Denitrifying

Water Sci Technol, 2003, 48(8), 135 - 41
The microbial community analysis of a 5-stage BNR process with step feed system; Lee HW et al.; The microbial communities of 5-stage BNR activated sludge samples were analyzed using fluorescence in-situ hybridization (FISH) and 16S rDNA characterization . The total cell numbers of each reactor were from 2.36 x 10(9) cells/ml to 2.83 x 10(9) cells/ml . From 56.5% to 62.0% of total DAPI cell counts were hybridized to the most bacterial specific probe EUB 338 . Among them, beta-proteobacteria were most dominant in each tank . The number of phosphate accumulating organisms (PAOs) was almost 50% of the total cell number in anoxic-1 tank, and these results indicate that this process has a high content of denitrifying phosphorus accumulating organisms (dPAOs) . In contrast with FISH, 16S rDNA analysis showed that dominant groups were the Cytophaga-Flavobacterium group and high G+C% gram-positive bacteria, which were determined as PAOs in anoxic-1 tank . The beta subclass Proteobacteria did not accumulate a large amount of polyphosphate . The overall results indicate that high G+C% gram-positive bacteria and the Cytophaga-Flavobacterium group might play a key role as dPAOs in this process.

Water Sci Technol, 2003, 48(8), 9 - 18
Nitrogen removal from digester supernatant via nitrite--SBR or SHARON?
Fux C, Lange K, Faessler A, Huber P, Grueniger B, Siegrist H.
Separate biological elimination of nitrogen from the digester supernatant of a municipal wastewater treatment plant (WWTP) was investigated in pilot and full-scale plants . Denitrification mainly via nitrite was achieved in a sequencing batch reactor (SBR) and a continuous flow reactor (CSTR or SHARON) . Suppression of nitrite oxidation in the SBR was feasible at short aerobic/anaerobic intervals allowing for immediate denitrification of the produced nitrite . Nitrate production could also be stopped by exposing the biomass to anaerobic conditions for 11 days . Temporarily high concentrations (up to 80 gNH3-Nm(-3)) of free ammonia could not be considered as the major reason for inhibiting nitrite oxidation . In a full-scale SBR plant 90% of the nitrogen load was denitrified in a total hydraulic retention time (HRT) of 1.6 days and with a sludge age between 15 and 20 days . Ethanol and methanol were used for denitrification . The specific average substrate consumption was 2.2 gCOD(dosed) g(-1)N(removed) with an effective biomass yield of 0.2 gCOD(biomass) g(-1)COD(dosed) . No dosing with base was required . In the SHARON process full nitrogen elimination was achieved only with a total HRT greater than 4 days at 29 degrees C . The overall costs were estimated at 1.4 euros kg(-1)N(removed) for the SBR and 1.63 euros kg(-1)N(removed) in SHARON mode, respectively . The SHARON process is simple in operation (CSTR) but the tank volume has to be significantly greater than in SBR.

FEMS Microbiol Lett, 2003 Dec 12, 229(2), 173 - 8
Site-directed mutagenesis of NnrR: a transcriptional regulator of nitrite and nitric oxide reductase in Rhodobacter sphaeroides; Laratta WP et al.; NnrR, a transcriptional activator and member of the CRP/FNR family of regulators, is responsible for controlling the expression of a number of denitrification genes in Rhodobacter sphaeroides 2.4.3 . The apparent effector for NnrR is nitric oxide, and in its presence NnrR activates expression of the nirK gene and the nor operon, encoding nitrite reductase and nitric oxide reductase, respectively . Whether nitric oxide directly interacts with NnrR to activate transcription is unknown . Other denitrifiers carry putative orthologs of NnrR . To gain insight into NnrR function, a number of conserved residues were mutagenized . The impact of these changes on NnrR function was assessed by monitoring expression of a nirK-lacZ fusion . In this way a region spanning from Tyr93 to Cys103 that contains residues critical for NnrR activity was identified.

Mikrobiologiia, 2003 Sep-Oct, 72(5), 699 - 706
{Distribution and physiology of microorganisms in petrochemical oily sludge of plant}; Nikitina EV et al.; The occurrence, vertical distribution, and the physiological state of microorganisms in a petrochemical oily sludge deposit were studied . The total number and the number of viable microbial cells at depths of 0.2 and 3 m were about 10 and 10(8) cells/g dry wt . sludge . Most microbial cells taken from the middle (1 m deep) and the bottom (3 m deep) sludge horizons showed a delayed colony-forming ability, which suggested that the cells occurred in a hypometabolic state . The relative number of microaerobic denitrifying microorganisms steeply increased with depth . The amount of microorganisms tolerant to 3, 5, and 10% NaCl and capable of growing at 7 and 40 degrees C varied from 10(2) to 10(8) CFU/g dry wt . sludge . Petrochemical oily sludge was found to maintain the growth of heterotrophs, among which the degraders of oily sludge and ten different individual polycyclic aromatic hydrocarbons were detected . The occurrence of highly adaptable microorganisms with an adequate metabolic potential in the petrochemical oily sludge deposit implies that its bioremediation is possible without introducing special microorganisms.

Biochemistry, 2003 Dec 23, 42(50), 14856 - 61
Time-resolved resonance Raman and time-resolved step-scan FTIR studies of nitric oxide reductase from Paracoccus denitrificans: comparison of the heme b3-FeB site to that of the heme-CuB in oxidases; Pinakoulaki E et al.; Time-resolved resonance Raman (TR(3)) and time-resolved step-scan (TRS(2)) FTIR spectroscopies have been used to probe the structural dynamics at the heme b(3) proximal and distal sites after carbon monoxide photolysis from fully reduced CO-bound nitric oxide reductase . The Raman spectra of the transient species exhibit structural differences relative to the equilibrium geometry of heme b(3) . The most significant of these is a shift of 8 cm(-1) to higher frequency of the 207 cm(-1) mode, and a shift of 7 cm(-1) to lower frequency of the nu(4) mode . Our results indicate that the 207 cm(-1) mode observed in the equilibrium-reduced heme b(3) originates from nu(Fe-His) . Its behavior in the photolytic transients indicates that the relaxed Fe-His state is not significantly populated . We suggest that relaxation along the tilt angle (theta) of the proximal histidine with respect to the heme plane and the out-of-plane displacement of the Fe (q) are coupled, and ligand binding and dissociation are accompanied by significant changes in the angular orientation of the His ligand . The results are compared to those obtained for the aa(3)-cytochrome c oxidase from Paracoccus denitrificans . The results are compared to those obtained for the aa(3)-cytochrome c oxidase from P . denitrificans . The TR(3) and TRS(2) FTIR data demonstrate significant alterations in the nature of the heme-protein dynamics between nitric oxide reductase and heme-copper oxidases resulting from specific structural differences in their respective hemepockets.

J Environ Sci Health A Tox Hazard Subst Environ Eng, 2003, 38(12), 2955 - 66
Simultaneous removal of volatile organic compounds (VOCs) and nitrogen: batch test; Jang A et al.; A batch test was conducted to investigate the effect of the chemical oxygen demand, (COD)/N ratio, nitrate, nitrite and temperature, on the microbial degradation of volatile organic compounds (VOCs), and the denitrification capability using VOCs-acclimatized and un-acclimatized cultures . The nitrite reduction rates differed with each reactor, as follows: 15.418 NO2-N mg MLVSS g(-1) h(-1) in the benzene and methanol (BM) reactor, 27.463 NO2-N mg MLVSS g(-1) h(-1) in the toluene and methanol (TM) reactor and 44.358 NO2-N mg MLVSS g(-1) h(-1) in the methanol (M) reactor . According to the COD/N ratio, the nitrate reduction rates of the BM and TM reactors acclimatized by VOCs changed in the ranges of 29.4-33.41 NO3-N mg MLVSS g(-1) day(-1) and 56.4-65.9 NO3-N mg MLVSS g(-1) day(-1), respectively . Thus, benzene was not effectively utilized as a carbon source . Conversely, toluene was utilized as a carbon source by the denitrifiers under substrate limited conditions . The specific denitrification rates were also greater in the TM reactor than those for both the substrate limited and unlimited conditions in the BM reactor.

Biochim Biophys Acta, 2003 Dec 8, 1607(2-3), 79 - 90
The mitochondrial and prokaryotic proton-translocating NADH:ubiquinone oxidoreductases: similarities and dissimilarities of the quinone-junction sites; Grivennikova VG et al.; The catalytic properties of the rotenone-sensitive NADH:ubiquinone reductase (Complex I) in bovine heart submitochondrial particles and in inside-out vesicles derived from Paracoccus denitrificans and Rhodobacter capsulatus were compared . The prokaryotic enzymes catalyze the NADH oxidase and NADH:quinone reductase reactions with similar kinetic parameters as those for the mammalian Complex I, except for lower apparent affinities for the substrates--nucleotides . Unidirectional competitive inhibition of NADH oxidation by ADP-ribose, previously discovered for submitochondrial particles, was also evident for tightly coupled P . denitrificans vesicles, thus suggesting that a second, NAD(+)-specific site is present in the simpler prokaryotic enzyme . The inhibitor sensitivity of the forward and reverse electron transfer reactions was compared . In P . denitrificans and Bos taurus vesicles different sensitivities to rotenone and Triton X-100 for the forward and reverse electron transfer reactions were found . In bovine heart preparations, both reactions showed the same sensitivity to piericidin, and the inhibition was titrated as a straight line . In P . denitrificans, the forward and reverse reactions show different sensitivity to piericidin and the titrations of both activities were curvilinear with apparent I(50) (expressed as mole of inhibitor per mole of enzyme) independent of the enzyme concentration . This behavior is explained by a model involving two different sites rapidly interacting with piericidin within the hydrophobic phase.

Acta Biotheor, 2003, 51(4), 295 - 315
Relations between bacterial biomass and carbon cycle in marine sediments: an early diagenetic model; Talin F et al.; A new model for early diagenetic processes has been developed through a new formula explicitly accounting for microbial population dynamics . Following a mechanistic approach based on enzymatic reactions, a new model has been proposed for oxic mineralisation and denitrification . It incorporates the dynamics of bacterial metabolism . We find a general formula for inhibition processes of which some other mathematical expressions are particular cases . Moreover a fast numerical algorithm has been developed . It allows us to perform simulations of different diagenetic models in non-steady states . We use this algorithm to compare our model to a classical one (Soetaert et al., 1996) . Dynamical evolutions of a perturbation of particulate organic carbon (POC) input are studied for both models . The results are very similar for stationary cases . But with variable inputs, the bacterial biomass dynamics brings about noticeable differences, and these are discussed.

Microbiology, 2003 Dec, 149(Pt 12), 3405 - 12
Fine-tuned regulation by oxygen and nitric oxide of the activity of a semi-synthetic FNR-dependent promoter and expression of denitrification enzymes in Paracoccus denitrificans; Mazoch J et al.; In Paracoccus denitrificans at least three fumarate and nitrate reductase regulator (FNR)-like proteins {FnrP, nitrite and nitric oxide reductases regulator (NNR) and NarR} control the expression of several genes necessary for denitrifying growth . To gain more insight into this regulation, beta-galactosidase activity from a plasmid carrying the lacZ gene fused to the Escherichia coli melR promoter with the consensus FNR-binding (FF) site was examined . Strains defective in the fnrP gene produced only very low levels of beta-galactosidase, indicating that FnrP is the principal activator of the FF promoter . Anoxic beta-galactosidase levels were much higher relative to those under oxic growth and were strongly dependent on the nitrogen electron acceptor used, maximal activity being promoted by N(2)O . Additions of nitrate or nitroprusside lowered beta-galactosidase expression resulting from an oxic to micro-oxic switch . These results suggest that the activity of FnrP is influenced not only by oxygen, but also by other factors, most notably by NO concentration . Observations of nitric oxide reductase (NOR) activity in a nitrite-reductase-deficient strain and in cells treated with haemoglobin provided evidence for dual regulation of the synthesis of this enzyme, partly independent of NO . Both regulatory modes were operative in the FnrP-deficient strain, but not in the NNR-deficient strain, suggesting involvement of the NNR protein . This conclusion was further substantiated by comparing the respective NOR promoter activities.

Appl Environ Microbiol, 2003 Dec, 69(12), 7002 - 8
Identification and distribution of insertion sequences of Paracoccus solventivorans; Bartosik D et al.; Three novel insertion sequences (ISs) (ISPso1, ISPso2, and ISPso3) of the soil bacterium Paracoccus solventivorans DSM 11592 were identified by transposition into entrapment vector pMEC1 . ISPso1 (1,400 bp) carries one large open reading frame (ORF) encoding a putative basic protein (with a DDE motif conserved among transposases {Tnps} of elements belonging to the IS256 family) with the highest levels of similarity with the hypothetical Tnps of Rhodospirillum rubrum and Sphingopyxis macrogoltabida . ISPso2 (832 bp) appeared to be closely related to ISPpa2 of Paracoccus pantotrophus DSM 11072 and IS1248 of Paracoccus denitrificans PdX22, both of which belong to the IS427 group (IS5 family) . These elements contain two overlapping ORFs and a putative frameshift motif (AAAAG) responsible for production of a putative transframe Tnp . ISPso3 (1,286 bp) contains a single ORF, whose putative product showed homology with Tnps of ISs classified as members of a distinct subgroup of the IS5 group of the IS5 family . The highest levels of similarity were observed with ISSsp126 of Sphingomonas sp . and IS1169 of Bacteroides fragilis . Analysis of the distribution of ISs of P . solventivorans revealed that ISPso2-like elements are the most widely spread of the elements in nine species of the genus PARACOCCUS: ISPso1 and ISPso3 are present in only a few paracoccal strains, which suggests that they were acquired by lateral transfer . Phylogenetic analysis of Tnps of the novel ISs and their closest relatives showed their evolutionary relationships and possible directions of lateral transfer between various bacterial hosts.

Int J Syst Evol Microbiol, 2003 Nov, 53(Pt 6), 1961 - 6
Thermomonas fusca sp . nov . and Thermomonas brevis sp . nov., two mesophilic species isolated from a denitrification reactor with poly(epsilon-caprolactone) plastic granules as fixed bed, and emended description of the genus Thermomonas; Mergaert J et al.; Previously, 22 aerobic Gram-negative bacteria were isolated from biofilms growing on granules of the synthetic polyester poly(epsilon-caprolactone); the granules were used as a fixed bed in a denitrification reactor . All the strains showed similar fatty acid profiles . The 16S rRNA gene sequences of five strains were phylogenetically related to Thermomonas spp . Repetitive extragenic palindromic DNA-PCR (REP-PCR) fingerprinting revealed four groups, and DNA hybridizations between representative strains showed that the strains belonged to two new species within the genus Thermomonas, for which the names Thermomonas fusca (type strain LMG 21737(T)=DSM 15424(T)) and Thermomonas brevis (type strain LMG 21746(T)=DSM 15422(T)) are proposed . Both species are able to grow at low temperatures, but not at 50 degrees C, and are non-haemolytic . Both species can be differentiated by several other phenotypic features from earlier described species of the genus Thermomonas . Cell extracts contain mainly branched fatty acids, with C(15 : 0) iso, C(17 : 1) iso omega9c, C(11 : 0) iso 3OH and C(11 : 0) iso as main constituents . The G+C content of the DNA of the novel species is between 67.6 and 68.7 mol%.

Int J Syst Evol Microbiol, 2003 Nov, 53(Pt 6), 1779 - 83
Thialkalivibrio nitratireducens sp . nov., a nitrate-reducing member of an autotrophic denitrifying consortium from a soda lake; Sorokin DY et al.; Strain ALEN 2(T) was isolated from a mixed culture capable of complete autotrophic denitrification with thiosulfate as electron donor at pH 10; the mixed culture was enriched from sediment from Lake Fazda (Wadi Natrun, Egypt), a hypersaline alkaline lake . The isolate had large, non-motile, coccoid or barrel-shaped cells with intracellular sulfur globules . The bacterium was obligately chemolithoautotrophic . It grew with reduced sulfur compounds aerobically and anaerobically with nitrate as electron acceptor, nitrate being reduced to nitrite . It was moderately halophilic and obligately alkaliphilic . On the basis of genetic analysis and its unique phenotype, strain ALEN 2(T) (=DSM 14787(T)=UNIQEM 213(T)) is proposed as the type strain of a novel species of the genus Thialkalivibrio, Thialkalivibrio nitratireducens.

Anal Biochem, 2004 Jan 1, 324(1), 45 - 51
Lucigenin and coelenterazine as superoxide probes in mitochondrial and bacterial membranes; Kervinen M et al.; The chemiluminescent superoxide indicators lucigenin and coelenterazine were compared in rat liver submitochondrial particles and cytoplasmic membranes from Paracoccus denitrificans . Qualitative monitoring is possible with both probes, but quantitative work with lucigenin is hampered by its dependence on one-electron reduction before the photon-emitting reaction . Therefore, calibration of measurements on complex I, capable of efficient lucigenin prereduction with reduced nicotinamide adenine dinucleotide, against xanthine oxidase, which in the presence of hypoxanthine is not able to reduce the probe to a significant rate compared to complex I, may give results in error by one order of magnitude . Coelenterazine, although susceptible of storage-dependent high background chemiluminescence, does not require prereduction and is thus a more reliable probe.

Rapid Commun Mass Spectrom, 2003, 17(23), 2597 - 604
Quantifying nitrate retention processes in a riparian buffer zone using the natural abundance of 15N in NO3-; Dhondt K et al.; Quantifying the relative importance of denitrification and plant uptake to groundwater nitrate retention in riparian zones may lead to methods optimising the construction of riparian zones for water pollution control . The natural abundance of 15N in NO3- has been shown to be an interesting tool for providing insights into the NO3- retention processes occurring in riparian zones . In this study, 15N isotope fractionation (variation in delta15N of the residual NO3-) due to denitrification and due to plant uptake was measured in anaerobic soil slurries at different temperatures (5, 10 and 15 degrees C) and in hydroponic systems with different plant species (Lolium perenne L., Urtica dioica L . and Epilobium hirsutum L.) . It was found that temperature had no significant effect on isotope fractionation during denitrification, which resulted in a 15N enrichment factor epsilonD of -22.5 +/- 0.6 per thousand . On the other hand, nitrate uptake by plants resulted in 15N isotope fractionation, but was independent of plant species, leading to a 15N enrichment factor epsilonP of -4.4 +/- 0.3 per thousand . By relating these two laboratory-defined enrichment factors to a field enrichment factor for groundwater nitrate retention during the growing season (epsilonR = -15.5 +/- 1.0 per thousand ), the contribution of denitrification and plant uptake to groundwater nitrate retention could be calculated . The relative importance of denitrification and plant uptake to groundwater nitrate retention in the riparian buffer zone was 49 and 51% during spring, 53 and 47% during summer, and 75 and 25% during autumn . During wintertime, high micropore dissolved organic carbon (DOC) concentrations and low redox potentials due to decomposition of the highly productive riparian vegetation probably resulted in a higher denitrification rate and favoured other nitrate retention processes such as nitrate immobilisation or dissimilatory nitrate reduction to ammonium (DNRA) . This could have biased the 15N isotope fractionation and led to a low 15N enrichment factor for groundwater nitrate retention during wintertime (-6.2 +/- 0.9 per thousand ) . In contradiction to what many other studies suggest, it is possible that due to plant decomposition during the winter period other nitrate transformation processes compete with denitrification .

Environ Microbiol, 2003 Nov, 5(11), 1192 - 202
Cell density related H2 consumption in relation to anoxic Fe(0) corrosion and precipitation of corrosion products by Shewanella oneidensis MR-1; De Windt W et al.; In the absence of oxygen, a protective H2 film is formed around an Fe(0) surface, inhibiting the electron flow from this surface . Our study of anoxic corrosion of Fe(0) beads revealed that, in the presence of Shewanella oneidensis MR-1, H2 removal and precipitation of Fe mineral particles on the cell surface are determining processes for corrosion . These two biologically mediated processes were governed by cell density . H2 removal by Shewanella oneidensis was detected at cell concentrations of 1.0 x 10(6) live cells ml-1 and higher and H2 was electron donor for denitrification of NO3- . The removal of the protective H2 layer from Fe(0) beads by Shewanella oneidensis, resulted in an increase of Fe release out of the Fe(0) beads from 153 +/- 25 mg l(-1) to 196 +/- 7 mg l-1 after 20 h . When the cell concentration exceeded 1.0 x 10(8) live cells ml-1, precipitation of iron minerals on the cell surface was characteristic for the greatest percentage of MR-1 cells, whereas micrometre-scale iron precipitates not associated with culturable cell biomass significantly decreased in number . Addition of supernatant of a corrosion assay with high cell concentration induced metabolic activity in a corrosion assay with low cell concentration, resulting in increased H2 consumption and Fe release from Fe(0) beads . Homoserine lactone-like molecules were detected in the supernatant by a bio-assay, suggesting the involvement of a quorum-sensing regulatory mechanism.

Acta Astronaut, 2003 Aug-Nov, 53(4-10), 249 - 57
Synthesis of biomass and utilization of plants wastes in a physical model of biological life-support system; Tikhomirov AA et al.; The paper considers problems of biosynthesis of higher plants' biomass and "biological incineration" of plant wastes in a working physical model of biological LSS . The plant wastes are "biologically incinerated" in a special heterotrophic block involving Californian worms, mushrooms and straw . The block processes plant wastes (straw, haulms) to produce soil-like substrate (SLS) on which plants (wheat, radish) are grown . Gas exchange in such a system consists of respiratory gas exchange of SLS and photosynthesis and respiration of plants . Specifics of gas exchange dynamics of high plants--SLS complex has been considered . Relationship between such a gas exchange and PAR irradiance and age of plants has been established . Nitrogen and iron were found to the first to limit plants' growth on SLS when process conditions are deranged . The SLS microflora has been found to have different kinds of ammonifying and denitrifying bacteria which is indicative of intensive transformation of nitrogen-containing compounds . The number of physiological groups of microorganisms in SLS was, on the whole, steady . As a result, organic substances--products of exchange of plants and microorganisms were not accumulated in the medium, but mineralized and assimilated by the biocenosis . Experiments showed that the developed model of a man-made ecosystem realized complete utilization of plant wastes and involved them into the intrasystem turnover . c2003 International Astronautical Federation . Published by Elsevier Science Ltd . All rights reserved.

J Exp Bot, 2004 Jan, 55(394), 11 - 25 Epub 2003 Nov 28.
Global aspects of C/N interactions determining plant-environment interactions; Raven JA et al.; The atomic C:N ratio in photolithotrophs is a function of their content of nucleic acids, proteins, lipids, polysaccharides, and other organic materials, and varies from about 5 in some protein-rich microalgae to much higher values in macroalgae and in higher plants with relatively more structural and energy storage materials . These differences in C:N ratios among organisms means that there is more N assimilation by photosynthetic organisms in the oceans than on land despite the near equality of global photosynthetic C assimilation rates in the two environments . Aquatic organisms obtain inorganic C and inorganic N from the surrounding water . Terrestrial photolithotrophs obtain inorganic C, dinitrogen (by diazotrophy) and some combined N from the atmosphere, with the remaining combined N coming from the soil . The nitrogen cost of growth (biomass production rate per unit plant N) varies with the C:N ratio and specific growth rate of the organism . The C:N ratio of plants can be increased with no, or minimal, decrease in growth rate by switching from N-containing to N-free solutes involved in, for example, UV-B screening or by reducing the content of particular proteins . The water cost of growth (water lost per unit biomass gain) in terrestrial plants is a function of N supply and of C supply; water cost is lower with higher N and C availability . Water supply is also important in determining denitrification rates on land and on N (and C) fluxes from terrestrial to aquatic systems.

Water Sci Technol, 2003, 48(6), 171 - 8
Simultaneous biodegradation of a phenol and 3,4-dimethylphenol mixture under denitrifying conditions; Puig-Grajales L et al.; Denitrification is a feasible alternative for the treatment of phenolic bearing-wastewaters . The aim of this study was to evaluate the biodegradability of phenolic compounds, as the only carbon and energy source in batch and continuous experiments, using nitrate as a final electron acceptor . Experiments in a continuous upward anaerobic sludge bed reactor demonstrated the possibility of biodegrading a mixture of phenol and 3,4-dimethylphenol at organic loads of 251.6 and 39.5 mg/L-d, respectively, at a COD/NO3(-)-N ratio of 2.57 . A nitrogen production efficiency of 86% was obtained according to the nitrate consumption . GC-MS analyses demonstrated that m-cresol was an intermediate of 3,4-dimethylphenol degradation in batch conditions, and had an inhibitory effect on phenol degradation.

Water Sci Technol, 2003, 48(6), 165 - 70
Biodegradation of octylphenol polyethoxylates by denitrification; Jimenez-Gonzalez A et al.; The biodegradation route of the octylphenol ethoxylates (OPEOs) by denitrification in an upflow anoxic sludge blanket (UASB) reactor was studied . An anaerobic sludge adapted to denitrifying conditions with acetate was adapted with increasing amounts of OPEOs and diminishing amounts of acetate until only 300 mg x L(-1) of OPEOs were fed . Only 70% of stoichiometric NO3- was fed so partial removal was expected . The total OPEOs fed was transformed with 70% COD removal . HPLC and GC-MS analyses showed that octylphenol (OP) was immediately formed but disappeared while other intermediates, the ethoxylated moieties; mono and diethoxylate (OPEO1 and OPEO2 respectively) led to the cleavage of the alkyl chain to form propylphenol triethoxylate (PPEO3) and heptylphenol diethoxylated (HPEO2) . These last two compounds are produced due to an attack to both sides of the molecule; the hydrophilic and the hydrophobic . These findings suggest three simultaneous routes of OPEO degradation.

Water Sci Technol, 2003, 48(6), 75 - 9
Evolution of the bacterial community during granules formation in denitrifying reactors followed by molecular, culture-independent techniques; Etchebehere C et al.; The microbial community in two acetate-fed denitrifying reactors, inoculated with methanogenic sludge, was monitored by 16S rDNA-based methods (SSCP and FISH) . Both reactors converged to similar, stable communities . The predominant organisms belonged to the genera Thauera, Paracoccus and Denitrobacter, detected both by molecular and culture-based methods.

Water Res, 2004 Jan, 38(1), 13 - 6
Evidence of anoxic methane oxidation coupled to denitrification; Islas-Lima S et al.; Denitrification using methane as sole electron donor under anoxic condition was investigated . Sludge produced by a denitrifying reactor using acetate as electron donor was put in contact with methane at partial pressures from 1.8 to 35.7kPa . Nitrate depletion and gaseous nitrogen production were measured . The denitrification rate was independent of the methane partial pressure when superior or equal to 8.8kPa . The nitrate depletion was asymptotic . A denitrification rate of 0.25g NO(3)(-)-Ng(-1) VSSd(-1) was observed at the onset of culturing, followed by a slower and lineal denitrification rate of 4.9x10(-3)g NO(3)(-)-Ng(-1) VSSd(-1) . Abiotic nitrate removal or the availability of another carbon source were discarded from control experiments made in the absence of methane or using sterilized inoculum.

Biotechnol Adv, 2003 Dec, 22(1-2), 135 - 59
Using ecotechnology to address water quality and wetland habitat loss problems in the Mississippi basin: a hierarchical approach; Day JW Jr et al.; Human activities are affecting the environment at continental and global scales . An example of this is the Mississippi basin where there has been a large scale loss of wetlands and water quality deterioration over the past century . Wetland and riparian ecosystems have been isolated from rivers and streams . Wetland loss is due both to drainage and reclamation, mainly for agriculture, and to isolation from the river by levees, as in the Mississippi delta . There has been a decline in water quality due to increasing use of fertilizers, enhanced drainage and the loss of wetlands for cleaning water . Water quality has deteriorated throughout the basin and high nitrogen in the Mississippi river is causing a large area of hypoxia in the Gulf of Mexico adjacent to the Mississippi delta . Since the causes of these problems are distributed over the basin, the solution also needs to be distributed over the basin . Ecotechnology and ecological engineering offer the only ecologically sound and cost-effective method of solving these problems . Wetlands to promote nitrogen removal, mainly through denitrification but also through burial and plant uptake, offer a sound ecotechnological solution . At the level of the Mississippi basin, changes in farming practices and use of wetlands for nitrogen assimilation can reduce nitrogen levels in the River . There are additional benefits of restoration of wetland and riverine ecosystems, flood control, reduction in public health threats, and enhanced wildlife and fisheries . At the local drainage basin level, the use of river diversions in the Mississippi delta can address both problems of coastal land loss and water quality deterioration . Nitrate levels in diverted river water are rapidly reduced as water flows through coastal watersheds . At the local level, wetlands are being used to treat municipal wastewater . This is a cost-effective method, which results in improved water quality, enhanced wetland productivity and increased accretion . The problems in the Mississippi basin serves as an example for other watersheds in the Gulf of Mexico . This is especially important in Mexico, where there is a strong need for economical solutions to ecological problems . The Usumacinta delta-Laguna de Terminos regional ecosystem is an example where ecotechnological approaches offer realistic solutions to environmental problems.

Biochemistry (Mosc), 2003 Oct, 68(10), 1101 - 8
Proton-translocating ATP-synthase of Paracoccus denitrificans: ATP-hydrolytic activity; Zharova TV et al.; Tightly coupled membranes of Paracoccus denitrificans catalyze oxidative phosphorylation but are incapable of ATP hydrolysis . The conditions for observation and registration of the venturicidin-sensitive ATPase activity of subbacterial particles derived from this organism are described . The ATP hydrolytic activity does not appear after prolonged incubation in the presence of pyruvate kinase and phosphoenol pyruvate (to remove ADP), EDTA (to remove Mg2+) and/or inorganic phosphate, whereas the activity dramatically increases after energization of the membranes . ATP hydrolysis by -activated ATPase is coupled with electric potential formation . Inorganic phosphate prevents and azide promotes a decline of the enzyme activity during ATP hydrolysis . The addition of uncouplers results in rapid and complete inactivation of ATPase . The -dependent ATPase activity increases upon dilution of the membranes . The results are discussed as evidence for the presence of distinct ATP-synthase and ATP-hydrolase states of F(o)F(1) complex in the coupling membranes (Vinogradov, A . D . (1999) Biochemistry (Moscow), 64, 1219-1229) . The proposal is made that part of the free energy released from oxidoreduction in the respiratory chain is used to maintain active conformation of the energy-transducing proteins.

J Biol Chem, 2004 Feb 6, 279(6), 5000 - 7 Epub 2003 Nov 10.
Assembly of respiratory complexes I, III, and IV into NADH oxidase supercomplex stabilizes complex I in Paracoccus denitrificans; Stroh A et al.; Stable supercomplexes of bacterial respiratory chain complexes III (ubiquinol:cytochrome c oxidoreductase) and IV (cytochrome c oxidase) have been isolated as early as 1985 (Berry, E . A., and Trumpower, B . L . (1985) J . Biol . Chem . 260, 2458-2467) . However, these assemblies did not comprise complex I (NADH:ubiquinone oxidoreductase) . Using the mild detergent digitonin for solubilization of Paracoccus denitrificans membranes we could isolate NADH oxidase, assembled from complexes I, III, and IV in a 1:4:4 stoichiometry . This is the first chromatographic isolation of a complete "respirasome." Inactivation of the gene for tightly bound cytochrome c552 did not prevent formation of this supercomplex, indicating that this electron carrier protein is not essential for structurally linking complexes III and IV . Complex I activity was also found in the membranes of mutant strains lacking complexes III or IV . However, no assembled complex I but only dissociated subunits were observed following the same protocols used for electrophoretic separation or chromatographic isolation of the supercomplex from the wild-type strain . This indicates that the P . denitrificans complex I is stabilized by assembly into the NADH oxidase supercomplex . In addition to substrate channeling, structural stabilization of a membrane protein complex thus appears as one of the major functions of respiratory chain supercomplexes.

Appl Environ Microbiol, 2003 Nov, 69(11), 6715 - 22
Aerobic denitrification of Pseudomonas aeruginosa monitored by online NAD(P)H fluorescence; Chen F et al.; Continuous cultures of Pseudomonas aeruginosa (ATCC 9027) maintained at different dissolved oxygen concentrations (DO) were studied for the effects of DO on various culture properties, especially aerobic respiration and denitrification . The DO was varied from 0 mg/liter (completely anoxic conditions) to 1.3 mg/liter and measured with optical sensors that could accurately determine very low DO based on oxygen-quenched luminescence . The strain was found to perform aerobic denitrification; while the specific rate decreased with increasing DO, denitrification persisted at approximately 1/8 of the maximum rate (1.7 mmol/g of cells/h) even at relatively high DO (1 to 1.3 mg/liter) . In the presence of nitrate, the culture's Monod half-rate saturation constant for O(2) was very small, <0.1 mg/liter . Aerobic denitrification appeared to function as an electron-accepting mechanism supplementary to or competitive with aerobic respiration . The shift of the culture's respiratory mechanism was also clearly detected with a fluorometer targeting intracellular NAD(P)H, i.e., the reduced forms of the NAD(P) coenzymes . Comparatively, the NAD(P)H fluorescence under the anoxic, denitrifying conditions (NFU(DN)) was highest, that under fully aerobic conditions (NFU(OX)) was lowest, and that under conditions in which both denitrification and aerobic respiration occurred (NFU) was intermediate . Representing a quantitative measure of the culture's "fractional approach" to the fully denitrifying state, the normalized fraction (NFU - NFU(OX))/(NFU(DN) - NFU(OX)) was correlated with DO and the calculated fraction of electrons accepted by denitrification . The NFU fraction decreased with increasing DO, following an empirical exponential relationship . The fraction of denitrification-accepted electrons increased with the NFU fraction: the increase was gradual and approximately linear at DO of >/==" BORDER="0">0.1 mg/liter but much sharper at lower DO . Online NAD(P)H fluorescence was demonstrated as a feasible technique for effective monitoring and quantitative description of the microaerobic state of microorganisms.

Appl Environ Microbiol, 2003 Nov, 69(11), 6447 - 54
Anaerobic ammonium oxidation measured in sediments along the Thames estuary, United Kingdom; Trimmer M et al.; Until recently, denitrification was thought to be the only significant pathway for N(2) formation and, in turn, the removal of nitrogen in aquatic sediments . The discovery of anaerobic ammonium oxidation in the laboratory suggested that alternative metabolisms might be present in the environment . By using a combination of (15)N-labeled NH(4)(+), NO(3)(-), and NO(2)(-) (and (14)N analogues), production of (29)N(2) and (30)N(2) was measured in anaerobic sediment slurries from six sites along the Thames estuary . The production of (29)N(2) in the presence of (15)NH(4)(+) and either (14)NO(3)(-) or (14)NO(2)(-) confirmed the presence of anaerobic ammonium oxidation, with the stoichiometry of the reaction indicating that the oxidation was coupled to the reduction of NO(2)(-) . Anaerobic ammonium oxidation proceeded at equal rates via either the direct reduction of NO(2)(-) or indirect reduction, following the initial reduction of NO(3)(-) . Whether NO(2)(-) was directly present at 800 micro M or it accumulated at 3 to 20 micro M (from the reduction of NO(3)(-)), the rate of (29)N(2) formation was not affected, which suggested that anaerobic ammonium oxidation was saturated at low concentrations of NO(2)(-) . We observed a shift in the significance of anaerobic ammonium oxidation to N(2) formation relative to denitrification, from 8% near the head of the estuary to less than 1% at the coast . The relative importance of anaerobic ammonium oxidation was positively correlated (P < 0.05) with sediment organic content . This report of anaerobic ammonium oxidation in organically enriched estuarine sediments, though in contrast to a recent report on continental shelf sediments, confirms the presence of this novel metabolism in another aquatic sediment system.

Environ Technol, 2003 Sep, 24(9), 1129 - 34
Nitrate removal from drinking water through the use of encapsulated microorganisms in alginate beads; Liu SX et al.; Biological treatment for removal of nitrate from drinking water is of great significance, as traditional physical and chemical methods could not effectively remove soluble nitrate . In this report immobilized microorganisms with co-immobilized calcium tartrate were used for reducing nitrate concentration (110 mg l(-1) NO3-N) in a model solution . The carbon source also functions as a stabilizing agent for the immobilization matrix . Experiments of denitrification showed a high nitrate removal rate while nitrite residual was at a concentration higher than expected . The nitrate concentration was reduced to nearly zero (0.2-1.4 mg l(-1)) after 3 days of operation . The calcium tartrate (4%, w/w) co-immobilized alginate beads had better nitrate removal performance than tartrate in solution . The nitrite-N residual concentration was approximately 1.1-2.9 mg l(-1) at the end of the experiments, showing the desirability of further denitrification . The stability of alginate beads was also tested both to evaluate their behaviors and investigate the efficacy of bead recycling . It was found that the beads could be used for 8-13 days consecutively without any structural deterioration and leaking of microbes.

Water Environ Res, 2003 Sep-Oct, 75(5), 434 - 43
Effects of oxygen on anoxic biodegradation of benzoate during continuous culture; Cinar O et al.; In this study, various amounts of oxygen were added to denitrifying chemostats receiving benzoate to mimic the input of oxygen to anoxic zones of biological nutrient removal systems . The effect of oxygen on the biodegradative capability of the mixed-microbial culture for benzoate was investigated . The anoxic benzoate biodegradative capability of the culture was not significantly changed as the mass flowrate of oxygen was increased to 40% of the input benzoate chemical oxygen demand (COD) mass flowrate, but was decreased approximately 70% when the mass flowrate of oxygen was increased to 70% of the input benzoate COD mass flowrate . The decrease in the anoxic benzoate biodegradative capability was due primarily to the loss of the denitrifying enzymes (measured by the anoxic pyruvate-degrading ability) and not to the loss of the key anoxic catabolic enzyme (benzoyl-coenzyme A reductase) . The proportional increase in the concentration of nitrate as the residual terminal electron acceptor and the lack of synthesis of aerobic ring-cleavage enzymes as the oxygen input to the chemostat was increased suggest that the mixed microbial culture preferred oxygen to nitrate as the terminal electron acceptor, but degraded benzoate using the anoxic metabolic pathway . The concentration of the mixed microbial culture increased as the oxygen input to the chemostat was increased, suggesting that the oxygen was used by cytochrome cbb3 rather than quinol oxidase because the energetic yield of cytochrome cbb3 is higher than that of quinol oxidase or the nitrogen oxide reductases.

Water Environ Res, 2003 Sep-Oct, 75(5), 406 - 11
Maintaining granulation in a denitrifying upflow sludge-blanket reactor treating groundwater with low hardness; Rouse JD et al.; Maintenance of denitrifying granular sludge for treating soft groundwater (total hardness = 75 mg calcium carbonate/L) in an upflow sludge-blanket reactor was demonstrated with complete removal of applied nitrate (20 mg N/L) over extended operation and a hydraulic residence time of 34 minutes . A high pH of approximately 9.0 was shown to be important for generation of mineral precipitation needed for production of heavy granular sludge with good retention characteristics . As a method of increasing precipitation potential, pH adjustment was determined to be more economically favorable than calcium or alkalinity supplementation . In addition, temporary increases in substrate loading were shown to be effective for enhancing biomass levels in a manageable granular sludge . The significance of biomass in promoting mineral precipitation was discussed.

Biotechnol Lett, 2003 Oct, 25(19), 1605 - 8
Isolation and characterization of sulfur-utilizing denitrifiers from the sulfur-oxidizing denitrification process; Tian D et al.; Of 14 potential sulfur-oxidizing strains, Pseudomonas sp . B21 and Agrobacterium sp . B19 were considered as denitrifiers . Under aerobic conditions, with S0 as electron donor, maximum cell growth rates were 0.022 (B21) and 0.043 h(-1) (B19) . Both grew optimally at pH 7.5 and 28 degrees C . When NO3-N was increased from 10 to 200 mg l(-1) the efficiency of nitrate removal of each strain gradually decreased, from 60 to 40% . Addition of suitable organic compounds (C/N < 4.2) increased the nitrate removal efficiencies of both strains, indicating their mixotrophic characters.

Water Res, 2003 Nov, 37(19), 4748 - 60
Degradation of microcystin in sediments at oxic and anoxic, denitrifying conditions; Holst T et al.; The potent toxin microcystin is frequently released during cyanobacterial blooms in eutrophic waters and may impose a risk to human health, when surface water is used for drinking water . For removal of microcystin in surface waters, infiltration through sediment is commonly used . In the present study, mineralization of 14C-labelled microcystin (accumulation of 14CO(2)) and concentration changes (protein phosphatase inhibition assay) demonstrated that indigenous microorganisms in the sediment of a water recharge facility were capable of degrading microcystin . At oxic or microaerophilic (<2% O(2)) conditions, microcystin added to sediment slurries at 70 microg l(-1) was reduced to <20 microg l(-1) in 1-2 weeks, and less than 3 microg l(-1) after 7 weeks . At anoxic conditions (<0.3% O(2)) and with addition of nitrate, the degradation was significantly stimulated, reducing microcystin from 100 to <20 microg l(-1) within 1 day . The simultaneous production of N(2)O in the samples suggests that the microcystin degradation was coupled to dissimilative nitrate reduction (denitrification) . Since aquifers and sediments beneath drinking water reservoirs often are anoxic, nitrate respiration may be an important process in removal and detoxification of microcystin.

