sustainable management; Xia LZ et al.; Six ponds of age 3 were selected 45 km north from Suzhou in the Tailake region, and research conducted on nitrogen and phosphorus cycling in P . vannanmei (Penaeus vannanme) ponds and M . nipponense (Macrobrachium nipponense) hatchery ponds under normal management . Two treatments each had three replications . The results confirmed that feed was the major path of nitrogen and phosphorus input, each accounted for 61.24% (193.81 kg ha(-1)) and 81.08% (45.20 kg ha(-1)) of the total nitrogen and phosphorus input for P . vannanme ponds; the values for M . nipponense ponds were 43.93% (86.31 kg ha(-1)) and 57.67% (14.61 kg ha(-1)), respectively . Water pumped into ponds contributed on average 83.57 kg ha(-1) nitrogen and 8.48 kg ha(-1) phosphorus for P . vannanmei ponds, and 87.48 kg ha(-1) nitrogen and 7.00 kg ha(-1) phosphorus for M . nipponense hatchery ponds . Shrimp harvest recovered 102.81 kg ha(-1) nitrogen (32.94% of the total nitrogen input) and 7.94 kg ha(-1) phosphorus (14.23% of the total phosphorus input) for P . vannanme ponds; and 43.94 kg ha(-1) nitrogen and 4.46 kg ha(-1) phosphorus for M . nipponense hatchery ponds . The sum of nitrogen losses through volatilization, denitrification and sedimentation was 173.62 and 122.39 kg ha(-1), 54.86% and 62.29% of the total nitrogen input for P . vannanme ponds and M . nipponense hatchery ponds, respectively . Sediment accumulated 41.46 and 14.63 kg ha(-1) phosphorus, 74.37% and 64.85% of the total phosphorus input for P . vannanm ponds and M . nipponense hatchery ponds . Draining and seeping caused 40.06 kg ha(-1) nitrogen (12.66% of total nitrogen input) and 6.36 kg ha(-1) phosphorus (11.40% of total phosphorus input) loss to the surrounding water from P . vannanme ponds in 114 days; 30.14 kg ha(-1) nitrogen (15.34% of the total input) and 4.45 kg ha(-1) phosphorus (17.57% of the total input) to channel water from M . nipponense hatchery ponds in 87 days, respectively . Countermeasures for sustainable pond management include improving feeds and feeding, sediment treatments, machine aerating, chemicals with no pollution, and integrated fish-shrimp cultivation . Management of water resources for pond and methods to reduce nitrogen and phosphorus loading into surrounding water from drainage are elucidated. Environ Geochem Health, 2004 Jun-Sep, 26(2-3), 179 - 86 Atmospheric deposition, mineralization and leaching of nitrogen in subtropical forested catchments, South China; Chen XY et al.; In recent years, China has conducted considerable research focusing on the emission and effects of sulphur (S) on human health and ecosystems . By contrast, there has been little emphasis on anthropogenic nitrogen (N) so far, even though studies conducted abroad indicate that long-range atmospheric transport of N and ecological effects (e.g . acidification of soil and water) may be significant . The Sino-Norwegian project IMPACTS, launched in 1999, has established monitoring sites at five forest ecosystems in the southern part of PR China to collect comprehensive data on air quality, acidification status and ecological effects . Here we present initial results about N dynamics at two of the IMPACTS sites located near Chongqing and Changsha, including estimation of atmospheric deposition fluxes of NOx and NHx and soil N transformations . Nitrogen deposition is high at both sites when compared with values from Europe and North America (25-38 kg ha(-1) yr(-1)) . About 70% of the deposited N comes as NH4, probably derived from agriculture . Leaching of N from soils is high and nearly all as NO3- . Transformation of N to NO3- in soils results in acidification rates that are high compared to rates found elsewhere . Despite considerable leaching of NO3- from the root zone of the soils, little NO3- appears in streamwater . This indicates that N retention or denitrification, both causing acid neutralization, may be important and probably occur in the groundwater and groundwater discharge zones . The soil flux density of mineral N, which is the sum of N deposition and N mineralization, and which is dominated by the N mineralization flux, may be a good indicator for leaching of NO3- in soils . However, this indicator seems site specific probably due to differences in land-use history and current N requirement. Water Sci Technol, 2004, 50(5), 67 - 72 Combined biological and physico-chemical treatment of baker's yeast wastewater including removal of coloured and recalcitrant to biodegradation pollutants; Gladchenko M et al.; The UASB reactor (35 degrees C) was quite efficient for removal of bulk COD (62-67%) even for such high strength and recalcitrant wastewater as the cultivation medium from the first separation process of baker'syeasts (the average organic loading rates varied from 3.7 to 10.3 g COD/l/d) . The aerobic-anoxic biofilter (20 degrees C) can