Biochemistry, 2003 Oct 28, 42(42), 12391 - 9
Electrochemical and FTIR spectroscopic characterization of the cytochrome bc1 complex from Paracoccus denitrificans: evidence for protonation reactions coupled to quinone binding; Ritter M et al.; The cytochrome bc(1) complex from Paracoccus denitrificans and soluble fragments of its cytochrome c(1) and Rieske ISP subunits are characterized by a combined approach of protein electrochemistry and FTIR difference spectroscopy . The FTIR difference spectra provide information about alterations in the protein upon redox reactions: signals from the polypeptide backbone, from the cofactors, and from amino acid side chains . We describe typical modes for conformational changes in the polypeptide and contributions of different secondary structure elements . Signals attributed to the different cofactors can be presented on the basis of selected potential steps . Modes associated with bound quinone are identified by comparison with spectra of quinone in solution at 1656, 1642, and 1610 cm(-1) and between 1494 and 1388 cm(-1), as well as at 1288 and 1262 cm(-1) . Signals originating from the quinone bound at the Q(o) site can be distinguished . On the basis of the infrared data, the total quinone concentration is determined to be 2.6-3.3 quinones per monomer, depending on preparation conditions . The balance of evidence supports the double-occupancy model . Interestingly, the amplitude of the band at 1746 cm(-1) increases with quinone content, reflecting a protonation reaction of acidic groups . In this context, the involvement of glutamates and/or aspartates in the vicinity of the Q(o) site is discussed on the basis of recently determined crystal structures.

J Bacteriol, 2003 Nov, 185(21), 6308 - 15
A mutant of Paracoccus denitrificans with disrupted genes coding for cytochrome c550 and pseudoazurin establishes these two proteins as the in vivo electron donors to cytochrome cd1 nitrite reductase; Pearson IV et al.; In Paracoccus denitrificans, electrons pass from the membrane-bound cytochrome bc(1) complex to the periplasmic nitrite reductase, cytochrome cd(1) . The periplasmic protein cytochrome c(550) has often been implicated in this electron transfer, but its absence, as a consequence of mutation, has previously been shown to result in almost no attenuation in the ability of the nitrite reductase to function in intact cells . Here, the hypothesis that cytochrome c(550) and pseudoazurin are alternative electron carriers from the cytochrome bc(1) complex to the nitrite reductase was tested by construction of mutants of P . denitrificans that are deficient in either pseudoazurin or both pseudoazurin and cytochrome c(550) . The latter organism, but not the former (which is almost indistinguishable in this respect from the wild type), grows poorly under anaerobic conditions with nitrate as an added electron acceptor and accumulates nitrite in the medium . Growth under aerobic conditions with either succinate or methanol as the carbon source is not significantly affected in mutants lacking either pseudoazurin or cytochrome c(550) or both these proteins . We concluded that pseudoazurin and cytochrome c(550) are the alternative electron mediator proteins between the cytochrome bc(1) complex and the cytochrome cd(1)-type nitrite reductase . We also concluded that expression of pseudoazurin is mainly controlled by the transcriptional activator FnrP.

Biochemistry, 2003 Oct 21, 42(41), 11968 - 81
Electron transfer complexes of cytochrome c peroxidase from Paracoccus denitrificans containing more than one cytochrome; Pettigrew GW et al.; According to the model proposed in previous papers {Pettigrew, G . W., Prazeres, S., Costa, C., Palma, N., Krippahl, L., and Moura, J . J . (1999) The structure of an electron-transfer complex containing a cytochrome c and a peroxidase, J . Biol . Chem . 274, 11383-11389; Pettigrew, G . W., Goodhew, C . F., Cooper, A., Nutley, M., Jumel, K., and Harding, S . E . (2003) Electron transfer complexes of cytochrome c peroxidase from Paracoccus denitrificans, Biochemistry 42, 2046-2055}, cytochrome c peroxidase of Paracoccus denitrificans can accommodate horse cytochrome c and Paracoccus cytochrome c(550) at different sites on its molecular surface . Here we use (1)H NMR spectroscopy, analytical ultracentrifugation, molecular docking simulation, and microcalorimetry to investigate whether these small cytochromes can be accommodated simultaneously in the formation of a ternary complex . The pattern of perturbation of heme methyl and methionine methyl resonances in binary and ternary solutions shows that a ternary complex can be formed, and this is confirmed by the increase in the sedimentation coefficient upon addition of horse cytochrome c to a solution in which cytochrome c(550) fully occupies its binding site on cytochrome c peroxidase . Docking experiments in which favored binary solutions of cytochrome c(550) bound to cytochrome c peroxidase act as targets for horse cytochrome c and the reciprocal experiments in which favored binary solutions of horse cytochrome c bound to cytochrome c peroxidase act as targets for cytochrome c(550) show that the enzyme can accommodate both cytochromes at the same time on adjacent sites . Microcalorimetric titrations are difficult to interpret but are consistent with a weakened binding of horse cytochrome c to a binary complex of cytochrome c peroxidase and cytochrome c(550) and binding of cytochrome c(550) to the cytochrome c peroxidase that is affected little by the presence of horse cytochrome c in the other site . The presence of a substantial capture surface for small cytochromes on the cytochrome c peroxidase has implications for rate enhancement mechanisms which ensure that the two electrons required for re-reduction of the enzyme after reaction with hydrogen peroxide are delivered efficiently.

FEMS Microbiol Lett, 2003 Sep 26, 226(2), 331 - 7
Aerobic and anaerobic nitrate and nitrite reduction in free-living cells of Bradyrhizobium sp . (Lupinus); Polcyn W et al.; Induction, energy gain, effect on growth, and interaction of nitrate and nitrite reduction of Bradyrhizobium sp . (Lupinus) USDA 3045 were characterized . Both nitrate and nitrite were reduced in air, although nitrite reduction was insensitive to ammonium inhibition . Anaerobic reduction of both ions was shown to be linked with energy conservation . A dissimilatory ammonification process was detected, which has not been reported in rhizobia so far . Nevertheless, anaerobic conversion of nitrate to ammonium was lower than 40%, which suggests the presence of an additional, nitrite reductase of denitrifying type . Nitrite toxicity caused a non-linear relationship between biomass produced and >2 mM concentrations of each N oxyanion consumed . At > or =5 mM initial concentrations of nitrate, a stoichiometric nitrite accumulation occurred and nitrite remained in the medium . This suggests an inhibition of nitrite reductase activity by nitrate, presumably due to competition with nitrate reductase for electron donors . Lowering of growth temperature almost completely diminished nitrite accumulation and enabled consumption as high as 10 mM nitrate, which confirms such a conclusion.

Huan Jing Ke Xue, 2003 Jul, 24(4), 109 - 12
{Chemical denitrification of nitrate from groundwater}; Zhang Y et al.; Batch experiments for catalytic reduction of nitrate from groundwater with Pd and/Cu catalysts were conducted . It was found that Pd-Cu combined catalysts at a ratio of 4 can maximize the nitrate reduction into nitrogen; above 80% total nitrogen removal efficiency was realized in this study . It was also found that the catalytic activity was affected by the amounts of catalysts, hydrogen flow rate and pressure, the initial concentration of nitrate . With an increasing in the amount of the catalysts, both nitrite and ammonia intermediates can be kept at a low level . A high flow rate and pressure of hydrogen was in favor of the catalytic reduction of nitrate, but not benefit for the selectivity . The catalytic reduction followed a first order kinetics in term of the initial nitrate concentration.

Appl Environ Microbiol, 2003 Oct, 69(10), 5941 - 9
Utilization of acyl-homoserine lactone quorum signals for growth by a soil pseudomonad and Pseudomonas aeruginosa PAO1; Huang JJ et al.; Acyl-homoserine lactones (AHLs) are employed by several Proteobacteria as quorum-sensing signals . Past studies have established that these compounds are subject to biochemical decay and can be used as growth nutrients . Here we describe the isolation of a soil bacterium, Pseudomonas strain PAI-A, that degrades 3-oxododecanoyl-homoserine lactone (3OC12HSL) and other long-acyl, but not short-acyl, AHLs as sole energy sources for growth . The small-subunit rRNA gene from strain PAI-A was 98.4% identical to that of Pseudomonas aeruginosa, but the soil isolate did not produce obvious pigments or AHLs or grow under denitrifying conditions or at 42 degrees C . The quorum-sensing bacterium P . aeruginosa, which produces both 3OC12HSL and C4HSL, was examined for the ability to utilize AHLs for growth . It did so with a specificity similar to that of strain PAI-A, i.e., degrading long-acyl but not short-acyl AHLs . In contrast to the growth observed with strain PAI-A, P . aeruginosa strain PAO1 growth on AHLs commenced only after extremely long lag phases . Liquid-chromatography-atmospheric pressure chemical ionization-mass spectrometry analyses indicate that strain PAO1 degrades long-acyl AHLs via an AHL acylase and a homoserine-generating HSL lactonase . A P . aeruginosa gene, pvdQ (PA2385), has previously been identified as being a homologue of the AHL acylase described as occurring in a Ralstonia species . Escherichia coli expressing pvdQ catalyzed the rapid inactivation of long-acyl AHLs and the release of HSL . P . aeruginosa engineered to constitutively express pvdQ did not accumulate its 3OC12HSL quorum signal when grown in rich media . However, pvdQ knockout mutants of P . aeruginosa were still able to grow by utilizing 3OC12HSL . To our knowledge, this is the first report of the degradation of AHLs by pseudomonads or other gamma-Proteobacteria, of AHL acylase activity in a quorum-sensing bacterium, of HSL lactonase activity in any bacterium, and of AHL degradation with specificity only towards AHLs with long side chains.

Nat Struct Biol, 2003 Nov, 10(11), 928 - 34 Epub 2003 Oct 05.
Structural and redox plasticity in the heterodimeric periplasmic nitrate reductase; Arnoux P et al.; The structure of the respiratory nitrate reductase (NapAB) from Rhodobacter sphaeroides, the periplasmic heterodimeric enzyme responsible for the first step in the denitrification process, has been determined at a resolution of 3.2 A . The di-heme electron transfer small subunit NapB binds to the large subunit with heme II in close proximity to the {4Fe-4S} cluster of NapA . A total of 57 residues at the N- and C-terminal extremities of NapB adopt an extended conformation, embracing the NapA subunit and largely contributing to the total area of 5,900 A(2) buried in the complex . Complex formation was studied further by measuring the variation of the redox potentials of all the cofactors upon binding . The marked effects observed are interpreted in light of the three-dimensional structure and depict a plasticity that contributes to an efficient electron transfer in the complex from the heme I of NapB to the molybdenum catalytic site of NapA.

J Bacteriol, 2003 Oct, 185(20), 6119 - 29
2-Oxoglutarate:NADP(+) oxidoreductase in Azoarcus evansii: properties and function in electron transfer reactions in aromatic ring reduction; Ebenau-Jehle C et al.; The conversion of {(14)C}benzoyl-coenzyme A (CoA) to nonaromatic products in the denitrifying beta-proteobacterium Azoarcus evansii grown anaerobically on benzoate was investigated . With cell extracts and 2-oxoglutarate as the electron donor, benzoyl-CoA reduction occurred at a rate of 10 to 15 nmol min(-1) mg(-1) . 2-Oxoglutarate could be replaced by dithionite (200% rate) and by NADPH ( approximately 10% rate); in contrast NADH did not serve as an electron donor . Anaerobic growth on aromatic compounds induced 2-oxoglutarate:acceptor oxidoreductase (KGOR), which specifically reduced NADP(+), and NADPH:acceptor oxidoreductase . KGOR was purified by a 76-fold enrichment . The enzyme had a molecular mass of 290 +/- 20 kDa and was composed of three subunits of 63 (gamma), 62 (alpha), and 37 (beta) kDa in a 1:1:1 ratio, suggesting an (alphabetagamma)(2) composition . The native enzyme contained Fe (24 mol/mol of enzyme), S (23 mol/mol), flavin adenine dinucleotide (FAD; 1.4 mol/mol), and thiamine diphosphate (0.95 mol/mol) . KGOR from A . evansii was highly specific for 2-oxoglutarate as the electron donor and accepted both NADP(+) and oxidized viologens as electron acceptors; in contrast NAD(+) was not reduced . These results suggest that benzoyl-CoA reduction is coupled to the complete oxidation of the intermediate acetyl-CoA in the tricarboxylic acid cycle . Electrons generated by KGOR can be transferred to both oxidized ferredoxin and NADP(+), depending on the cellular needs . N-terminal amino acid sequence analysis revealed that the open reading frames for the three subunits of KGOR are similar to three adjacently located open reading frames in Bradyrhizobium japonicum . We suggest that these genes code for a very similar three-subunit KGOR, which may play a role in nitrogen fixation . The alpha-subunit is supposed to harbor one FAD molecule, two {4Fe-4S} clusters, and the NADPH binding site; the beta-subunit is supposed to harbor one thiamine diphosphate molecule and one further {4Fe-4S} cluster; and the gamma-subunit is supposed to harbor the CoA binding site . This is the first study of an NADP(+)-specific KGOR . A similar NADP(+)-specific pyruvate oxidoreductase, which contains all domains in one large subunit, has been reported for the mitochondrion of the protist Euglena gracilis and the apicomplexan Cryptosporidium parvum.

J Environ Sci Health A Tox Hazard Subst Environ Eng, 2003, 38(10), 2425 - 33
Sludge dewatering using macrophytes in a small wastewater treatment system: a case study of a pilot scale plant in northern Italy; Barbieri A et al.; A study was conducted on a system consisting of two beds of 12 m2 each, planted with reeds and filled with sludge from an activated sludge plant dimensioned for 3000 p.e . During 2001, 10 kg of TS/m2 were loaded in each bed using sludge with a dry matter content of 3% during winter and of 0.5% in summer . The aim of the study was to evaluate parameters such as: COD, TSS, P-PO4(3-), N-NO2-, N-NO3-, N-NH4+ in percolation water, the dewatering capability of the two beds and to estimate N mass balance . The observations on dewatering process showed that dried sludge reached a content of 23% TS in winter and of 30% TS in summer . During summertime the plant did not release any percolation flow; in the rest of the year the concentrations of COD, TSS, P-PO4(3-), N-NO2-, N-NH4- remained within bounds of the European directive (91/271/CEE) . The N mass balance proved that 90% of N was removed by the denitrification process.

J Environ Sci Health A Tox Hazard Subst Environ Eng, 2003, 38(10), 2191 - 9
The role of DPB at anoxic stage in a novel package type BNR process with batch settler; Shin HS et al.; In this research, package type BNR process was developed to remove nutrients as well as suspended solids from domestic sewage . The effect of HRT reduction of settler on the removal of pollutants as well as the role of DPBs at anoxic reactor were investigated . The proposed package system was composed of sludge denitrification tank, anaerobic, anoxic, oxic, and batch settler . This system could remove nitrogen and phosphorus effectively at low COD/N ratio and also remove SS more effectively than other ordinary BNR system having the conventional settlers . The removal efficiency of total nitrogen (T-N) in optimal condition was about 75.1% under the TCOD/TKN ratio as low as 5.7 . The average concentration of the effluent TCOD, ortho-P, NH4+-N, NO3- -N and SS in the package type BNR system of 2 h settler retention time were 15.6 . 1.38, 1.4, 10.3 and 3.9 mg/L, respectively . In anoxic state, denitrifying dephosphatation, that is uptaking phosphate by using nitrate-nitrogen as an electron acceptor was observed . The removed NO3- -N concentration by denitrifying dephosphatation was 1.62 mg NO3- -N/mg PO4(3-)-P.

J Environ Sci Health A Tox Hazard Subst Environ Eng, 2003, 38(10), 2169 - 77
Denitrification in tertiary filtration: application of an up-flow filter; Farabegoli G et al.; The present paper shows the results obtained through an experimental work performed at the wastewater treatment plant of Rome, aimed at studying the performances of a tertiary filter regarding combined removal of suspended solids, COD, and nitrates . The up-flow sand filter was fed by the effluent coming from the secondary settling tank of the plant . The filter bed height was of 80 cm of silica sand . After a start up period, a study of particulate and soluble COD removal process was made, to establish the need of methanol in the denitrification process . Total COD removal efficiency was 60% on average, 55% due by soluble COD removal and 5% by particulate one . In the last phase of the experimental activity methanol was fed as carbon source, sodium sulfite was supplied to produce anoxic environment within the filter and the denitrification efficiency was studied . Nitrates removal rates after an acclimation period of 10 days increased up to 60%, with an effluent NO3-N of 8 mg/L . Denitrification rate was 2.4 kg/m3 d for water temperatures of 25 degrees C . Regarding methanol demand and biologic kinetics, the biomass yield coefficient was 0.3 kg(COD-X)/kg(me) . Consequently 2.7 kg of methanol was required per kilogram of denitrified nitrogen.

J Contam Hydrol, 2003 Oct, 66(1-2), 79 - 91
Accumulation of nitrite in denitrifying barriers when phosphate is limiting; Hunter WJ; Permeable in situ denitrifying barriers can remove nitrate from groundwater . Barriers may be constructed by filling an excavated area with a porous mixture of sand, fine gravel, and substrate or by the injection of a nonaqueous phase substrate into an aquifer . The substrate stimulates the development of a denitrifying microbial community by providing an electron donor . The objective of this study was to determine the ability of denitrifying barriers to function under low-phosphate conditions . Sand columns injected with a soybean oil emulsion were used as laboratory models of denitrifying barriers . When a natural groundwater containing 17 mg l(-1) nitrate-N and 0.009 mg l(-1) phosphate-P was pumped through the columns, only a small amount of nitrate was removed from the water and, in some effluent fractions, 52% to 88% of the influent nitrate had converted to nitrite . Nitrite also accumulated when the phosphate concentration of the groundwater was increased to 0.040 or 0.080 mg l(-1) phosphate-P . Only when a 0.160 mg l(-1) phosphate-P supplement was added to the groundwater was there a loss of nitrate without a large accumulation of nitrite . The addition of solid calcium phosphate or rock phosphate to the sand columns was found to provide adequate phosphate for denitrification in short-term studies . These studies point out the need to ensure that adequate phosphate is present in denitrifying barriers especially when such barriers are used beneath phosphate-binding soils.

J Contam Hydrol, 2003 Oct, 66(1-2), 59 - 77
Nitrate-consuming processes in a petroleum-contaminated aquifer quantified using push-pull tests combined with 15N isotope and acetylene-inhibition methods; Schurmann A et al.; Nitrate consumption in aquifers may result from several biogenic and abiotic processes such as denitrification, assimilatory NO3- reduction, dissimilatory NO3- reduction to ammonium (DNRA), or abiotic NO3- (or NO2-) reduction . The objectives of this study were to investigate the fate of NO3- in a petroleum-contaminated aquifer, and to assess the feasibility of using single-well push-pull tests (PPTs) in combination with 15N isotope and C2H2 inhibition methods for the quantification of processes contributing to NO3- consumption . Three consecutive PPTs were performed in a monitoring well of a heating oil-contaminated aquifer in Erlen, Switzerland . For each test, we injected 500 l of test solution containing 0.5 mM Br- as conservative tracer and either 0.5 mM unlabeled NO3- or approximately 0.3 mM 15N-labeled NO3- as reactant . Test solutions were sparged during preparation and injection with either N2, Ar or 10% C2H2 in Ar . After an initial incubation period of 1.5-3.2 h, we extracted the test solution/groundwater mixtures from the same location and measured concentrations of relevant species including Br-, NO3-, NO2-, N2O, N2, and NH4+ . In addition, we determined the 15N contents of N2, N2O, NH4+, and suspended biomass from 15N/14N isotope-ratio measurements . Average total test duration was 50.5 h . First-order rate coefficients (k) were computed from measured NO3- consumption, N2-15N production and N2O-15N production . From measured NO3- consumption we obtained nearly identical estimates of k for all PPTs with small 95% confidence intervals, indicating good reproducibility and accuracy for the tests . Estimates of k from N2-15N production and N2O-15N production indicated that denitrification accounted for only 46-49% of observed NO3- consumption . Production of N2-15N in the presence of C2H2 was observed during one of the tests, which may be an indicator for abiotic NO3- reduction . Moreover, 15N isotope analyses confirmed occurrence of assimilatory NO3- reduction (0.58 at.% 15N in suspended biomass) and to a smaller extent DNRA (up to 4 at.% 15N in NH4+) . Our results indicated that the combination of PPTs, 15N-isotope and C2H2 inhibition methods provided improved information on denitrification as well as alternative fates of NO3- in this aquifer.

Water Res, 2003 Jul, 37(13), 3070 - 8
A dual isotope approach to identify denitrification in groundwater at a river-bank infiltration site; Fukada T et al.; The identification of denitrification in the Torgau sand and gravel aquifer, Germany, was carried out by a dual isotope method of measuring both the delta 15N and delta 18O in NO3- . Samples were prepared by an anion exchange resin method (Silva et al., J . Hydrol . 228 (2000) 22) with a modification to the AgNO3-drying process from a freeze-drying to an oven-drying method . The occurrence of denitrification in the aquifer was confirmed by comparing the reduction of dissolved oxygen, dissolved organic carbon and NO3- concentrations with the dual isotope signatures . High nitrate concentrations were associated with low delta 15N and delta 18O values, and vice versa . The denitrification accords with a Rayleigh equation with calculated enrichment factors of epsilon = -13.62@1000 for delta 15N and epsilon = -9.80@1000 for delta 18O . The slope of the straight-line relationship between the delta 15N and delta 18O data demonstrated that the enrichment of the heavy nitrogen isotope was higher by a factor of 1.3 compared with the heavy oxygen isotope . It is concluded that the identification of this factor is a useful means for confirming denitrification in future groundwater studies.

Biochim Biophys Acta, 2003 Sep 30, 1606(1-3), 95 - 103
Active/de-active transition of respiratory complex I in bacteria, fungi, and animals; Maklashina E et al.; Mammalian complex I (NADH:ubiquinone oxidoreductase) exists as a mixture of interconvertible active (A) and de-activated (D) forms . The A-form is capable of NADH:quinone-reductase catalysis, but not the D-form . Complex I from the bacterium Paracoccus denitrificans, by contrast, exists only in the A-form . This bacterial complex contains 32 fewer subunits than the mammalian complex . The question arises therefore if the structural complexity of complex I from higher organisms correlates with its ability to undergo the A/D transition . In the present study, it was found that complex I from the bacterium Escherichia coli and from non-vertebrate organisms (earthworm, lobster, and cricket) did not show the A/D transitions . Vertebrate organisms (carp, frog, chicken), however, underwent similar A/D transitions to those of the well-characterized bovine complex I . Further studies showed that complex I from the lower eukaryotes, Neurospora crassa and Yarrowia lipolytica, exhibited very distinct A/D transitions with much lower activation barriers compared to the bovine enzyme . The A/D transitions of complex I as they relate to structure and regulation of enzymatic activity are discussed.

J Biol Inorg Chem, 2003 Nov, 8(8), 843 - 54 Epub 2003 Sep 23.
X-ray structure of methanol dehydrogenase from Paracoccus denitrificans and molecular modeling of its interactions with cytochrome c-551i; Xia ZX et al.; The X-ray structure of methanol dehydrogenase (MEDH) from Paracoccus denitrificans (MEDH-PD) was determined at 2.5 A resolution using molecular replacement based on the structure of MEDH from Methylophilus methylotrophus W3A1 (MEDH-WA) . The overall structures from the two bacteria are similar to each other except that the former has a longer C-terminal tail in each subunit and shows local differences in several insertion regions . The "X-ray sequence" of the segment alphaGly444-alphaLeu452 was established, including one insertion and seven replacements compared with the reported sequence . The primary electron acceptor of MEDH-PD is cytochrome c-551i (Cyt c551i) . Based on the crystal structure of MEDH-PD and of the published structure of Cyt c551i, their interactions were investigated by molecular modeling . As a guide and starting point, the covalently attached cytochrome and PQQ domains of the alcohol dehydrogenase from Pseudomonas putida HK5 (ADH2B) were used . In the modeling, two molecules of Cyt c551i could be accommodated in their interaction with the MEDH heterotetramer in accordance with the two-fold molecular symmetry of the latter . Two models are proposed, in both of which electrostatic and hydrogen bonding interactions make major contributions to inter-protein binding . One of these models involves salt bridges from alphaArg99 of MEDH to the heme propionic acids of Cyt c551i and the other involves salt bridges from alphaArg426 of MEDH to Glu112 of Cyt c551i . Both involve salt bridges from alphaLys93 of MEDH to Asp75 of Cyt c551i . The size and nature of the cytochrome/quinoprotein heterodimer interfaces and calculations of electronic coupling and electron transfer rates favor one of these models over the other.

J Food Prot, 2003 Sep, 66(9), 1727 - 32
Evaluation of several modifications of an ecometric technique for assessment of media performance; Kornacki JL et al.; Recovery of Listeria monocytogenes 101M, Jonesia denitrificans, salmonellae, and Pediococcus sp . NRRL B-2354 across nine media was evaluated with three modified versions of an ecometric method . Two approaches involved the use of broth cultures (10(8) to 10(9) CFU/ml) of individual strains and either large (10-microl) or small (1-microl) presterilized plastic loops . The third approach involved precultured slants and the inoculation of media with presterilized plastic inoculating needles (10(4) CFU per needle) . Absolute growth indices (AGIs) were compared . No significant differences (P < 0.05) between methods were found when tryptic soy agar supplemented with 0.6% yeast extract (TSAYE) was used for the recovery of L . monocytogenes, J . denitrificans, Pediococcus sp . NRRL B-2354, and Salmonella spp . However, the small loop-broth technique recovered significantly fewer Salmonella enterica Typhimurium DT104 and Salmonella Senftenberg 775W cells than the other two techniques did . The performance of each individual bacterial strain on each of nine media was assayed . The recovery of L . monocytogenes was excellent (AGI > 4.8) with TSAYE, PALCAM, modified Oxford medium (MOX), and Baird-Parker agar and slight with modified PRAB (AGI = 0.4) and deMan Rogosa Sharpe (MRS) agar (< 0.1), and the organism was not recovered with the remaining media (modified lysine iron agar {MLIA}, xylose lysine desoxycholate {XLD} agar, and xylose lysine tergitol 4 {XLT4} agar) . The recovery of J . denitrificans with TSAYE and MOX was excellent, significantly better than that achieved with PALCAM (AGI = 3.0), but the organism was not recovered with Baird-Parker agar or with the other media tested . The recovery of Pediococcus sp . NRRL B-2354 was excellent with TSAYE and modified PRAB medium > Baird-Parker agar > acidified MRS agar, but the organism was not recovered with any of the other media tested . The best recovery of S . enterica Typhimurium DT104 was achieved with TSAYE > MLIA > or = XLD agar > or = XLT4 agar > Baird-Parker > PALCAM, MOX, acidified MRS agar, modified PRAB, and MRS agar . The best recovery of Salmonella Senftenberg 775W was achieved with TSAYE, MLIA, and XLD agar > XLT4 agar, but the organism was not recovered with the other media evaluated.

J Microbiol Methods, 2003 Oct, 55(1), 41 - 50
Detection methods for the expression of the dissimilatory copper-containing nitrite reductase gene (DnirK) in environmental samples; Metz S et al.; In situ assays, based on monoclonal antibodies (mAbs), were developed to study the microbial expression of the bacterial dissimilatory copper-containing nitrite reductase gene (DnirK), one of the key enzymes involved in denitrification, in different ecosystems . With a combination of an anti-DnirK mAb and phylogenetic oligonucleotide probes, it is possible to bring structural and functional aspects of microbial communities together . To perform a double labelling, yielding a high signal strength for both the oligonucleotide and the antibody, cells have to be labelled with the oligonucleotide first followed by immunostaining . When the labelling sequence was changed, the accessibility for the oligonucleotide was reduced if high amounts of DnirK were expressed . Using flow cytometry, it was possible to sort bacterial cells, which were stained by the antibody, from nonlabelled cells . This technique provides means for a detailed analysis of populations, which express DnirK genes in the environment, including structural aspects of a community and detailed promoter studies . Using the immunostaining approach, it was possible to identify bacteria, which have the DnirK system expressed, in samples from a wastewater sewage treatment plant as well as in samples from the rhizosphere of wheat roots . Furthermore, expression studies using an Ochrobactrum anthropi strain were carried out to investigate the correlation between N(2)O production rates and DnirK expression in batch cultures, which had been shifted from aerobic to anaerobic conditions . As expected, expression of DnirK was the highest during periods with the greatest synthesis rates for N(2)O . However, the amount of expressed enzyme was not reduced in the cells, although the N(2)O production rates dropped in the cultures 12 h after the shift from aerobic to anaerobic conditions.

Sci Total Environ, 2003 Oct 1, 314-316, 397 - 409
LOIS in-stream water quality modelling . Part 2 . Results and scenarios; Boorman DB; The catchment and river modelling undertaken within the UK Land Ocean Interaction Study programme represents the most extensive consistent exercise of this type undertaken in the UK . The calibrated model provides a quantitative assessment of the water and chemical fluxes transported by the rivers Derwent, Yorkshire Ouse, Wharfe, Aire, Don and Trent into the Humber estuary . These fluxes have been partitioned according to source (diffuse or point) for each basin, and a number of determinands . Total nitrogen from diffuse sources ranged from 92% on the largely agricultural Derwent to 38% from the industrial Don catchment . Even on catchments dominated by diffuse sources, point sources can contribute most during summer months . A quantitative assessment of the effect of in-stream processes suggests that denitrification can remove up to 40% of the total nitrogen entering the river system . A climate change scenario representing the 2050s revealed major changes in flow patterns, with all rivers showing a faster return to zero soil moisture deficit in the autumn and increased winter runoff, but a mixed picture of enhanced and reduced summer flows . Many associated fluxes may also increase, but long-term mean concentrations reduce because of the increased flows . The effect of the scenario on a river ecosystem classification was minor . Fertilizer reduction scenarios showed that even a large reduction in N applied as fertilizer (50%) resulted in much smaller reductions in the load delivered to the Humber estuary (6-16%).

Int J Syst Evol Microbiol, 2003 Sep, 53(Pt 5), 1427 - 33
Gordonia sihwensis sp . nov., a novel nitrate-reducing bacterium isolated from a wastewater-treatment bioreactor; Kim KK et al.; A nitrate-reducing bacterium, strain SPR2(T), was isolated from a sulphur-oxidizing, autotrophic denitrification reactor used for advanced treatment of wastewater from the lake of Sihwa, Korea . The strain was aerobic but could grow under anaerobic conditions . It was Gram-positive, exhibited rough white colonies on complex nutrient agar, produced elementary branching hyphae that fragmented into rod/coccus-like elements and showed chemotaxonomic markers that were consistent with classification in the genus Gordonia, i.e . meso-diaminopimelic acid, arabinose and galactose in whole-cell hydrolysates, N-glycolylmuramic acid in the peptidoglycan wall, unbranched saturated and monounsaturated fatty acids plus tuberculostearic acid (TBSA), mycolic acids that comprised 48-56 carbon atoms and MK-9(H(2)) as the predominant menaquinone . The 16S rDNA sequence of strain SPR2(T) showed highest similarity to Gordonia amicalis DSM 44461(T) and Gordonia hydrophobica DSM 44015(T), with values of 98.2 and 97.9 %, respectively . These values were far below 99.5 % (usually found at the intraspecies level) and they were in the range that separates species at the intrageneric level . The separate phylogenetic position of SPR2(T) was supported by differences in fatty acid and mycolic acid compositions and in carbon utilization tests that distinguished strain SPR2(T) from all known Gordonia species . Combined genotypic and phenotypic data show that strain SPR2(T) merits recognition as a novel species within the genus Gordonia, for which the name Gordonia sihwensis sp . nov . is proposed; the type strain is SPR2(T) (=DSM 44576(T)=NRRL B-24155(T)).

Int J Syst Evol Microbiol, 2003 Sep, 53(Pt 5), 1271 - 6
Phylogenetic and physiological characterization of a heterotrophic, chemolithoautotrophic Thiothrix strain isolated from activated sludge; Rossetti S et al.; The sheathed filamentous bacterium known as strain CT3, isolated by micromanipulation from an activated sludge treatment plant in Italy, is a member of the genus Thiothrix in the gamma-Proteobacteria according to 16S rDNA sequence analysis . The closest phylogenetic neighbours of strain CT3 are strains I and Q(T), which were also isolated from activated sludge and belong to the species Thiothrix fructosivorans . These strains have respectively 99.2 and 99.4 % similarity to CT3 by 16S rDNA sequence comparison . CT3 shows 63-67 % DNA-DNA hybridization with strain I, which is the only currently viable strain of T . fructosivorans . CT3 is the second strain in the genus Thiothrix that has been shown to be capable of growing autotrophically with reduced sulfur compounds as the sole energy source; autotrophy was also confirmed in strain I . The first reported chemolithoautotrophic isolate of this genus was a strain of 'Thiothrix ramosa' that was isolated from a hydrogen sulfide spring and is morphologically distinguishable from all other described strains of Thiothrix, including CT3 . CT3 is an aerobic organism that is non-fermentative, not capable of denitrification and able to grow heterotrophically . Autotrophy in the genus Thiothrix should be investigated more fully to better define the taxonomy of this genus.

Int J Syst Evol Microbiol, 2003 Sep, 53(Pt 5), 1247 - 51
Bosea minatitlanensis sp . nov., a strictly aerobic bacterium isolated from an anaerobic digester; Ouattara AS et al.; A strictly aerobic, mesophilic bacterium, strain AMX 51(T), was isolated from anaerobic digester sludge . Cells were Gram-negative, motile, non-sporulating, straight to curved rods with one polar flagellum . The isolate had phenotypic traits of the genus Bosea, including cellular fatty acid and substrate utilization profiles . Physiological characteristics and antibiotic susceptibility were determined . Phylogenetic analysis revealed that strain AMX 51(T) was a member of the alpha-Proteobacteria, most closely related to Bosea thiooxidans DSM 9653(T) (similarity of 98.88 %) . Methylobacterium organophilum JCM 2833(T), Methylobacterium mesophilicum JCM 2829(T), Afipia clevelandensis DSM 7315(T), Afipia felis DSM 7326(T), Afipia broomeae DSM 7327(T), Blastobacter denitrificans LMG 8443(T) and Bradyrhizobium japonicum DSM 30131(T) showed significant 16S rRNA gene sequence similarities to strain AMX 51(T) . The DNA G+C composition of strain AMX 51(T) was 68.5 mol% . DNA-DNA hybridization analysis revealed 44.2 and 15.1 % relatedness between strain AMX 51(T) and the respective type strains of Bosea thiooxidans and A . felis . Overall results suggest that strain AMX 51(T) (=DSM 13099(T)=ATCC 700918(T)=CIP 106457(T)) represents a novel species of the genus Bosea; the name Bosea minatitlanensis sp . nov . is proposed.

Biochemistry, 2003 Sep 23, 42(37), 10896 - 903
Chemical and kinetic reaction mechanisms of quinohemoprotein amine dehydrogenase from Paracoccus denitrificans; Sun D et al.; Quinohemoprotein amine dehydrogenase (QHNDH) possesses a cysteine tryptophylquinone (CTQ) prosthetic group that catalyzes the oxidative deamination of primary amines . In addition to CTQ, two heme c cofactors are present in QHNDH that mediate the transfer of the substrate-derived electrons from CTQ to an external electron acceptor . Steady-state kinetic assays yielded relatively small k(cat) values (<6 s(-1)), and the rate-limiting step appears to be the interprotein electron transfer from heme in QHNDH to the external electron acceptor . Transient kinetic studies of the CTQ-dependent reduction of heme in QHNDH by amine substrates yielded different rate constants for different substrates (72, 190, and 162 s(-1) for methylamine, butylamine, and benzylamine, respectively) . Deuterium kinetic isotope effect (KIE) values of 5.3, 3.9, and 8.5 were observed, respectively, for the reactions of methylamine, butylamine, and benzylamine . These results suggest that the abstraction of a proton from the alpha-methylene group of the substrate, which occurs concomitant with CTQ reduction, is the rate-limiting step in the CTQ-dependent reduction of hemes in QHNDH by these amine substrates . In contrast, the reaction of 2-phenylethylamine with QHNDH does not exhibit a significant KIE ((H)k(3)/(D)k(3) = 1.05) and exhibits a much smaller rate constant of 16 s(-1) . This suggests that for 2-phenylethylamine, the rate-limiting step in the single-turnover reaction is either hydrolysis of the imine reaction intermediate from CTQ or product release prior to intraprotein electron transfer . Analysis of the products of the reactions of QHNDH with chiral deuterated 2-phenylethylamines demonstrated that the enzyme abstracts the pro-S proton of the substrate in a highly stereospecific manner . Inspection of the crystal structure of phenylhydrazine-inhibited QHNDH suggests that Asp33(gamma) is the residue that performs the proton abstraction . On the basis of these results, kinetic and chemical reaction mechanisms for QHNDH are proposed and discussed in the context of the crystal structure of the enzyme.

J Biol Chem, 2003 Nov 21, 278(47), 47269 - 74 Epub 2003 Sep 11.
An engineered CuA Amicyanin capable of intermolecular electron transfer reactions; Jones LH et al.; The type I copper center of amicyanin was replaced with a binuclear CuA center . To create this model CuA protein, a portion of the amino acid sequence that contains three of the ligands to the native type I copper center of Paracoccus denitrificans amicyanin was replaced with the corresponding portion of sequence that provides five ligands for the CuA center of cytochrome c oxidase from P . denitrificans . UV-visible and electron paramagnetic resonance spectroscopy confirm that the engineered protein as isolated possesses the mixed-valence Cu1.5Cu1.5 (purple) CuA center . Comparison of the spectroscopic properties of this CuA amicyanin with those of the CuA centers of other natural and engineered CuA proteins suggests that the spectroscopic features may be dictated more by the protein host than the sequence of the CuA loop . Novel reactions for a simple CuA model protein are also described . In contrast to other natural and engineered CuA proteins, the fully reduced CuA amicyanin may be reoxidized by molecular oxygen to the mixed-valence state . It is also shown that CuA amicyanin can serve as an electron donor and an electron acceptor for other redox proteins . The mixed-valence form accepts electrons from cytochromes c-551i and c-550 from P . denitrificans . The fully reduced form donates electrons to native and P94F amicyanin . The function as either an electron donor or acceptor is consistent with the measured redox potential of CuA amicyanin of +273 mV . These data indicate that this CuA amicyanin will be a particularly useful model protein for structure-function studies of reactivity and the electron transfer properties of the CuA redox center.

Environ Toxicol Chem, 2003 Sep, 22(9), 1993 - 7
Growth and nitrite and nitrous oxide accumulation of Paracoccus denitrificans ATCC 19367 in the presence of selected pesticides; Saez F et al.; The effects of the application of eight pesticides (aldrin, lindane, dimetoate, methylparathion, methidation, atrazine, simazine, and captan) on growth, respiratory activity (as CO2 production), denitrifying activity (as N2O released), and nitrite accumulation in the culture medium by Paracoccus denitrificans strain ATCC 19367 were studied . The fungicide captan totally inhibited growth and biological activity of P . denitrificans, while the rest of the tested pesticides delayed the growth and CO2 release of P . denitrificans but did not drastically affect the bacterial growth or respiratory capacity after 96 h of culture . The denitrifying activity of P . denitrificans ATCC 19367 (as N2O released) was negatively affected by all tested pesticides . The release of N2O was strongly inhibited by several organochlorinated and organophosphorated insecticides (aldrin, lindane, dimetoate, and methidation), which led to high accumulation of nitrite in the surrounding medium . Atrazine decreased N2O release after 48 h of culture because of negative effects on growth, and methylparathion and simazine delayed the onset of N2O release by P . denitrificans . These three pesticides reduced the accumulation of NO2- compared to unamended control cultures.

Appl Environ Microbiol, 2003 Sep, 69(9), 5354 - 63
Characterization of microbial communities in gas industry pipelines; Zhu XY et al.; Culture-independent techniques, denaturing gradient gel electrophoresis (DGGE) analysis, and random cloning of 16S rRNA gene sequences amplified from community DNA were used to determine the diversity of microbial communities in gas industry pipelines . Samples obtained from natural gas pipelines were used directly for DNA extraction, inoculated into sulfate-reducing bacterium medium, or used to inoculate a reactor that simulated a natural gas pipeline environment . The variable V2-V3 (average size, 384 bp) and V3-V6 (average size, 648 bp) regions of bacterial and archaeal 16S rRNA genes, respectively, were amplified from genomic DNA isolated from nine natural gas pipeline samples and analyzed . A total of 106 bacterial 16S rDNA sequences were derived from DGGE bands, and these formed three major clusters: beta and gamma subdivisions of Proteobacteria and gram-positive bacteria . The most frequently encountered bacterial species was Comamonas denitrificans, which was not previously reported to be associated with microbial communities found in gas pipelines or with microbially influenced corrosion . The 31 archaeal 16S rDNA sequences obtained in this study were all related to those of methanogens and phylogenetically fall into three clusters: order I, Methanobacteriales; order III, Methanomicrobiales; and order IV, Methanosarcinales: Further microbial ecology studies are needed to better understand the relationship among bacterial and archaeal groups and the involvement of these groups in the process of microbially influenced corrosion in order to develop improved ways of monitoring and controlling microbially influenced corrosion.

Appl Environ Microbiol, 2003 Sep, 69(9), 5216 - 21
Biotransformation of 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane (CL-20) by denitrifying Pseudomonas sp . strain FA1; Bhushan B et al.; The microbial and enzymatic degradation of a new energetic compound, 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane (CL-20), is not well understood . Fundamental knowledge about the mechanism of microbial degradation of CL-20 is essential to allow the prediction of its fate in the environment . In the present study, a CL-20-degrading denitrifying strain capable of utilizing CL-20 as the sole nitrogen source, Pseudomonas sp . strain FA1, was isolated from a garden soil . Studies with intact cells showed that aerobic conditions were required for bacterial growth and that anaerobic conditions enhanced CL-20 biotransformation . An enzyme(s) involved in the initial biotransformation of CL-20 was shown to be membrane associated and NADH dependent, and its expression was up-regulated about 2.2-fold in CL-20-induced cells . The rates of CL-20 biotransformation by the resting cells and the membrane-enzyme preparation were 3.2 +/- 0.1 nmol h(-1) mg of cell biomass(-1) and 11.5 +/- 0.4 nmol h(-1) mg of protein(-1), respectively, under anaerobic conditions . In the membrane-enzyme-catalyzed reactions, 2.3 nitrite ions (NO(2)(-)), 1.5 molecules of nitrous oxide (N(2)O), and 1.7 molecules of formic acid (HCOOH) were produced per reacted CL-20 molecule . The membrane-enzyme preparation reduced nitrite to nitrous oxide under anaerobic conditions . A comparative study of native enzymes, deflavoenzymes, and a reconstituted enzyme(s) and their subsequent inhibition by diphenyliodonium revealed that biotransformation of CL-20 is catalyzed by a membrane-associated flavoenzyme . The latter catalyzed an oxygen-sensitive one-electron transfer reaction that caused initial N denitration of CL-20.

FEMS Microbiol Lett, 2003 Aug 29, 225(2), 263 - 9
Isolation and characterization of novel halotolerant and/or halophilic denitrifying bacteria with versatile metabolic pathways for the degradation of trimethylamine; Kim SG et al.; Four denitrifying bacteria capable of degrading trimethylamine under both aerobic and denitrifying conditions were newly isolated from coastal sediments and wastewater contaminated by marine water . All strains were in alpha-Proteobacteria . Strain GP43 was classified as a member of genus Paracoccus, and strain PH32, PH34 and GRP21 were novel organisms with remote phylogenetic position from other genus alpha-Proteobacteria . Among these four strains were the halophilic strains PH32, PH34 and GRP21, which did not grow in the absence of sodium chloride in culture medium . Cells grown under denitrifying conditions possessed trimethylamine dehydrogenase while cells grown aerobically possessed two different enzymes for oxidation of trimethylamine, trimethylamine dehydrogenase and trimethylamine monooxygenase . The newly isolated strain PH32, PH34 and GRP21 may be the first halophilic bacteria to degrade trimethylamine under denitrifying conditions.

J Mol Biol, 2003 Sep 12, 332(2), 369 - 83
Complete nucleotide sequence of pHG1: a Ralstonia eutropha H16 megaplasmid encoding key enzymes of H(2)-based ithoautotrophy and anaerobiosis; Schwartz E et al.; The self-transmissible megaplasmid pHG1 carries essential genetic information for the facultatively lithoautotrophic and facultatively anaerobic lifestyles of its host, the Gram-negative soil bacterium Ralstonia eutropha H16 . We have determined the complete nucleotide sequence of pHG1 . This megaplasmid is 452,156 bp in size and carries 429 potential genes . Groups of functionally related genes form loose clusters flanked by mobile elements . The largest functional group consists of lithoautotrophy-related genes . These include a set of 41 genes for the biosynthesis of the three previously identified hydrogenases and of a fourth, novel hydrogenase . Another large cluster carries the genetic information for denitrification . In addition to a dissimilatory nitrate reductase, both specific and global regulators were identified . Also located in the denitrification region is a set of genes for cytochrome c biosynthesis . Determinants for several enzymes involved in the mineralization of aromatic compounds were found . The genes for conjugative plasmid transfer predict that R.eutropha forms two types of pili . One of them is related to the type IV pili of pathogenic enterobacteria . pHG1 also carries an extensive "junkyard" region encompassing 17 remnants of mobile elements and 22 partial or intact genes for phage-type integrase . Among the mobile elements is a novel member of the IS5 family, in which the transposase gene is interrupted by a group II intron.

Environ Monit Assess, 2003 Sep, 87(2), 133 - 44
Monitoring the impact of dissolved oxygen and nitrite on anoxic biofilm in continuous denitrification process; Jang A et al.; An anoxic biofilm involved in continuous denitrification process was monitored to investigate the effect of different concentrations of influent dissolved oxygen (DO) or nitrite on the biofilm . Microelectrode measurements evidenced nitrate removal activity of biofilm . When different concentrations of DO were applied to the reactor, generally decreased concentrations of DO were observed as bed depth increased from the bottom of the reactor . Greatest decrease of the DO was observed in the lower 20% of the bed depth . Nitrate removal efficiency was inversely proportional to influent DO concentrations (8.3-11.9 DO mg L(-1)) or nitrite loading rates (0-5.5 N-NO2- kg m(-3) day(-1)) employed in this study . Nitrite loading rates to achieve more than 90% of nitrate removal efficiency were 1.46 N-NO2- kg m(-3) day(-1) or less at pH 7.5 and 0.34 N-NO2- kg m(-3) day(-1) or less at pH 6.8 . Nitrate removal efficiency was 63% or more within the lower 20% of the bed depth at the nitrite loading rates that allowed more than 90% of nitrate removal efficiency of the reactor . The results of this study provide first quantitative data that nitrate removal performance of an anoxic biofilm is inhibited by DO or nitrite, reported to be a limiting factor in the suspended biological denitrification process.

Ann Chim, 2003 Jul-Aug, 93(7-8), 621 - 30
Improvement of denitrification rates in confined zones of fractured subsoils under continuous wastewater injection; Carrieri C et al.; An attempt to improve the natural biodegradation rate in a fractured aquifer (Nardo (Le), Southern Italy) subject since '91 to the injection of 12,000 m3/d of treated municipal wastewater, has been carried out through tests on pilot plants . The lab experiments showed that a complete nitrogen removal can be realized after 10 d of infiltration in fractures by adding whey in the influent waste . The lab results have been used in a mathematical model in order to simulate ground water quality changes caused by the addition of whey in the injecting water . The analytical parameters of ground water sampled in monitoring wells located at different distances from the injection site, have been compared with mathematical model results . Moreover for preserving such resource from pollution, the mathematical model allowed two areas, around the injection site to be drawn . These areas, where withdrawals would be prohibited, are required to avoid infections by pathogens and bacteria in ground water due to incomplete biodegradation processes . The experimental results can be useful to identify standardized methodology for artificial ground water recharge which could be required for aquifer clean up (Water Frame Directive 2000/60).

J Environ Sci Health A Tox Hazard Subst Environ Eng, 2003 Sep, 38(9), 1703 - 15
Pilot-scale evaluation of select nitrate removal technologies; Darbi A et al.; Due to the extensive application of artificial nitrogen-based fertilizers and animal manure on land, many water agencies face problems of increasing concentrations of nitrate in groundwater . The contamination of groundwater by nitrate may pose a significant public health problem . The threat of methemoglobinemia is well documented and reflected in the US drinking water standard of 10 mg/L as nitrate-nitrogen . Approximately 45% of Saskatchewan's population use groundwater for drinking purposes, out of which, approximately 23% (230,000) are rural residents . The water used is made available from over 48,000 privately owned wells in regions where there is an extensive application of chemical fertilizers . Biological denitrification, ion exchange, and reverse osmosis (RO) processes were selected for a field study . Field studies were conducted on these processes . The sulfur/limestone autotrophic denitrification (SLAD) process was selected to achieve biological removal of nitrate from groundwater . The feasibility of the system was evaluated under anaerobic conditions . An ion exchange study was conducted using Ionac A554 which is a strong anion exchange resin . In the case of groundwater containing low sulfate concentrations, A554 offered high nitrate removal . However, the disposal of regenerant brine can be a problem . A reverse osmosis unit with Filmtec membrane elements (FT30-Element Family) was used in the study on nitrate removal . The unit effluent average nitrate concentration was less than the maximum allowable concentration.

J Biol Chem, 2003 Nov 21, 278(47), 46734 - 40 Epub 2003 Aug 23.
Different interaction modes of two cytochrome-c oxidase soluble CuA fragments with their substrates; Maneg O et al.; Cytochrome-c oxidase is the terminal enzyme in the respiratory chains of mitochondria and many bacteria and catalyzes the formation of water by reduction of dioxygen . The first step in the cytochrome oxidase reaction is the bimolecular electron transfer from cytochrome c to the homobinuclear mixed-valence CuA center of subunit II . In Thermus thermophilus a soluble cytochrome c552 acts as the electron donor to ba3 cytochrome-c oxidase, an interaction believed to be mainly hydrophobic . In Paracoccus denitrificans, electrostatic interactions appear to play a major role in the electron transfer process from the membrane-spanning cytochrome c552 . In the present study, soluble fragments of the CuA domains and their respective cytochrome c electron donors were analyzed by stopped-flow spectroscopy to further characterize the interaction modes . The forward and the reverse electron transfer reactions were studied as a function of ionic strength and temperature, in all cases yielding monoexponential time-dependent reaction profiles in either direction . From the apparent second-order rate constants, equilibrium constants were calculated, with values of 4.8 and of 0.19, for the T . thermophilus and P . denitrificans c552 and CuA couples, respectively . Ionic strength strongly affects the electron transfer reaction in P . denitrificans indicating that about five charges on the protein interfaces control the interaction, when analyzed according to the Bronsted equation, whereas in the T . thermophilus only 0.5 charges are involved . Overall the results indicate that the soluble CuA domains are excellent models for the initial electron transfer processes in cytochrome-c oxidases.

J Contam Hydrol, 2003 Sep, 65(3-4), 245 - 68
Biogeochemistry and isotope geochemistry of a landfill leachate plume; van Breukelen BM et al.; The biogeochemical processes were identified which improved the leachate composition in the flow direction of a landfill leachate plume (Banisveld, The Netherlands) . Groundwater observation wells were placed at specific locations after delineating the leachate plume using geophysical tests to map subsurface conductivity . Redox processes were determined using the distribution of solid and soluble redox species, hydrogen concentrations, concentration of dissolved gases (N(2), Ar, and CH(4)), and stable isotopes (delta15N-NO(3), delta34S-SO(4), delta13C-CH(4), delta2H-CH(4), and delta13C of dissolved organic and inorganic carbon (DOC and DIC, respectively)) . The combined application of these techniques improved the redox interpretation considerably . Dissolved organic carbon (DOC) decreased downstream in association with increasing delta13C-DOC values confirming the occurrence of degradation . Degradation of DOC was coupled to iron reduction inside the plume, while denitrification could be an important redox process at the top fringe of the plume . Stable carbon and hydrogen isotope signatures of methane indicated that methane was formed inside the landfill and not in the plume . Total gas pressure exceeded hydrostatic pressure in the plume, and methane seems subject to degassing . Quantitative proof for DOC degradation under iron-reducing conditions could only be obtained if the geochemical processes cation exchange and precipitation of carbonate minerals (siderite and calcite) were considered and incorporated in an inverse geochemical model of the plume . Simulation of delta13C-DIC confirmed that precipitation of carbonate minerals happened.

Water Environ Res, 2003 Jul-Aug, 75(4), 355 - 67
Engineered bioretention for removal of nitrate from stormwater runoff; Kim H et al.; A bioretention unit is a simple, plant- and soil-based, low-impact treatment and infiltration facility for treating stormwater runoff in developed areas . Nitrate, however, is not attenuated in conventional bioretention facilities . Thus, this study systematically evaluated a reengineered concept of bioretention for nitrate removal via microbial denitrification, which incorporates a continuously submerged anoxic zone with an overdrain . Experimental studies were performed in four phases . In the first two phases, column studies demonstrated that, overall, newspaper is the best solid-phase electron-donor substrate for denitrification out of the set studied (alfalfa, leaf mulch compost, newspaper, sawdust, wheat straw, wood chips, and elemental sulfur) based on superior nitrate removal and effluent water quality . The nitrate loading and hydraulic loading studies in the second phase provided design information . In the third phase, system viability after 30- and 84-day dormant periods was evaluated in column studies, demonstrating that newspaper-supported biological denitrification should be effective under conditions of intermittent loadings . Finally, in the fourth phase, pilot-scale bioretention studies demonstrated the effectiveness of the proposed design, showing nitrate plus nitrite mass removals of up to 80% . These results indicate that engineered bioretention for the removal of nitrogen from stormwater runoff has the potential for successful application as an urban stormwater treatment practice.

J Environ Qual, 2003 Jul-Aug, 32(4), 1474 - 80
Electron affinity coefficients of nitrogen oxides and biodegradation kinetics in denitrification of contaminated stream water; Kim SH et al.; During the dry season in Korea, rivers become more vulnerable to contamination by biochemical oxygen demand (BOD) and nitrogen . It is hypothesized that the natural characteristics of the streams in Korea allow the contaminated water to be treated at the tributaries . Down-stream river water quality in Korea may be improved by spraying the contaminated stream water from the tributaries over the surrounding floodplains . The consequent water filtration through the soil could remove the contaminants through aerobic and denitrifying reactions . In this study, the kinetics parameters of the denitrifying reaction in floodplain filtration were determined using contaminated stream water . For the electron donor the Monod kinetics was used, while the competitive Michaelis-Menten model was employed for the electron acceptors . The parameters to the competitive Michaelis-Menten model were found using continuous denitrifying reactions, instead of the batch reactions employed in previous studies, to match the conditions needed to apply the competitive Michaelis-Menten kinetics . From the result, it was found that continuous reactions as well as batch reactions could be used to determine the affinity coefficients in denitrification . The results of this study also showed that the affinity coefficient of NO2, using continuous reactions, was similar to that of other studies in the literature found via batch reactions, whereas the affinity coefficient of N2O was much larger than that acquired with batch reactions . The parameters obtained in this study will be used in future work to simulate the contaminant behaviors during floodplain filtration using a mathematical model.

J Environ Sci Health A Tox Hazard Subst Environ Eng, 2003 Aug, 38(8), 1489 - 97
The effect of nitrate and different substrates on enhanced biological phosphorus removal in sequencing batch reactors; Yagci NO et al.; Enhanced biological phosphorus removal (EBPR) requires an anaerobic-aerobic sequence and short chain fatty acids, namely acetate . It is also known that the presence of nitrate in the anaerobic phase inhibits EBPR . This study describes a lab-scale experimentation carried out to study the effect of different substrates on EBPR and behaviour of PAOs under anoxic conditions in a sequencing batch reactor operated using synthetic wastewater . Experimental data show that the EBPR performance is significantly affected by glucose rich influent . Low COD/TKN ratios caused lower phosphorus removal performance since nitrate entering the anaerobic zone consumes substrate for denitrification . The results also show that anoxic phosphate uptake took place together with nitrate reduction when there was no external substrate . However, the uptake rate under anoxic conditions was lower than that under aerobic conditions.

Water Sci Technol, 2003, 47(12), 141 - 8
Optimisation of Noosa BNR plant to improve performance and reduce operating costs; Thomas M et al.; Noosa WWTP is publicly owned and privately operated by Australian Water Services . The process includes primary sedimentation, raw sludge fermentation, biological nutrient removal (BNR), sand filtration and ultraviolet (UV) disinfection . An innovative feature of the plant is the supplementary carbon dosing facility to avoid the use of metal salts (alum or ferric) for phosphorus removal . The average flow treated during 2000 was 9.0 ML/d . The annual 50 percentile effluent quality requirements for nutrients are total N < 5 mg/L and total P < 1 mg/L . The objectives of this project were to: determine the cause of variability in phosphorus removal; develop a strategy to control the variability in phosphorus removal; and minimise the operating cost of supplementary carbon dosing while achieving the effluent quality requirements . An investigation of chemical and microbiological parameters was implemented and it was concluded that there were several factors causing variability in phosphorus removal, rather than a single cause . The following four major causes were identified, and the control strategies that were adopted resulted in the plant achieving annual 50 percentile effluent total P = 0.37 mg/L and total N = 3.0 mg/L during 2001 . First, phosphorus removal was limited by the available VFA supply due to consumption of VFA by other organisms competing with phosphate accumulating organisms (PAO), and due to diurnal variations in the sewage VFA and phosphate concentrations . Therefore, supplementary carbon dosing was essential to make allowance for competing reactions . Second, increasing the fermenter VFA yield via supplementary carbon dosing with molasses was found to be an effective and economic way of ensuring reliable phosphorus removal . Third, nitrate in the RAS resulted in consumption of VFA by denitrifying bacteria, particularly with process configurations where the RAS was recycled directly into the anaerobic zone . Incorporating a RAS denitrification zone into the process rectified this problem . Finally, glycogen accumulating organisms (GAO) were observed in BNR sludge samples, and consumption of VFA by GAO appeared to cause decreased phosphorus removal . Better phosphorus removal was obtained using VFA derived from the fermenter than dosing an equivalent amount of acetic acid . It was hypothesized that GAO have a competitive advantage to use acetate and PAO have a competitive advantage to use propionate, butyrate or some other soluble COD compound in the fermenter effluent . Contrary to popular belief, acetate may not be the optimum VFA for biological phosphorus removal . The competition between PAO and GAO for different VFA species under anaerobic conditions requires further investigation in order to control the growth of GAO and thereby improve reliability of biological phosphorus removal processes.

Water Sci Technol, 2003, 47(12), 81 - 8
Coarse media filtration--an alternative to settling in wastewater treatment; Odegaard H et al.; In this paper coarse media filtration has been analysed as an alternative to the traditional settling in primary, secondary and tertiary treatment of wastewater . Various filter media configurations were evaluated for enhanced primary filtration . It was found that a dual media configuration based on Kaldnes biofilm media (1 and K2modified) were most suitable when taking both separation efficiency as well as filter run time into consideration . SS removal efficiencies of around 75% were achieved in the dual Kaldnes primary filter at around 20 m/h without any chemical addition and around 85% at low dosage (1-2 mg/l) of a high MW cationic polymer FO4440SH . In the latter case COD was removed by around 70% . Further experiments were carried out on a multi-media Kaldnes-Filtralite-Sand (KFS) filter for enhanced primary treatment as well as for secondary filtration directly downstream of a high-rate moving bed biofilm reactor, resulting in an extremely compact secondary treatment process . The secondary KFS-filter gave SS-removal efficiencies around 90% (effluent SS < 15 mg/l) and filter run times of around 24 hrs at filtration rates of 10 m/h (sludge loading rates of around 1 kg/m2h) when a small dose (2 mg/l) of polymer was used . It is also demonstrated that the primary filter may also be utilised as a pre-denitrification reactor . A denitrification rate of 1.5 kg NO3-N(equiv.)/m3d was achieved when the filter was operated at a filtration rate of 5 m/h.

Water Sci Technol, 2003, 48(1), 95 - 102
Improved nutrient removal using in situ continuous on-line sensors with short response time; Ingildsen P et al.; Nutrient sensors that can be located directly in the activated sludge processes are gaining in number at wastewater treatment plants . The in situ location of the sensors means that they can be located close to the processes that they aim to control and hence are perfectly suited for automatic process control . Compared to the location of automatic analysers in the effluent from the sedimentation reactors the in situ location means a large reduction in the response time . The settlers typically work as a first-order delay on the signal with a retention time in the range of 4-12 hours depending on the size of the settlers . Automatic process control of the nitrogen and phosphorus removal processes means that considerable improvements in the performance of aeration, internal recirculation, carbon dosage and phosphate precipitation dosage can be reached by using a simple control structure as well as simple PID controllers . The performance improvements can be seen in decreased energy and chemicals consumption and less variation in effluent concentrations of ammonium, total nitrogen and phosphate . Simple control schemes are demonstrated for the pre-denitrification and the post precipitation system by means of full-scale plant experiments and model simulations.

Water Sci Technol, 2003, 48(1), 87 - 94
Enhanced biological phosphorus removal process implemented in membrane bioreactors to improve phosphorous recovery and recycling; Lesjean B et al.; The enhanced biological phosphorus removal (EBPR) process was adapted to membrane bioreactor (MBR) technology . One bench-scale plant (BSP, 200-250 L) and two pilot plants (PPs, 1,000-3,000 L each) were operated under several configurations, including pre-denitrification and post-denitrification without addition of carbon source, and two solid retention times (SRT) of 15 and 26 d . The trials showed that efficient Bio-P removal can be achieved with MBR systems, in both pre- and post-denitrification configurations . EBPR dynamics could be clearly demonstrated through batch-tests, on-line measurements, profile analyses, P-spiking trials, and mass balances . High P-removal performances were achieved even with high SRT of 26 d, as around 9 mgP/L could be reliably removed . After stabilisation, the sludge exhibited phosphorus contents of around 2.4%TS . When spiked with phosphorus (no P-limitation), P-content could increase up to 6%TS . The sludge is therefore well suited to agricultural reuse with important fertilising values . Theoretical calculations showed that increased sludge age should result in a greater P-content . This could not be clearly demonstrated by the trials . This effect should be all the more significant as the influent is low in suspended solids.

Water Sci Technol, 2003, 48(1), 77 - 85
A proposed sustainable BNR plant with the emphasis on recovery of COD and phosphate; Hao XD et al.; Water problems have to be solved in an integrated way, and sustainability has become a major issue . For this reason, developing more sustainable wastewater treatment processes is needed . New discoveries and good understanding on microbial conversions of nitrogen and phosphorus make more sustainable processes possible . New options for decentralized sustainable sanitation are generally compared to conventional sewage systems, we think that for a proper comparison also innovative centralized treatment schemes should be evaluated . In this article, a more sustainable WWTP is proposed for municipal wastewater treatment, mainly based on the principles of denitrifying dephosphatation and anaerobic ammonium oxidation (ANAMMOX) . The proposed system consists of a first stage of the A/B process in which maximal sludge production is achieved . In this way, COD is regained as sludge for methanation . The following BCFS and CANON processes can remove N and P with minimal or no COD need . As a potential fertiliser, struvite can easily be removed from the sludge water by adding magnesium compounds . A case study is done on the basis of the mass balance over the proposed plant . The effluent from the system has a good quality to be recycled . This could also make a contribution to meeting the world's water needs and lessening the impact on the world's water environment . Since all the separate units are already applied or tested on pilot-scale, no problems for technical implementation are foreseen.

Water Sci Technol, 2003, 48(1), 37 - 46
Nutrients in urine: energetic aspects of removal and recovery; Maurer M et al.; The analysis of different removal and recovery techniques for nutrients in urine shows that in many cases recovery is energetically more efficient than removal and new-production from natural resources . Considering only the running electricity and fossil energy requirements for the traditional way of wastewater treatment and fertiliser production, the following specific energy requirements can be calculated: 45 MJ kg(-1)N for denitrification in a WWTP, 49 MJ kg(-1)p for P-precipitation in a WWTP, 45 MJ kg(-1)N for N-fertiliser and 29 MJ kg(-1)p for P-fertiliser production . These numbers are higher than the values derived for thermal volume reduction of urine (35 MJ kg(-1)N for eliminating 90% water) or production of struvite (102 MJ kg(-1)N, including 2.2 kg P) . Considering only the electricity and fossil energy for the traditional way of wastewater treatment and fertiliser production, the energy value of 1 PE urine is 0.87 MJ PE(-1) d(-1) (fertiliser value: 0.44, wastewater treatment: 0.43 MJ PE(-1) d(-1)) . A more detailed life cycle assessment (LCA) of the entire urine collection system, including the required materials and the environmental burden, support the energy analysis . The LCA compares conventional denitrification in a wastewater treatment plant with collecting urine in households, reducing the volume by evaporation and using it as a multi-nutrient fertiliser . The primary energy consumption for recovery and reuse of urine, including the nutrients N, P and K, is calculated with 65 MJ kg(-1)N, compared with 153 MJ kg(-1)N derived for the conventional 'recycling over the atmosphere'.

Acta Crystallogr D Biol Crystallogr, 2003 Sep, 59(Pt 9), 1551 - 6 Epub 2003 Aug 19.
Structure of the phenylhydrazine adduct of the quinohemoprotein amine dehydrogenase from Paracoccus denitrificans at 1.7 A resolution; Datta S et al.; The 109 kDa quinohemoprotein amine dehydrogenase (QHNDH) from Paracoccus denitrificans contains a novel redox cofactor, cysteine tryptophylquinone (CTQ) . This cofactor is derived from a pair of gene-encoded amino acids by post-translational modification and was previously identified and characterized within an 82-residue subunit by chemical methods and crystallographic analysis at 2.05 A resolution . It contains an orthoquinone moiety bound to the indole ring and catalyzes the oxidation of aliphatic and aromatic amines through formation of a Schiff-base intermediate involving one of the quinone O atoms . This paper reports the structural analysis of the complex of QHNDH with the enzyme inhibitor phenylhydrazine determined at 1.70 A resolution . The phenylhydrazone product is attached to the C6 position, identifying the O6 atom of CTQ as the site of Schiff-base formation as postulated by analogy to another amine-oxidizing enzyme, methylamine dehydrogenase . Furthermore, the inner N atom closest to the phenyl ring of phenylhydrazine forms a hydrogen bond to gammaAsp33 in the complex, lending support to the hypothesis that this residue serves as the active-site base for proton abstraction during catalysis.

J Environ Sci Health A Tox Hazard Subst Environ Eng, 2003 Jul, 38(7), 1269 - 84
Comparison of two combined bioelectrochemical and sulfur autotrophic denitrification processes for drinking water treatment; Wang H et al.; Two combined bioelectrochemical and sulfur autotrophic denitrification (CBSAD) processes for the treatment of nitrate contaminated drinking water were studied in this article, the main difference between the two processes was whether the limestone was packed in the reactor . In these processes, the sulfur denitrification was carried out in the lower part (Sulfur Part) of the reactors while the bioelectrochemical hydrogen denitrification in the upper part (Bioelectrochemical Part) . Sulfur Part of one reactor was packed with elemental sulfur and limestone while no limestone was packed in Sulfur Part of the other, the former reactor is referred to as RSL and the latter as RS . The denitrification results of the two reactors were compared under different conditions, from which it can be concluded that the minimum current of RSL was about 2 mA higher than that of RS . However, at the same hydraulic retention time (HRT) and minimum current, the nitrate removal of both reactors was higher than 90% while no nitrite was accumulated in the effluent . Ca2+ concentration in Sulfur Part effluent of RSL was increased because of the packed limestone, which led to the requirement of Ca2+ removal in Bioelectrochemical Part . The effluent sulfate concentration of RSL was higher than that of RS . When current was lower than 3 mA, the effluent pH value of RSL was about 0.6 higher than that of RS . However, the effluent pH of two parts of both reactors was about neutral under optimum operation conditions . The optimum operation condition of RSL was 1.9-4h HRT under 1.5-14 mA minimum current, while that of RSL was 1.9-5 h under 3-16.5 mA . The effluent quality of RS was better than that of RSL.

Huan Jing Ke Xue, 2003 May, 24(3), 126 - 31
{Nitrogen transformation during co-composting of flower wastes and cattle manure}; Zhang X et al.; Nitrogen transformation during co-composting of flower wastes and cattle manure were studied in pilot scale . The first stage was aerobic static bed of composting based on temperature feedback and control via aeration rate regulation, which lasted about 20d . The second stage was window composting, with a period of about 40d . Changes of composting of total nitrogen (TN), organic nitrogen, inorganic nitrogen, NH4(+)-N and NO3(-)-N were studied . Results show that ammoniation and denitrifcation were serious at the beginning of first stage . The total nitrogen losses was about 41.98% during 60 days of composting, most of which was losses of organic nitrogen contributing about 99.95% at first stage . Losses of N were governed mainly by volatilization of ammonia as the pile temperatures and pH values were high . It is hoping to reduce nitrogen losses by effective aeration rate regulation, improving C/N of raw materials and addition of acidic amendment . On the part of material of low C/N and high nitrate, it is not appropriate for NH4(+)-N < or = 0.04% and NH4/NO3 < or = 0.16 as maturity indicators.

FEBS Lett, 2003 Aug 14, 549(1-3), 39 - 42
Unidirectional effect of lauryl sulfate on the reversible NADH:ubiquinone oxidoreductase (Complex I); Grivennikova VG et al.; Lauryl sulfate inhibits the Deltamu;(H)(+)-dependent reverse electron transfer reactions catalyzed by NADH:ubiquinone oxidoreductase (Complex I) in coupled bovine heart submitochondrial particles and in vesicles derived from Paracoccus denitrificans . The inhibitor affects neither NADH oxidase (coupled or uncoupled) nor NADH:ferricyanide reductase and succinate oxidase activities at the concentrations that selectively prevent the succinate-supported, rotenone-sensitive NAD(+) or ferricyanide reduction . Possible uncoupling effects of the inhibitor are ruled out: in contrast to oligomycin and gramicidin, which increases and decreases the rate of the reverse electron transfer, respectively, in parallel with their coupling and uncoupling effects, lauryl sulfate does not affect the respiratory control ratio . A mechanistic model for the unidirectional effect of lauryl sulfate on the Complex I catalyzed oxidoreduction is proposed.

Biophys Chem, 2003 Jul 1, 104(3), 617 - 22
Steady-state kinetic analysis of substrate pair cycling between two enzymes: application to a mediated electron transport between the cytoplasmic membrane and the periplasmic nitrite reductase of Paracoccus denitrificans; Kucera I et al.; An extended kinetic model is presented for the process catalysed by two enzymes mutually connected by the cycling of two reversibly interconvertible chemically relative species . Expressions are derived for the steady-state velocity, limiting velocity (V) and the half-saturation concentration of the cycling substrate (A(0.5)) . It is shown that the velocity depends on the total concentration of cycling substrate hyperbolically if both enzymes have equal activities . Based on these theoretical considerations, an experimental comparison was made between pseudoazurin and cytochrome c(550) as physiological electron transfer mediators for nitrite reduction in an in vitro reconstituted part of the respiratory chain of Paracoccus denitrificans . Pseudoazurin exhibited 1.7-fold higher V and 14-fold higher A(0.5) than cytochrome c(550) under the experimental conditions used (20 mM Tris chloride, pH 7.3, 30 degrees C).

Water Res, 2003 Sep, 37(16), 4011 - 7
Quantification of denitrification potential in carbonaceous trickling filters; Biesterfeld S et al.; Biofilm samples from a carbonaceous trickling filter (TF) were evaluated in bench scale reactors to determine their maximum potential denitrification rates . Intact, undisturbed biofilms were placed into 0.6 L bench-scale reactors filled with sterilized, primary clarifier effluent spiked with nitrate to a final concentration of 16-18 mg/L as N . Dissolved oxygen concentrations were maintained between 2 and 4 mg/L in the bulk aqueous phase . Nitrate loss from the reactors was monitored over a 5h period . Denitrification rates of 3.09-5.55 g-N/m(2)day were observed with no initial lag period . This suggests that the capacity for denitrification is inherent in the biofilm and that denitrification can take place even when oxygen is present in the bulk aqueous phase . There were no significant differences in denitrification rates per unit area of media (g-N/m(2)day) either between (a) . experimental runs or (b) . sampling locations over the trickling filter . This suggests that denitrification potentials are uniform over the entire volume of the full-scale TF . For wastewater treatment plants with TFs that currently nitrify downstream, this approach may be used to meet less stringent permitted discharge concentrations and may allow some facilities to postpone or eliminate construction of additional unit processes for denitrification.

Water Sci Technol, 2003, 47(11), 203 - 9
The effect of low sludge age on wastewater fractionation (S(S), S(I)); Haider S et al.; In lab-scale experiments at the 2-stage activated sludge pilot plant of Vienna's central WWTP it is shown that the wastewater soluble COD concentration, which is inert to a sludge with SRT < 1 d (SI(A)) is about double compared to the S(I) concentration in sludge with SRT > 10 d (SI(B)) . Unexpectedly the ratio of SI(A)/SI(B) is independent of the sludge age between SRTs of 0.4 and 1.0 days . The difference between the two S(I) fractions is soluble COD that is readily biodegradable by the sludge with SRT > 10 d . However, it is degraded at a lower maximum growth rate . These results comply with earlier results gained with different methods and at different WWTPs . It is hypothesised that very low sludge ages result in a selection of fast growing bacteria, which can utilise only part of the S(S) in the raw wastewater . The other part of S(S) therefore remains in the wastewater and can thus be utilised for enhanced denitrification in the second stage . It is still unknown beyond which sludge age the soluble inert COD SI(A) starts to decrease, finally reaching the value SI(B) for low loaded systems (SRT > 5 days) . From this point on S(I) and S(S) are assumed only to depend on the wastewater composition and not on the sludge age . The assumption of the Activated Sludge Model No.1 that the biodegradable fractions can be modelled as a single substrate and by a single removal kinetic (one Monod term) appears not to be applicable for low sludge ages . Some suggestions for mathematical modelling, design and operation of 2-stage activated sludge systems are given.

Water Sci Technol, 2003, 47(11), 109 - 14
Controlling nitrogen removal using redox and ammonium sensors; Cecil D; At the Ejby Molle WWTP (300,000 PE) operated by the Odense Water Ltd . a system of aeration control which combines ammonium concentration and redox potential has proven itself as a method of reducing nitrogen levels in the effluent . The nitrogen removal process at this plant proceeds in parallel aeration tanks that alternate between periods of aeration and denitrification . At the same time influent is redirected from one tank to the other such that influent and effluent is primarily to and from the tank where denitrification is going on . The aeration stops and the tanks open and close when the ammonium concentration reaches its set point . Aeration is restarted again when redox potential drops to its low set point or when ammonium reaches its upper set point . The average total nitrogen concentration in the effluent from the Ejby Molle WWTP went from 4.7 down to 2.1 mgN/l after this control system was implemented . Not all of this reduction can be explained by better control . However we believe that at least 1 mgN/L of the reduction and probably more was the result of the control system based on the combination of ammonium and redox sensors.

Water Sci Technol, 2003, 47(11), 53 - 9
Enhanced nitrogen removal in SBRs bypassing nitrate generation accomplished by multiple aerobic/anoxic phase pairs; Katsogiannis AN et al.; A lab-scale SBR was used for the study of nitrogen removal from a synthetic wastewater with an ammonium-nitrogen concentration of 50 mg/L . The react phase of the reactor operation was divided into three sets of consecutive aerobic and anoxic periods with a duration ratio of 1:3 (20 min aerobic and 1 h anoxic phase) . Under these operating conditions, nitrogen removal was achieved via nitrite i.e . no nitratification (oxidation of nitrite to nitrate) and hence no denitratification (reduction of nitrate to nitrite) was taking place in the aerobic and anoxic phase, respectively . This was attributed to the suppression of the nitrite-oxidizers activity due to the short aerobic phase duration . This presumption was supported by the ever decreasing amount of nitrate-nitrogen generated in the react phase during the transient, even when the activated sludge of the reactor was supplemented with additional nitrite-oxidizers . On the other hand, denitrification was mainly based on stored carbon sources, as long as the organic carbon (provided in the form of acetate) was never accumulated during the anoxic/anaerobic fill phase of the reactor operation.

Water Sci Technol, 2003, 47(11), 9 - 15
Evaluation of influent prefermentation as a unit process upon biological nutrient removal; McCue T et al.; The objective of this NSF sponsored research was to provide a controlled comparison of identical continuous flow biological nutrient removal (BNR) processes both with and without prefermentation in order to provide a stronger, more quantitative, technical basis for design engineers to determine the potential benefits of prefermentation to EBPR in treating domestic wastewater . Specifically, this paper focused upon the potential impacts of primary influent prefermentation upon BNR processes treating septic domestic wastewater . This study can be divided into two distinct phases--an initial bench-scale phase which treated septic P-limited (TCOD:TP>40) wastewater and a subsequent pilot-scale phase which treated septic COD-limited (TCOD:TP<40) wastewater . The following conclusions can be drawn from the results obtained to date . Prefermentation increased both RBCOD, SBCOD and VFA content of septic domestic wastewater . Prefermentation resulted in increased biological P removal for a highly septic, non-P limited (TCOD:TP<40:1) wastewater . However, in septic, P-limited (TCOD:TP>40:1) wastewater, changes in net P removal due to prefermentation were suppressed by limited P availability, even though P release and PHA content were affected . Prefermentation increased specific anoxic denitrification rates for both COD and P-limited wastewaters, and in the pilot (COD-limited) study also coincided with greater system N removal.

Microbiology, 2003 Aug, 149(Pt 8), 1971 - 9
Physiological role of S-formylglutathione hydrolase in C(1) metabolism of the methylotrophic yeast Candida boidinii; Yurimoto H et al.; The methylotrophic yeast Candida boidinii exhibits S-formylglutathione hydrolase activity (FGH, EC 3.1.2.12), which is involved in the glutathione-dependent formaldehyde oxidation pathway during growth on methanol as the sole carbon source . The structural gene, FGH1, was cloned from C . boidinii, and its predicted amino acid sequence showed more than 60 % similarity to those of FGHs from Paracoccus denitrificans and Saccharomyces cerevisiae, and human esterase D . FGH from C . boidinii contained a C-terminal tripeptide, SKL, which is a type I peroxisome-targeting signal, and a bimodal distribution of FGH between peroxisomes and the cytosol was demonstrated . The FGH1 gene was disrupted in the C . boidinii genome by one-step gene disruption . The fgh1Delta strain was still able to grow on methanol as a carbon source under methanol-limited chemostat conditions with low dilution rates (D<0.05 h(-1)), conditions under which a strain with disruption of the gene for formaldehyde dehydrogenase (another enzyme involved in the formaldehyde oxidation pathway) could not survive . These results suggested that FGH is not essential but necessary for optimal growth on methanol . This is believed to be the first report of detailed analyses of the FGH1 gene in a methylotrophic yeast strain.

Mikrobiologiia, 2003 May-Jun, 72(3), 370 - 3
{The reduction of Cr(VI) by bacteria of the genus Pseudomonas}; Dmitrienko GN et al.; Non-nitrate-reducing collection bacteria from the genus Pseudomonas were found to be able to use hexavalent chromium as a terminal electron acceptor . The reduction of Cr(VI) was accompanied by an increase in the cell biomass . At the Cr(VI) concentrations in the medium lower than 15 mg/l, the non-nitrate-reducing pseudomonads reduced Cr(VI) less efficiently than did denitrifying pseudomonads . In contrast, at the Cr(VI) concentrations higher than 30 mg/l, Cr(VI) was reduced more efficiently by the non-nitrate-reducing pseudomonads than by the denitrifying pseudomonads.

FEMS Microbiol Lett, 2003 Aug 8, 225(1), 161 - 5
Chloride dependence of growth in bacteria; Roessler M et al.; Chloride is an abundant anion on earth but studies analyzing a possible function of chloride in prokaryotes are scarce . To address the question, we have tested 44 different Gram-negative and Gram-positive bacteria for a chloride dependence or chloride stimulation of growth . None required chloride for growth at their optimal growth (salt) conditions . However, in hyperosmotic media containing high concentrations of Na+, 11 bacteria (Aeromonas hydrophila, Bacillus megaterium, Bacillus subtilis, Corynebacterium glutamicum, Escherichia coli, Paracoccus denitrificans, Proteus mirabilis, Proteus vulgaris, Staphylococcus aureus, Thermus thermophilus, and Vibrio fischeri) had a strict chloride dependence for growth or were significantly stimulated by chloride . These data show that chloride is essential for growth at high salt (Na+) concentrations in various species of the domain Bacteria.