be used for removal of remaining BOD and ammonia from strong nitrogenous anaerobic effluents; however, it suffered from COD-deficiency to fulfil denitrification requirements . To balance the COD/N ratio, some bypass of raw wastewater should be added to the biofilter feed . The application of iron chloride coagulation for post-treatment of aerobic effluents may fulfil the discharge limits (even for colour mainly exerted by hardly biodegradable melanoidins) under iron concentrations around 200 mg/l. Water Sci Technol, 2004, 50(5), 51 - 8 Heavy metal pollution from Russian landfill leachates and its elimination together with other contaminants; Kalyuzhnyi S et al.; Systematic monitoring of raw leachates (RL) from the operating landfill "Khmet'yevo" during December, 2001--June, 2002 with regard to heavy metals (HM) revealed that these RL were moderately contaminated with Fe, Zn, Pb and Cd (Cu is present in non-dangerous concentrations) . This contamination depends on season--the winter leachates are less polluted compared to the summer ones . For removal of HM together with removal of bulk COD, the UASB reactors were applied where, besides elimination of the major part of organic matter, concomitant precipitation of HM in the form of insoluble sulphides inside the sludge bed occurred due to development of the process of biological sulphate reduction . Both removal processes were quite efficient even during operation under submesophilic and psychrophilic conditions (20-10 degrees C) . The subsequent submesophilic aerobic-anoxic treatment of submesophilic anaerobic effluents led to only 75% of total inorganic N removal due to COD deficiency for denitrification created by a too efficient anaerobic step . On the contrary, psychrophilic anaerobic effluents (richer in COD compared to the submesophilic ones) were more suitable for subsequent aerobic-anoxic treatment giving the total N removal of 95 and 92% at 20 and 10 degrees C, respectively . The final effluent is approaching the current national standards for direct discharge of treated wastewater. J Environ Sci (China), 2004, 16(4), 674 - 7 Experimental study on the simultaneous desulfurization and denitrification by duct injection; Zhao Y et al.; The highly active absorbent with oxidization based on fly ash, lime and additive was prepared . Experiments of simultaneous desulfurization and denitrification were carried out using fixture bed and duct injection . The influencial factors for the absorptive capacity of the absorbent were studied . The absorptive capacities of 120.7 mg for SO2 and 43.7 mg for NOx were achieved at a Ca/(S + N) molar ratio 1.2, respectively, corresponding removal efficiencies of 87% and 76%, while spent absorbent appeared in the form of dry powder . The optimal temperature and humidity of flue gas treated with this process were shown to be approximately 50 degrees C, and 5% respectively . The mechanism of removal for SO2 and NOx was investigated . In comparison with traditional dry FGD, this process appears to have lower cost, less complicated configuration and simpler disposal of used absorbent . The valuable references can be provided for industrial application by this process . The foreground of application will be vast in China and in the world. J Environ Sci (China), 2004, 16(4), 548 - 52 Effect of carbon source and nitrate concentration on denitrifying phosphorus removal by DPB sludge; Wang YY et al.; Effect of added carbon source and nitrate concentration on the denitrifying phosphorus removal by DPB sludge was systematically studied using batch experiments, at the same time the variation of ORP was investigated . Results showed that the denitrifying and phosphorus uptake rate in anoxic phase increased with the high initial anaerobic carbon source addition . However once the initial COD concentration reached a certain level, which was in excess to the PHB saturation of poly-P bacteria, residual COD carried over to anoxic phase inhibited the subsequent denitrifying phosphorus uptake . Simultaneously, phosphate uptake continued until all nitrate was removed, following a slow endogenous release of phosphate . High nitrate concentration in anoxic phase increased the initial denitrifying phosphorus rate . Once the nitrate was exhausted, phosphate uptake changed to release . Moreover, the time of this turning point occurred later with the higher nitrate addition . On the other hand, through on-line monitoring the variation of the ORP with different initial COD concentration, it was found ORP could be used as a control parameter for phosphorus release, but it is impossible to utilize ORP for controlling the denitrificaion and anoxic phosphorus uptake operations. Crit Rev Biotechnol, 2004, 24(2-3), 97 - 124 Phytoremediation--a novel and promising approach for environmental clean-up; Suresh B et al.; Phytoremediation is an eco friendly approach for