J Bacteriol, 2003 Aug, 185(16), 4920 - 9
Benzoate-coenzyme A ligase from Thauera aromatica: an enzyme acting in anaerobic and aerobic pathways; Schuhle K et al.; In the denitrifying member of the beta-Proteobacteria Thauera aromatica, the anaerobic metabolism of aromatic acids such as benzoate or 2-aminobenzoate is initiated by the formation of the coenzyme A (CoA) thioester, benzoyl-CoA and 2-aminobenzoyl-CoA, respectively . Both aromatic substrates were transformed to the acyl-CoA intermediate by a single CoA ligase (AMP forming) that preferentially acted on benzoate . This benzoate-CoA ligase was purified and characterized as a 57-kDa monomeric protein . Based on V(max)/K(m), the specificity constant for 2-aminobenzoate was 15 times lower than that for benzoate; this may be the reason for the slower growth on 2-aminobenzoate . The benzoate-CoA ligase gene was cloned and sequenced and was found not to be part of the gene cluster encoding the general benzoyl-CoA pathway of anaerobic aromatic metabolism . Rather, it was located in a cluster of genes coding for a novel aerobic benzoate oxidation pathway . In line with this finding, the same CoA ligase was induced during aerobic growth with benzoate . A deletion mutant not only was unable to grow anaerobically on benzoate or 2-aminobenzoate, but also aerobic growth on benzoate was affected . This suggests that benzoate induces a single benzoate-CoA ligase . The product of benzoate activation, benzoyl-CoA, then acts as inducer of separate anaerobic or aerobic pathways of benzoyl-CoA, depending on whether oxygen is lacking or present.

Biotechnol Prog, 2003 Jul-Aug, 19(4), 1323 - 8
Biological reduction of nitric oxide in aqueous Fe(II)EDTA solutions; van der Maas P et al.; The reduction of nitric oxide (NO) in aqueous solutions of Fe(II)EDTA is one of the core processes in BioDeNOx, an integrated physicochemical and biological technique for NO(x)() removal from industrial flue gases . NO reduction in aqueous solutions of Fe(II)EDTA (20-25 mM, pH 7.2 +/- 0.2) was investigated in batch experiments at 55 degrees C . Reduction of NO to N(2) was found to be biologically catalyzed with nitrous oxide (N(2)O) as an intermediate . Various sludges from full-scale denitrifying and anaerobic reactors were capable to catalyze NO reduction under thermophilic conditions . The NO reduction rate was not affected by the presence of ethanol or acetate . EDTA-chelated Fe(II) was found to be a suitable electron donor for the biological reduction of nitric oxide to N(2), with the concomitant formation of Fe(III)EDTA . In the presence of ethanol, EDTA-chelated Fe(III) was reduced to Fe(II)EDTA . This study strongly indicates that redox cycling of FeEDTA plays an important role in the biological denitrification process within the BioDeNOx concept.

Int J Syst Evol Microbiol, 2003 Jul, 53(Pt 4), 1085 - 91
Sterolibacterium denitrificans gen . nov., sp . nov., a novel cholesterol-oxidizing, denitrifying member of the beta-Proteobacteria; Tarlera S et al.; A bacterial strain (Chol-1S(T)) that is able to oxidize cholesterol to CO2 and reduce nitrate to dinitrogen was enriched and isolated from an upflow sludge bed (USB) anoxic reactor that treats sanitary landfill leachate from the city of Montevideo, Uruguay . Cells of strain Chol-1S(T) were gram-negative, rod-shaped to slightly curved, measured 0.5-0.6 x 1.0-1.3 microm and were motile by a single polar flagellum . Strain Chol-1S(T) grew optimally at 30-32 degrees C and pH 7.0, with a doubling time of 44-46 h when cholesterol was used as the sole carbon and energy source . The metabolism of strain Chol-1S(T) was strictly respiratory, with oxygen or nitrate as the terminal electron acceptor . The presence of ubiquinone Q-8 as the sole respiratory lipoquinone indicated that strain Chol-1S(T) belonged to the beta-subclass of the Proteobacteria . Phosphatidylethanolamine was the predominant polar lipid and the G + C content of the DNA was 65.3 mol% . The fatty acid profile of strain Chol-1S(T), cultivated under denitrifying conditions by using a defined mineral medium supplemented with cholesterol, was characterized by the following major components: summed feature 4 (C16:1 omega7c and/or iso C15:0 2-OH), C16:0, C18:1 omega7c and hydroxy acid C10:0 3-OH . Minor components included C10:0, C11:0, C12:0, C14:0, C15:0, C19:0, C19:0 10-methyl and hydroxylated acids C8:0 3-OH and C16:0 3-OH . Analysis of the 16S rDNA sequence showed that strain Chol-1S(T) represents a separate lineage within the Thauera, Azoarcus, Zoogloea and Rhodocyclus assemblage of the beta-Proteobacteria . Strain Chol-1S(T) had highest sequence similarity (96.5%) with strain 72Chol, a denitrifying beta-Proteobacterium . On the basis of polyphasic evidence, strain Chol-1S(T) (=DSM 13999T=ATCC BAA-354T) is proposed as the type strain of Sterolibacterium denitrificans gen . nov., sp . nov.

Environ Monit Assess, 2003 Sep, 87(1), 1 - 31
Evaluation of ground water denitrification at a biosolids disposal site; Oertel AO et al.; A study was conducted at a sanitary sewage sludge (biosolids) disposal site in Springfield, Illinois, U.S.A . to determine if biological denitrification played a significant factor in attenuation of ground water nitrate values . The site selected for this study is a 23 ha (57 acre) dedicated biosolids disposal facility located adjacent to a 75.7 million liter per day (20 million gallons per day) municipal treatment plant that uses anaerobic solids stabilization for treatment of generated biosolids material . Biosolids have been disposed of by fixed-point spray applicators at the site since 1976, which has caused ground water nitrate levels to increase significantly above background levels . A method was developed using a conservative chemical tracer to simulate the biosolids application process and monitor the ground water directly beneath the simulated disposal site . Results demonstrated a net decline of nitrates that could not be attributed to dilution alone . While the monitoring methodology developed for this study did not directly estimate the denitrification rate, a rate for overall nitrate reduction was calculated that could be considered to take into account all transport and reduction mechanisms such as denitrification, advection, dispersion and dilution.

Biotechnol Bioeng, 2003 Sep 20, 83(6), 627 - 37
Cometabolism of Cr(VI) by Shewanella oneidensis MR-1 produces cell-associated reduced chromium and inhibits growth; Middleton SS et al.; Microbial reduction is a promising strategy for chromium remediation, but the effects of competing electron acceptors are still poorly understood . We investigated chromate (Cr(VI)) reduction in batch cultures of Shewanella oneidensis MR-1 under aerobic and denitrifying conditions and in the absence of an additional electron acceptor . Growth and Cr(VI) removal patterns suggested a cometabolic reduction; in the absence of nitrate or oxygen, MR-1 reduced Cr(VI), but without any increase in viable cell counts and rates gradually decreased when cells were respiked . Only a small fraction (1.6%) of the electrons from lactate were transferred to Cr(VI) . The 48-h transformation capacity (Tc) was 0.78 mg (15 micromoles) Cr(VI) reduced . {mg protein}(-1) for high levels of Cr(VI) added as a single spike . For low levels of Cr(VI) added sequentially, Tc increased to 3.33 mg (64 micromoles) Cr(VI) reduced . {mg protein}(-1), indicating that it is limited by toxicity at higher concentrations . During denitrification and aerobic growth, MR-1 reduced Cr(VI), with much faster rates under denitrifying conditions . Cr(VI) had no effect on nitrate reduction at 6 microM, was strongly inhibitory at 45 microM, and stopped nitrate reduction above 200 microM . Cr(VI) had no effect on aerobic growth at 60 microM, but severely inhibited growth above 150 microM . A factor that likely plays a role in Cr(VI) toxicity is intracellular reduced chromium . Transmission electron microscopy (TEM) and electron energy loss spectroscopy (EELS) of denitrifying cells exposed to Cr(VI) showed reduced chromium precipitates both extracellularly on the cell surface and, for the first time, as electron-dense round globules inside cells .

C R Biol, 2003 Apr, 326(4), 391 - 9
Effect of organic and mineral fertilizers on N-use by wheat under different irrigation frequencies; Ichir LL et al.; A field trial was established in Errachidia, southern Morocco, to investigate the interaction between wheat residue management and mineral 15N-labelled ammonium sulphate, under different irrigation treatments, applied to wheat (Triticum durum var . Karim) . In treatments I1, I2, I3 and I4, plots were irrigated every 10, 15, 21 and 30 days . Each plot contained three sub-plots that received three fertilization treatments: T1 received 42 kg N ha-1 of ammonium sulphate before seedling, 42 kg N ha-1 of ammonium sulphate labelled with 9.764 at % 15N excess at tillering and 84 N kg ha-1 of ammonium sulphate at flowering; T2 received 42 kg N ha-1 of ammonium sulphate labelled with 9.764 at % 15N excess at seedling, 42 kg N ha-1 at tillering and 42 kg N ha-1 at flowering; T3 received 4800 kg ha-1 of wheat residue labelled with 1.504 at % 15N excess and 42 kg N ha-1 of ammonium sulphate before seedling and 42 kg N ha-1 of ammonium sulphate at flowering . Nitrogen fertilization with 168 kg N ha-1 did no significantly increase grain and straw yields in comparison to the 126 kg N ha-1 application . The combination of the organic input and supplementary application of mineral fertilizer N has been found as a more attractive management option . For all irrigation treatments, the % recovery of N in the whole plant was higher in plants that received 15N at tillering (63%, 49% respectively for irrigation intervals between 10 and 30 d) than in plants that received 15N just after seeding (28% for irrigation each 10- and 30-d intervals) . For the irrigation treatment each 10 and 15 days, the 15N was mainly recovered by the grain for all fertilization treatments, whereas for irrigation treatment each 30 days, the grain and straw recovered nearly equal amounts of fertilizer . For grain and straw of wheat, nitrogen in the plant derived from the fertilizer was low, while most of the N was derived from the soil for all irrigation and fertilization treatments . The % nitrogen in the plant derived from the fertilizer values showed no significant difference between the different plant parts . The results suggested a dominant influence of moisture availability on the fertilizer N uptake by wheat . Under dry conditions the losses of N can be allotted to denitrification and volatilisation.

Biochemistry, 2003 Jul 29, 42(29), 8809 - 17
ATR-FTIR spectroscopy of the P(M) and F intermediates of bovine and Paracoccus denitrificans cytochrome c oxidase; Iwaki M et al.; The structures of P(M) and F intermediates of bovine and Paracoccus denitrificans cytochrome c oxidase were investigated by perfusion-induced attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy . Transitions from the "fast" oxidized state to the P(M) or F states were initiated by perfusion with buffer containing either CO/oxygen or H(2)O(2) . Intermediates were quantitated by simultaneous monitoring of visible absorption changes in the protein film . For both bovine and P . denitrificans oxidase, the major features of the IR difference spectrum of P(M) were similar when produced by CO/oxygen or by H(2)O(2) treatments . These IR difference spectra were distinctly different from the IR difference spectrum of F that formed with extended treatment with H(2)O(2) . Some IR bands could be assigned tentatively to perturbations of heme a(3) ring modes and substituents, and these perturbations were greater in P(M) than in F . Other bands could be assigned to surrounding protein changes . Strong perturbation of the environment of a carboxylic acid, most likely E-242 (bovine numbering), occurred in P(M) and relaxed back in F . A second redox-sensitive carboxylic acid was also perturbed in the bovine P(M) intermediate . Further consistent signatures of P(M) in both oxidases that were absent in F were strong negative bands at 1547 and 1313 cm(-1) in bovine oxidase (1542 and 1314 cm(-1) in P . denitrificans) and a positive band at approximately 1519 cm(-1) . From comparison with available IR data on model compounds, it is suggested that these reflect changes in the covalent tyrosine-histidine ligand to Cu(B) . These findings are discussed in relation to the oxidase catalytic cycle.

Environ Technol, 2003 Jun, 24(6), 693 - 702
Denitrification and phosphorus release under anoxic conditions in an anoxic-anaerobic-aerobic BNR process; Ko KB et al.; An anoxic-anaerobic-aerobic biologial nutrient removal process was used in this study . The kinetic aspects of denitrification and phosphorus release under anoxic conditions were investigated by conducting a pilot-scale plant operation under various SRTs (solids retention times), HRTs (hydraulic retention times) and internal recycle ratios . The process was capable of completely denitrifying the NOx- -N (the sum of NO2- -N and NO3- -N) in the nitrified recycle, resulting in an NOx- -N concentration of less than 1.0 mg l(-1) N in the anoxic zones . Denitrification and phosphorus release were accomplished due to abundant organic substrates in the anoxic zone at the head end of the process and achieved approximately equivalent rates with respect to influent SCOD loading in the zone . Phosphorus release continued without any nitrate inhibition due to low NOx- -N concentrations of less than 2.0 mg l(-1) N in the anaerobic zone.

Water Res, 2003 Sep, 37(15), 3767 - 75
Combined bioelectrochemical and sulfur autotrophic denitrification for drinking water treatment; Wang H et al.; A combined bioelectrochemical and sulfur autotrophic denitrification process for drinking water treatment was put forward and investigated extensively in this paper . In this new process, the bioelectrochemical denitrification was carried out in the upper part of the reactor while sulfur denitrification in the lower part . The H+ produced in Sulfur Part could be consumed by hydrogen denitrification in Bioelectrochemical Part . Therefore, the limestone for pH adjustment in Sulfur Part was not necessary in this combined process, which avoided the problem of hardness increase . The sulfate accumulation in this combined reactor was less than that of the sulfur limestone autotrophic denitrification system . The effluent from two parts was kept neutral at optimum operation conditions . When the influent nitrate was 30 mg-N/L, the reactor could be operated efficiently at the hydraulic retention time ranged from 1.9 to 5h (corresponding minimum current was 16-3 mA), i.e . the effluent NO3(-)-N removal ranged from 90% to 100% without nitrite accumulation and the effluent sulfate concentration was lower than 170 mg/L . The maximum volume-loading rate of the reactor was 0.381 kg NO3(-)-N/(m3d) . The biomass and scanning electron microscope micrographs of Sulfur Part were also analyzed.

Chemosphere, 2003 Sep, 52(9), 1553 - 8
Basic processes in phytoremediation and some applications to air pollution control; Morikawa H et al.; In this short review, basic processes for phytoremediation and plant enzymes that are potentially useful in phytoremediation are briefly summarized . The remaining part describes the applications of the basic processes to decontaminate pollutants in the environments that have been done in our laboratory . Our work includes (1) analysis of the capability of various naturally occurring plants to decontaminate atmospheric nitrogen dioxide and selection of nitrogen-dioxide-philic plants that grow with nitrogen dioxide as the sole nitrogen source, (2) production of transgenic plants to improve plants' capability to decontaminate atmospheric nitrogen dioxide, and (3) analysis of the denitrification process in plants to innovate a gas--gas-converting plants that convert nitrogen dioxide to nitrogen gas.

Analyst, 2003 Jun, 128(6), 724 - 7
Development of dimethyl sulfoxide biosensor using a mediator immobilized enzyme electrode; Cheng HC et al.; A mediator immobilized dimethyl sulfoxide (DMSO) sensor using DMSO reductase (DMSO-R) was constructed . Methyl viologen (MV) was used as a mediator and immobilized on a glassy carbon (GC) electrode with Nafion polymer . DMSO-R from Rhodobacter sphaeroides f . sp . denitrificans was retained by a dialysis film on the modified GC electrode . The amperometric signal in response to DMSO was observed . The linear range of the calibration curve for DMSO was between 0 and 600 microM . The response time was within 100 s and the relative standard deviation was 4% at 200 microM DMSO (n = 4) . To eliminate the background noise derived from oxygen in samples, the glucose oxidase-catalase retained DMSO sensor was also examined.

Water Sci Technol, 2003, 47(10), 101 - 8
Biological nutrient removal from meat processing wastewater using a sequencing batch reactor; Thayalakumaran N et al.; Meat processing effluents are rich in nutrients (nitrogen: 75-200 mg L(-1) and phosphorus: 20-40 mg L(-1)) and COD (800-2,000 mg L(-1)) after primary treatment . A laboratory scale sequencing batch reactor (SBR) was operated for the treatment of a beef processing effluent from slaughtering and boning operations . An effective SBR cycle was found for removal of COD, nitrogen and phosphorus at 22 degrees C . The solid retention time was 15 days while the hydraulic retention time (HRT) was 2.5 days . The total nitrogen in the wastewater was reduced to less than 10 mg L(-1), while the total phosphorus decreased to less than 1.0 mg L(-1) . The residual effluent soluble COD was found to be non-biodegradable as reflected by no further soluble COD removal following prolonged aeration . Removal of biodegradable soluble COD, ammonia nitrogen and soluble phosphate phosphorus of greater than 99% was achieved in the SBR . Good prediction of ammonia and nitrate nitrogen removal was obtained using IWA Activated Sludge Model . The operating cycle is shown to be appropriate to achieve simultaneous removal of COD and nutrients from the meat processing wastewater . Alkalinity and pH have an inverse relationship during the initial anaerobic and aerobic stages due to production and stripping of CO2 . Use of a low level of DO in the final aerobic stage ensured complete ammonia removal and enhanced denitrification.

FEMS Microbiol Lett, 2003 Jul 15, 224(1), 61 - 5
Sequence analysis of a gene product synthesized by Xanthomonas sp . during growth on natural rubber latex; Jendrossek D et al.; Xanthomonas sp . secretes an extracellular protein (Mr approximately 70+/-5 kDa) during growth on purified natural rubber {poly(1,4-cis-isoprene)} but not during growth on water-soluble carbon sources such as glucose or gluconate . A 1.3 kbp DNA fragment coding for an internal part of the structural gene of the 70 kDa protein was amplified by nested polymerase chain reaction (PCR) using amino acid sequence information obtained after Edman degradation of selected trypsin-generated peptides of the purified 70 kDa protein . The PCR product was used as a DNA probe to clone the complete structural gene from genomic DNA of Xanthomonas sp . The sequenced DNA contained a 2037 bp open reading frame which coded for a polypeptide of 678 amino acids (Mr 74.6 kDa) and which included the features of the N-terminal signal peptidase cleavage site (Mr approximately 72.9 kDa for the mature protein) . Analysis of the amino acid sequence revealed the presence of two heme binding motifs (CXXCH) and a approximately 20 amino acids long sequence that is conserved in the Paracoccus denitrificans and Pseudomonas aeruginosa diheme cytochrome c peroxidases (CCPs) . This region includes a histidine residue (H519 in Xanthomonas sp . and H265 and H271 in the Pseudomonas strains, respectively) that is essential for activity in CCPs and that is also conserved in other bacterial oxidases . Blast analysis confirmed the relatedness of the 70 kDa protein to heme-containing oxidases and suggested that it is a member of a new family of relatively large (approximately 500 to approximately 1000 amino acids) extracellular proteins with so far unknown function being only far related in amino acid sequence to P . denitrificans and P . aeruginosa CCPs.

Appl Environ Microbiol, 2003 Jul, 69(7), 4159 - 66
Reductive dehalogenation of brominated phenolic compounds by microorganisms associated with the marine sponge Aplysina aerophoba; Ahn YB et al.; Marine sponges are natural sources of brominated organic compounds, including bromoindoles, bromophenols, and bromopyrroles, that may comprise up to 12% of the sponge dry weight . Aplysina aerophoba sponges harbor large numbers of bacteria that can amount to 40% of the biomass of the animal . We postulated that there might be mechanisms for microbially mediated degradation of these halogenated chemicals within the sponges . The capability of anaerobic microorganisms associated with the marine sponge to transform haloaromatic compounds was tested under different electron-accepting conditions (i.e., denitrifying, sulfidogenic, and methanogenic) . We observed dehalogenation activity of sponge-associated microorganisms with various haloaromatics . 2-Bromo-, 3-bromo-, 4-bromo-, 2,6-dibromo-, and 2,4,6-tribromophenol, and 3,5-dibromo-4-hydroxybenzoate were reductively debrominated under methanogenic and sulfidogenic conditions with no activity observed in the presence of nitrate . Monochlorinated phenols were not transformed over a period of 1 year . Debromination of 2,4,6-tribromophenol, and 2,6-dibromophenol to 2-bromophenol was more rapid than the debromination of the monobrominated phenols . Ampicillin and chloramphenicol inhibited activity, suggesting that dehalogenation was mediated by bacteria . Characterization of the debrominating methanogenic consortia by using terminal restriction fragment length polymorphism (TRFLP) and denaturing gradient gel electrophoresis analysis indicated that different 16S ribosomal DNA (rDNA) phylotypes were enriched on the different halogenated substrates . Sponge-associated microorganisms enriched on organobromine compounds had distinct 16S rDNA TRFLP patterns and were most closely related to the delta subgroup of the proteobacteria . The presence of homologous reductive dehalogenase gene motifs in the sponge-associated microorganisms suggested that reductive dehalogenation might be coupled to dehalorespiration.

Appl Environ Microbiol, 2003 Jul, 69(7), 3938 - 44
Use of a green fluorescent protein-based reporter fusion for detection of nitric oxide produced by denitrifiers; Yin S et al.; To determine if green fluorescent protein could be used as a reporter for detecting nitric oxide production, gfp was fused to nnrS from Rhodobacter sphaeroides 2.4.3 . nnrS was chosen because its expression requires nitric oxide . The presence of the fusion in R . sphaeroides 2.4.3 resulted in a significant increase in fluorescent intensity of the cells, but only when nitrite reductase was active . Cells lacking nitrite reductase activity and consequently the ability to generate nitric oxide were only weakly fluorescent when grown under denitrification-inducing conditions . One of the R . sphaeroides strains unable to generate nitric oxide endogenously was used as a reporter to detect exogenously produced nitric oxide . Incubation of this strain with sodium nitroprusside, a nitric oxide generator, significantly increased its fluorescence intensity . Mixing of known denitrifiers with the reporter strain also led to significant increases in fluorescence intensity, although the level varied depending on the denitrifier used . The reporter was tested on unknown isolates capable of growing anaerobically in the presence of nitrate, and one of these was able to induce expression of the fusion . Analysis of the 16S rRNA gene sequence of this isolate placed it within the Thauera aromatica subgroup, which is known to contain denitrifiers . These experiments demonstrate that this green fluorescent protein-based assay provides a useful method for assessing the ability of bacteria to produce nitric oxide.

Appl Microbiol Biotechnol, 2003 Jul, 62(1), 53 - 60 Epub 2003 Mar 06.
A novel enzyme, D-3-hydroxyaspartate aldolase from Paracoccus denitrificans IFO 13301: purification, characterization, and gene cloning; Liu JQ et al.; A novel enzyme, D-3-hydroxyaspartate aldolase (D-HAA), catalyzing the conversion of D-3-hydroxyaspartate to glyoxylate plus glycine, was purified to homogeneity from Paracoccus denitrificans IFO 13301 . D-HAA is strictly D-specific as to the alpha-position, whereas the enzyme does not distinguish between threo and erythro forms at the beta-position . In addition to D-3-hydroxyaspartate, the enzyme also acts on d-threonine, D-3-3,4-dihydroxyphenylserine, D-3-3,4-methylenedioxyphenylserine, and D-3-phenylserine . The D-HAA gene was cloned and sequenced . The gene contains an open reading frame consisting of 1,161 nucleotides corresponding to 387 amino acid residues . The predicted amino acid sequence displayed 35% and 22% identity with that of the D-threonine aldolase of Arthrobacter sp . DK-38 and Alcaligenes xylosoxidan IFO 12669, respectively . This is the first paper reporting both a purified enzyme with D-3-hydroxyaspartate aldolase activity and also its gene cloning.

Water Res, 2003 Aug, 37(14), 3463 - 71
A new method for characterizing denitrifying phosphorus removal bacteria by using three different types of electron acceptors; Hu JY et al.; This study investigated the characteristics of denitrifying phosphorus removal bacteria by using three different types of electron acceptors as well as the positive role of nitrite in phosphorus removal process . Denitrifying phosphorous removal bacteria was enriched under anaerobic-anoxic (A/A) condition . To understand A/A sludge better, sludge from two other sources were also studied . These include sludges obtained from a lab-scale anaerobic-anoxic-aerobic (A/A/O) system and a local sewage treatment plant . Three types of possible electron acceptors (oxygen, nitrate and nitrite) were examined for their roles in phosphorus uptake . The results obtained indicated that oxygen, nitrate and nitrite were able to act as electron acceptors successfully . This observation suggested that in addition to the two well-accepted groups of phosphorus removal bacteria (one can only utilize oxygen to take up phosphorus, P(O), while the other can use both oxygen and nitrate, P(ON)), a new group of phosphorus removal bacteria, P(ON(n)), which could use oxygen, nitrate or nitrite to take up phosphorus was identified . The relative population of these three types of bacteria could be calculated from results obtainable from phosphorus uptake batch experiments with either oxygen or nitrate or nitrite as electron acceptor . The results obtained in this study showed that A/A sludge had similar phosphorus removal performance as the A/A/O sludge . However, it has better denitrifying phosphorus removal capability, which was demonstrated by the relative population of the three groups of bacteria . The results also suggested that nitrite was not an inhibitor to phosphorus removal process . Instead, it is an alternative electron acceptor to oxygen or nitrate.

Biosci Biotechnol Biochem, 2003 May, 67(5), 1115 - 20
Denitrification of nitrate by the fungus Cylindrocarpon tonkinense; Watsuji TO et al.; The denitrifying fungus Cylindrocarpon tonkinense was thought to be able to denitrify only nitrite (NO2-) but not nitrate (NO3-) to form nitrous oxide (N2O) . Here we found, however, that C . tonkinense can denitrify NO3- under certain conditions . Presence of ammonium (NH3+) in addition to NO3- and the use of a fermentable sugar as an electron donor were key conditions for inducing the denitrifying activity . Such induction accompanied a remarkable increase in the intracellular level of the enzyme activities related to NO3- metabolism . These activities contained assimilatory type NADPH (or NADH)-dependent NO3- reductase (aNar), dissimilatory nitrite reductase (dNir), and nitric oxide reductase (P450nor), but did not contain ubiquinol-dependent, dissimilatory NO3- reductase (dNar) . The denitrification was inhibited by tungstate, an inhibitor of Nar . These results demonstrated occurrence of a novel type of denitrification in C . tonkinense, in which assimilatory type Nar is possibly involved.

Biosci Biotechnol Biochem, 2003 May, 67(5), 1109 - 14
A possible role of NADPH-dependent cytochrome P450nor isozyme in glycolysis under denitrifying conditions; Watsuji TO et al.; The denitrifying fungus Cylindrocarpon tonkinense contains two isozymes of cytochrome P450nor . One isozyme, P450nor1, uses NADH specifically as its electron donor whereas the other isozyme P450nor2 prefers NADPH to NADH . Here we show that P450nor1 is localized in both cytosol and mitochondria, like P450nor of Fusarium oxysporum, while P450nor2 is exclusively in cytosol . We also found that the addition of glucose as a carbon source to the culture media leads to the production of much more P450nor2 in the fungal cells than a non-fermentable substrate (glycerol or acetate) does . These results suggest that the NADP-dependent pentose phosphate cycle acts predominantly in C . tonkinense as the glycolysis pathway under the denitrifying conditions, which was confirmed by the observation that glucose induced enzyme activities involved in the cycle . These results showed that P450nor2 should act as the electron sink under anaerobic, denitrifying conditions to regenerate NADP+ for the pentose phosphate cycle.

Ital J Biochem, 2003 Mar, 52(1), 33 - 6
The proton pump of the mitochondrial bc1 complex; Cocco T et al.; The molecular mechanism of the proton pump activity by the respiratory chain bc1 complex is still unknown . This group has proposed since long time that protonation/deprotonation events in the apoproteins of the complex are cooperatively linked to the oxido-reduction reactions at the quinone catalytic centre . Protolytic residues in the apoproteins can thus provide proton transfer pathways between the bulk aqueons phases and the redox centre . A series of experiments has been carried out aimed at demonstrating a role of particular complex subunits in the pump process . In this paper recent results are reviewed which have evidenced a definite role of polypeptide carboxyl residues in the proton pump mechanism . In particular, experiments carried out with both the bovine and P . denitrificans purified enzymes have indicated a specific involvement of aspartic residue(s) in the Rieske Fe/S protein in the proton pump function.

Water Sci Technol, 2003, 47(9), 129 - 35
Biodegradation of the energetic compound TNT through a multiple-stage treatment approach; Davel J et al.; Biodegradation of the energetic compound 2,4,6-trinitrotoluene (TNT) and its intermediate 2,4,6-triaminotoluene (TAT) was investigated in this study . From previous investigations, a relationship between the biological utilization of ethanol as co-substrate for the reduction of TNT under anaerobic conditions was proposed using an anaerobic fluidized-bed reactor (AFBR) . In this study, the theoretical co-substrate requirement for reduction of TNT to TAT was further investigated through the systematic lowering of the ethanol loading to the reactor . Near complete reduction to TAT was observed up to a critical ethanol loading point, as well as the production of methane from the limited excess available ethanol . Once ethanol deficient loading conditions were established, the increased presence of incompletely reduced degradation intermediates, such as 2,4-diamino-6-nitrotoluene, and even TNT, was observed . The cessation of methanogenesis confirmed that no excess ethanol was available . Degradation of the TAT intermediate in the reactor effluent was investigated using two second-stage reactors under oxidizing conditions . The first was an aerobic activated sludge reactor, and the second was a denitrifying fluidized-bed reactor (DenFBR) . The aerobic reactor was successful in lowering the chemical oxygen demand (COD), but complete removal of TAT was not accomplished . Because of TAT polymerization and auto-oxidation under aerobic conditions, it was difficult to confirm to what extent of TAT removal was biological . In the DenFBR, incompletely reduced TNT intermediates were not successfully degraded, but strong evidence existed for the degradation of TAT . This is the first known report of second stage degradation of TAT under denitrifying conditions.

Arch Microbiol, 2003 Aug, 180(2), 127 - 33 Epub 2003 Jun 24.
Complete denitrification in coculture of obligately chemolithoautotrophic haloalkaliphilic sulfur-oxidizing bacteria from a hypersaline soda lake; Sorokin DY et al.; Eight anaerobic enrichment cultures with thiosulfate as electron donor and nitrate as electron acceptor were inoculated with sediment samples from hypersaline alkaline lakes of Wadi Natrun (Egypt) at pH 10; however, only one of the cultures showed stable growth with complete nitrate reduction to dinitrogen gas . The thiosulfate-oxidizing culture subsequently selected after serial dilution developed in two phases . Initially, nitrate was mostly reduced to nitrite, with a coccoid morphotype prevailing in the culture . During the second stage, nitrite was reduced to dinitrogen gas, accompanied by mass development of thin motile rods . Both morphotypes were isolated in pure culture and identified as representatives of the genus Thioalkalivibrio, which includes obligately autotrophic sulfur-oxidizing haloalkaliphilic species . Nitrate-reducing strain ALEN 2 consisted of large nonmotile coccoid cells that accumulated intracellular sulfur . Its anaerobic growth with thiosulfate, sulfide, or polysulfide as electron donor and nitrate as electron acceptor resulted in the formation of nitrite as the major product . The second isolate, strain ALED, was able to grow anaerobically with thiosulfate as electron donor and nitrite or nitrous oxide (but not nitrate) as electron acceptor . Overall, the action of two different sulfur-oxidizing autotrophs resulted in the complete, thiosulfate-dependent denitrification of nitrate under haloalkaliphilic conditions . This process has not yet been demonstrated for any single species of chemolithoautotrophic sulfur-oxidizing haloalkaliphiles.

Izv Akad Nauk Ser Biol, 2003 May-Jun, (3), 365 - 70
{Denitrification potential and CO2 emission in the northern forest soils of the Enisei meridian (the Siberian IGBP transect)}; Meniailo OV et al.; To estimate the probable contribution of northern forest soils to the global budget of greenhouse microgases, the cryogenic soils along the Yenisei meridian have been studied with respect to their potential denitrification and carbon mineralization activities . It is shown that the forest soils of the boreal zone have a high denitrification potential and, under conditions of a high nitrate nitrogen content, may be a source of nitrogen oxide emission . A significant correlation is observed between N2O and CO2 emissions (r = 0.85, p < 0.001).

J Contam Hydrol, 2003 Jul, 64(3-4), 169 - 90
Experimental study and steady-state simulation of biogeochemical processes in laboratory columns with aquifer material; Amirbahman A et al.; Packed bed laboratory column experiments were performed to simulate the biogeochemical processes resulting from microbially catalyzed oxidation of organic matter . These included aerobic respiration, denitrification, and Mn(IV), Fe(III) and SO(4) reduction processes . The effects of these reactions on the aqueous- and solid-phase geochemistry of the aquifer material were closely examined . The data were used to model the development of alkalinity and pH along the column . To study the independent development of Fe(III)- and SO(4)-reducing environments, two columns were used . One of the columns (column 1) contained small enough concentrations of SO(4) in the influent to render the reduction of this species unimportant to the geochemical processes in the column.The rate of microbially catalyzed reduction of Mn(IV) changed with time as evidenced by the variations in the initial rate of Mn(II) production at the head of the column . The concentration of Mn in both columns was controlled by the solubility of rhodochrosite (MnCO(3(S))).In the column where significant SO(4) reduction took place (column 2), the concentration of dissolved Fe(II) was controlled by the solubility of FeS . In column 1, where SO(4) reduction was not important, maximum dissolved Fe(II) concentrations were controlled by the solubility of siderite (FeCO(3(S))) . Comparison of solid-phase and aqueous-phase data suggests that nearly 20% of the produced Fe(II) precipitates as siderite in column 1 . The solid-phase analysis also indicates that during the course of experiment, approximately 20% of the total Fe(III) hydroxides and more than 70% of the amorphous Fe(III) hydroxides were reduced by dissimilatory iron reduction.The most important sink for dissolved S(-II) produced by the enzymatic reduction of SO(4) was its direct reaction with solid-phase Fe(III) hydroxides leading initially to the formation of FeS . Compared to this pathway, precipitation as FeS did not constitute an important sink for S(-II) in column 2 . In this column, the total reacted S(-II) estimated from the concentration of dissolved sulfur species was in good agreement with the produced Cr(II)-reducible sulfur in the solid phase . Solid-phase analysis of the sulfur species indicated that up to half of the originally produced FeS may have possibly transformed to FeS(2).

J Bacteriol, 2003 Jul, 185(13), 3978 - 82
Bradyrhizobium japonicum NnrR, a denitrification regulator, expands the FixLJ-FixK2 regulatory cascade; Mesa S et al.; In Bradyrhizobium japonicum, a gene named nnrR was identified which encodes a protein with high similarity to FNR/CRP-type transcriptional regulators . Mutant strains carrying an nnrR null mutation were unable to grow anaerobically in the presence of nitrate or nitrite, and they lacked both nitrate and nitrite reductase activities . Anaerobic activation of an nnrR'-'lacZ fusion required FixLJ and FixK(2) . In turn, N oxide-mediated induction of nir and nor genes encoding nitrite and nitric oxide reductase, respectively, depended on NnrR . Thus, NnrR expands the FixLJ-FixK(2) regulatory cascade by an additional control level which integrates the N oxide signal required for maximal induction of the denitrification genes.

J Bacteriol, 2003 Jul, 185(13), 3753 - 63
Identification and characterization of transposable elements of Paracoccus pantotrophus; Bartosik D et al.; We studied diversity and distribution of transposable elements residing in different strains (DSM 11072, DSM 11073, DSM 65, and LMD 82.5) of a soil bacterium Paracoccus pantotrophus (alpha-Proteobacteria) . With application of a shuttle entrapment vector pMEC1, several novel insertion sequences (ISs) and transposons (Tns) have been identified . They were sequenced and subjected to detailed comparative analysis, which allowed their characterization (i.e., identification of transposase genes, terminal inverted repeats, as well as target sequences) and classification into the appropriate IS or Tn families . The frequency of transposition of these elements varied and ranged from 10(-6) to 10(-3) depending on the strain . The copy number, localization (plasmid or chromosome), and distribution of these elements in the Paracoccus species P . pantotrophus, P . denitrificans, P . methylutens, P . solventivorans, and P . versutus were analyzed . This allowed us to distinguish elements that are common in paracocci (ISPpa2, ISPpa3--both of the IS5 family--and ISPpa5 of IS66 family) as well as strain-specific ones (ISPpa1 of the IS256 family, ISPpa4 of the IS5 family, and Tn3434 and Tn5393 of the Tn3 family), acquired by lateral transfer events . These elements will be of a great value in the design of new genetic tools for paracocci, since only one element (IS1248 of P . denitrificans) has been described so far in this genus.

Biochemistry, 2003 Jun 24, 42(24), 7318 - 25
MauG, a novel diheme protein required for tryptophan tryptophylquinone biogenesis; Wang Y et al.; The biosynthesis of methylamine dehydrogenase (MADH) from Paracoccus denitrificans requires four genes in addition to those that encode the two structural protein subunits . None of these gene products have been previously isolated . One of these, mauG, exhibits sequence similarity to diheme cytochrome c peroxidases and is required for the synthesis of the tryptophan tryptophylquinone (TTQ) prosthetic group of MADH . A system was developed for the homologous expression of MauG in P . denitrificans . Its signal sequence was correctly processed, and it was purified from the periplasmic cell fraction . The protein contains two covalent c-type hemes, as predicted from the deduced sequence . EPR spectroscopy reveals that the protein as isolated possesses about equal amounts of one high-spin heme with axial symmetry and one low-spin heme with rhombic symmetry . The low-spin heme contains a major and minor component suggesting a small degree of heme heterogeneity . The high-spin heme and the major low-spin heme component each exhibit resonances that are atypical of c-type hemes and dissimilar to those reported for diheme cytochrome c peroxidases . MauG exhibited only very weak peroxidase activity when assayed with either c-type cytochromes or o-dianisidine as an electron donor . Fully reduced MauG was shown to bind carbon monoxide and could be reoxidized by oxygen . The relevance of these unusual properties of MauG is discussed in the context of its role in TTQ biogenesis.

ScientificWorldJournal, 2001 Oct 23, 1 Suppl 2, 597 - 604
Estimated historical and current nitrogen balances for Illinois; David MB et al.; The Midwest has large riverine exports of nitrogen (N), with the largest flux per unit area to the Mississippi River system coming from Iowa and Illinois . We used historic and current data to estimate N inputs, outputs, and transformations for Illinois where human activity (principally agriculture and associated landscape drainage) have had a dominant impact . Presently, approximately 800,000 Mg of N is added each year as fertilizer and another 420,000 Mg is biologically fixed, primarily by soybean (Glycine max L . Merr.) . These annual inputs are greater than exports in grain, which results in surplus N throughout the landscape . Rivers within the state export approximately 50% of this surplus N, mostly as nitrate, and the remainder appears to be denitrified or temporarily incorporated into the soil organic matter pool . The magnitude of N losses for 1880, 1910, 1950, and 1990 are compared . Initial cultivation of the prairies released large quantities of N (approximately 500,000 Mg N year(-1)), and resulted in riverine N transport during the late 19th century that appears to have been on the same order of magnitude as contemporary N losses . Riverine flux was estimated to have been at a minimum in about 1950, due to diminished net mineralization and low fertilizer inputs . Residual fertilizer N from corn (Zea mays L.), biological N fixed by soybean, short-circuiting of soil water through artificial drainage, and decreased cropping-system diversity appear to be the primary sources for current N export.

ScientificWorldJournal, 2001 Oct 30, 1 Suppl 2, 114 - 21
Controlled release urea as a nitrogen source for spring wheat in Western Canada: yield, grain N content, and N use efficiency; Haderlein L et al.; Controlled release nitrogen (N) fertilizers have been commonly used in horticultural applications such as turf grasses and container-grown woody perennials . Agrium, a major N manufacturer in North and South America, is developing a low-cost controlled release urea (CRU) product for use in field crops such as grain corn, canola, wheat, and other small grain cereals . From 1998 to 2000, 11 field trials were conducted across western Canada to determine if seed-placed CRU could maintain crop yields and increase grain N and N use efficiency when compared to the practice of side-banding of urea N fertilizer . CRU was designed to release timely and adequate, but not excessive, amounts of N to the crop . Crop uptake of N from seed-placed CRU was sufficient to provide yields similar to those of side-banded urea N . Grain N concentrations of the CRU treatments were higher, on average, than those from side-banded urea, resulting in 4.2% higher N use efficiency across the entire N application range from 25 to 100 kg ha(-1) . Higher levels of removal of N in grain from CRU compared to side-banded urea can result in less residual N remaining in the soil, and limit the possibility of N losses due to denitrification and leaching.

ScientificWorldJournal, 2001 Nov 21, 1 Suppl 2, 750 - 7
Nutrient management programs, nitrogen fertilizer practices, and groundwater quality in Nebraska's Central Platte Valley (U.S.), 1989-1998; Daberkow S et al.; Given the societal concern about groundwater pollution from agricultural sources, public programs have been proposed or implemented to change farmer behavior with respect to nutrient use and management . However, few of these programs designed to change farmer behavior have been evaluated due to the lack of detailed data over an appropriate time frame . The Central Platte Natural Resources District (CPNRD) in Nebraska has identified an intensively cultivated, irrigated area with average groundwater nitrate-nitrogen (N) levels about double the EPA"s safe drinking water standard . The CPNRD implemented a joint education and regulatory N management program in the mid-1980s to reduce groundwater N . This analysis reports N use and management, yield, and groundwater nitrate trends in the CPNRD for nearly 3000 continuous-corn fields from 1989 to 1998, where producers faced limits on the timing of N fertilizer application but no limits on amounts . Groundwater nitrate levels showed modest improvement over the 10 years of this analysis, falling from the 1989-1993 average of 18.9 to 18.1 mg/l during 1994-1998 . The availability of N in excess of crop needs was clearly documented by the CPNRD data and was related to optimistic yield goals, irrigation water use above expected levels, and lack of adherence to commercial fertilizer application guidelines . Over the 10-year period of this analysis, producers reported harvesting an annual average of 9729 kg/ha, 1569 kg/ha (14%) below the average yield goal . During 1989-1998, producers reported annually applying an average of 162.5 kg/ha of commercial N fertilizer, 15.7 kg/ha (10%) above the guideline level . Including the N contribution from irrigation water, the potential N contribution to the environment (total N available less estimated crop use) was estimated at 71.7 kg/ha . This is an estimate of the nitrates available for denitrification, volatilization, runoff, future soil N, and leaching to groundwater . On average, between 1989-1993 and 1994-1998, producers more closely followed CPNRD N fertilizer recommendations and increased their use of postemerge N applications--an indication of improved synchrony between N availability and crop uptake.

ScientificWorldJournal, 2001 Nov 06, 1 Suppl 2, 632 - 41
Global pollution of surface waters from point and nonpoint sources of nitrogen; van Drecht G et al.; Global 0.5- by 0.5-degree resolution estimates are presented on the fate of nitrogen (N) stemming from point and nonpoint sources, including plant uptake, denitrification, leaching from the rooting zone, rapid flow through shallow groundwater, and slow flow through deep groundwater to riverine systems . Historical N inputs are used to describe the N flows in groundwater . For nonpoint N sources (agricultural and natural ecosystems), calculations are based on local hydrology, climate, geology, soils, climate and land use combined with data for 1995 on crop production, N inputs from N fertilizers and animal manure, and estimates for ammonia emissions, biological N fixation, and N deposition . For point sources, our estimates are based on population densities and human N emissions, sanitation, and treatment . The results provide a first insight into the magnitude of the N losses from soil-plant systems and point sources in various parts of the world, and the fate of N during transport in atmosphere, groundwater, and surface water . The contribution to the river N load by anthropogenic N pollution is dominant in many river basins in Europe, Asia, and North Africa . Our model results explain much of the variation in measured N export from different world river basins.

ScientificWorldJournal, 2001 Nov 27, 1 Suppl 2, 605 - 14
A model study on the role of wetland zones in lake eutrophication and restoration; Janse JH et al.; Shallow lakes respond in different ways to changes in nutrient loading (nitrogen, phosphorus) . These lakes may be in two different states: turbid, dominated by phytoplankton, and clear, dominated by submerged macrophytes . Both states are self-stabilizing; a shift from turbid to clear occurs at much lower nutrient loading than a shift in the opposite direction . These critical loading levels vary among lakes and are dependent on morphological, biological, and lake management factors . This paper focuses on the role of wetland zones . Several processes are important: transport and settling of suspended solids, denitrification, nutrient uptake by marsh vegetation (increasing nutrient retention), and improvement of habitat conditions for predatory fish . A conceptual model of a lake with surrounding reed marsh was made, including these relations . The lake-part of this model consists of an existing lake model named PCLake . The relative area of lake and marsh can be varied . Model calculations revealed that nutrient concentrations are lowered by the presence of a marsh area, and that the critical loading level for a shift to clear water is increased . This happens only if the mixing rate of the lake and marsh water is adequate . In general, the relative marsh area should be quite large in order to have a substantial effect . Export of nutrients can be enhanced by harvesting of reed vegetation . Optimal predatory fish stock contributes to water quality improvement, but only if combined with favourable loading and physical conditions . Within limits, the presence of a wetland zone around lakes may thus increase the ability of lakes to cope with nutrients and enhance restoration . Validation of the conclusions in real lakes is recommended, a task hampered by the fact that, in the Netherlands, many wetland zones have disappeared in the past.

ScientificWorldJournal, 2001 Nov 21, 1 Suppl 2, 35 - 41
Nitrogen balance of effluent irrigated silage cropping systems in southern Australia; Smith CJ et al.; The nitrogen (N) balance in a double-cropped, effluent spray irrigation system was examined for several years in southern Australia . The amounts of N added by irrigation, removed in the crop, and lost by ammonia (NH3) volatilisation, denitrification, and leaching were measured . Results from the project provide pig producers with the knowledge necessary to evaluate the efficiency of such systems for managing N, and enable sustainable effluent reuse practices to be developed . Oats were grown through the winter (May to November) without irrigation, and irrigated maize was grown during the summer/autumn (December to April) . Approximately 18 mm of effluent was applied every 3 days . The effluent was alkaline (pH 8.3) and the average ammoniacal-N (NH4+ + NH3) concentration was 430 mg N/l (range: 320 to 679 mg N/l) . Mineral N in the 0- to 1.7-m layer tended to increase during the irrigation season and decrease during the winter/spring . About 2000 kg N/ha was found in the profile to a depth of 2 m in October 2000 . N removed in the aboveground biomass (oats + maize) was 590 and 570 kg N/ha/year, equivalent to 25% of the applied N . Average NH3 volatilisation during the daytime (6:00 to 19:00) was 2.74 kg N/ha, while volatilisation at night (19:00 to 6:00) was 0.4 kg N/ha, giving a total of 3.1 kg N/ha/day . This represents approximately 12% of the N loading, assuming that these rates apply throughout the season . The balance of the N accumulated in the soil profile during the irrigation season, as 15N-labelled N studies confirmed . The high recovery of the 15N-labelled N, and the comparable distribution of 15N and Br in the soil profile, implied that there was little loss of N by denitrification, even though the soil was wet enough for leaching of both tracers.

J Biol Chem, 2003 Sep 19, 278(38), 35861 - 8 Epub 2003 Jun 10.
Purification and characterization of the MQH2:NO oxidoreductase from the hyperthermophilic archaeon Pyrobaculum aerophilum; de Vries S et al.; The membrane-bound NO reductase from the hyperthermophilic denitrifying archaeon Pyrobaculum aerophilum was purified to homogeneity . The enzyme displays MQH2:NO oxidoreductase (qNOR) activity, consists of a single subunit, and contains heme and nonheme iron in a 2:1 ratio . The combined results of EPR, resonance Raman, and UV-visible spectroscopy show that one of the hemes is bis-His-coordinated low spin (gz = 3.015; gy = 2.226; gx = 1.45), whereas the other heme adopts a high spin configuration . The enzyme also contains one nonheme iron center, which in the oxidized enzyme is antiferromagnetically coupled to the high spin heme . This binuclear high spin heme/nonheme iron center is EPR-silent and the site of NO reduction . The reduced high spin heme is bound to a neutral histidine and can bind CO to form of a low spin complex . The oxidized high spin heme binds NO, yielding a ferric nitrosyl complex, the intermediate causing the commonly found substrate inhibition in NO reductases (Ki(NO) = 7 microm) . The qNOR as present in the membrane is, in contrast to the purified enzyme, quite thermostable, incubation at 100 degrees C for 86 min leading to 50% inhibition . The pure enzyme lacks heme b and instead contains stoichiometric amounts of hemes Op1 and Op2, ethenylgeranylgeranyl and hydroxyethylgeranylgeranyl derivatives of heme b, respectively . The archaeal qNOR is the first example of a NO reductase, which contains modified hemes reminiscent of cytochrome bo3 and aa3 oxidases . This report is the first describing the purification and structural and spectroscopic properties of a thermostable NO reductase.

Appl Biochem Biotechnol, 2003 Apr-Jun, 109(1-3), 253 - 62
Combined biologic (anaerobic-aerobic) and chemical treatment of starch industry wastewater; Sklyar V et al.; A combined biologic and chemical treatment of high-strength (total chemical oxygen demand {CODtot} up to 20 g/L), strong nitrogenous (total N up to 1 g/L), and phosphoric (total P up to 0.4 g/L) starch industry wastewater was investigated at laboratory-scale level . As a principal step for COD elimination, upflow anaerobic sludge bed reactor performance was investigated at 30 degrees C . Under hydraulic retention times (HRTs) of about 1 d, when the organic loading rates were higher than 15 g of COD/(L.d), the CODtot removal varied between 77 and 93%, giving effluents with a COD/N ratio of 4-5:1, approaching the requirements of subsequent denitrification . The activated sludge reactor operating in aerobic-anoxic regime (HRT of about 4 d, duration of aerobic and anoxic phases of 30 min each) was able to remove up to 90% of total nitrogen and up to 64% of COD tot from the anaerobic effluents under 17-20 degrees C . The coagulation experiments with Fe(III) showed that 1.4 mg of resting hardly biodegradable COD and 0.5 mg of phosphate (as P) could be removed from the aerobic effluents by each milligram of iron added.

Wei Sheng Yan Jiu, 2003 Mar, 32(2), 95 - 7
{Study on denitrification using different carbon sources}; Tan Y et al.; In this study, bench scale tests were conducted to study the potentials of immobilized denitrifier to reduce nitrate in the presence of 4 different carbon sources: glucose, cane sugar, methanol and acetic acid . The results showed that the carbon sources can be used by the immobilized bacteria as exogenous carbon sources . While using methanol, the average denitrifying velocity was lower than the others . Dissimilatory reduction to ammonium was not significant and accounted for less than 5% of reduced nitrate . By a 6-hour hydraulic residence time, the denitrification rates were higher than 96% . The nitrate enriched water of S lake was also treated by the immobilized denitrifier to study the character of denitrification, especially on the using of natural carbon sources as electron donotor . The results showed that more than 90% of the nitrate in the water could be reduced by the immobilized bacteria, and more than 20% of the natural carbon sources in the water could be used by the immobilized cells.

Biotechnol Prog, 2003 May-Jun, 19(3), 1019 - 21
Nitrate removal in aquariums by immobilized pseudomonas; Tal Y et al.; Biological denitrification of nitrate to nitrogen gas was examined in a freshwater and a marine aquarium . Nitrate removal in the aquarium water was accomplished with denitrifiers immobilized in a freeze-dried, alginate-starch matrix . Starch served as a bacterial carbon source and cellular matrix-strengthening filler . Freeze-dried beads were placed in canisters through which nitrate-rich aquarium water was recirculated . The freshwater aquarium (100 L) contained goldfish (Carassius auratus) at a total biomass of 390 g, whereas cichlids (Oreochromis mossambicus) were kept at a similar stocking density in the marine aquarium . Denitrification resulted in low ambient nitrate concentrations in both aquariums . The specific nitrate removal rate of the freshwater beads was significantly higher (50 microg of NO(3)-N/bead/day) than that of seawater beads (5 microg of NO(3)-N/bead/day) . Differences in ambient nitrate concentrations between both aquariums and diffusion limitation of nitrate to the active denitrification sites within the beads might explain these observed differences.

Appl Environ Microbiol, 2003 Jun, 69(6), 3476 - 83
Nitric oxide reductase (norB) genes from pure cultures and environmental samples; Braker G et al.; A PCR-based approach was developed to recover nitric oxide (NO) reductase (norB) genes as a functional marker gene for denitrifying bacteria . norB database sequences grouped in two very distinct branches . One encodes the quinol-oxidizing single-subunit class (qNorB), while the other class is a cytochrome bc-type complex (cNorB) . The latter oxidizes cytochrome c, and the gene is localized adjacent to norC . While both norB types occur in denitrifying strains, the qnorB type was also found in a variety of nondenitrifying strains, suggesting a function in detoxifying NO . Branch-specific degenerate primer sets detected the two norB types in our denitrifier cultures . Specificity was confirmed by sequence analysis of the norB amplicons and failure to amplify norB from nondenitrifying strains . These primer sets also specifically amplified norB from freshwater and marine sediments . Pairwise comparison of amplified norB sequences indicated minimum levels of amino acid identity of 43.9% for qnorB and 38% for cnorB . Phylogenetic analysis confirmed the existence of two classes of norB genes, which clustered according to the respective primer set . Within the qnorB cluster, the majority of genes from isolates and a few environmental clones formed a separate subcluster . Most environmental qnorB clones originating from both habitats clustered into two distinct subclusters of novel sequences from presumably as yet uncultivated organisms . cnorB clones were located on separate branches within subclusters of genes from known organisms, suggesting an origin from similar organisms.

Appl Environ Microbiol, 2003 Jun, 69(6), 3152 - 7
Aerobic denitrifying bacteria that produce low levels of nitrous oxide; Takaya N et al.; Most denitrifiers produce nitrous oxide (N(2)O) instead of dinitrogen (N(2)) under aerobic conditions . We isolated and characterized novel aerobic denitrifiers that produce low levels of N(2)O under aerobic conditions . We monitored the denitrification activities of two of the isolates, strains TR2 and K50, in batch and continuous cultures . Both strains reduced nitrate (NO(3)(-)) to N(2) at rates of 0.9 and 0.03 micro mol min(-1) unit of optical density at 540 nm(-1) at dissolved oxygen (O(2)) (DO) concentrations of 39 and 38 micro mol liter(-1), respectively . At the same DO level, the typical denitrifier Pseudomonas stutzeri and the previously described aerobic denitrifier Paracoccus denitrificans did not produce N(2) but evolved more than 10-fold more N(2)O than strains TR2 and K50 evolved . The isolates denitrified NO(3)(-) with concomitant consumption of O(2) . These results indicated that strains TR2 and K50 are aerobic denitrifiers . These two isolates were taxonomically placed in the beta subclass of the class Proteobacteria and were identified as P . stutzeri TR2 and Pseudomonas sp . strain K50 . These strains should be useful for future investigations of the mechanisms of denitrifying bacteria that regulate N(2)O emission, the single-stage process for nitrogen removal, and microbial N(2)O emission into the ecosystem.

Biosci Biotechnol Biochem, 2003 Apr, 67(4), 937 - 9
Formate-forming fungal catabolic pathway to supply electrons to nitrate respiration; Kuwazaki S et al.; Pyruvate was catabolized anaerobically by resting cells of Fusarium oxysporum to form formate and acetate . Addition of nitrate decreased the accumulation of formate in the medium with concomitant formation of nitrite and N2O . The results suggested a unique metabolic pathway that occurs in fungi mediated by pyruvate-formate lyase to supply electrons via formate to fungal denitrification.

J Biol Chem, 2003 Aug 22, 278(34), 31584 - 92 Epub 2003 Jun 03.
Nitric oxide formation by Escherichia coli . Dependence on nitrite reductase, the NO-sensing regulator Fnr, and flavohemoglobin Hmp; Corker H et al.; Nitric oxide (NO) is a key signaling and defense molecule in biological systems . The bactericidal effects of NO produced, for example, by macrophages are resisted by various bacterial NO-detoxifying enzymes, the best understood being the flavohemoglobins exemplified by Escherichia coli Hmp . However, many bacteria, including E . coli, are reported to produce NO by processes that are independent of denitrification in which NO is an obligatory intermediate . We demonstrate using an NO-specific electrode that E . coli cells, grown anaerobically with nitrate as terminal electron acceptor, generate significant NO on adding nitrite . The periplasmic cytochrome c nitrite reductase (Nrf) is shown, by comparing Nrf+ and Nrf- mutants, to be largely responsible for NO generation . Surprisingly, an hmp mutant did not accumulate more NO but, rather, failed to produce detectable NO . Anaerobic growth of the hmp mutant was not stimulated by nitrate, and the mutant failed to produce periplasmic cytochrome(s) c, leading to the hypothesis that accumulating NO in the absence of Hmp inactivates the global anaerobic regulator Fnr by reaction with the {4Fe-4S}2+ cluster (Cruz-Ramos, H., Crack, J., Wu, G., Hughes, M . N., Scott, C., Thomson, A . J., Green, J., and Poole, R . K . (2002) EMBO J . 21, 3235-3244) . Fnr thus failed to up-regulate nitrite reductase . The model is supported by the inability of an fnr mutant to generate NO and by the restoration of NO accumulation to hmp mutants upon introducing a plasmid encoding Fnr* (D154A) known to confer activity in the presence of oxygen . A cytochrome bd-deficient mutant retained NO-generating activity . The present study reveals a critical balance between NO-generating and -detoxifying activities during anaerobic growth.

Acta Crystallogr D Biol Crystallogr, 2003 May, 59(Pt 5), 835 - 42 Epub 2003 Apr 25.
The structure of glyceraldehyde 3-phosphate dehydrogenase from Alcaligenes xylosoxidans at 1.7 A resolution; Antonyuk SV et al.; The enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the Gram-negative denitrifying bacterial species Alcaligenes xylosoxidans was purified and crystallized as a contaminant protein during purification of nitrous oxide reductase . This is the first structure of a GAPDH from a denitrifying species . The crystal structure was solved at 1.7 A resolution by molecular replacement using the structure of GAPDH from Bacillus stearothermophilus as a starting model . The quality of the structure enabled the amino-acid sequence of the A . xylosoxidans GAPDH to be assigned . The structure is that of the apo-enzyme, lacking the NAD+ cofactor and with the active-site residue Cys154 oxidized . The global structure of the enzyme has a homotetrameric quaternary structure similar to that observed for its bacterial and eukaryotic counterparts . The essential role of Cys154 in the enzyme activity has been confirmed . In monomer O two half-occupancy sulfate ions were found at the active site, which are analogous to the substrate and the "attacking" phosphate seen in B . stearothermophilus . One half-occupancy sulfate ion is also located in the substrate-binding site of monomer P.

Appl Microbiol Biotechnol, 2003 Nov, 63(1), 68 - 74 Epub 2003 May 27.
Anaerobic oxidation of 2-chloroethanol under denitrifying conditions by Pseudomonas stutzeri strain JJ; Dijk JA et al.; A bacterium that uses 2-chloroethanol as sole energy and carbon source coupled to denitrification was isolated from 1,2-dichloroethane-contaminated soil . Its 16 S rDNA sequence showed 98% similarity with the type strain of Pseudomonas stutzeri (DSM 5190) and the isolate was tentatively identified as Pseudomonas stutzeri strain JJ . Strain JJ oxidized 2-chloroethanol completely to CO(2) with NO(3)(- )or O(2) as electron acceptor, with a preference for O(2) if supplied in combination . Optimum growth on 2-chloroethanol with nitrate occurred at 30 degrees C with a mu(max) of 0.14 h(-1) and a yield of 4.4 g protein per mol 2-chloroethanol metabolized . Under aerobic conditions, the mu(max) was 0.31 h(-1) . NO(2)(-) also served as electron acceptor, but reduction of Fe(OH)(3), MnO(2), SO(4)(2-), fumarate or ClO(3)(-) was not observed . Another chlorinated compound used as sole energy and carbon source under aerobic and denitrifying conditions was chloroacetate . Various different bacterial strains, including some closely related Pseudomonas stutzeri strains, were tested for their ability to grow on 2-chloroethanol as sole energy and carbon source under aerobic and denitrifying conditions, respectively . Only three strains, Pseudomonas stutzeri strain LMD 76.42, Pseudomonas putida US2 and Xanthobacter autotrophicus GJ10, grew aerobically on 2-chloroethanol . This is the first report of oxidation of 2-chloroethanol under denitrifying conditions by a pure bacterial culture.

Biochem Soc Trans, 2003 Jun, 31(Pt 3), 553 - 7
A two-faced molecule offers NO explanation: the proximal binding of nitric oxide to haem; Lawson DM et al.; Cytochrome c ' (cyt c ') is found in the periplasmic space of denitrifying bacteria where it is thought to mediate the transfer of NO between the nitrogen-cycle enzymes dissimilatory nitrite reductase and nitric oxide reductase . It contains a 5-coordinate (5c) His-ligated haem that shares spectroscopic and ligand-binding properties with the haem group in the sensory domain of soluble guanylate cyclase (sGC) . The latter is an extremely important enzyme involved in the control of vasodilation and blood clotting . Curiously, the enzyme is activated up to 200-fold by the binding of NO to the haem, whereas the binding of CO gives rise to only a mild stimulation of activity . Through X-ray crystallography we have studied NO and CO binding to cyt c ' . CO binds to the distal face to give a 6-coordinate (6c) adduct . By contrast, NO binding gives rise to a 5c adduct through the displacement of the proximal His, to give a novel and unexpected proximal binding mode for NO . These results are also supported by a range of spectroscopies . In the absence of a crystal structure for sGC we propose that cyt c ' provides a structural model for the haem domain of this enzyme and thereby helps to explain the differential effects of NO and CO on its activity.

Biotechnol Bioeng, 2003 Jul 20, 83(2), 140 - 8
Identification and comparison of aerobic and denitrifying polyphosphate-accumulating organisms; Zeng RJ et al.; Two laboratory-scale sequencing batch reactors (SBRs) were operated for enhanced biological phosphorus removal (EBPR) in alternating anaerobic-aerobic or alternating anaerobic-anoxic modes, respectively . Polyphosphate-accumulating organisms (PAOs) were enriched in the anaerobic-aerobic SBR and denitrifying PAOs (DPAOs) were enriched in the anaerobic-aerobic SBR . Fluorescence in situ hybridization (FISH) demonstrated that the well-known PAO, "Candidatus Accumulibacter phosphatis" was abundant in both SBRs, and post-FISH chemical staining with 4,6-diamidino-2-phenylindol (DAPI) confirmed that they accumulated polyphosphate . When the anaerobic-anoxic SBR enriched for DPAOs was converted to anaerobic-aerobic operation, aerobic uptake of phosphorus by the resident microbial community occurred immediately . However, when the anaerobic-aerobic SBR enriched for PAOs was exposed to one cycle with anoxic rather than aerobic conditions, a 5-h lag period elapsed before phosphorus uptake proceeded . This anoxic phosphorus-uptake lag phase was not observed in the subsequent anaerobic-aerobic cycle . These results demonstrate that the PAOs that dominated the anaerobic-aerobic SBR biomass were the same organisms as the DPAOs enriched under anaerobic-anoxic conditions .

Water Res, 2003 Jul, 37(12), 2953 - 9
Continuous biotransformation and removal of nitrophenols under denitrifying conditions; Karim K et al.; The effect of COD/NO(3)(-)-N ratio on the biotransformation and removal of 2-nitrophenol (2-NP), 4-nitrophenol (4-NP), and 2,4-dinitrophenol (2,4-DNP) was studied in bench scale upflow anaerobic sludge blanket (UASB) reactors . Sodium acetate and sodium nitrate were used as electron donor (substrate) and electron acceptor, respectively . Nitrate nitrogen loading was increased from 0.098 to 0.6 kg/m(3)d in order to keep COD/NO(3)(-)-N ratio as 20.8, 14.3, 9.8, 5.0, 4.0 and 3.33 . Throughout the study, input nitrophenolic concentration and hydraulic retention time (HRT) were kept constant as 30 mg/l and 24h, respectively . 2-Aminophenol (2-AP), 4-aminophenol (4-AP) and 2-amino,4-nitrophenol (2-A,4-NP) were found as the major intermediate metabolite of 2-NP, 4-NP and 2,4-DNP, respectively . Removal of all the three nitrophenols increased with lowering of COD/NO(3)(-)-N ratio . However, nitrophenols removal got adversely affected when COD/NO(3)(-)-N ratio was reduced below 5 . Maximum removal achieved were 91.63%, 90.17% and 86.10% for 2-NP, 4-NP and 2,4-DNP, respectively at a COD/NO(3)(-)-N ratio of 5 . Simultaneous denitrification and methanogenesis was observed in all the reactors throughout the study.

J Biochem (Tokyo), 2003 Apr, 133(4), 461 - 5
Hybrid respiration in the denitrifying mitochondria of Fusarium oxysporum; Takaya N et al.; Induction of the mitochondrial nitrate-respiration (denitrification) system of the fungus Fusarium oxysporum requires the supply of low levels of oxygen (O(2)) . Here we show that O(2) and nitrate (NO(3)(-)) respiration function simultaneously in the mitochondria of fungal cells incubated under hypoxic, denitrifying conditions in which both O(2) and NO(3)(-) act as the terminal electron acceptors . The NO(3)(-) and nitrite (NO(2)(-)) reductases involved in fungal denitrification share the mitochondrial respiratory chain with cytochrome oxidase . F . oxysporum cytochrome c(549) can serve as an electron donor for both NO(2)(-) reductase and cytochrome oxidase . We are the first to demonstrate hybrid respiration in respiring eukaryotic mitochondria.

Biochemistry, 2003 May 27, 42(20), 6005 - 12
Interaction of cytochrome c with cytochrome c oxidase: an NMR study on two soluble fragments derived from Paracoccus denitrificans; Wienk H et al.; The functional interactions between the various components of the respiratory chain are relatively short-lived, thus allowing high turnover numbers but at the same time complicating the structural analysis of the complexes . Chemical shift mapping by NMR spectroscopy is a useful tool to investigate such transient contacts, since it can monitor changes in the electron-shielding properties of a protein as the result of temporary contacts with a reaction partner . In this study, we investigated the molecular interaction between two components of the electron-transfer chain from Paracoccus denitrificans: the engineered, water-soluble fragment of cytochrome c(552) and the Cu(A) domain from the cytochrome c oxidase . Comparison of {(15)N,(1)H}-TROSY spectra of the {(15)N}-labeled cytochrome c(552) fragment in the absence and in the presence of the Cu(A) fragment showed chemical shift changes for the backbone amide groups of several, mostly uncharged residues located around the exposed heme edge in cytochrome c(552) . The detected contact areas on the cytochrome c(552) surface were comparable under both fully reduced and fully oxidized conditions, suggesting that the respective chemical shift changes represent biologically relevant protein-protein interactions.

Microb Ecol, 2003 Jul, 46(1), 113 - 21 Epub 2003 May 13.
Genetic characterization of the nitrate reducing community based on narG nucleotide sequence analysis; Cheneby D et al.; The ability of facultative anerobes to respire nitrate has been ascribed mainly to the activity of a membrane-bound nitrate reductase encoded by the narGHJI operon . Respiratory nitrate reduction is the first step of the denitrification pathway, which is considered as an important soil process since it contributes to the global cycling of nitrogen . In this study, we employed direct PCR, cloning, and sequencing of narG gene fragments to determine the diversity of nitrate-reducing bacteria occurring in soil and in the maize rhizosphere . Libraries containing 727 clones in total were screened by restriction fragment analysis . Phylogenetic analysis of 128 narG sequences separated the clone families into two main groups that represent the Gram-positive and Gram-negative nitrate-reducing bacteria . Novel narG lineages that branch distinctly from all currently known membrane bound nitrate-reductase encoding genes were detected within the Gram-negative branch . All together, our results revealed a more complex nitrate-reducing community than did previous culture-based studies . A significant and consistent shift in the relative abundance of the nitrate-reducing groups within this functional community was detected in the maize rhizosphere . Thus a substantially higher abundance of the dominant clone family and a lower diversity index were observed in the rhizosphere compared to the unplanted soil, suggesting that a bacterial group has been specifically selected within the nitrate-reducing community . Furthermore, restriction fragment length polymorphism analysis of cloned narG gene fragments proved to be a powerful tool in evaluating the structure and the diversity of the nitrate-reducing community and community shifts therein.

Microb Ecol, 2003 Jul, 46(1), 12 - 21 Epub 2003 May 13.
Bacterial diversity in a marine methanol-fed denitrification reactor at the montreal biodome, Canada; Labbe N et al.; The bacterial biota of a methanol-fed denitrification reactor used to treat seawater at the Montreal Biodome were investigated using culture-dependent and molecular biology methods . The microbiota extracted from the reactor carriers were cultivated on three media . Three isolate types were recovered and their 16S ribosomal DNA (rDNA) genes were determined . The analysis showed that the isolate types were related to alpha-Proteobacteria . They are members of the Hyphomicrobium and Paracoccus genera and the Phyllobacteriaceae family . Uncultured bacteria were identified through a 16S rDNA library generated from total DNA extracted from the microbiota . Clones were screened for different restriction profiles and for different DGGE (denaturing gradient gel electrophoresis) migration profiles . More than 70% of clones have the same restriction profile, and the sequence of representative clones showed a relation with the Methylophaga members of the Piscirickettsia family (gamma-Proteobacteria) . Sequences from other profiles were related to bacterial species involved in denitrification . The number of species in the denitrification reactor was estimated at 15 . Bacterial colonization on newly added carriers in the denitrification reactor was monitored by PCR-DGGE . The DGGE migration profiles evolved during the first 5 weeks and then remained essentially unchanged . PCR-DGGE was also used to monitor the microbial profiles in various aquarium locations . As expected, bacterial populations differed from one location to another, except for the sand and trickling filters which presented similar DGGE migration profiles.

Bioresour Technol, 2003 Mar, 87(1), 75 - 80
Biological denitrification in a continuous-flow pilot bioreactor containing immobilized Pseudomonas butanovora cells; Kesseru P et al.; Pseudomonas butanovora, a novel denitrifying bacterium, was immobilized in composite beads and filled into a reactor system . The pilot bioreactor average denitrification activity was at ethanol-C:nitrate-N ratios of 3:1 and 1.5:1 0.88 and 0.54 kg NO3(-)-Nm(-3) d(-1), respectively . The denitrification was stable in spite of the relatively low hydraulic retention times of 2.47 and 3 h . The nitrate content of the influent was almost completely reduced at the first level of the bioreactor and the nitrite formed underwent reduction in the upper part of the reactor . The experimentally determined optimum ethanol-C:nitrate-N ratio was 1.41 +/- 0.41 . In consequence of the aerobic conditions, the acetic acid produced by the oxygenation of ethanol was also detectable in the reactor effluent . The pH of the effluent (7.58) never exceeded the acceptable maximum (8.5) . The nitrate removal efficiency of the cells was nearly 1000% at both C:N ratios, and the nitrite content of the effluent was around the prescribed limit throughout the continuous operation . This continuous-flow pilot bioreactor containing immobilized P . butanovora cells proved an efficient denitrification system with a relatively low retention time.

J Bacteriol, 2003 May, 185(10), 3167 - 78
Energy-generating enzymes of Burkholderia cepacia and their interactions with macrophages; Punj V et al.; We previously demonstrated that several clinical and environmental isolates of Burkholderia cepacia secreted ATP-utilizing enzymes to the medium; the secretion of these enzymes by cystic fibrosis lung isolate strain 38 was shown to be greatly enhanced in the presence of alpha(2)-macroglobulin . Fractionation of the growth medium of cystic fibrosis isolate strain 71 belonging to genomovar I demonstrated the presence of two additional proteins, homologues of Pseudomonas aeruginosa azurin and cytochrome c(551), which are normally involved in electron transfer during denitrification . A Q-Sepharose column flowthrough fraction of the growth medium of B . cepacia strain 71 enriched with the azurin and cytochrome c(551) homologues triggered apoptosis in macrophages and mast cells, leading to their death . Incubation of the Q-Sepharose column flowthrough fraction with antiazurin and anti-cytochrome c(551) antibodies greatly reduced cell death . We cloned and hyperexpressed a gene from B . cepacia strain 71 that encodes the homologue of P . aeruginosa azurin . Such azurin homologues were detected in the growth medium of several strains belonging to genomovars I, III, and VI but not in the growth medium of strains belonging to other genomovars . The growth medium of the strains that elaborated the azurin homologue had high cytotoxicity towards macrophages . Purified azurin homologue was shown to induce apoptosis in macrophages in a caspase-dependent manner and was localized in both the cytosol and nucleus when incubated with or microinjected into macrophages . This is an interesting example of the interaction of a bacterial protein normally involved in cellular energetics with macrophages to effect their cell death.

Water Res, 2003 May, 37(10), 2435 - 41
Heterotroph anoxic yield in anoxic aerobic activated sludge systems treating municipal wastewater; Muller A et al.; As input to the steady state design and kinetic simulation models for the activated sludge system, the correct value for the heterotroph anoxic yield is essential to provide reliable estimates for the system denitrification potential . This paper examines activated sludge anoxic yield values in the literature, and presents experimental data quantifying the value . In the literature, in terms of the structure of ASM1 and similar models, theoretically it has been shown that the anoxic yield should be reduced to approximately 0.79 the value of the aerobic yield . This theoretical value is validated with data from corresponding aerobic OUR and anoxic nitrate time profiles in a batch fed laboratory scale long sludge age activated sludge system treating municipal wastewater . The value also is in close agreement with values in the literature measured with both artificial substrates and municipal wastewater . Thus, it is concluded that, in ASM1 and similar models, for an aerobic yield of 0.67mg COD/mg COD, the anoxic yield should be about 0.53 mg COD/mg COD . Including such a lower anoxic yield in ASM1 and similar models will result in a significant increase in denitrification potential, due to increased denitrification with wastewater RBCOD as substrate . In terms of the structure of ASM3, for the proposed substrate storage yields and the aerobic yield of 0.63 mg COD/mg COD, experimental data indicate that the corresponding anoxic yield should be about 0.42 mg COD/mg COD . This is significantly lower than the proposed value of 0.54 mg COD/mg COD, and requires further investigation.

Water Res, 2003 May, 37(10), 2394 - 8
Denitrification at low temperatures using a suspended carrier biofilm process; Welander U et al.; The denitrification process was studied in a stirred lab-scale suspended carrier biofilm reactor at low temperatures (3-20 degrees C) . The reactor was filled to 50% with Kaldnes K1 carriers . The denitrification rate showed only a rather weak dependence on the temperature, the rate at 3 degrees C being approximately 55% of that at 15 degrees C . The maximum denitrification rate obtained at 15 degrees C was 2.7 g NO(x)(-)-Nm(-2)carrier d (-1) . The maximum denitrification rate at 3 degrees C during an 8-day period was found to be constant . During the 8 days, the hydraulic retention time was approximately 1.5h and the inlet NO(3)(-)-N concentration was 30 mg x l(-1).

J Contam Hydrol, 2003 Apr-May, 62-63, 361 - 80
Chemical analyses of pore water from boreholes USW SD-6 and USW WT-24, Yucca Mountain, Nevada; Yang IC et al.; Analyses of pore water extracted from cores of boreholes USW SD-6 in the central part and USW WT-24 in the northern part of Yucca Mountain, Nevada, show significant vertical and lateral variations in dissolved-ion concentrations . Analyses of samples of only a few milliliters of pore water extracted by uniaxial or triaxial compression and by ultracentrifugation methods from adjacent core samples are generally in agreement, within the analytical error of 10% to 15% . However, the values of silica for water obtained by ultracentrifugation are consistently lower than values for water obtained by compression . The larger concentrations probably are due to localized pressure solution of silicate minerals during compression . The shallower water from core in borehole USW SD-6 was extracted from nonwelded units collectively referred to as the Paintbrush Tuff nonwelded (PTn) . The deeper water was from core in both boreholes USW SD-6 and USW WT-24 in the nonwelded units referred to as the Calico Hills nonwelded (CHn) . Significant differences in mean dissolved-ion concentrations in pore water between the PTn and CHn are (1) decreases in Ca, Mg, SO(4), and NO(3) and (2) increases in HCO(3) and (Na+K)/(Ca+Mg) ratios . The decrease in NO(3) and the increase in HCO(3) could be the result of denitrification through the oxidation of organic matter . The decrease in Ca and associated increase in (Na+K)/(Ca+Mg) is the result of ion exchange with zeolites in the CHn in borehole USW WT-24 . This effect is not nearly as pronounced in borehole USW SD-6, probably reflecting a smaller amount of zeolitization of the CHn in USW SD-6 . Geochemical calculations using the PHREEQC code indicate that the pore water from both boreholes USW SD-6 and USW WT-24 is uniformly undersaturated in anhydrite, gypsum, and amorphous silica, but supersaturated in quartz and chalcedony . The saturation of calcite, aragonite, sepiolite, and dolomite is more variable from sample to sample.

Sci Total Environ, 2003 May 20, 307(1-3), 191 - 201
Natural denitrification in the Kakamigahara groundwater basin, Gifu prefecture, central Japan; Mohamed MA et al.; Although nitrate is recognized as the most common groundwater contaminant due to growing anthropogenic sources, such as agriculture in particular, its adverse effects on human and animal health are debatable . The current issue, however, is to control and reduce nitrate contamination with regards to the long residence time of groundwater within aquifers . Denitrification has recently been recognized for its ability to reduce high nitrate concentrations in groundwater . The Kakamigahara groundwater basin, Gifu prefecture, Japan, witnessed rising levels of nitrate (>12 mg/l NO(3)-N) originating from agricultural sources . Chemical analyses for the determination of major constituents of groundwater and delta(15)N of residual nitrate were performed on representative groundwater samples in order to fulfill two main objectives . One is to investigate the current situation of nitrate groundwater pollution . The second objective is to determine whether the denitrification is a potential natural mechanism, which eliminates nitrate pollution in the Kakamigahara aquifer . Agricultural nitrate contamination of groundwater was obvious from characteristically high concentrations of Ca(2+), Mg(2+), NO(3)(-) and SO(4)(2-) . High nitrate concentrations were found on the eastern side of the basin in association with vegetable cultivation fields, and decreased gradually towards the west of the basin along the direction of groundwater flow . The decrease of nitrate concentration was conveniently coupled with increase of HCO(3)(-) (the heterotrophic denitrification product), pH and delta(15)N of residual nitrate (due to isotopic fractionation) from east to west . Therefore, denitrification in situ is continuously removing nitrate from the Kakamigahara groundwater system.

J Environ Qual, 2003 Mar-Apr, 32(2), 727 - 35
Denitrification in constructed wetlands used for treatment of swine wastewater; Hunt PG et al.; Constructed wetland treatment of swine wastewater probably involves substantial denitrification . Our objective was to assess denitrification and denitrification enzyme activity (DEA) in such wetlands in relation to plant communities, N loading, carbon or nitrogen limitations, and water depth . Two wetland cells each 3.6 m wide and 33.5 m long were connected in series . One set of cells was planted with rushes and bulrushes, including soft rush (Juncus effusus L.), softstem bulrush {Schoenoplectus tabernaemontani (K.C . Gmel.) Pallal, American bulrush {Schoenoplectus americanus (Pers.) Volkart ex Schinz & R . Keller}, and woolgrass bulrush {Scirpus cyperinus (L.) Kunth} . Another set was planted with bur-reeds and cattails, including American bur-reed (Sparganium americanum Nutt.), broadleaf cattail (Typha latifolia L.), and narrowleaf cattail (Typha angustifolia L.) . The sets will be referred to herein as bulrush and cattail wetlands, respectively . Denitrification and DEA were measured via the acetylene inhibition method in intact soil cores and disturbed soil samples that were taken during four years (1994-1997) . Although DEA in the disturbed samples was greater than denitrification in the core samples, the measurements were highly correlated (r2 > or = 0.82) . The DEA was greater in the bulrush wetlands than the cattail wetlands, 0.516 and 0.210 mg N kg(-1) soil h(-1), respectively; and it increased with the cumulative applied N . The DEA mean was equivalent to 9.55 kg N ha(-1) d(-1) in the bulrush wetlands . We hypothesized and confirmed that DEA was generally limited by nitrate rather than carbon . Moreover, we determined that one of the most influential factors in DEA was wetland water depth . In bulrush wetlands, the slope and r2 values of the control treatment were -0.013 mg N kg(-1) soil h(-1) mm(-1) depth and r2 = 0.89, respectively . Results of this investigation indicate that DEA can be very significant in constructed wetlands used to treat swine wastewater.