remediation of contaminated soil and water using plants . Phytoremediation is comprised of two components, one by the root colonizing microbes and the other by plants themselves, which degrade the toxic compounds to further non-toxic metabolites . Various compounds, viz . organic compounds, xenobiotics, pesticides and heavy metals, are among the contaminants that can be effectively remediated by plants . Plant cell cultures, hairy roots and algae have been studied for their ability to degrade a number of contaminants . They exhibit various enzymatic activities for degradation of xenobiotics, viz . dehalogenation, denitrification leading to breakdown of complex compounds to simple and non-toxic products . Plants and algae also have the ability to hyper accumulate various heavy metals by the action of phytochelatins and metallothioneins forming complexes with heavy metals and translocate them into vacuoles . Molecular cloning and expression of heavy metal accumulator genes and xenobiotic degrading enzyme coding genes resulted in enhanced remediation rates, which will be helpful in making the process for large-scale application to remediate vast areas of contaminated soils . A few companies worldwide are also working on this aspect of bioremediation, mainly by transgenic plants to replace expensive physical or chemical remediation techniques . Selection and testing multiple hyperaccumulator plants, protein engineering ofphytochelatin and membrane transporter genes and their expression would enhance the rate of phytoremediation, making this process a successful one for bioremediation of environmental contamination . Recent years have seen major investments in the R&D, which have also resulted in competition of filing patents by several companies for economic gains . The details of science & technology related to phytoremediation have been discussed with a focus on future trends and prospects of global relevance. Water Res, 2004 Nov, 38(19), 4262 - 70 Biofilm and biomass characteristics in high-performance fluidized-bed biofilm reactors; Rabah FK et al.; Two laboratory-scale fluidized-bed biofilm reactors (FBBRs) were used to investigate the biomass concentration and the biofilm characteristics in a high-performance FBBR used for the denitrification of exceptionally high-nitrate wastewater (1000 mg N/L) . Reported correlations by other workers for predicting the biomass concentration in FBBR were examined for their validity in comparison with the experimental results of this study and the best set of applicable correlations was recommended . The effects of the two main operational parameters, the superficial velocity and nitrogen loading rate on the biomass concentration in the FBBR were also studied . Correlations for the drag coefficient and the expansion index from the literature, together with the biofilm dry density correlation produced from this study were found to produce the best prediction of the FBBR biomass concentration compared to other reported correlations . The average biomass concentration in the FBBR decreased with the increase of the superficial velocity in the range of 45-65 m/h at all applied nitrogen loadings (i.e . 6, 8, 12 and 16 kg N/m3bedd). Biochemistry, 2004 Oct 26, 43(42), 13487 - 95 NO reductase from Bacillus azotoformans is a bifunctional enzyme accepting electrons from menaquinol and a specific endogenous membrane-bound cytochrome c551; Suharti et al.; Bacillus azotoformans is a Gram-positive denitrifying soil bacterium, which is capable of respiring nitrate, nitrite, nitric oxide, and nitrous oxide under anaerobic conditions . It contains a unique menaquinol-dependent nitric oxide reductase (qCu(A)NOR) with a Cu(A) center in its small subunit . The qCu(A)NOR exhibits menaquinol-dependent NO reductase activity, whereas reduced horse heart cytochrome c was inactive . Here we describe the purification of three membrane-bound c cytochromes from B . azotoformans . Their apparent molecular masses on SDS-PAGE are approximately 11 kDa . At neutral pH, these c cytochromes are negatively charged and the E(m) for all is close to 150 mV . Only one of these c cytochromes, which exhibits an alpha-band maximum at 551 nm, acts as a direct electron donor to qCu(A)NOR . Further investigation demonstrated that this cytochrome c(551) possesses two lipoyl moieties, which presumably function to anchor it to the membrane . Steady-state kinetic studies reveal that cytochrome c(551) is a noncompetitive inhibitor of NO reduction when menaquinol is used as an electron donor . This finding points to the presence of two different electron donation pathways in qCu(A)NOR . The ability of qCu(A)NOR to accept electrons from both menaquinol and cytochrome c(551) might be related to the regulation of the rate of NO reduction especially as a defense mechanism of B . azotoformans against the toxicity of NO . Growth experiments