J Environ Qual, 2003 Mar-Apr, 32(2), 711 - 26
Nutrient transport in a restored riparian wetland; Vellidis G et al.; We determined the water quality effect of a restored forested riparian wetland adjacent to a manure application area and a heavily fertilized pasture in the Georgia Coastal Plain . The buffer system was managed based on USDA recommendations and averaged 38 m in width . Water quality and hydrology data were collected from 1991-1999 . A nitrate plume in shallow ground water with concentrations exceeding 10 mg NO3-N L(-1) moved into the restored forested riparian wetland . Along most of the plume front, concentrations were less than 4 mg NO3-N L(-1) within 25 m . Two preferential flow paths associated with past hydrologic modifications to the site allowed the nitrate plume to progress further into the restored forested riparian wetland . Surface runoff total N, dissolved reactive phosphorus (DRP), and total P concentrations averaged 8.63 mg N L(-1), 1.37 mg P L(-1), and 1.48 mg P L(-1), respectively, at the field edge and were reduced to 4.18 mg N L(-1), 0.31 mg P L(-1), and 0.36 mg P L(-1), respectively, at the restored forested riparian wetland outlet . Water and nutrient mass balance showed that retention and removal rates for nitrogen species ranged from a high of 78% for nitrate to a low of 52% for ammonium . Retention rates for both DRP and total P were 66% . Most of the N retention and removal was accounted for by denitrification . Mean annual concentrations of total N and total P leaving the restored forested riparian wetland were 1.98 mg N L(-1) and 0.24 mg P L(-1), respectively.

J Environ Qual, 2003 Mar-Apr, 32(2), 642 - 53
Evaluation of nitrate nitrogen fluxes from a tile-drained watershed in central Iowa; Tomer MD et al.; Nitrate N fluxes from tile-drained watersheds have been implicated in water quality studies of the Mississippi River basin, but actual NO3-N loads from small watersheds during long periods are poorly documented . We evaluated discharge and NO3-N fluxes passing the outlet of an Iowa watershed (5134 ha) and two of its tile-drained subbasins (493 and 863 ha) from mid-1992 through 2000 . The cumulative NO3-N load from the catchment was 168 kg ha(-1), and 176 and 229 kg ha(-1) from the subbasins . The outlet had greater total discharge (1831 mm) and smaller flow-weighted mean NO3-N concentration (9.2 mg L(-1)) than the subbasins, while the larger subbasin had greater discharge (1712 vs . 1559 mm) and mean NO3-N concentration (13.4 vs . 11.3 mg L(-1)) than the smaller subbasin . Concentrations exceeding 10 mg L(-1) were common, but least frequent at the outlet . Nitrate N was generally not diluted by large flows, except during 1993 flooding . The outlet showed smaller NO3-N concentrations at low flows . Relationships between discharge and NO3-N flux showed log-log slopes near 1.0 for the subbasins, and 1.2 for the outlet, considering autocorrelation and measurement-error effects . We estimated denitrification of subbasin NO3-N fluxes in a hypothetical wetland using published data . Assuming that temperature and NO3-N supply could limit denitrification, then about 20% of the NO3-N would have been denitrified by a wetland constructed to meet USDA-approved criteria . The low efficiency results from the seasonal timing and NO3-N content of large flows . Therefore, agricultural and wetland best management practices (BMPs) are needed to achieve water quality goals in tile-drained watersheds.

Huan Jing Ke Xue, 2003 Jan, 24(1), 113 - 6
{Study on nitrogen removal treating agriculture wastewater in subsurface constructed wetland}; Zhang R et al.; Nitrogen removal in subsurface constructed wetland treating agriculture non-pointed wastewater was studied in pilot-scale . The experimental results showed that the removal rates of TN increases with HRT increasing . The removal rates of TN achieved 60% in macrophyte bed system when the nominal hydraulic retention time was 5 days . Removal of TN followed first-order plug flow kinetics . Rate constant of Non-plant bed, Phragmitas communis bed and Zizania caduciflora bed was 0.14, 0.26 and 0.20 d-1 respectively . Denitrification was the main removal mechanism of total nitrogen, the amount of TN that can be removed by harvesting were above 15% compared to the loading into constructed wetland system.

Biosens Bioelectron, 2003 May, 18(5-6), 735 - 9
Amperometric dimethyl sulfoxide sensor using dimethyl sulfoxide reductase from Rhodobacter sphaeroides; Abo M et al.; An amperometric dimethyl sulfoxide (DMSO) sensor was constructed based on DMSO reductase (DMSO-R) . DMSO-R from Rhodobacter sphaeroides f . sp . denitrificans was immobilized by BSA-glutaraldehyde cross-linking at the surface of a glassy carbon electrode . Mediators were added to the sample solution in a free form . Several mediators (methyl viologen (MV), benzyl viologen (BV), neutral red (NR), safranin T (ST), FMN, phenazine methosulfate (PMS)), which can donate electrons to DMSO-R, were examined with the DMSO-R immobilized electrode . Among them MV was selected as a model mediator because of its wide linear response range and fast response time . The response current was effected by the measurement temperature but hardly effected by the pH of the sample solution . The response current was increased with the measurement temperature up to 50 degrees C . A response current was observed at 1 microM DMSO and the response time was 20 s under the optimum conditions . The response was observed for approximately 2 weeks . By the reduction of Schiff base in the cross-linking layer the response range became narrower but most of the response current was retained at 300 microM of DMSO for more than 5 weeks.

Water Sci Technol, 2003, 47(5), 211 - 6
Monitoring the influence of toxic compounds on microbial denitrifying biofilm processes; Li J et al.; Microelectrode measurements were conducted to obtain nitrate, pH and redox potential profiles within anoxic denitrifying biofilms . The influence of a toxic organic compound (acid orange 7) on biofilm microprofiles was also monitored using microelectrodes . The data provide evidence that the denitrifying biofilms were stratified into an anoxic layer and an anaerobic layer . The anaerobic zone might provide a niche for the biodegradation of recalcitrant organic compounds in biofilms . It was found that acid orange 7 and its biodegradation byproducts had only a slight impact on biofilm nitrate, pH and redox potential profiles.

Water Sci Technol, 2003, 47(5), 175 - 9
Extraction of exopolymers from biofilms: the protective effect of glutaraldehyde; Azeredo J et al.; The extraction of the exopolymeric matrix is a prerequisite to properly assessing the composition of the biofilm . Several extraction methods have already been developed, however, no universal method has yet been adopted because the compromise between high yields of extraction and minimum cell lysis is difficult to establish . In fact, most of the extraction methods promote leakage of intracellular material . The most common extraction methods, Dowex resin and sonication, were assayed in biofilms of Pseudomonas fluorescens and Alcaligenes denitrificans submitted to a pre-treatment with glutaraldehyde (GTA) . The assessment of ATP released after extraction was used as a criterion of cell lysis . The results showed that GTA is a protective agent against cell lysis . The pre-treatment with GTA is particularly useful combined with sonication.

Water Sci Technol, 2003, 47(5), 61 - 7
A technique using a membrane flow cell to determine average mass transfer coefficients and tortuosity factors in biofilms; Garcia Lopez LA et al.; Average mass transfer coefficients of an inert compound (LiCI) within denitrifying biofilms were monitored during biofilm growth in a membrane flow cell under different flow conditions, until the biofilm reached (pseudo-) steady state . Average effective diffusivities were found to increase with the decrease tortuosity factors of the biofilm matrix . The lowest tortuosity factor corresponded the highest liquid velocity.

Mikrobiologiia, 2003 Jan-Feb, 72(1), 117 - 25
{Biological activity in the modern and buried soils of the St.Petersburg's historical center}; Rusakov AV et al.; Biological activity in the urban modern and medieval soils of St . Petersburg was determined using soil samples taken from sections located at the historical center of this city nearby the Kazan Cathedral, the Twelve Colleges building (now the main building of St . Petersburg State University), and on the site where the Swedish fortress Nienshants formerly existed . The studied parameters of biological activity included the microbial transformation rate of organic matter under aerobic and anaerobic conditions, the intensity of denitrification and nitrogen fixation, and the amount of microbial biomass . This investigation is the first attempt to comparatively study modern urban anthropogenically impacted soils and buried soils that had formed the soil cover of this region before St . Petersburg was founded . The major microbiological and physicochemical parameters of the soils were subjected to correlation analysis.

Water Res, 2003 Apr, 37(8), 1953 - 71
Experimental and model assisted investigation of an operational strategy for the BPR under low influent concentrations; Kruhne U et al.; The behaviour of a pilot scale biological phosphorus removal process (BPR) of the alternating type was investigated during periods of low influent concentrations and increased hydraulic load . A process disturbance of this type result in an increase in the phosphate concentration level in the anoxic/aerobic reactors and in the plant effluent shortly after the influent wastewater returns to normal strength . The accumulation of phosphorus in the system was avoided by the addition of an external carbon source either to the influent or to the effluent from the anaerobic reactor in form of sodium acetate . With the help of such an addition, the internal carbon storage compounds could be maintained at a high level, which is shown by poly-hydroxy-alcanoates (PHA) measurements . Several levels of acetate addition were investigated experimentally in order to determine a minimal amount of internally stored carbon, which could ensure the stabilization of BPR during such dynamic influent conditions . Furthermore reduction of aeration time during periods of low influent concentrations was investigated . It was observed that BPR was stabilized by combining a reduction of aeration time with carbon source addition, which maintained the internal stored carbon at a higher level . This combined control action resulted in a desired high BPR activity when the normal strength of the influent wastewater was re-established . The failure of the BPR process was sometimes observed even when comparatively high concentrations of PHA could be detected and an identification of a minimal PHA level was not possible . During this investigation an extended version of the activated sludge model No . 2 (ASM2), which includes denitrification by phosphate accumulating organisms, is used for the detailed analysis of the experiments . The model predicted the phosphorus build-up after the process disturbance as well as the performance during the stabilized experiments . Assisted by the model, the investigations indicate that a PHA limitation is not the only factor affecting the recovery of the BPR process during periods of low influent concentrations.

Biochemistry, 2003 Apr 22, 42(15), 4534 - 43
Characterization and topology of the membrane domain Nqo10 subunit of the proton-translocating NADH-quinone oxidoreductase of Paracoccus denitrificans; Kao MC et al.; The proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans is composed of 14 different subunits (Nqo1-Nqo14) . Of these, seven subunits (Nqo7, Nqo8, and Nqo10-14) which are equivalent to the mitochondrial DNA-encoded subunits of complex I constitute the membrane segment of the enzyme complex; the remaining subunits make up the peripheral part of the enzyme . We report here on the biochemical characterization and heterologus expression of the Nqo10 subunit . The Nqo10 subunit could not be extracted from the Paracoccus membranes by NaI or alkaline treatment, which is consistent with the presumed membrane localization . By using the maltose-binding protein (MBP) fusion system, the Nqo10 subunit was overexpressed in Escherichia coli . The MBP-fused Nqo10 was expressed in membrane fractions of the host cell and was extractable by Triton X-100 . The extracted fusion protein was then isolated by one-step affinity purification through an amylose column . By using immunochemical methods in conjunction with cysteine-scanning mutagenesis and chemical modification techniques, the topology of the Nqo10 subunit expressed in E . coli membranes was determined . The data indicate that the Nqo10 subunit consists of five transmembrane segments with the N- and C-terminal regions facing the periplasmic and cytoplasmic sides of the membrane, respectively . In addition, the data also suggest that the proposed topology of the MBP-fused Nqo10 subunit expressed in E . coli membranes is consistent with that of the Nqo10 subunit in the native Paracoccus membranes . From the experimentally determined topology together with computer prediction programs, a topological model for the Nqo10 subunit is proposed.

Biodegradation, 2002, 13(5), 353 - 60
Effects of alternative carbon sources on biological transformation of nitrophenols; Karim K et al.; The removal of nitrophenols under denitrifying conditions was studied in bench-scale upflow anaerobic sludge blanket (UASB) reactors (R1, R2, R3 and R4) using three different carbon sources . Initially acetate was used as carbon source (substrate) in all the four reactors followed by glucose and methanol . Reactor R1 was kept as control and R2, R3, R4 were fed with 30 mg/l concentration of 2-nitrophenol (2-NP), 4-nitrophenol (4-NP), and 2,4-dinitrophenol (2,4-DNP), respectively . Throughout the study the hydraulic retention time (HRT) and COD/NO3-N ratio were kept as 24 h and 10, respectively . 2-Aminophenol (2-AP), 4-aminophenol (4-AP) and 2-amino,4-nitrophenol (2-A,4-NP) were found as the major intermediate metabolites of 2-NP, 4-NP and 2,4-DNP degradation, respectively . Methanol was found to be a better carbon source for 4-NP and 2,4-DNP degradation as compared to acetate and glucose, while 2-NP degradation was not influenced much by the change of substrate . Nitrate nitrogen removal was always more than 99% . COD removal efficiency of the nitrophenol fed reactors varied from 85.7% to 97.7% . The oxidation-reduction potential (ORP) inside the reactors dropped, up to -300 mv, with glucose as carbon source . As the reactors were switched over to methanol, ORP increased to -190 mv . The granular sludge developed inside the reactors was light brown in colour when acetate and glucose were used as substrate, which turned dark brown to black at the end of methanol run . Biomass yield in terms of volatile suspended solids was observed as 0.15, 0.089 and 0.14 g per gram of COD removal for acetate, glucose and methanol, respectively.

Chemosphere, 2003 Feb, 50(6), 725 - 32
Nitrate distribution and denitrification in the saturated zone of paddy field under rice/wheat rotation; Zhu JG et al.; Nitrate concentration in well water collected from the wells near farm houses was investigated in the Taihu Lake basin (TBL) of China . Nitrate-N content of the well water ranged from 0.1 to 23 mgNl(-1), and 41% exceeded the criteria (10 mg Nl(-1)) . It was found that the difference in well conditions, especially the depth of the well, was the main cause of the difference in the nitrate concentration of well water, i.e . it was higher in shallow well and lower in deeper well . A recommendation was made for local farmers to drill wells deeper than 10 m in order to reduce the risk of high ingestion of nitrate-N in their drinking water . Nitrate distribution and denitrification in the saturated zone of a paddy field under rice/wheat rotation in the TBL were studied . Porous pipes were installed in triplicate at depths of 1.5, 2.0, 2.5, 3.5 and 5 m respectively to collect the soil solution samples . Results showed that nitrate was the predominant N form in soil solution of saturated zone, and it increased from 1.5 to 2.5 m depth, and decreased from 2.5 to 5 m depth . N2O captured in the soil solution was very high comparing with N2O content in air . N2O content was positively correlated with nitrate concentrations in the soil profile . These results indicate that nitrate leached into saturated zone was mainly transformed via denitrification processes . Comparing the sum of inorganic nitrogen with the total nitrogen in soil solution samples collected from those wells at the field, some soluble organic nitrogen was found about 1-2 mg N l(-1) in average.

J Gen Appl Microbiol, 2002 Dec, 48(6), 299 - 308
Diaphorobacter nitroreducens gen nov, sp nov, a poly(3-hydroxybutyrate)-degrading denitrifying bacterium isolated from activated sludge; Khan ST et al.; Three denitrifying strains of bacteria capable of degrading poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) were isolated from activated sludge and characterized . All of the isolates had almost identical phenotypic characteristics . They were motile gram-negative rods with single polar flagella and grew well with simple organic compounds, as well as with PHB and PHBV, as carbon and energy sources under both aerobic and anaerobic denitrifying conditions . However, none of the sugars tested supported their growth . The cellular fatty acid profiles showed the presence of C16:1omega7cis and C16:0 as the major components and of 3-OH-C10:0 as the sole component of hydroxy fatty acids . Ubiquinone-8 was detected as the major respiratory quinone . A 16S rDNA sequence-based phylogenetic analysis showed that all the isolates belonged to the family Comamonadaceae, a major group of beta-Proteobacteria, but formed no monophyletic cluster with any previously known species of this family . The closest relative to our strains was an unidentified bacterium strain LW1 (=DSM 13225) (99.9% similarity), reported previously as a 1-chloro-4-nitrobenzene degrading bacterium . DNA-DNA hybridization levels among the new isolates were more than 60%, whereas those between our isolates and strain DSM 13225 were less than 50% . The G+C content of genomic DNA of the new strains was 64 to 65 mol% . Based on these results, we concluded that the PHBV-degrading denitrifying isolates should be classified as a new genus and a new species, for which we propose the name Diaphorobacter nitroreducens . The type strain is strain NA10B (=JCM 11421=CIP 107294) . We also propose to classify strain DSM 13225 as a genospecies of Diaphorobacter.

Nature, 2003 Apr 10, 422(6932), 608 - 11
Anaerobic ammonium oxidation by anammox bacteria in the Black Sea; Kuypers MM et al.; The availability of fixed inorganic nitrogen (nitrate, nitrite and ammonium) limits primary productivity in many oceanic regions . The conversion of nitrate to N2 by heterotrophic bacteria (denitrification) is believed to be the only important sink for fixed inorganic nitrogen in the ocean . Here we provide evidence for bacteria that anaerobically oxidize ammonium with nitrite to N2 in the world's largest anoxic basin, the Black Sea . Phylogenetic analysis of 16S ribosomal RNA gene sequences shows that these bacteria are related to members of the order Planctomycetales performing the anammox (anaerobic ammonium oxidation) process in ammonium-removing bioreactors . Nutrient profiles, fluorescently labelled RNA probes, 15N tracer experiments and the distribution of specific 'ladderane' membrane lipids indicate that ammonium diffusing upwards from the anoxic deep water is consumed by anammox bacteria below the oxic zone . This is the first time that anammox bacteria have been identified and directly linked to the removal of fixed inorganic nitrogen in the environment . The widespread occurrence of ammonium consumption in suboxic marine settings indicates that anammox might be important in the oceanic nitrogen cycle.

Nature, 2003 Apr 10, 422(6932), 606 - 8
N2 production by the anammox reaction in the anoxic water column of Golfo Dulce, Costa Rica; Dalsgaard T et al.; In oxygen-depleted zones of the open ocean, and in anoxic basins and fjords, denitrification (the bacterial reduction of nitrate to give N2) is recognized as the only significant process converting fixed nitrogen to gaseous N2 . Primary production in the oceans is often limited by the availability of fixed nitrogen such as ammonium or nitrate, and nitrogen-removal processes consequently affect both ecosystem function and global biogeochemical cycles . It was recently discovered that the anaerobic oxidation of ammonium with nitrite--the 'anammox' reaction, performed by bacteria--was responsible for a significant fraction of N2 production in some marine sediments . Here we show that this reaction is also important in the anoxic waters of Golfo Dulce, a 200-m-deep coastal bay in Costa Rica, where it accounts for 19-35% of the total N2 formation in the water column . The water-column chemistry in Golfo Dulce is very similar to that in oxygen-depleted zones of the oceans--in which one-half to one-third of the global nitrogen removal is believed to occur . We therefore expect the anammox reaction to be a globally significant sink for oceanic nitrogen.

Biochim Biophys Acta, 2003 Apr 11, 1647(1-2), 381 - 9
Catching catalysis in the act: using single crystal kinetics to trap methylamine dehydrogenase reaction intermediates; Pearson AR et al.; Methylamine dehydrogenase (MADH) is produced by a range of gram-negative methylotrophic and autotrophic bacteria, and allows the organisms to utilise methylamine as the sole source of carbon . The enzyme catalyses the oxidation of methylamine to formaldehyde and ammonia, leaving it in a two-electron reduced state . To complete the catalytic cycle, MADH is reoxidised via an electron transfer (ET) chain . The redox center in the enzyme is the organic cofactor tryptophan tryptophylquinone (TTQ) derived from the posttranslational modification of two Trp residues in the protein . This cofactor has spectral features in the visible region, which change during catalytic turnover, defining spectrally distinct reaction intermediates that reflect the electronic state of the TTQ . In the case of the Paracoccus denitrificans enzyme the physiologic ET chain involves the protein redox partner amicyanin (a blue copper protein) . A stable binary (MADH/amicyanin) complex can be formed, and its crystal structure has been solved to 2.5 A resolution by Chen et al . {Biochemistry 21 (1992) 4959} . These crystals were shown to be competent for catalysis and ET by Merli et al . {J . Biol . Chem . 271 (1996) 9177} using single crystal polarised absorption spectroscopy . Through a novel combination of single crystal visible microspectrophotometry, X-ray crystallography and freeze-trapping, we have trapped reaction intermediates of the enzyme in complex with its physiological redox partner amicyanin in the crystalline state . We will present data confirming that catalysis and ET in the binary complex crystals can be tracked by single crystal visible microspectrophotometry . We will also show that the reaction pathway is unperturbed by the presence of cryoprotectant solution, enabling direct freeze-trapping of reaction intermediates within the crystal . We will present new data demonstrating that the binary complex crystals are also capable of exhibiting UV light-dependent oxidase activity, as observed in solution {Biochim . Biophys . Acta 1364 (1998) 297}.

Biochim Biophys Acta, 2003 Apr 11, 1647(1-2), 337 - 42
Catalysis and electron transfer in protein crystals: the binary and ternary complexes of methylamine dehydrogenase with electron acceptors; Ferrari D et al.; Polarized absorption microspectrophotometry has been used to detect catalysis and intermolecular electron transfer in single crystals of two multiprotein complexes: (1) the binary complex between Paracoccus denitrificans methylamine dehydrogenase, which contains tryptophan-tryptophylquinone (TTQ) as a cofactor, and its redox partner, the blue copper protein amicyanin; (2) the ternary complex between the same two proteins and cytochrome c-551i . Continuous wave electron paramagnetic resonance has been used to compare the state of copper in polycrystalline powders of the two systems . While catalysis and intermolecular electron transfer from reduced TTQ to copper are too fast to be accessible to our measurements, heme reduction occurs over a period of several minutes . The observed rate constant is about four orders of magnitude lower than in solution . The analysis of the temperature dependence of this apparent constant provides values for the parameters H(AB), related to electronic coupling between the two centers, and lambda, the reorganizational energy, that are compatible with electron transfer being the rate-determining step . From these parameters and the known distance between copper and heme, it is possible to calculate the parameter beta, which depends on the nature of the intervening medium, obtaining a value typical of electron transfer across a protein matrix . These findings suggest that the ternary complex in solution might achieve a higher efficiency than the rigid crystal structure thanks to an as yet unidentified role of protein dynamics.

Biochim Biophys Acta, 2003 Apr 11, 1647(1-2), 289 - 96
Redox properties of quinohemoprotein amine dehydrogenase from Paracoccus denitrificans; Fujieda N et al.; Paracoccus denitrificans produces two primary enzymes for the amine oxidation, tryptophan-tryptophylquinone (TTQ)-containing methylamine dehydrogenase (MADH) and quinohemoprotein amine dehydrogenase (QH-AmDH) . QH-AmDH has a novel cofactor, cysteine tryptophylquinone (CTQ) and two hemes c . In this work, the redox potentials of three redox centers in QH-AmDH were determined by a mediator-assisted continuous-flow column electrolytic spectroelectrochemical technique . Kinetics of the electron transfer from QH-AmDH to three kinds of metalloproteins, amicyanin, cytochrome c(550), and horse heart cytochrome c were examined on the basis of the theory of mediated-bioelectrocatalysis . All these metalloproteins work as a good electron acceptor of QH-AmDH and donate the electron to the terminal oxidase of P . denitrificans, which was revealed by reconstitution of the respiratory chain . These properties are in marked contrast with those of MADH, which shows high specificity to amicyanin . These electron transfer kinetics are discussed in terms of thermodynamics and structural property.

Environ Pollut, 2003, 121(3), 389 - 99
Acid rain and nitrogen deposition in a sub-tropical watershed (Piracicaba): ecosystem consequences; Krusche AV et al.; High levels of wet N and acidic deposition were measured in southeast Brazil . In this study we addressed the sensitivity of water bodies and soils to acidification and N deposition in the Piracicaba River basin (12,400 km2) . Average acid neutralization capacity (ANC) at 23 river sampling sites varied from 350 to 1800 microeq l(-1) . Therefore, rivers and streams in the Piracicaba basin are well buffered, if the lower limit of 200 microeq l(-1) is assumed as an indication of poorly buffered waters . ANC is increased by untreated wastewaters discarded into rivers and streams of the region . Average NO3 concentrations varied from 20 to 70 microeq l(-1) . At the most polluted river sites, NO3 concentration is not highest, however, probably due to NO3 reduction and denitrification . Most of the nitrogen in streams is also provided by wastewaters and not by wet deposition . The majority of the soils in the basin, however, are acidic with a low base cation content and high aluminum concentration . Therefore, soils in this basin are poorly buffered and, in areas of forest over sandy soils, acidification may be a problem.

FEMS Microbiol Lett, 2003 Mar 28, 220(2), 261 - 9
Properties of the periplasmic nitrate reductases from Paracoccus pantotrophus and Escherichia coli after growth in tungsten-supplemented media; Gates AJ et al.; Paracoccus pantotrophus grown anaerobically under denitrifying conditions expressed similar levels of the periplasmic nitrate reductase (NAP) when cultured in molybdate- or tungstate-containing media . A native PAGE gel stained for nitrate reductase activity revealed that only NapA from molybdate-grown cells displayed readily detectable nitrate reductase activity . Further kinetic analysis showed that the periplasmic fraction from cells grown on molybdate (3 microM) reduced nitrate at a rate of V(max)=3.41+/-0.16 micromol {NO(3)(-)} min(-1) mg(-1) with an affinity for nitrate of K(m)=0.24+/-0.05 mM and was heat-stable up to 50 degrees C . In contrast, the periplasmic fraction obtained from cells cultured in media supplemented with tungstate (100 microM) reduced nitrate at a much slower rate, with much lower affinity (V(max)=0.05+/-0.002 micromol {NO(3)(-)} min(-1) mg(-1) and K(m)=3.91+/-0.45 mM) and was labile during prolonged incubation at >20 degrees C . Nitrate-dependent growth of Escherichia coli strains expressing only nitrate reductase A was inhibited by sub-mM concentrations of tungstate in the medium . In contrast, a strain expressing only NAP was only partially inhibited by 10 mM tungstate . However, none of the above experimental approaches revealed evidence that tungsten could replace molybdenum at the active site of E . coli NapA . The combined data show that tungsten can function at the active site of some, but not all, molybdoenzymes from mesophilic bacteria.

Biochemistry, 2003 Apr 8, 42(13), 3966 - 78
Effects of multiple ligand binding on kinetic isotope effects in PQQ-dependent methanol dehydrogenase; Hothi P et al.; The reaction of PQQ-dependent methanol dehydrogenase (MDH) from Methylophilus methylotrophus has been studied by steady-state and stopped-flow kinetic methods, with particular reference to multiple ligand binding and the kinetic isotope effect (KIE) for PQQ reduction . Phenazine ethosulfate (PES; an artificial electron acceptor) and cyanide (a suppressant of endogenous activity), but not ammonium (an activator of MDH), compete for binding at the catalytic methanol-binding site . Cyanide does not activate turnover in M . methylotrophus MDH, as reported previously for the Paracoccus denitrificans enzyme . Activity is dependent on activation by ammonium but is inhibited at high ammonium concentrations . PES and methanol also influence the stimulatory and inhibitory effects of ammonium through competitive binding . Reaction profiles as a function of ammonium and PES concentration differ between methanol and deuterated methanol, owing to force constant effects on the binding of methanol to the stimulatory and inhibitory ammonium binding sites . Differential binding gives rise to unusual KIEs for PQQ reduction as a function of ammonium and PES concentration . The observed KIEs at different ligand concentrations are independent of temperature, consistent with their origin in differential binding affinities of protiated and deuterated substrate at the ammonium binding sites . Stopped-flow studies indicate that enzyme oxidation is not rate-limiting at low ammonium concentrations (<4 mM) during steady-state turnover . At higher ammonium concentrations (>20 mM), the low effective concentration of PES in the active site owing to the competitive binding of ammonium lowers the second-order rate constant for enzyme oxidation, and the oxidative half-reaction becomes more rate limiting . A sequential stopped-flow method is reported that has enabled, for the first time, a detailed study of the reductive half-reaction of MDH and comparison with steady-state data . The limiting rate of PQQ reduction (0.48 s(-1)) is less than the steady-state turnover number, and the observed KIE in stopped-flow studies is unity . Although catalytically active, we propose reduction of the oxidized enzyme generated in stopped-flow analyses is gated by conformational change or ligand exchange . Slow recovery from this trapped state on mixing with methanol accounts for the slow reduction of PQQ and a KIE of 1 . This study emphasizes the need for caution in using inflated KIEs, and the temperature dependence of KIEs, as a probe for hydrogen tunneling.

Environ Technol, 2003 Feb, 24(2), 161 - 9
Process control and design considerations for methanol-induced denitrification in a sequencing batch reactor; Louzeiro NR et al.; The primary goal of this research was to determine the effect of methanol-induced denitrification on volatile suspended solids production, settleability, and oxidation-reduction potential in a full-scale sequencing batch reactor . Batch tests were also conducted to determine the influence of mixing and acclimatization on the denitrification of wastewater with methanol . The observed sludge production in the full-scale sequencing batch reactor with methanol addition was 0.21 kg volatile suspended solids l(-1) methanol, versus the calculated stoichiometric sludge production of 0.17 kg volatile suspended solids l(-1) methanol . The settleability in the full-scale sequencing batch reactor, measured by the sludge volume index, increases linearly with increasing denitrification rate . The total change in the oxidation-reduction potential magnitude during a sequencing batch reactor cycle increased linearly with increasing denitrification rate . A minimum of 55% increase in the denitrification rate was observed in a batch reactor with methanol addition and a sludge acclimatized to methanol addition, compared to a batch reactor with methanol addition and a non-acclimatized sludge . The non-acclimatized batch reactor had a negligible denitrification rate without methanol addition . However, significant denitrification rates were observed in the acclimatized batch reactors without methanol addition, potentially caused by microbial storage or an increased population of denitrifiers that scavenge any available carbon . A completely mixed batch reactor, with sludge acclimatized to methanol addition during the anoxic cycle, had an increase in the denitrification rate ranging from 660%, without methanol addition, to 200%, with a methanol dosage of 12.7 mg l(-1), compared to the unmixed batch reactor with an acclimatized sludge . Therefore, mixing appears to be critical to the denitrification process, to realize the best kinetic performance.

Int J Syst Evol Microbiol, 2003 Jan, 53(Pt 1), 147 - 52
Alicycliphilus denitrificans gen . nov., sp . nov., a cyclohexanol-degrading, nitrate-reducing beta-proteobacterium; Mechichi T et al.; A facultatively denitrifying bacterium, strain K601T, was isolated at 30 degrees C from a municipal sewage plant on cyclohexanol as sole carbon source and nitrate as electron acceptor . Under aerobic conditions this strain used acetate, fumarate, lactate, pyruvate, crotonate, indole, glucose, vanillate, 4-hydroxybenzoate, m-cresol, o-cresol and p-cresol . Under denitrifying conditions the strain used cyclohexanol, cyclohexanone, 1,3-cyclohexanedione, 2-cyclohexenone, 1,3-cyclohexanediol (cis and trans), monocarboxylic acids (C2-C7), adipate, pimelate, 5-oxocaproate, citrate, 2-oxoglutarate, succinate, malate, crotonate, lactate, pyruvate and fumarate . Cells were short rods, 0.6 microm wide and 1-2 microm long, motile, non-spore-forming, Gram-negative, and catalase- and oxidase-positive . Strain K601T used nitrate, nitrite and oxygen as electron acceptors, but not sulfate, sulfite or fumarate . The DNA G+C content of strain K601T was 66 mol% . Phylogenetic analysis, based on 16S rDNA sequencing, showed that strain K601T represents a separate lineage of the family Comamonadaceae in the beta-subclass of Proteobacteria . Based on the high 16S rDNA sequence divergence and phenotypic characteristics, the name Alicycliphilus denitrificans gen . nov., sp . nov . is proposed for this strain . The type strain is K60IT (=DSM 14773T =CIP 107495T).

Acta Crystallogr D Biol Crystallogr, 2003 Apr, 59(Pt 4), 677 - 87 Epub 2003 Mar 25.
1.3 A X-ray structure of an antibody Fv fragment used for induced membrane-protein crystallization; Essen LO et al.; The antibody Fv fragment 7E2 has previously been employed in the induced crystallization of the integral membrane protein cytochrome c oxidase from Paracoccus denitrificans . The 1.3 A X-ray structure of the uncomplexed antibody fragment reveals conserved water networks on the surfaces of the framework regions . A novel consensus motif for water coordination, XX(S/T), is found along the edges of the beta-sandwich, where a water molecule forms hydrogen bonds to the carbonyl O atom of a residue at position N and the OG hydroxyl groups of conserved serines or threonines at position N + 2 . Multiple conformations were found in the hydrophobic core for residues IleL21, LeuL33 and the disulfide bridges . An internal water molecule that is compatible with only one of the three packing states of the V(L) core suggests local 'breathing' of the variable domain . TrpH47, a conserved key residue of the V(H)/V(L) interface, is crucially involved in the formation of the antigen-binding site by adopting a novel conformation that specifically stabilizes the non-canonical CDR-L3 loop . Finally, a comparison with 7E2-cytochrome c oxidase complexes demonstrates that binding of this membrane-bound antigen proceeds without major conformational changes of the 7E2 antibody fragment.

Int J Syst Evol Microbiol, 2003 Jan, 53(Pt 1), 323 - 9
Caldithrix abyssi gen . nov., sp . nov., a nitrate-reducing, thermophilic, anaerobic bacterium isolated from a Mid-Atlantic Ridge hydrothermal vent, represents a novel bacterial lineage; Miroshnichenko ML et al.; A novel, moderately thermophilic, strictly anaerobic, mixotrophic bacterium, designated strain LF13T, was isolated from a deep-sea hydrothermal chimney sample that was collected at a vent site at 14 degrees 45' N, 44 degrees 59' W on the Mid-Atlantic Ridge . Cells were Gram-negative, thin, non-motile rods of variable length . Strain LF13T grew optimally at pH 6.8-7.0 and 60 degrees C with 2.5% (w/v) NaCl . It grew chemo-organoheterotrophically, fermenting proteinaceous substrates, pyruvate and Casamino acids . The strain was able to grow by respiration, utilizing molecular hydrogen (chemolithoheterotrophically) or acetate as electron donors and nitrate as an electron acceptor . Ammonium was formed in the course of denitrification . One-hundred milligrams of yeast extract per litre were required for growth of the strain . The G + C content of the genomic DNA of strain LF13T was 42.5 mol% . Neither 16S rDNA sequence similarity values nor phylogenetic analysis unambiguously related strain LF13T with members of any recognized bacterial phyla . On the basis of 16S rDNA sequence comparisons, and in combination with physiological and morphological traits, a novel genus, Caldithrix, is proposed, with strain LF13T (= DSM 13497T =VKM B-2286T) representing the type species, Caldithrix abyssi.

Appl Microbiol Biotechnol, 2003 Apr, 61(2), 103 - 9 Epub 2003 Jan 14.
Application of polyhydroxyalkanoates for denitrification in water and wastewater treatment; Hiraishi A et al.; Application of polyhydroxyalkanoates (PHAs) and related biodegradable polymers has gained momentum in various areas of biotechnology . A promising application that started appearing in the past decade is the use of PHAs as the solid substrate for denitrification of water and wastewater . This type of denitrification, termed here "solid-phase denitrification", has several advantages over the conventional system supplemented with liquid organic substrate . PHAs serve not only as constant sources of reducing power for denitrification but also as solid matrices favorable for development of microbial films . In addition, in contrast to conventional processes, the use of PHAs has no potential risk of release of dissolved organic carbon with the resultant deterioration of effluent water quality . If the production cost of PHAs can be brought down, its application to the denitrification process will become economically more promising . A number of PHA-degrading denitrifying bacteria have been isolated and characterized from activated sludge and continuous flow-bed reactors for denitrification with PHAs . Most of these isolates have been assigned phylogenetically to members of beta-Proteobacteria, especially those of the family Comamonadaceae . The metabolic and regulatory relationships between PHA degradation and denitrification, and the interactive relationship between PHA-degrading cells and the solid surface structure are important subjects awaiting future studies, which would provide a new insight into our comprehensive understanding of the solid-phase denitrification process.

Biochemistry, 2003 Mar 25, 42(11), 3224 - 30
Understanding quinone cofactor biogenesis in methylamine dehydrogenase through novel cofactor generation; Pearson AR et al.; Cofactors made from constitutive amino acids in proteins are now known to be relatively common . A number of these involve the generation of quinone cofactors, such as topaquinone in the copper-containing amine oxidases, and lysine tyrosylquinone in lysyl oxidase . The biogenesis of the quinone cofactor tryptophan tryptophylquinone (TTQ) in methylamine dehydrogenase (MADH) involves the post-translational modification of two constitutive Trp residues (Trp(beta)(57) and Trp(beta)(108) in Paracoccus denitrificans MADH) . The modifications for generating TTQ are the addition of two oxygens to the indole ring of Trp(beta)(57) and the formation of a covalent cross-link between Cepsilon3 of Trp(beta)(57) and Cdelta1 of Trp(beta)(108) . The order in which these events occur is unknown . To investigate the role Trp(beta)(108) may play in this process, this residue was mutated to both a His (betaW108H) and a Cys (betaW108C) residue . For each mutant, the majority of the protein that was isolated was inactive and exhibited weaker subunit-subunit interactions than native MADH . Analysis by mass spectrometry suggested that the inactive protein was a biosynthetic intermediate with only one oxygen atom incorporated into Trp(beta)(57) and no cross-link with residue beta108 . However, in each mutant preparation, a small percentage of the mutant enzyme was active and appears to possess a functional tryptophylquinone cofactor . In the case of betaW108C, this cofactor may be identical to cysteine tryptophylquinone, recently described in the bacterial quinohemoprotein amine dehydrogenase . In betaW108H, the active cofactor is presumably a histidine tryptophylquinone, which has not been previously described, and represents the synthesis of a novel quinone protein cofactor.

Water Sci Technol, 2003, 47(3), 163 - 6
Parasite contamination of liquid sludge from urban wastewater treatment plants; Schwartzbrod J et al.; This study was performed on sludge samples from 20 wastewater treatment plants located in the north west of France with capacities of 1,000-20,000 inhabitant equivalents . The types of treatment studied were activated sludge low charge with and without denitrification . Respectively, 110 samples of fresh sludge and 84 samples of discharged sludge for spreading were analysed . Globally 78.6% of samples contained helminth eggs belonging to the cestodes (6.1%) and nematodes (93.9%) . Most of the nematode eggs detected were viable with 135 positive samples . The distribution, according to genera, indicated a high prevalence of Toxocara eggs (77.4%) followed by Capillaria (13.2%), Trichuris (8.1%) and Ascaris (1.3%) . For viable nematode eggs, the concentrations detected ranged from < 1 to 28/4 gDM for fresh sludge and from < 1 to 9.6/4 gDM for discharged sludge.

J Environ Sci Health A Tox Hazard Subst Environ Eng, 2003 Feb, 38(2), 339 - 52
Denitrifying characteristics of the multiple stages enhanced biological nutrient removal process with external carbon sources; Chou YJ et al.; This research investigated denitrifying activity of activated sludge with three external carbon sources (sodium acetate, methanol and glucose) via a series of batch experiments . Activated sludge used was cultivated in a multiple stages enhanced biological nutrient removal (EBNR) process that exhibited high removal efficiency of effective carbon, nitrogen, and phosphorus . Results showed type of external carbon source had a significant influence on specific nitrate utilization rate, nitrite accumulation, adaptive time of microorganisms, and nitrate removal efficiency . Sodium acetate addition resulted in high phosphate concentration in effluent; meanwhile methanol caused increasing turbidity and carbon breakthrough problem . When glucose was fed to be the external carbon source, accumulative nitrite concentration was higher than that with sodium acetate or methanol addition . When sodium acetate, methanol and glucose were used to be the electron donor, average dosages for nitrate elimination were 6.97, 5.85, and 5.65 mg-COD/mg-N, respectively . Because the final polyhydroxyalkanoates (PHAs) concentrations contained within the biomass were more than the original level and no phosphate re-release was observed, glycogen-accumulating organisms (GAOs) might exist in the multiple stages EBNR process and increased carbon dosage for further nitrate removal.

J Biol Chem, 2003 May 23, 278(21), 18761 - 6 Epub 2003 Mar 13.
Direct detection of Fe(IV){double bond}O intermediates in the cytochrome aa3 oxidase from Paracoccus denitrificans/H2O2 reaction; Pinakoulaki E et al.; We report the first evidence for the formation of the "607- and 580-nm forms" in the cytochrome oxidase aa3/H2O2 reaction without the involvement of tyrosine 280 . The pKa of the 607-580-nm transition is 7.5 . The 607-nm form is also formed in the mixed valence cytochrome oxidase/O2 reaction in the absence of tyrosine 280 . Steady-state resonance Raman characterization of the reaction products of both the wild-type and Y280H cytochrome aa3 from Paracoccus denitrificans indicate the formation of six-coordinate low spin species, and do not support, in contrast to previous reports, the formation of a porphyrin pi-cation radical . We observe three oxygen isotope-sensitive Raman bands in the oxidized wild-type aa3/H2O2 reaction at 804, 790, and 358 cm-1 . The former two are assigned to the Fe(IV){double bond}O stretching mode of the 607- and 580-nm forms, respectively . The 14 cm-1 frequency difference between the oxoferryl species is attributed to variations in the basicity of the proximal to heme a3 His-411, induced by the oxoferryl conformations of the heme a3-CuB pocket during the 607-580-nm transition . We suggest that the 804-790 cm-1 oxoferryl transition triggers distal conformational changes that are subsequently communicated to the proximal His-411 heme a3 site . The 358 cm-1 mode has been found for the first time to accumulate with the 804 cm-1 mode in the peroxide reaction . These results indicate that the mechanism of oxygen reduction must be reexamined.

Environ Toxicol Chem, 2003 Mar, 22(3), 540 - 4
Biotransformation of 3,5-dibromo-4-hydroxybenzonitrile under denitrifying, Fe(III)-reducing, sulfidogenic, and methanogenic conditions; Knight VK et al.; Bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) is a halogenated aromatic nitrile herbicide used on a variety of crops for the postemergence control of annual broad-leaved weeds . The anaerobic biodegradability of bromoxynil and its aerobic transformation product, 3,5-dibromo-4-hydroxybenzoate, were examined in enrichment cultures established with anaerobic sediment under denitrifying, Fe(III)-reducing, sulfidogenic, and methanogenic conditions . Bromoxynil (100 microM) was depleted in 20 to 30 d in the methanogenic, sulfidogenic, and Fe(IIi)-reducing enrichments but was stable under denitrifying conditions . The 3,5-dibromo-4-hydroxybenzoate (100 microM) was depleted within 20 to 35 d under all four anaerobic conditions . Both compounds were stable in sterile controls . Bromoxynil and 3,5-dibromo-4-hydroxybenzoate were readily utilized upon respiking of the cultures . During utilization of bromoxynil, stoichiometric release of bromide was observed with transient accumulation of metabolites identified as bromocyanophenol, cyanophenol, and phenol . Bromoxynil heptanoate and octanoate were rapidly hydrolyzed to bromoxynil, which was further degraded . These results indicate that bromoxynil and 3,5-dibromo-4-hydroxybenzoate are degraded under different anaerobic conditions . Anaerobic degradation of bromoxynil proceeds via reductive debromination to 4-cyanophenol, which is further transformed to phenol and can ultimately be degraded to carbon dioxide.

J Clin Microbiol, 2003 Mar, 41(3), 1289 - 94
Paracoccus yeeii sp . nov . (formerly CDC group EO-2), a novel bacterial species associated with human infection; Daneshvar MI et al.; CDC eugonic oxidizer group 2 (EO-2) is a group of unclassified gram-negative bacterial strains isolated from various human sources . As determined by biochemical tests and analyses of fatty acid compositions, these organisms form a homogeneous group that appears to be distinct from but related to other Paracoccus species . Molecular studies were performed on a set of 13 EO-2 strains from various clinical sources and geographic locations in the United States and Canada to determine their relationship to the Paracoccus genus . Control strains were Paracoccus denitrificans ATCC 17741(T), P . versutus ATCC 25364(T), P . aminophilus ATCC 49673(T), P . solventivorans ATCC 700252(T), and Psychrobacter immobilis ATCC 43116(T), which are phenotypically similar to EO-2 . Nearly complete (1,500-base) 16S rRNA gene sequencing of eight EO-2 strains showed a high level of sequence similarity (>99.3%) within the group, and a BLAST search of GenBank placed the EO-2 cluster in close proximity to Paracoccus species (95 to 97% similarity) . DNA-DNA hybridization studies of 13 of the EO-2 strains showed all to be related at the species level, with >70% relatedness under stringent conditions and a divergence within the group of less than 2% . None of the Paracoccus control strains hybridized at >54% with any of the EO-2 strains . These results indicate that EO-2 represents a new Paracoccus species, the first isolated from human clinical specimens . A new species, Paracoccus yeeii, is proposed for the EO-2 strains . The type strain of P . yeeii is CDCG1212 (ATCC BAA-599 and CCUG 46822), isolated in Pennsylvania from dialysate of a 77-year-old male with peritonitis.

Appl Environ Microbiol, 2003 Mar, 69(3), 1866 - 70
Anaerobic mineralization of quaternary carbon atoms: isolation of denitrifying bacteria on pivalic acid (2,2-dimethylpropionic acid); Probian C et al.; The degradability of pivalic acid was established by the isolation of several facultative denitrifying strains belonging to Zoogloea resiniphila, to Thauera and Herbaspirillum, and to Comamonadaceae, related to {Aquaspirillum} and Acidovorax, and of a nitrate-reducing bacterium affiliated with Moraxella osloensis . Pivalic acid was completely mineralized to carbon dioxide . The catabolic pathways may involve an oxidation to dimethylmalonate or a carbon skeleton rearrangement, a putative 2,2-dimethylpropionyl coenzyme A mutase.

Appl Environ Microbiol, 2003 Mar, 69(3), 1655 - 61
N2O-producing microorganisms in the gut of the earthworm Aporrectodea caliginosa are indicative of ingested soil bacteria; Ihssen J et al.; The main objectives of this study were (i) to determine if gut wall-associated microorganisms are responsible for the capacity of earthworms to emit nitrous oxide (N(2)O) and (ii) to characterize the N(2)O-producing bacteria of the earthworm gut . The production of N(2)O in the gut of garden soil earthworms (Aporrectodea caliginosa) was mostly associated with the gut contents rather than the gut wall . Under anoxic conditions, nitrite and N(2)O were transient products when supplemental nitrate was reduced to N(2) by gut content homogenates . In contrast, nitrite and N(2)O were essentially not produced by nitrate-supplemented soil homogenates . The most probable numbers of fermentative anaerobes and microbes that used nitrate as a terminal electron acceptor were approximately 2 orders of magnitude higher in the earthworm gut than in the soil from which the earthworms originated . The fermentative anaerobes in the gut and soil displayed similar physiological functionalities . A total of 136 N(2)O-producing isolates that reduced either nitrate or nitrite were obtained from high serial dilutions of gut homogenates . Of the 25 representative N(2)O-producing isolates that were chosen for characterization, 22 isolates exhibited >99% 16S rRNA gene sequence similarity with their closest cultured relatives, which in most cases was a soil bacterium, most isolates were affiliated with the gamma subclass of the class Proteobacteria or with the gram-positive bacteria with low DNA G+C contents, and 5 isolates were denitrifiers and reduced nitrate to N(2)O or N(2) . The initial N(2)O production rates of denitrifiers were 1 to 2 orders of magnitude greater than those of the nondenitrifying isolates . However, most nondenitrifying nitrate dissimilators produced nitrite and might therefore indirectly stimulate the production of N(2)O via nitrite-utilizing denitrifiers in the gut . The results of this study suggest that most of the N(2)O emitted by earthworms is due to the activation of ingested denitrifiers and other nitrate-dissimilating bacteria in the gut lumen.

Can J Microbiol, 2002 Dec, 48(12), 1089 - 98
Ralstonia basilensis M91-3, a denitrifying soil bacterium capable of using s-triazines as nitrogen sources; Stamper DM et al.; The purpose of this study was to characterize the phylogenetic and phenotypic traits of M91-3, a soil bacterium capable of mineralizing atrazine (2-chloro-4-N-isopropyl-6-N-ethyl-s-triazine) . The isolate was identified as Ralstonia basilensis based on 99.5% homology of the 16S rRNA sequence and various chemotaxonomic data . The isolate used atrazine as the sole source of energy, carbon, and nitrogen . It could also use several other s-triazines as nitrogen sources . Ralstonia basilensis M91-3 was capable of denitrification, which was confirmed by gas chromatographic analysis of nitrous oxide under acetylene blockage conditions.

Huan Jing Ke Xue, 2002 Nov, 23(6), 105 - 7
{Denitrification of drinking water with an active carbon-electrochemical biological reactor}; Qu J et al.; A packed bed electrochemical biological reactor was studied and utilized for denitrification of drinking water . Using active carbon as the filter, graphite plate as anode, and active carbon fibre as cathodes, an optimum reductive condition in the reactor was established for denitrification of nitrate . The test results proved that both nitrate and nitrite in the water could be removed effectively . At the case of 40 mL/h inflow, 40 mg/L of NO3-N, and 14 mA of current density, a 100% denitrification rate was achieved and no nitrite was detected . Because there is no any additional pollution in the treating process, this method is suggested to be a safety process for drinking water denitrification.

J Bacteriol, 2003 Mar, 185(6), 1895 - 902
Operon structure and regulation of the nos gene region of Pseudomonas stutzeri, encoding an ABC-Type ATPase for maturation of nitrous oxide reductase; Honisch U et al.; The synthesis of a functional nitrous oxide reductase requires an assembly apparatus for the insertion of the prosthetic copper . Part of the system is encoded by maturation genes located in Pseudomonas stutzeri immediately downstream of the structural gene for the enzyme . We have studied the transcriptional organization and regulation of this region and found a nosDFYL tatE operon structure . In addition to a putative ABC transporter, consisting of NosD, NosF, and NosY, the operon encodes a Cu chaperone, NosL, and a component of the Tat translocon, TatE . The nosD operon was activated in response to anaerobiosis and nitrate denitrification . The membrane-bound regulator NosR was required for operon expression; in addition, DnrD, a regulator of the Crp-Fnr family, enhanced expression under anaerobic conditions . This establishes a likely signal transduction sequence of NO --> DnrD --> nosR/NosR --> nosD operon . DnrD-dependent expression was also observed for the nnrS operon (located immediately downstream of the nosD operon), which encodes a putative heme-Cu protein (NnrS) and a member of the short-chain dehydrogenase family (ORF247) . The NosF protein, encoded within the nosD operon, exhibits sequence similarity to ABC-type ATPases . It was fused to the Escherichia coli maltose-binding protein and overexpressed in soluble form . The fusion protein was purified and shown to have ATPase activity . NosF is the first maturation factor for which a catalytic function has been demonstrated in vitro.

Bioresour Technol, 2003 Jul, 88(3), 215 - 9
Effect of organic carbon shock loading on endogenous denitrification in sequential batch reactors; Rodriguez Mora F et al.; This work was focused on the performance evaluation of sequential batch reactors (SBR) treating sewage, through a process of endogenous biological denitrification . Different operational conditions were carried out, and the behaviour under the effects of organic shock loading was examined . Three laboratory scale reactors were operated simultaneously and fed with similar wastewater . The substratum was molasses and nitrate, as carbon and nitrogen sources, respectively . The three reactors were operated during different aeration periods (0, 15 and 30 min) . Sudden changes (shock loading) in organic matter concentration were performed during the experiment . Thus, influent load was quickly increased threefold in relation to the original concentration . Results indicated that SBR reactors withstand adequately moderate shock loading . With regard to substratum degradation, nitrate elimination achieved was approximately 80%, while denitrification rate was approximately 0.87 mgg(-1)h(-1).

Biochim Biophys Acta, 2003 Mar 6, 1557(1-3), 119 - 24
Passive penetration of nitrate through the plasma membrane of Paracoccus denitrificans and its potentiation by the lipophilic tetraphenylphosphonium cation; Kucera I; Previously, it has been shown that treatment of Paracoccus denitrificans cells with phenylglyoxal inhibits the methyl-viologen-linked nitrate reductase activity by blocking the nitrate transporter . This inhibition disappears if tetraphenylphosphonium cation (TPP(+)) is added to the assay medium . In the present paper, the following evidence suggests that the effect of TPP(+) results from an increased transmembrane anion permeability and not from transporter reactivation or cell lysis . (1) Beside nitrate, TPP(+) also mediated the utilisation of chlorate, which normally lacks access to the cytoplasm . (2) The TPP(+) pathway had about hundred-times higher K(m) values for nitrate and chlorate than nitrate reductase in Triton X-100 permeabilised cells . (3) Although the uncoupler CCCP alone failed to overcome the PG block, it stimulated the operation of the TPP(+) pathway . (4) The method of continuous variations allowed the transport stoichiometry TPP(+)/NO(3)(-) to be determined as 3, indicating charge compensation for nitrate movement and the subsequent transmembrane two-electron redox reaction . Anion uptake was also measured independently from passive swelling of uncoupled spheroplasts in iso-osmotic solutions of ammonium salts . The permeability to nitrate lay in the permeability sequence Cl(-)<NO(3)(-)<ClO(4)(-)<SCN(-) and was further enhanced by TPP(+).

Plant Cell Physiol, 2003 Feb, 44(2), 212 - 6
An abundant periplasmic protein of the denitrifying phototroph Rhodobacter sphaeroides f . sp . denitrificans is PstS, a component of an ABC phosphate transport system; Matsuzaki M et al.; To understand a physiological role of an abundant 34-kDa periplasmic protein in the denitrifying phototroph Rhodobacter sphaeroides f . sp . denitrificans grown in a medium containing malate as the carbon source, the gene for the protein was isolated . The deduced amino acid sequence of the protein had a sequence similarity of 66.2% to that of PstS from Sinorhizobium meliloti . The downstream sequence of the Rhodobacter pstS contained five genes similar to pstCAB and phoUB, and its upstream sequence contained a putative regulatory sequence that is analogous to the Pho box involved in phosphate-limitation-induced gene expression in Escherichia coli . Both the amount of the PstS and the pstS promoter-driven expression of lacZ activity increased about two-fold in response to phosphate limitation . This is the first isolation of pst genes encoding proteins of an ABC phosphate transporter system from phototrophic bacteria.

J Biol Chem, 2003 May 2, 278(18), 15514 - 22 Epub 2003 Feb 24.
Characterization of cluster N5 as a fast-relaxing {4Fe-4S} cluster in the Nqo3 subunit of the proton-translocating NADH-ubiquinone oxidoreductase from Paracoccus denitrificans; Yano T et al.; The NADH-quinone oxidoreductase from Paracoccus denitrificans consists of 14 subunits (Nqo1-14) and contains one FMN and eight iron-sulfur clusters . The Nqo3 subunit possesses fully conserved 11 Cys and 1 His in its N-terminal region and is considered to harbor three iron-sulfur clusters; however, only one binuclear (N1b) and one tetranuclear (N4) were previously identified . In this study, the Nqo3 subunit containing 1x{2Fe-2S} and 2x{4Fe-4S} clusters was expressed in Escherichia coli . The second {4Fe-4S}(1+) cluster is detected by EPR spectroscopy below 6 K, exhibiting very fast spin relaxation . The resolved EPR spectrum of this cluster is broad and nearly axial . The subunit exhibits an absorption-type EPR signal around g approximately 5 region below 6 K, most likely arising from an S = 3/2 ground state of the fast-relaxing {4Fe-4S}(1+) species . The substitution of the conserved His(106) with Cys specifically affected the fast-relaxing {4Fe-4S}(1+) cluster, suggesting that this cluster is coordinated by His(106) . In the cholate-treated NDH-1-enriched P . denitrificans membranes, we observed EPR signals arising from a {4Fe-4S} cluster below 6 K, exhibiting properties similar to those of cluster N5 detected in other complex I/NDH-1 and of the fast-relaxing {4Fe-4S}(1+) cluster in the expressed Nqo3 subunit . Hence, we propose that the His-coordinated {4Fe-4S} cluster corresponds to cluster N5.

Water Res, 2003 Apr, 37(7), 1551 - 6
Effects of pH and precipitation on autohydrogenotrophic denitrification using the hollow-fiber membrane-biofilm reactor; Lee KC et al.; Experiments carried out in a hollow-fiber, membrane-biofilm reactor (HFMBR) showed that the optimum pH for autotrophic denitrification was in the range 7.7-8.6, with the maximum efficiency at 8.4 . Increasing the pH above 8.6 caused a significant decrease in nitrate removal rate and a dramatic increase in nitrite accumulation . The pH rose by 1.2 units when a large buffer was not added, suggesting that some field applications may require pH control . Precipitation of Ca(2+) occurred in every experiment . Precipitation was the largest sink for carbonate, and it also offset alkalinity production by denitrification . Although the alkalinity increased in most cases, systems with a high carbonate buffer and high pH accentuated precipitation, and the net change in alkalinity was negative . The long-term success of field applications of the HFMBR may depend upon the interactions among calcium concentration, total carbonate concentration, pH, and alkalinity changes .

Water Res, 2003 Mar, 37(6), 1401 - 5
Model experiments on the microbial removal of chromium from contaminated groundwater; Vainshtein M et al.; A bacterial consortium with representatives of sulfate-reducing and denitrifying bacteria was selectively enriched . Model experiments under microaerobic conditions showed that it precipitated chromium from Cr (VI)-containing waters (area of a former electroplating factory, Leipzig, Germany) by two different mechanisms: by sulfate reduction and precipitation as sulfide, and by some direct reduction . Sulfate reduction needed fatty acids as organic substrates and resulted at the first stage in no sulfide accumulation . In the absence of the fatty acids but with straw as organic substrate, the direct reduction of chromium was observed without sulfate reduction . In this case Cr (VI)-reduction rate correlated with that of the denitrification.

Water Res, 2003 Mar, 37(6), 1239 - 51
Kinetic modeling of a mixed culture of Pseudomonas denitrificans and Bacillus subtilis under aerobic and anoxic operating conditions; Marazioti C et al.; The kinetics of biological denitrification have been studied and several models, with varying degree of complexity, to be used for design purposes have been presented in the recent years . However, most of these kinetic studies were performed with mixed (and not well defined) microbial systems, such as activated sludge . In the present work, kinetic experiments were carried out in order to study the dynamic characteristics of a defined mixed culture of the denitrifiers Pseudomonas denitrificans and Bacillus subtilis under anoxic and aerobic conditions in a defined synthetic medium involving a mixture of organic substrates, in the presence of nitrates and/or nitrites . Denitrification was assumed to occur by the consecutive reduction of nitrates to nitrites and then to nitrogen gas without accumulation of intermediate gaseous products . The behavior of these defined mixed cultures was predicted using a kinetic model based on the kinetic models that have already been developed for each bacterium separately and the predictions were compared with the results from mixed culture experiments . The overall mathematical model that was developed and validated in the present work is capable of describing the behavior of the mixed culture in the above conditions, i.e . the nitrates and nitrites reduction kinetics, the cell growth, and the organic carbon utilization rates.

Biochemistry, 2003 Feb 25, 42(7), 2046 - 55
The electron transfer complexes of cytochrome c peroxidase from Paracoccus denitrificans; Pettigrew GW et al.; We have used microcalorimetry and analytical ultracentrifugation to test the model proposed in Pettigrew et al . {(1999) J . Biol . Chem . 274, 11383-11389} for the binding of small cytochromes to the cytochrome c peroxidase of Paracoccus denitrificans . Both methods reveal complexity in behavior due to the presence of a monomer/dimer equilibrium in the peroxidase . In the presence of either Ca(2+), or higher ionic strength, this equilibrium is shifted to the dimer . Experiments to study complex formation with redox partners were performed in the presence of Ca(2+) in order to simplify the equilibria that had to be considered . The results of isothermal titration calorimetry reveal that the enzyme can bind two molecules of horse cytochrome c with K(d) values of 0.8 microM and 2.5 microM (at 25 degrees C, pH 6.0, I = 0.026) but only one molecule of Paracoccus cytochrome c-550 with a K(d) of 2.8 microM, molar binding ratios confirmed by ultracentrifugation . For both horse cytochrome c and Paracoccus cytochrome c-550, the binding is endothermic and driven by a large entropy change, a pattern consistent with the expulsion of water molecules from the interface . For horse cytochrome c, the binding is weakened 3-fold at I = 0.046 M due to a smaller entropy change, and this is associated with an increase in enzyme turnover . In contrast, neither the binding of cytochrome c-550 nor its oxidation rate is affected by raising the ionic strength in this range . We propose that, at low ionic strength, horse cytochrome c is trapped in a nonproductive orientation on a broad capture surface of the peroxidase.

Chemosphere, 2003 Apr, 51(2), 143 - 52
Biodegradation of chlorinated solvents in a water unsaturated topsoil; Borch T et al.; In order to investigate topsoils as potential sinks for chlorinated solvents from the atmosphere, the degradation of trichloromethane (CHCl(3)), 1,1,1-trichloroethane (CH(3)CCl(3)), tetrachloromethane (CCl(4)), trichloroethene (C(2)HCl(3)) and tetrachloroethene (C(2)Cl(4)) was studied in anoxic laboratory experiments designed to simulate denitrifying conditions in water unsaturated topsoil . Active denitrification was demonstrated by measuring the release of 15N in N(2) to the headspace from added 15N labeled nitrate . The degradation of chlorinated aliphatic compounds was followed by measuring their concentrations in the headspace above the soil.The headspace concentrations of all the chlorinated solvents except CH(3)CCl(3) were significantly (P<or=0.05) lower after 41 days in biologically active batches as compared to sterile batches . For the compounds with significantly decreasing headspace concentrations, the decline was the least for CHCl(3) within the 41 days of incubation . The headspace concentrations of trichloro- and tetrachloroethene decreased more than 50% during the first 20 days with no considerable indication of abiotic transformation . While slow abiotic removal was observed, tetrachloromethane was completely biotransformed after 16 days . Based on the results in this study, we conclude that anaerobic topsoils are potential sinks for these contaminants, and that a natural attenuation potential exists, even in water unsaturated topsoils.

Biotechnol Bioeng, 2003 Apr 20, 82(2), 238 - 50
The effect of dissolved oxygen on PHB accumulation in activated sludge cultures; Third KA et al.; Nitrogen removal from wastewater is often limited by the availability of reducing power to perform denitrification, especially when treating wastewaters with a low carbon:nitrogen ratio . In the increasingly popular sequencing batch reactor (SBR), bacteria have the opportunity to preserve reducing power from incoming chemical oxygen demand (COD) as poly-beta-hydroxybutyrate (PHB) . The current study uses laboratory experiments and mathematical modeling in an attempt to generate a better understanding of the effect of oxygen on microbial conversion of COD into PHB . Results from a laboratory SBR with acetate as the organic carbon source showed that the aerobic acetate uptake process was oxygen-dependent, producing higher uptake rates at higher dissolved oxygen (DO) supply rates . However, at the lower DO supply rates (k(L)a 6 to 16 h(-1), 0 mg L(-1) DO), a higher proportion of the substrate was preserved as PHB than at higher DO supply rates (k(L)a 30, 51 h(-1), DO >0.9 mg L(-1)) . Up to 77% of the reducing equivalents available from acetate were converted to PHB under oxygen limitation (Y(PHB/Ac) 0.68 Cmol/Cmol), as opposed to only 54% under oxygen-excess conditions (Y(PHB/Ac) 0.48 Cmol/Cmol), where a higher fraction of acetate was used for biomass growth . It was calculated that, by oxygen management during the feast phase, the amount of PHB preserved (1.4 Cmmol L(-1) PHB) accounted for an additional denitrification potential of up to 18 mg L(-1) nitrate-nitrogen . The trends of the effect of oxygen (and hence ATP availability) on PHB accumulation could be reproduced by the simulation model, which was based on biochemical stoichiometry and maximum rates obtained from experiments . Simulated data showed that, at low DO concentrations, the limited availability of adenosine triphosphate (ATP) prevented significant biomass growth and most ATP was used for acetate transport into the cell . In contrast, high DO supply rates provided surplus ATP and hence higher growth rates, resulting in decreased PHB yields . The results suggest that oxygen management is crucial to conserving reducing power during the feast phase of SBR operation, as excessive aeration rates decrease the PHB yield and allow higher biomass growth .

Syst Appl Microbiol, 2002 Dec, 25(4), 572 - 83
Phenotypic and genotypic diversity of rhizobia nodulating Pterocarpus erinaceus and P . lucens in Senegal; Sylla SN et al.; A total of fifty root nodules isolates of fast-growing and slow growing rhizobia from Pterocarpus ennaceus and Pterocarpus lucens respectively native of sudanean and sahelian regions of Senegal were characterized . These isolates were compared to representative strains of known rhizobial species . Twenty-two new isolates were slow growers and twenty-eight were fast growers . A polyphasic approach was performed including comparative total protein sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) profile analysis; 16S rDNA and 16S-23S rDNA intergenic spacer (IGS) sequence analysis . By SDS-PAGE the slow growing isolates grouped in one major cluster containing reference strains of Bradyrhizobium sp . including strains isolated in Africa, in Brazil and in New Zealand . Most of the fast-growing rhizobia grouped in four different clusters or were separate strains related to Rhizobium and Mesorhizobium strains . The 16S rDNA and 16S-23S rDNA IGS sequences analysis showed accurately the differentiation of fast growing rhizobia among the Rhizobium and Mesorbizobium genospecies . The representative strains of slow growing rhizobia were identified as closely related to Bradyrbizobium elkanii and Bradyrhizobium japonicum . Based on 16S rDNA sequence analysis, one slow growing strain (ORS199) was phylogenetically related to Bradyrbizobium sp . (Lupinus) and Blastobacter denitrificans . This position of ORS 199 was not confirmed by IGS sequence divergence . We found no clear relation between the diversity of strains, the host plants and the ecogeographical origins.

Water Sci Technol, 2003, 47(1), 297 - 302
Setting-up a control simulation strategy for a sequencing batch reactor (SBR): application to municipal wastewater; Casellas M et al.; The use of a simulation model for setting up a control strategy for a sequencing batch reactor necessary for treating municipal wastewater is described . The model used is the ASM no 1 model . The objective of the pollution control treatment is the removal of carbon and nitrogen; the optimisation is concerned with the improvement in the biological removal of nitrogen . After experimental identification of the initialisation variables, the model enables different SBR control scenarios to be tested (time variation for each process) leading to the total elimination of nitrogen . The best simulation was tested in a laboratory reactor . On that scale, it was noted that denitrification is an endogenous process . Lastly, the control strategy was tested on a semi-industrial pilot working in a pollution control plant . Other control scenarios can be devised and tested by simulation, in order to improve the productivity of the reactor.

Water Sci Technol, 2003, 47(1), 237 - 44
Nitrate removal by simultaneous sulfur utilizing autotrophic and heterotrophic denitrification under different organics and alkalinity conditions: batch experiments; Oh SE et al.; The effect of various organic compounds were tested using lab-scale batch reactors . At sufficient alkalinity, the initial nitrate nitrogen concentration of 100 mg/L was completely reduced in all batch reactors . Sulfate production decreased by the addition of organics . The concentration range of organics used in this experiment did not inhibit autotrophic denitrification except for propionate . Propionate inhibited autotrophic denitrification a little, indicated by a lower sulfate production rate . Biomass in suspension increased with higher initial organic concentrations, showing higher DOC consumption . As the concentration of organics increased, alkalinity increased accordingly . Under the conditions of low alkalinity, in the case of a control reactor without organics, only about 30% of the initial nitrate was reduced . With half the theoretically required dosage of methanol, the denitrification rates increased slightly . When ethanol, acetate, and propionate were used, denitrification went to completion . When excess organics was added, however, sulfate production was significantly decreased . Interestingly, even when small amounts of organics were added, autotrophic denitrification was promoted as indicated by the sulfate production.

Water Sci Technol, 2003, 47(1), 159 - 65
Nutrient removal using anaerobically fermented leachate of food waste in the BNR process; Lee CY et al.; Nutrients removal efficiencies highly depend on the presence of biodegradable organic carbon in the biological nutrient removal (BNR) process but most domestic wastewater in Korea has shown a low C/N ratio and has a small amount of biodegradable COD (chemical oxygen demand) . On the other hand, about 11,577 tons of food waste that contains a lot of organic material has been produced in Korea per day . The feasibility and applicability of anaerobically fermented leachate of food waste (AFLFW) as an external carbon source was examined in the laboratory-scale BNR process at different operation conditions with synthetic wastewater and domestic sewage . As the addition of AFLFW increased, the average removal efficiencies of SCOD, T-N, T-P changed from 96%, 60%, and 2% to 90%, 77%, and 67%, respectively . From anoxic nitrate utilization tests, it was observed that once the readily biodegradable COD (especially VFAs) was depleted, the denitrification rate reduced from 8.2 mg NO3-N/g VSS/hr to 0.7 mg NO3-N/g VSS/hr . From the molecular size distribution test, it was concluded that about 60% of soluble COD in effluent, which was considered to originate from AFLFW, had a large molecular size (> 30 kDa) that was not used by microorganisms.

Adv Space Res, 2003, 31(1), 63 - 8
New problems to be solved for establishing closed life support system; Nitta K; New test bed facilities such as Bioplex and CEEF have been constructed to test the new advanced technologies for solving the various problems as follows, (1) how to develop air content stabilization technologies with gas balance between the generation and the absorption by living organisms, (2) how to solve the mismatching between the assimilation rate of autotrophic organisms and the respiration rate of heterotrophic organisms, (3) how to balance the speed of the waste decomposition with the absorption speed of nutrient components in the sequential plant cultivation, (4) how to develop new nutrient adjusting subsystems for each plant species, (5) how to compensate the denitrification during the waste decomposition and anaerobic microbes in the nutrient solution . c2002 COSPAR . Published by Elsevier Science Ltd . All rights reserved.

Microbiology, 2003 Jan, 149(Pt 1), 229 - 37
Characterization of a nitrate-respiring bacterial community using the nitrate reductase gene (narG) as a functional marker; Gregory LG et al.; Bacterial cultures capable of reducing nitrate to nitrite, or of complete denitrification, were established from 5, 10, 15 and 20 cm depths of a freshwater sediment . Taxonomic analysis of the 56 isolates using 16S rRNA gene sequences revealed an unexpected species richness, which included representatives of the gamma-Proteobacteria, Bacillus spp., Staphylococcus spp . and members of the ACTINOBACTERIA: Gram-positive species tended to predominate in the lower depths of the sediment, where there was evidence of active sulphate respiration . Sequences (from the narG gene) potentially encoding the catalytic subunit of the membrane-associated nitrate reductase were successfully amplified from 46 of the isolates, using a nested PCR with four degenerate primers . NarG sequences clustered into three major groupings that were supported by alternative phylogenetic analyses . The NarG sequences from Gram-positive isolates (according to rRNA gene phylogeny) clustered together within sequences from the low-G+C Gram-positive bacteria . However, this cluster also included two sequences from members of the genus PSEUDOMONAS: Another group contained mostly NarG sequences from the Proteobacteria (according to rRNA gene phylogeny), but also included five sequences from Gram-positive species . The third group of NarG sequences contained three sequences from Gram-positive species . Thus, the NarG-derived phylogeny is not entirely consistent with 16S rRNA-based taxonomy, precluding the use of the narG gene as a taxonomically useful tool for the characterization of nitrate-respiring bacteria . Total DNA was also extracted from the four depth intervals of the sediment sample and used in similar narG amplifications . Most sequences amplified directly from environmental DNA clustered in the Gram-negative group, and none was in the predominantly Gram-positive group . The study also revealed a degree of spatial organization of a nitrate-respiring community in terms of both microbiology and narG sequences.

Microbiology, 2003 Jan, 149(Pt 1), 29 - 36
Transcriptional regulation of the nos genes for nitrous oxide reductase in Pseudomonas aeruginosa; Arai H et al.; The genes for nitrous oxide (N(2)O) reduction, nosRZDFYL, are clustered on the chromosome of Pseudomonas aeruginosa . Promoter assays using transcriptional fusions to lacZ revealed that the structural gene for nitrous oxide reductase, nosZ, is transcribed with the upstream nosR gene . The nosR gene product is not required for the activity of the nosR promoter . A sequence similar to the consensus FNR-binding motif was found 41.5 bp upstream from the major transcriptional start point of nosR . Mutation of the motif significantly reduced the promoter activity . DNR, an FNR-related transcriptional regulator required for the expression of denitrification genes in P . aeruginosa, is necessary for the transcription of nosR, indicating that the motif is recognized by DNR . Nitrite (NO-2), nitric oxide (NO) and NO-generating reagents induced nosR promoter activity, but N(2)O did not . The NO-2-induced nosR promoter activity was reduced by mutation of the NO-2 reductase gene . However, a low concentration of NO-2 induced the promoter activity in a NO reductase mutant . These results indicate that NO is the inducer molecule for transcription of the nos genes.

Arch Biochem Biophys, 2003 Feb 15, 410(2), 230 - 7
Characterization of the interaction of Rhodobacter capsulatus cytochrome c peroxidase with charge reversal mutants of cytochrome c(2); Koh M et al.; Steady-state kinetics for the reaction of Rhodobacter capsulatus bacterial cytochrome c peroxidase (BCCP) with its substrate cytochrome c(2) were investigated . The Rb . capsulatus BCCP is dependent on calcium for activation as previously shown for the Pseudomonas aeruginosa BCCP and Paracoccus denitrificans enzymes . Furthermore, the activity shows a bell-shaped pH dependence with optimum at pH 7.0 . Enzyme activity is greatest at low ionic strength and drops off steeply as ionic strength increases, resulting in an apparent interaction domain charge product of -13 . All cytochromes c(2) show an asymmetric distribution of surface charge, with a concentration of 14 positive charges near the exposed heme edge of Rb . capsulatus c(2) which potentially may interact with approximately 6 negative charges, localized near the edge of the high-potential heme of the Rb . capsulatus BCCP . To test this proposal, we constructed charge reversal mutants of the 14 positively charged residues located on the front face of Rb . capsulatus cytochrome c(2) and examined their effect on steady-state kinetics with BCCP . Mutated residues in Rb . capsulatus cytochrome c(2) that showed the greatest effects on binding and enzyme activity are K12E, K14E, K54E, K84E, K93E, and K99E, which is consistent with the site of electron transfer being located at the heme edge . We conclude that a combination of long-range, nonspecific electrostatic interactions as well as localized salt bridges between, e.g., cytochrome c(2) K12, K14, K54, and K99 with BCCP D194, D241, and D6, account for the observed kinetics.

Appl Environ Microbiol, 2003 Feb, 69(2), 1159 - 71
Oligonucleotide microarray for the study of functional gene diversity in the nitrogen cycle in the environment; Taroncher-Oldenburg G et al.; The analysis of functional diversity and its dynamics in the environment is essential for understanding the microbial ecology and biogeochemistry of aquatic systems . Here we describe the development and optimization of a DNA microarray method for the detection and quantification of functional genes in the environment and report on their preliminary application to the study of the denitrification gene nirS in the Choptank River-Chesapeake Bay system . Intergenic and intragenic resolution constraints were determined by an oligonucleotide (70-mer) microarray approach . Complete signal separation was achieved when comparing unrelated genes within the nitrogen cycle (amoA, nifH, nirK, and nirS) and detecting different variants of the same gene, nirK, corresponding to organisms with two different physiological modes, ammonia oxidizers and denitrifying halobenzoate degraders . The limits of intragenic resolution were investigated with a microarray containing 64 nirS sequences comprising 14 cultured organisms and 50 clones obtained from the Choptank River in Maryland . The nirS oligonucleotides covered a range of sequence identities from approximately 40 to 100% . The threshold values for specificity were determined to be 87% sequence identity and a target-to-probe perfect match-to-mismatch binding free-energy ratio of 0.56 . The lower detection limit was 10 pg of DNA (equivalent to approximately 10(7) copies) per target per microarray . Hybridization patterns on the microarray differed between sediment samples from two stations in the Choptank River, implying important differences in the composition of the denitirifer community along an environmental gradient of salinity, inorganic nitrogen, and dissolved organic carbon . This work establishes a useful set of design constraints (independent of the target gene) for the implementation of functional gene microarrays for environmental applications.

Appl Environ Microbiol, 2003 Feb, 69(2), 760 - 8
Anaerobic degradation of ethylbenzene by a new type of marine sulfate-reducing bacterium; Kniemeyer O et al.; Anaerobic degradation of the aromatic hydrocarbon ethylbenzene was studied with sulfate as the electron acceptor . Enrichment cultures prepared with marine sediment samples from different locations showed ethylbenzene-dependent reduction of sulfate to sulfide and always contained a characteristic cell type that formed gas vesicles towards the end of growth . A pure culture of this cell type, strain EbS7, was isolated from sediment from Guaymas Basin (Gulf of California) . Complete mineralization of ethylbenzene coupled to sulfate reduction was demonstrated in growth experiments with strain EbS7 . Sequence analysis of the 16S rRNA gene revealed a close relationship between strain EbS7 and the previously described marine sulfate-reducing strains NaphS2 and mXyS1 (similarity values, 97.6 and 96.2%, respectively), which grow anaerobically with naphthalene and m-xylene, respectively . However, strain EbS7 did not oxidize naphthalene, m-xylene, or toluene . Other compounds utilized by strain EbS7 were phenylacetate, 3-phenylpropionate, formate, n-hexanoate, lactate, and pyruvate . 1-Phenylethanol and acetophenone, the characteristic intermediates in anaerobic ethylbenzene degradation by denitrifying bacteria, neither served as growth substrates nor were detectable as metabolites by gas chromatography-mass spectrometry in ethylbenzene-grown cultures of strain EbS7 . Rather, (1-phenylethyl)succinate and 4-phenylpentanoate were detected as specific metabolites in such cultures . Formation of these intermediates can be explained by a reaction sequence involving addition of the benzyl carbon atom of ethylbenzene to fumarate, carbon skeleton rearrangement of the succinate moiety (as a thioester), and loss of one carboxyl group . Such reactions are analogous to those suggested for anaerobic n-alkane degradation and thus differ from the initial reactions in anaerobic ethylbenzene degradation by denitrifying bacteria which employ dehydrogenations.

Wei Sheng Yan Jiu, 2002 Feb, 31(1), 19 - 21
{Study on the denitrification of drinking water with upflow anaerobic sludge blanket reactor}; Tan Y et al.; The characteristics of denitrification was investigated with a pilot scale upflow anaerobic sludge blanket (UASB) reactor at room temperature . The results showed that when brewery waste degrading sludge was used as seed, the starting process was completed within 7 weeks, with hydraulic residence time shortened from 11.1 h to 4.7 h, COD/N/P = 200/5/1 and influent NO3-(-)N concentration increased from 5 mg/L to 100 mg/L . After the process starting, the most probable number {n(MPN)} of denitrifying bacteria was 60 folds and the maximum velocity of CH4 produced was 10 folds higher than before . The removal efficiency of No3-(-)N was 99%, C/N ratio and pH value were investigated as effect factors . When C/N > = or 1.0, the NO3(-)-N removal efficiencies were not different from those of C/N < 1.0 groups significantly . The pH value could meet the discharge standards.

FEBS Lett, 2003 Jan 30, 535(1-3), 166 - 70
Sequence analysis reveals new membrane anchor of reaction centre-bound cytochromes possibly related to PufX; Hucke O et al.; Most of the bacterial photosynthetic reaction centres known to date contain a cytochrome subunit with four covalently bound haem groups . In the case of Blastochloris viridis, this reaction centre subunit is anchored in the membrane by a lipid molecule covalently attached to the cysteine which forms the N-terminus of the mature protein after processing by a signal peptidase . We show that posttranslational N-terminal cleavage of the cytochrome subunit does not occur in the aerobic photosynthetic bacterium Roseobacter denitrificans . From sequence analysis of the resulting elongated N-terminus it follows that a transmembrane helix is anchoring the reaction centre-bound cytochrome in the membrane . Comparative sequence analysis strongly suggests that all cytochrome subunits lacking the lipid coupling cysteine share this structural feature . Comparison of the N-terminal segment of the cytochrome subunit of Roseobacter denitrificans with the sequences of the PufX proteins from Rhodobacter sphaeroides and Rhodobacter capsulatus suggests a phylogenetic relation.

Water Res, 2003 Mar, 37(5), 1005 - 14
A central fission pathway in alkylphenol ethoxylate biodegradation; Franska M et al.; A representative alkylphenol Triton X-100 (having 9.5 oxyethylene subunits) was treated over 40 days under the conditions of the continuous flow activated sludge simulation test in a plant with aeration and denitrification chambers . Treated sewage was separated by sequential extraction with ethyl acetate and chloroform . The extracts were analysed by high-performance liquid chromatography-electrospray ionisation-mass spectrometry (HPLC-ESI-MS) . Single ion chromatograms of the chloroform extracts showed the presence of neutral, mono- and di-carboxylated poly(ethylene glycols) . This is evidence of the central fission of alkylphenol ethoxylates (APE) . Simultaneously, the APE having omega-carboxylated oxyethylene chains were identified . This is the evidence that apart from central fission, the omega-oxidation oxyethylene chain pathway also occurs.