in batch culture indeed show that B . azotoformans is highly NO tolerant, in contrast to, for example, Paracoccus denitrificans that has a monofunctional cytochrome c-dependent NOR . We propose that the menaquinol pathway, which has a 4-fold greater maximal activity than the pathway via cytochrome c(551), is used for NO detoxification, whereas electron donation via the endogenous cytochrome c involves the cytochrome b(6)f complex serving the bioenergetic needs of the organism. J Bacteriol, 2004 Nov, 186(21), 7312 - 26 Characterization of nutrient-induced dispersion in Pseudomonas aeruginosa PAO1 biofilm; Sauer K et al.; The processes associated with early events in biofilm formation have become a major research focus over the past several years . Events associated with dispersion of cells from late stage biofilms have, however, received little attention . We demonstrate here that dispersal of Pseudomonas aeruginosa PAO1 from biofilms is inducible by a sudden increase in carbon substrate availability . Most efficient at inducing dispersal were sudden increases in availability of succinate > glutamate > glucose that led to approximately 80% reductions in surface-associated biofilm biomass . Nutrient-induced biofilm dispersion was associated with increased expression of flagella (fliC) and correspondingly decreased expression of pilus (pilA) genes in dispersed cells . Changes in gene expression associated with dispersion of P . aeruginosa biofilms were studied by using DNA microarray technology . Results corroborated proteomic data that showed gene expression to be markedly different between biofilms and newly dispersed cells . Gene families that were upregulated in dispersed cells included those for flagellar and ribosomal proteins, kinases, and phage PF1 . Within the biofilm, genes encoding a number of denitrification pathways and pilus biosynthesis were also upregulated . Interestingly, nutrient-induced dispersion was associated with an increase in the number of Ser/Thr-phosphorylated proteins within the newly dispersed cells, and inhibition of dephosphorylation reduced the extent of nutrient-induced dispersion . This study is the first to demonstrate that dispersal of P . aeruginosa from biofilms can be induced by the addition of simple carbon sources . This study is also the first to demonstrate that dispersal of P . aeruginosa correlates with a specific dispersal phenotype. J Bacteriol, 2004 Nov, 186(21), 7161 - 74 The completely sequenced plasmid pEST4011 contains a novel IncP1 backbone and a catabolic transposon harboring tfd genes for 2,4-dichlorophenoxyacetic acid degradation; Vedler E et al.; The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterium Achromobacter xylosoxidans subsp . denitrificans strain EST4002 contains plasmid pEST4011 . This plasmid ensures its host a stable 2,4-D(+) phenotype . We determined the complete 76,958-bp nucleotide sequence of pEST4011 . This plasmid is a deletion and duplication derivative of pD2M4, the 95-kb highly unstable laboratory ancestor of pEST4011, and was self-generated during different laboratory manipulations performed to increase the stability of the 2,4-D(+) phenotype of the original strain, strain D2M4(pD2M4) . The 47,935-bp catabolic region of pEST4011 forms a transposon-like structure with identical copies of the hybrid insertion element IS1071::IS1471 at the two ends . The catabolic regions of pEST4011 and pJP4, the best-studied 2,4-D-degradative plasmid, both contain homologous, tfd-like genes for complete 2,4-D degradation, but they have little sequence similarity other than that . The backbone genes of pEST4011 are most similar to the corresponding genes of broad-host-range self-transmissible IncP1 plasmids . The backbones of the other three IncP1 catabolic plasmids that have been sequenced (the 2,4-D-degradative plasmid pJP4, the haloacetate-catabolic plasmid pUO1, and the atrazine-catabolic plasmid pADP-1) are nearly identical to the backbone of R751, the archetype plasmid of the IncP1 beta subgroup . We show that despite the overall similarity in plasmid organization, the pEST4011 backbone is sufficiently different (51 to 86% amino acid sequence identity between individual backbone genes) from the backbones of members of the three IncP1 subgroups (the alpha, beta, and gamma subgroups) that it belongs to a new IncP1subgroup, the delta subgroup . This conclusion was also supported by a phylogenetic analysis of the trfA2, korA, and traG gene products of different IncP1 plasmids. J Microbiol Methods, 2004 Dec, 59(3), 327 - 35 Quantification of denitrifying bacteria in soils by nirK gene targeted real-time PCR; Henry S et al.; Denitrification, the reduction of nitrate to nitrous oxide or dinitrogen, is the major biological mechanism by which fixed nitrogen returns to the atmosphere from soil and water . Microorganisms capable of denitrification are widely distributed in the environment but little is known about their abundance since quantification is performed using fastidious and time-consuming MPN-based approaches . We used real-time PCR to quantify the denitrifying nitrite reductase gene (nirK), a key enzyme of the denitrifying pathway catalyzing the reduction of soluble nitrogen oxide to gaseous form . The real-time PCR assay was linear over 7 orders of magnitude and sensitive down to 10(2) copies by assay . Real-time PCR analysis of different soil samples showed nirK densities of 9.7x10(4) to 3.9x10(6) copies per gram of soil . Soil real-time PCR products were cloned and sequenced . Analysis of 56 clone sequences revealed that all cloned real-time PCR products exhibited high similarities to previously described nirK . However, phylogenetic analysis showed that most of environmental sequences are not related to nirK from cultivated denitrifiers. J Mol Evol, 2004 Aug, 59(2), 158 - 76 Phylogenetic analysis of proteins associated in the four major energy metabolism systems: photosynthesis, aerobic respiration, denitrification, and sulfur respiration; Tomiki T et al.; The four electron transfer energy metabolism systems, photosynthesis, aerobic respiration, denitrification, and sulfur respiration, are thought to be evolutionarily related because of the similarity of electron transfer patterns and the existence of some homologous proteins . How these systems have evolved is elusive . We therefore conducted a comprehensive homology search using PSI-BLAST, and phylogenetic analyses were conducted for the three homologous groups (groups 1-3) based on multiple alignments of domains defined in the Pfam database . There are five electron transfer types important for catalytic reaction in group 1, and many proteins bind molybdenum . Deletions of two domains led to loss of the function of binding molybdenum and ferredoxin, and these deletions seem to be critical for the electron transfer pattern changes in group 1 . Two types of electron transfer were found in group 2, and all its member proteins bind siroheme and ferredoxin . Insertion of the pyridine nucleotide disulfide oxidoreductase domain seemed to be the critical point for the electron transfer pattern change in this group . The proteins belonging to group 3 are all flavin enzymes, and they bind flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) . Types of electron transfer in this group are divergent, but there are two common characteristics . NAD(P)H works as an electron donor or acceptor, and FAD or FMN transfers electrons from/to NAD(P)H . Electron transfer functions might be added to these common characteristics by the addition of functional domains through the evolution of group 3 proteins . Based on the phylogenetic analyses in this study and previous studies, we inferred the phylogeny of the energy metabolism systems as follows: photosynthesis (and possibly aerobic respiration) and the sulfur/nitrogen assimilation system first diverged, then the sulfur/nitrogen dissimilation system was produced from the latter system. Water Sci Technol, 2004, 50(4), 341 - 9 Anoxic control of odour and corrosion from sewer networks; Yang W et al.; Anoxic processes can effectively control odour and corrosion in sewer networks . However, the absence of fundamental knowledge on the kinetics of anoxic transformation of sewage prevents the engineering applications of anoxic control in sewers . This paper focuss on a basic understanding of the anoxic transformations needed for a conceptual simulation of the water phase processes . Experiments conducted in batch reactors have shown that nitrite builds up in wastewater during denitrification . Part of the nitrate-reducing biomass is capable of utilizing nitrite after nitrate is depleted . Compared with aerobic transformation, anoxic processes have low values of maximum growth rate of the biomass and also a low endogenous respiration rate . Heterotrophic yield determined under anoxic conditions, at level of 0.25 mmol e-eq (mmol e-eq)(-1), accounted for less than 40% of the corresponding aerobic values. Bioresour Technol, 2005 Feb, 96(3), 351 - 8 Piggery wastewater characterisation for biological nitrogen removal process design; Boursier H et al.; In intensive farming areas, the design of biological nitrogen removal plants for piggery wastewater requires the determination of the chemical oxygen demand (COD) fractions of the effluent . For this purpose, an experimental procedure was developed to quantify the inert soluble (SI) and particulate (XI) COD fractions, as well as the readily (SS) and the slowly (XS) biodegradable COD fractions . For the four wastewaters tested, the SI and the XI fractions were equal to 3-4 g O(2)l(-1) and 17-28 g O(2)l(-1), respectively, which resulted in a total inert fraction of 42-84% of total COD . The SS and the XS fractions were very variable, r