Water Res, 2003 Mar, 37(5), 983 - 92
Degradation of 2,4,6-trinitrotoluene by immobilized horseradish peroxidase and electrogenerated peroxide; Beom Lee K et al.; This paper presents horseradish peroxidase (HRP)-catalyzed removal of 2,4,6-trinitrotoluene (TNT) by an electrochemical packed-bed flow reactor operated in a circulating batch mode with the help of in situ generated hydrogen peroxide . HRP immobilized on the reticulated vitreous carbon electrode was prepared for the cyclic voltammetry of 2,4,6-TNT . Effects of pH and temperature on the TNT electroreduction in 0.2M phosphate buffer saturated with oxygen were examined . HRP immobilized carbon electrode was capable of catalyzing the oxidation and detoxification of 44 microM TNT in aqueous solution under optimized conditions . The removal rate of TNT for the electroenzymatic method was much greater than for electrochemical and biochemical methods . Stoichiometric and kinetic studies indicated that the hydrogen peroxide was utilized more effectively in the electroenzymatic method . Denitrification as intermediate reaction was also investigated.

J Bacteriol, 2003 Feb, 185(3), 887 - 96
Requirements for Cu(A) and Cu-S center assembly of nitrous oxide reductase deduced from complete periplasmic enzyme maturation in the nondenitrifier Pseudomonas putida; Wunsch P et al.; Bacterial nitrous oxide (N(2)O) reductase is the terminal oxidoreductase of a respiratory process that generates dinitrogen from N(2)O . To attain its functional state, the enzyme is subjected to a maturation process which involves the protein-driven synthesis of a unique copper-sulfur cluster and metallation of the binuclear Cu(A) site in the periplasm . There are seven putative maturation factors, encoded by nosA, nosD, nosF, nosY, nosL, nosX, and sco . We wanted to determine the indispensable proteins by expressing nos genes from Pseudomonas stutzeri in the nondenitrifying organism Pseudomonas putida . An in silico study of denitrifying bacteria revealed that nosL, nosX (or a homologous gene, apbE), and sco, but not nosA, coexist consistently with the N(2)O reductase structural gene and other maturation genes . Nevertheless, we found that expression of only three maturation factors (periplasmic protein NosD, cytoplasmic NosF ATPase, and the six-helix integral membrane protein NosY) together with nosRZ in trans was sufficient to produce catalytically active holo-N(2)O reductase in the nondenitrifying background . We suggest that these obligatory factors are required for Cu-S center assembly . Using a mutational approach with P . stutzeri, we also studied NosA, the Cu-containing outer membrane protein previously thought to have Cu insertase function, and ScoP, a putative membrane-anchored chaperone for Cu(A) metallation . Both of these were found to be dispensable elements for N(2)O reductase biosynthesis . Our experimental and in silico data were integrated in a model of N(2)O reductase maturation.

Water Res, 2003 Feb, 37(4), 914 - 20
Addition of trace metals increases denitrification rate in closed marine systems; Labbe N et al.; We investigated the effect of trace metals (Fe, Mn, Cu, Zn and Mo) on the denitrification unit at the Montreal Biodome . Two dosages of the five trace metals were tested on a denitrifying bacterial population which was extracted from the denitrification unit and cultured in 250 mL chemostats with artificial seawater . The low dosage showed a 20% increase in the denitrification rate whereas the high dosage had a more pronounced effect with a 250% increase . No increase in bacterial growth was observed, suggesting that the trace metals had an effect on the denitrification activity . When the trace metals were tested separately, only iron had a significant effect similar to the increase in the denitrification rate observed when the five trace metals were added . The combination of Fe and Mn caused a small but significant increase compared to the five trace metals . We then tested the effect of adding Fe, Mn and Cu to the denitrification unit at the Montreal Biodome . A high dosage of these trace metals showed a 250% increase in the denitrification rate, which went from 200 to 700 g NO(x)-N/d . Our results showed that the addition of trace metals is crucial for denitrification activities.

Water Res, 2003 Feb, 37(4), 853 - 63
Factors influencing deterioration of denitrification by oxygen entering an anoxic reactor through the surface; Plosz BG et al.; The purpose of the paper is to examine the factors that influence the deterioration of denitrification in open anoxic reactors . For this investigation an ASM 1-based simulation model was developed and successfully applied to fit data from batch experiments carried out in lab-scale reactor vessels (uncovered and covered) using both clarified domestic wastewater and synthetic wastewater . Applying the verified model, simulation studies were performed to investigate the effects of available denitrifiable substrate, biomass concentration, oxygen transfer rate, and temperature on deterioration of denitrification in open anoxic reactors . It has been shown that oxygen entering an anoxic reactor through the surface may not just affect denitrification metabolically, but also kinetically, due to increased dissolved oxygen (DO) concentration exerting an inhibitory effect on the denitrification rate . When the exogenous substrate concentration in the reactor vessel is high enough for a high consumption rate, the DO concentration is kept low . The higher the biomass concentration, and thereby the consumption rate of endogenous substrate, the lower the DO concentration during the low-rate denitrification phase . At low substrate removal rates, decreasing temperature will cause the DO concentration in anoxic vessels to increase . The results suggest that assuring removal of available exogenous carbon source at high rate by staging of open anoxic bioreactors may significantly improve denitrification efficiency.

Water Sci Technol, 2002, 46(11-12), 121 - 5
Reduction of sludge by ozone treatment and production of carbon source for denitrification; Ahn KH et al.; The feasibility of ozone treatment of municipal sludge for sludge reduction and carbon source production has been investigated . Significant accumulation of solubilized organics and unsettlable micro-solids (UMS) was observed at relatively low ozone dosages while mineralization became dominant at higher dosages . Batch denitrification experiments showed that the solubilized organics and the UMS could be utilized as carbon sources for nitrogen removal . In terms of overall sludge reduction, 54% reduction of the total sludge mass could be achieved by ozone treatment at 0.2 g-O3/g-MLSS.

Water Sci Technol, 2002, 46(11-12), 99 - 104
Denitrification of industrial wastewater with sulfur and limestone packed column; Nugroho R et al.; An autotrophic denitrification system was developed for nitrate contaminated industrial wastewater whose C/N ratio was very low . The microbes containing Thiobacillus denitrificans as a dominant species were attached on the surface of granular elemental sulfur packed in a column . Elemental sulfur was used as an electron donor for autotrophic denitrification . The granules of limestone were mixed with the granular sulfur to moderate the decrease of alkalinity during autotrophic denitrification . The stoichiometry and basic kinetics of denitrification were studied in column runs . The effects of minerals such as phosphorus on treatment performance were clarified . The wastewater from steel production plants was treated by the present biofilm process . Low extent of nitrogen removal was caused by the lack of minerals.

Water Sci Technol, 2002, 46(11-12), 93 - 8
Characterization of microbial community in nitrogen removal process of metallurgic wastewater by PCR-DGGE; Yoshie S et al.; The metallurgic wastewater generated from the processes of recovering precious metals from industrial wastes contains high concentrations of nitrogen compounds such as ammonia and nitric acid and of salts such as sodium chloride and sodium sulfate . Biological nitrogen removal from this wastewater was attempted by a circulating bioreactor system equipped with an anoxic packed bed and an aerobic fluidized bed . The anoxic packed bed of this system was found to effectively remove nitrite and nitrate from the wastewater by denitrification at a removal ratio of 97% . As a result of denitrification activity tests at various NaCl concentrations, the sludge obtained from the anoxic packed bed exhibited accumulation of nitrite at 5.0 and 8.4% NaCl concentrations, suggesting that the reduction of nitrite is the key step in the denitrification pathway under hypersaline conditions . The microbial community analysis by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA (rDNA) fragments revealed that the community diversity varied in accordance with water temperature, nitrate-loading rate and ionic strength . When particular major DGGE bands were excised, reamplified and directly sequenced, the dominant species in the anoxic packed bed were affiliated with the beta and gamma subclasses of the class Proteobacteria such as Alcaligenes defragrans and Pseudomonas spp., respectively.

Water Sci Technol, 2002, 46(11-12), 57 - 62
Packed bed columns for high rate nitrogen and carbon removals; Ong SL et al.; Two packed bed column (PB) systems, namely anoxic-anoxic and anoxic-oxic were investigated for treatment of wastewater containing high concentrations of nitrogen (N) and COD . The anoxic-anoxic PB removal rates 6.70 kg N/m3 x d and 26.02 kg COD/m3 x d, respectively . The responding removal rates of the anoxic-oxic PB system were 7.41 kg N/m3 and 28.00 kg COD/m3 x d, respectively . The N and COD removal efficiencies of anoxic anoxic PB system were in the range of 99.2-100% and 97.2-98.8%, respectively . The corresponding removal efficiencies of anoxic-oxic PB system were 97.5-100% and 98.6-99.4%, respectively . These findings showed that a PB system consisting of anoxic-oxic columns in series has a high capacity to remove nitrogenous and carbonaceous compounds even though the influent to the anoxic stage was oxygenated . Better system stability in terms of denitrification was, however, obtained with the anoxic-anoxic system.

Water Sci Technol, 2002, 46(11-12), 39 - 44
Nitrate removal rate in a continuous column denitrification reactor using hydrogen generated by electrolysis with carbon anodes and stainless cathodes; Dadang S et al.; An autotrophic continuous denitrification process, using hydrogen generated by electrolysis with activated carbon anodes, was experimentally demonstrated to be an effective nitrate removal process . Several fixed bed columns with polypropylene packing and honeycomb shaped activated carbon anodes and stainless rod cathodes were set in a thermostat chamber at 30 degrees C, and potassium nitrate enriched tap water as influent was supplied at various flow rates and electric currents . Although the anode is in the same column where microbial biomass grows, sufficient nitrate removal was observed . For example, almost complete removal of nitrate and nitrite was observed at a hydraulic retention time (HRT) as short as 1.8 h . A model assuming successive denitrification reactions and plug-flow process, nitrate reduction rate = k1 {NO3-} {H2}, and nitrite reduction rate = k2 {NO2-} {H2}(1.5) was proposed . Calculated results with k1 = 1.3 mmol(-1) h(-1) and k2 = 3.3 mmol(-1.5) x h(-1) agreed well with all the experimental results.

Mol Microbiol, 2003 Jan, 47(2), 549 - 59
Induction of apoptosis in macrophages by Pseudomonas aeruginosa azurin: tumour-suppressor protein p53 and reactive oxygen species, but not redox activity, as critical elements in cytotoxicity; Goto M et al.; Azurin is a copper-containing protein involved in electron transfer during denitrification . We reported recently that purified azurin demonstrates cytotoxicity to macrophages by forming a complex with the tumour-suppressor protein p53, thereby stabilizing it and enhancing its function as an inducer of proapoptotic activity (Yamada, T., Goto, M., Punj, V., Zaborina, O., Kimbara, K., Das Gupta, T . K., and Chakrabarty, A . M . 2002, Infect Immun70: 7054-7062) . It is, however, not known whether the oxidoreductase (redox) activity of azurin or the involvement of copper is important for its cytotoxicity . We have isolated apo-azurin devoid of copper and site-directed mutants that are redox negative because of either replacement of a cysteine residue (Cys-112) involved in co-ordination with copper or mutational replacement of two methionine residues (Met-44 and Met-64) that are present in the hydrophobic patch of azurin and allow interaction of azurin with its redox partner cytochrome c551 . We demonstrate that, although the wild type (wt) and the Cys-112 Asp mutant azurin can form complexes with the tumour-suppressor protein p53 and generate high levels of reactive oxygen species (ROS), the redox-negative Met-44LysMet-64Glu mutant azurin is defective in complex formation with p53, generates low levels of ROS and lacks appreciable cytotoxicity towards macrophages . Thus, complex formation with p53 and ROS generation, rather than azurin redox activity, are important in the cytotoxic action of azurin towards macrophages.

Appl Environ Microbiol, 2003 Jan, 69(1), 107 - 12
Isolation and characterization of a sulfur-oxidizing chemolithotroph growing on crude oil under anaerobic conditions; Kodama Y et al.; Molecular approaches have shown that a group of bacteria (called cluster 1 bacteria) affiliated with the epsilon subclass of the class Proteobacteria constituted major populations in underground crude-oil storage cavities . In order to unveil their physiology and ecological niche, this study isolated bacterial strains (exemplified by strain YK-1) affiliated with the cluster 1 bacteria from an oil storage cavity at Kuji in Iwate, Japan . 16S rRNA gene sequence analysis indicated that its closest relative was Thiomicrospira denitrificans (90% identity) . Growth experiments under anaerobic conditions showed that strain YK-1 was a sulfur-oxidizing obligate chemolithotroph utilizing sulfide, elemental sulfur, thiosulfate, and hydrogen as electron donors and nitrate as an electron acceptor . Oxygen also supported its growth only under microaerobic conditions . Strain YK-1 could not grow on nitrite, and nitrite was the final product of nitrate reduction . Neither sugars, organic acids (including acetate), nor hydrocarbons could serve as carbon and energy sources . A typical stoichiometry of its energy metabolism followed an equation: S(2-) + 4NO(3)(-) --> SO(4)(2-) + 4NO(2)(-) (Delta G(0) = -534 kJ mol(-1)) . In a difference from other anaerobic sulfur-oxidizing bacteria, this bacterium was sensitive to NaCl; growth in medium containing more than 1% NaCl was negligible . When YK-1 was grown anaerobically in a sulfur-depleted inorganic medium overlaid with crude oil, sulfate was produced, corresponding to its growth . On the contrary, YK-1 could not utilize crude oil as a carbon source . These results suggest that the cluster 1 bacteria yielded energy for growth in oil storage cavities by oxidizing petroleum sulfur compounds . Based on its physiology, ecological interactions with other members of the groundwater community are discussed.

Protein Expr Purif, 2003 Jan, 27(1), 42 - 8
Expression of a fully functional cd1 nitrite reductase from Pseudomonas aeruginosa in Pseudomonas stutzeri; Arese M et al.; Nitrite reductases are redox enzymes catalysing the one electron reduction of nitrite to nitrogen monoxide (NO) within the bacterial denitrification process . We have cloned the gene for cd(1) nitrite reductase (Pa-nirS) from Pseudomonas aeruginosa into the NiRS(-) strain MK202 of Pseudomonas stutzeri and expressed the enzyme under denitrifying conditions . In the MK202 strain, denitrification is abolished by the disruption of the endogenous nitrite reductase gene; thus, cells can be grown only in the presence of oxygen . After complementation with Pa-nirS gene, cells supplemented with nitrate can be grown in the absence of oxygen . The presence of nitrite reductase was proven in vivo by the demonstration of NO production, showing that the enzyme was expressed in the active form, containing both heme c and d(1) . A purification procedure for the recombinant PaNir has been developed, based on the P . aeruginosa purification protocol; spectroscopic analysis of the purified protein fully confirms the presence of the d(1) heme cofactor . Moreover, the functional characterisation of the recombinant NiR has been carried out by monitoring the production of NO by the purified NiR enzyme in the presence of nitrite by an NO electrode . The full recovery of the denitrification properties in the P . stutzeri MK202 strain by genetic complementation with Pa-NiR underlines the high homology between enzymes of nitrogen oxianion respiration . Our work provides an expression system for cd(1) nitrite reductase and its site-directed mutants in a non-pathogenic strain and is a starting point for the in vivo study of recombinant enzyme variants.

Int J Syst Evol Microbiol, 2002 Nov, 52(Pt 6), 2211 - 7
Shewanella denitrificans sp . nov., a vigorously denitrifying bacterium isolated from the oxic-anoxic interface of the Gotland Deep in the central Baltic Sea; Brettar I et al.; Three strains of denitrifying estuarine bacteria, OS217(T), 05220 and OS226, were characterized for their physiological and biochemical features, fatty acid profiles and their phylogenetic position based on 16S rDNA sequences . The strains were isolated from the oxic-anoxic interface of an anoxic basin of the central Baltic Sea . Phylogenetic analyses of the 16S rDNA sequences revealed a clear affiliation with members of the genus Shewanella of the gamma-Proteobacteria . The closest sequence similarity was seen with Shewanella baltica, Shewanella putrefaciens and Shewanella frigidimarina (95-96%) . The dominant fatty acids were 16:1omega7c, 15:0 iso, 16:0 and 13:0 iso . The G+C content of the DNA ranged from 46.8 to 48.1 mol% . The strains were unpigmented, polarly flagellated, mesophilic, facultatively anaerobic and able to use nitrate, nitrite and sulphite as electron acceptors . Growth was observed at salinities from 0 to 6%, with an optimum between 1 and 3% . According to their morphology, physiology, fatty acid composition and 16S rRNA sequences, the described bacteria fitted well into the genus Shewanella, but could be easily distinguished from the Shewanella species described to date . Because of their capacity for vigorous denitrification, the name Shewanella denitrificans sp . nov . is suggested for the Baltic isolates, for which the type strain is OS217(T) (= DSM 15013(T) = LMG 21692(T)).

Int J Syst Evol Microbiol, 2002 Nov, 52(Pt 6), 2183 - 90
Pseudomonas chloritidismutans sp . nov., a non-denitrifying, chlorate-reducing bacterium; Wolterink AF et al.; A Gram-negative, facultatively anaerobic, rod-shaped, dissimilatory chlorate-reducing bacterium, strain AW-1(T), was isolated from biomass of an anaerobic chlorate-reducing bioreactor . Phylogenetic analysis of the 16S rDNA sequence showed 100% sequence similarity to Pseudomonas stutzeri DSM 50227 and 98.6% sequence similarity to the type strain of P . stutzeri (DSM 5190(T)) . The species P . stutzeri possesses a high degree of genotypic and phenotypic heterogeneity . Therefore, eight genomic groups, termed genomovars, have been proposed based upon deltaTm values, which were used to evaluate the quality of the pairing within heteroduplexes formed by DNA-DNA hybridization . In this study, DNA-DNA hybridization between strain AW-1(T) and P . stutzeri strains DSM 50227 and DSM 5190(T) revealed respectively 80.5 and 56.5% similarity . DNA-DNA hybridization between P . stutzeri strains DSM 50227 and DSM 5190(T) revealed 48.4% similarity . DNA-DNA hybridization indicated that strain AW-1(T) is not related at the species level to the type strain of P . stutzeri . However, strain AW-1(T) and P . stutzeri DSM 50227 are related at the species level . The physiological and biochemical properties of strain AW-1(T) and the two P . stutzeri strains were compared . A common characteristic of P . stutzeri strains is the ability to denitrify . However, in growth experiments, strain AW-1(T) could use only chlorate or oxygen as an electron acceptor and not nitrate, perchlorate or bromate . Strain AW-1(T) is the first chlorate-reducing bacterium described that does not possess another oxyanion-reduction pathway . Cell extracts of strain AW-1(T) showed chlorate and bromate reductase activities but not nitrate reductase activity . P . stutzeri strains DSM 50227 and DSM 5190(T) could use nitrate or oxygen as an electron acceptor, but not chlorate . Chlorate reductase activity, in addition to nitrate reductase activity, was detected in cell extracts of both P . stutzeri strains . Chlorite dismutase activity was absent in extracts of both P . stutzeri strains but was present in extracts of strain AW-1(T) . Based on the hybridization experiments and the physiological and biochemical data, it is proposed that strain AW-1(T) be classified as a novel species of Pseudomonas, Pseudomonas chloritidismutans sp . nov . The type strain is strain AW-1(T) (= DSM 13592(T) = ATCC BAA-443(T)).

Int J Syst Evol Microbiol, 2002 Nov, 52(Pt 6), 2155 - 61
Pseudoxanthomonas taiwanensis sp . nov., a novel thermophilic, N2O-producing species isolated from hot springs; Chen MY et al.; Two novel thermophilic bacterial strains, with an optimum growth temperature of between 50 and 60 degrees C, were isolated from the Chi-ban Hot Springs in eastern Taiwan . Strains CB-225 and CB-226(T) were aerobic, thermophilic, non-sporulating, yellow-pigmented heterotrophic organisms . These strains exhibited an unusual denitrification reaction, reducing nitrite, but not nitrate, with the production of N2O only . On the basis of a phylogenetic analysis of 16S rDNA sequences, DNA-DNA similarity data, morphological, physiological and biochemical characteristics, and fatty acid compositions, it was found that the novel strains belonged to the genus Pseudoxanthomonas and represented a novel species within this genus, for which the name Pseudoxanthomonas taiwanensis is proposed; the type strain is CB-226(T) (= ATCC BAA-404(T) = CCRC 17172(T)) . P . taiwanensis differs from the only member of the genus Pseudoxanthomonas, the mesophilic species Pseudoxanthomonas broegbernensis, in that it exhibits a higher growth temperature and different morphological characteristics, such as the absence of polar flagella.

Arch Biochem Biophys, 2003 Jan 15, 409(2), 327 - 34
Inhibition by phenylglyoxal of nitrate transport in Paracoccus denitrificans: a comparison with the effect of a protonophorous uncoupler; Kucera I; The amino acid modifier phenylglyoxal (PG) gradually inactivated the methyl viologen-coupled nitrate reductase activity of the anoxically grown whole cells of Paracoccus denitrificans . A double log plot of the pseudo-first-order inactivation rate constant versus PG concentration was linear with a mean slope of 1.4 (0.1M sodium phosphate) or 0.87 (0.1M sodium borate) . Phenylglyoxalation of cells lowered the limiting velocity (V), while hardly affecting the apparent half-saturation concentration (K(m)) of nitrate . Nitrate afforded no protection against inactivation . The inhibition by PG could be removed by the detergent Triton X-100 or by the lipid-soluble tetraphenylphosphonium countercation, suggesting that PG exerts its effect at the level of nitrate transport . Based on studies with membrane potential- and pH-sensitive fluorescent probes, the inhibition was shown not to be due to changes in the electrochemical gradient of hydrogen ions . Both K(m) and V values for nitrate uptake increased in a hyperbolic fashion in response to exogenously added nitrite . Nitrite promoted a bypass of the inhibition caused by low concentrations of the proton-conducting agent carbonyl cyanide m-chlorophenylhydrazone (CCCP), but was almost ineffective in the case of the PG block . These results are rationalized in terms of two nitrate import pathways that are comparably inhibited by PG and differ in their sensitivities to CCCP . A simplified kinetic model for phenylglyoxalation is proposed to account for the observed nonintegral reaction orders.

Arch Biochem Biophys, 2003 Jan 15, 409(2), 315 - 26
Biotin sulfoxide reductase: Tryptophan 90 is required for efficient substrate utilization; Pollock VV et al.; Rhodobacter sphaeroides f . sp . denitrificans biotin sulfoxide reductase (BSOR) catalyzes the reduction of d-biotin d-sulfoxide to biotin and contains the molybdopterin guanine dinucleotide (MGD) cofactor as its sole prosthetic group . Comparison of the primary sequences of BSOR and the closely related enzyme dimethyl sulfoxide reductase (DMSOR) indicated a number of conserved residues, including an active-site tryptophan residue (W90), which has been suggested to be involved in hydrogen bonding to the oxo group on the Mo(VI) center in BSOR . Site-directed mutagenesis has been used to replace tryptophan 90 in BSOR with phenylalanine, tyrosine, and alanine residues to examine the role of this residue in catalysis . All three BSOR mutant proteins were purified to homogeneity and contained MGD . The mutant proteins retained very limited activity toward the oxidizing substrates tested, with W90F retaining the most activity (3.4% of wild type) . All three W90 mutant proteins exhibited greatly reduced k(cat) values compared to that of the wild-type enzyme, which was accompanied by little change in K(mapp) . In addition, the mutant proteins had perturbed visible absorption and circular dichroism spectra suggesting different oxidation states of the Mo center . Purified samples of wild-type BSOR did not exhibit electron paramagnetic resonance (EPR) signals indicating a Mo(VI) center . After redox-cycling, partially reduced samples of wild-type BSOR revealed a proton-split S=1/2 Mo(V) resonance (g(1,2,3)=1.999, 1.981, 1.967; A(1,2,3)=1.40, 1.00, 1.05 mT) analogous to that observed in DMSOR . In contrast, EPR studies of the purified W90 mutant proteins revealed distinct S=1/2 Mo(V) resonances that were resistant to both oxidation and reduction, indicating that the Mo was trapped in the intermediate Mo(V) oxidation state . These results strongly suggest that W90 in BSOR plays a critical role in catalysis by serving as a hydrogen bond donor to the oxo group on the Mo(VI) center.

J Gen Appl Microbiol, 1998 Feb, 44(1), 19 - 26
Production of ubiquinone-10 using bacteria; Yoshida H et al.; Among the bacterial strains known to contain ubiquinone-10, three strains, Agrobacterium tumefaciens KY-3085 (ATCC4452), Paracoccus denitrificans KY-3940 (ATCC19367) and Rhodobacter sphaeroides KY-4113 (FERM-P4675), were selected as excellent producers of this ubiquinone . The ubiquinone-10 production by the Agrobacterium and Rhodobacter strains was affected by aeration . An ethionine-resistant mutant (M-37) derived from A . tumefaciens KY-3085 promoted increased production of ubiquinone-10 (20% higher than the parent) . Another Agrobacterium mutant (AU-55), which was induced by the successive addition of four genetic markers, showed a tolerance to the suppression of ubiquinone-10 production caused by aeration, and the fermentation time for production was remarkably shortened . The amount of ubiquinone-10 produced by this Agrobacterium mutant reached 180 mg/l in a 58 h culture . A green mutant (carotenoid-deficient mutant, Co-22-11) derived from R . sphaeroides KY-4113 produced 350 mg/l of ubiquinone-10 under culturing conditions with a limited supply of air, the ubiquinone-10 content being 8.7 mg/g-dry cell . In this case, the amount and content corresponded to 2.8 and 3.6 times larger than those given by the wild-type strain, respectively . A multiple-layer structure of cell membrane was observed in the highly ubiquinone-10 accumulating cell of the green mutant by electron microscopy . The amount of ubiquinone-10 produced by P . denitrificans was much lower than those of the other two strains.

Biodegradation, 2002, 13(3), 163 - 70
Nitrite reduction by a mixed culture under conditions relevant to shortcut biological nitrogen removal; Chung J et al.; Dissimilative reduction of nitrite by nitrite-acclimated cells was investigated in a batch reactor under various environmental conditions that can be encountered in shortcut biological nitrogen removal (SBNR: ammonia to nitrite and nitrite to nitrogen gas) . The maximum specific nitrite reduction rate was as much as 4.3 times faster than the rate of nitrate reduction when individually tested, but the reaction was inhibited in the presence of nitrate when the initial nitrate concentration was greater than approximately 25 mg-N/1 or the initial NO3(-)N/NO2(-)-N ratio was larger than 0.5 . Nitrite reduction was also inhibited by nitrite itself when the concentration was higher than that to which the cells had been acclimated . Therefore, it was desirable to avoid excessively high nitrite and nitrate concentrations in a denitrification reactor . Nitrite reduction, however, was not affected by an alkaline pH (in the range of 7-9) or a high concentration of FA (in the range of 16-39 mg/l), which can be common in SBNR processes . The chemical oxygen demand (COD) requirement for nitrite reduction was approximately 22-38% lower than that for nitrate reduction, demonstrating that the SBNR process can be economical . The specific consumption, measured as the ratio of COD consumed to nitrogen removed, was affected by the availability of COD and the physiological state of the cells . The ratio increased when the cells grew rapidly and were storing carbon and electrons.

Biotechnol Bioeng, 2003 Feb 20, 81(4), 397 - 404
Enrichment of denitrifying glycogen-accumulating organisms in anaerobic/anoxic activated sludge system; Zeng RJ et al.; Denitrifying glycogen-accumulating organisms (DGAO) were successfully enriched in a lab-scale sequencing batch reactor (SBR) running with anaerobic/anoxic cycles and acetate feeding during the anaerobic period . Acetate was completely taken up anaerobically, which was accompanied by the consumption of glycogen and the production of poly-beta-hydroxy-alkanoates (PHA) . In the subsequent anoxic stage, nitrate or nitrite was utilized as electron acceptor for the oxidation of PHA, resulting in glycogen replenishment and cell growth . The above phenotype showed by the enrichment culture demonstrates the existence of DGAO . Further, it was found that the anaerobic behavior of DGAO could be predicted well by the anaerobic GAO model of Filipe et al . (2001) and Zeng et al . (2002a) . The final product of denitrification during anoxic stage was mainly nitrous oxide (N(2)O) rather than N(2) . The data strongly suggests that N(2)O production may be caused by the inhibition of nitrous oxide reductase by an elevated level of nitrite accumulated during denitrification . The existence of these organisms is a concern in biological nutrient removal systems that typically have an anaerobic/anoxic/aerobic reactor sequence since they are potential competitors to the polyphosphate-accumulating organisms .

Environ Sci Technol, 2002 Nov 15, 36(22), 4729 - 34
Short-term changes in bacterial community fingerprints and potential activities in an alfisol supplemented with solid waste leachates; Poly F et al.; We investigated the effect on soil functioning of adding leachates from municipal solid waste incinerator (MSWI) ashes to laboratory columns (bare soil) and to field experimental plots (bare soil or grass cover) . Leachate of MSWI-solidified air pollution control residues (SAPCr) contained more salts but less heavy metals than did MSWI-bottom ash (BA) leachate . Leachate-supplemented soils (BA soil, SAPCr soil) and control (water added) soil (W) were analyzed after 30 days . Potential denitrifying activity (PDA) and potential N2 fixation (acetylene reduction assay, ARA) were measured in controlled conditions . PDA was significantly lower in SAPCr soil than in W soil, both in the laboratory (-45%) and in bare soil in the field (-31%) . ARA values were lower in bare SAPCr soil (-54%) and in bare BA (-25%) soil . Both activities remained unaffected by leachate supplementation in soil under permanent grass cover . Automated ribosomal intergenic spacer analysis (A-RISA) fingerprints and RFLP of nifH gene pools were used to assess changes in the structure of bacterial community . Multivariate analysis of these fingerprints revealed that SAPCr leachate had a stronger effect than BA leachate on the total and N2-fixing bacterial communities . Similar results were obtained for laboratory and bare soil field plots, but leachates did not affect nifH gene pools from soil under permanent grass cover . The stronger impact of SAPCr leachate on both structure of bacterial communities and activities supports the conclusion that observed effects would result from the abundance of salts rather than from heavy metal toxicity.

Biochem Biophys Res Commun, 2003 Jan 3, 300(1), 36 - 40
Characterization of two Cu-containing protein fragments obtained by limited proteolysis of Hyphomicrobiumdenitrificans A3151 nitrite reductase; Yamaguchi K et al.; The unusual Hyphomicrobium denitrificans nitrite reductase containing two type 1 Cu sites and one type 2 Cu site (MW, 50 kDa) has been proteolyzed to two protein fragments (14 and 35 kDa) with subtilisin . The visible absorption, CD, and EPR spectra of these proteins imply that the blue 14-kDa protein fragment has one type 1 Cu site, which is axially elongated trigonal bipyramidal, and the green 35-kDa protein fragment has one type 1 Cu site having a flattened tetrahedral geometry with one type 2 Cu site . The 35-kDa fragment shows the nitrite reduction activity a little higher than to that of native HdNIR . The redox potentials of the 14- and 35-kDa fragments are +345 and +353mV vs . NHE at pH 7.0, respectively . Moreover, the intermolecular electron transfer rate constant of the 35-kDa fragment from an electron donor, cognate cytochrome c(550), is nearly the same as that of the native enzyme.

Water Sci Technol, 2002, 46(10), 89 - 96
The AF-BNR-SCP process as a way to reduce global sludge production: comparison with classical approaches on a full scale basis; Battistoni P et al.; The paper presents a comparison between the performances of two full scale wastewater treatment plants operating in Italy, considering the mass balances including P treatments, and results coming from an analysis of 16 similar plants in Europe and USA, in order to evaluate sludge overproduction due to chemical P removal adoption . Specific production of 9.5 and 12.5 kgTS/P.E.y were found for a BNR and denitrification plant scheme respectively . These results were compared, on a mass balance basis, with the performances coming from the adoption of the integrated waste/wastewater cycles, in which OFMSW fermentation is used as C source to promote BNR performances and P removal from anaerobic supernatants as struvite crystals . ASM2 simulations are used to verify the advantages coming from this approach in terms of sludge reduction . A complete mass balance of the process is carried out, and it is shown that this last process allows us to achieve the lowest sludge production among the processes considered, coupling this with the economic benefits coming from OFMSW disposal and struvite crystallisation.

Water Sci Technol, 2002, 46(10), 71 - 7
Ozonation of wastewater sludge for reduction and recycling; Ahn KH et al.; An ozone treatment system was introduced as an alternative method for municipal sludge treatment and disposal . A pilot-scale facility was built to investigate the feasibility of the ozonation for sludge reduction and recycle . The system consists of three main parts; advanced wastewater treatment, sludge ozone treatment and belt press dewatering . Ozonation of wastewater sludge resulted in mass reduction by mineralization as well as volume reduction by improvement of dewatering characteristics . The supernatant of the ozonated sludge, consisting of solubilized organics and micro-particles, proved to be an effective carbon source for denitrification . A simple economic assessment reveals that the ozonation process can be more economical than incineration for sludge treatment and disposal at small- and medium-sized wastewater treatment plants.

Exp Eye Res, 2002 Dec, 75(6), 669 - 76
Vasodilatory effects of nipradilol, an alpha- and beta-adrenergic blocker with nitric oxide releasing action, in rabbit ciliary artery; Yoshitomi T et al.; Nipradilol is a new antiglaucoma ophthalmic agent used in Japan . Topical application of nipradilol is reported to increase ocular blood flow . To investigate the action of this drug, we studied the effect of nipradilol on the isolated rabbit ciliary artery . Under the dissecting microscope, ciliary arteries were prepared from rabbit eyes and mounted on a myograph system . The effects of nipradilol on the isolated rabbit ciliary artery were investigated using isometric tension recording methods . Nipradilol provoked a dose-dependent (10 microM-1m M) relaxation in ciliary arteries that were pre-contracted with high-K solutions (K(+): 100.7 m M) . It also inhibited the amplitude of smooth muscle contraction evoked by field stimulation . Nipradilol was more effective in relaxing phenylephrine-induced contraction (EC(50): 21.6+/-16.3 microM) compared to high-K solution-induced contractions (EC(50): 230+/-130 microM) . Application of N(w)-nitro- L -arginine methylester (300 microM), a nitric oxide (NO) synthase inhibitor, or denudations of endothelium by rubbing the inner surface with a scalp hair did not affect this relaxation . However, NO scavenger carboxy-PTIO (1m M) or methylene blue (10 microM), a guanylate cyclase inhibitor, inhibited the nipradilol-induced relaxation . These results indicate that nipradilol relaxes the rabbit ciliary artery by two different mechanisms . First, the relaxation is due to the NO produced by denitrification of nipradilol itself . Second, nipradilol may act as an alpha-adrenergic antagonist . These actions of nipradilol may explain the mechanisms of increased ocular blood flow in vivo.

J Environ Qual, 2002 Nov-Dec, 31(6), 1858 - 67
Environmental and agronomic implications of water table and nitrogen fertilization management; Elmi AA et al.; Nitrate (NO3-) pollution of surface and subsurface waters has become a major problem in agricultural ecosystems . Field trials were conducted from 1996 to 1998 at St-Emmanuel, Quebec, Canada, to investigate the combined effects of water table management (WTM) and nitrogen (N) fertilization on soil NO3- level, denitrification rate, and corn (Zea mays L.) grain yield . Treatments consisted of a combination of two water table treatments: free drainage (FD) with open drains at a 1.0-m depth from the soil surface and subirrigation (SI) with a design water table of 0.6 m below the soil surface, and two N fertilizer (ammonium nitrate) rates: 120 kg N ha(-1) (N120) and 200 kg N ha(-1) (N200) . Compared with FD, SI reduced NO3(-)-N concentrations in the soil profile by 37% in spring 1997 and 2% in spring 1998; and by 45% in fall 1997 and 19% in fall 1998 (1 mg NO3(-)-N L(-1) equals approximately 4.43 mg NO3- L(-1)) . The higher rate of N fertilization resulted in greater levels of NO3(-)-N in the soil solution . Denitrification rates were higher in SI than in FD plots, but were unaffected by N rate . The N200 rate produced higher yields than N120 in 1996 and 1997, but not 1998 . Corn yields in SI plots were 7% higher than FD plots in 1996 and 3% higher in 1997, but 25% lower in 1998 because the SI system was unable to drain the unusually heavy June rains, resulting in waterlogging . These findings suggest that SI can be used as an economical means of reducing NO3- pollution without compromising crop yields during normal growing seasons.

Environ Toxicol Chem, 2002 Dec, 21(12), 2631 - 9
Effect of ethanol and methyl-tert-butyl ether on monoaromatic hydrocarbon biodegradation: response variability for different aquifer materials under various electron-accepting conditions; Ruiz-Aguilar GM et al.; Aquifer microcosms were used to determine how ethanol and methyl-tert-butyl ether (MtBE) affect monoaromatic hydrocarbon degradation under different electron-accepting conditions commonly found in contaminated sites experiencing natural attenuation . Response variability was investigated by using aquifer material from four sites with different exposure history . The lag phase prior to benzene, toluene, ethylbenzene, and xylenes (BTEX) and ethanol degradation was typically shorter in microcosms with previously contaminated aquifer material, although previous exposure did not always result in high degradation activity . Toluene was degraded in all aquifer materials and generally under a broader range of electron-accepting conditions compared to benzene, which was degraded only under aerobic conditions . The MtBE was not degraded within 100 d under any condition, and it did not affect BTEX or ethanol degradation patterns . Ethanol was often degraded before BTEX compounds and had a variable effect on BTEX degradation as a function of electron-accepting conditions and aquifer material source . An occasional enhancement of toluene degradation by ethanol occurred in denitrifying microcosms with unlimited nitrate; this may be attributable to the fortuitous growth of toluene-degrading bacteria during ethanol degradation . Nevertheless, experiments with flow-through aquifer columns showed that this beneficial effect could be eclipsed by an ethanol-driven depletion of electron acceptors, which significantly inhibited BTEX degradation and is probably the most important mechanism by which ethanol could hinder BTEX natural attenuation . A decrease in natural attenuation could increase the likelihood that BTEX compounds reach a receptor as well as the potential duration of exposure.

J Biol Inorg Chem, 2003 Jan, 8(1-2), 75 - 82 Epub 2002 Aug 29.
Metal-ligand interactions in perturbed blue copper sites: a paramagnetic (1)H NMR study of Co(II)-pseudoazurin; Fernandez CO et al.; Pseudoazurin is an electron transfer copper protein, a member of the cupredoxin family . The protein is frequently found in denitrifying bacteria, where it is the electron donor of nitrite reductase . The copper at the active site is coordinated to His40, Cys78, His81 and Met86 in a distorted tetragonal geometry . We have recorded and assigned the (1)H NMR spectra of Co(II)-substituted pseudoazurin from Achromobacter cycloclastes . The (1)H NMR spectrum of Co(II)-pseudoazurin closely resembles that of Co(II)-rusticyanin, reflecting an altered conformation for the Met-Co(II)-Cys moiety in both proteins, compared to Co(II)-azurin, amicyanin and stellacyanin . The electron spin density onto the Sgamma(Cys) is larger in Co(II)-pseudoazurin compared to Co(II)-rusticyanin . Instead, the Co(II)-Met interaction is similar in both derivatives . Hence, the different metal-ligand interactions might be independently modulated by the protein structure . The present work also shows that the electron spin density onto the Co(II)-S(cys) bond is sensibly smaller than the Cu(II)-S(cys) . Notwithstanding, NMR data on Co(II)-substituted blue copper proteins can be safely extrapolated to native Cu(II) proteins.

J Biol Inorg Chem, 2003 Jan, 8(1-2), 29 - 37 Epub 2002 Jul 13.
Ca2+ and the bacterial peroxidases: the cytochrome c peroxidase from Pseudomonas stutzeri; Timoteo CG et al.; The production of cytochrome c peroxidase (CCP) from Pseudomonas ( Ps.) stutzeri (ATCC 11607) was optimized by adjusting the composition of the growth medium and aeration of the culture . The protein was isolated and characterized biochemically and spectroscopically in the oxidized and mixed valence forms . The activity of Ps . stutzeri CCP was studied using two different ferrocytochromes as electron donors: Ps . stutzeri cytochrome c(551) (the physiological electron donor) and horse heart cytochrome c . These electron donors interact differently with Ps . stutzeri CCP, exhibiting different ionic strength dependence . The CCP from Paracoccus ( Pa.) denitrificans was proposed to have two different Ca(2+) binding sites: one usually occupied (site I) and the other either empty or partially occupied in the oxidized enzyme (site II) . The Ps . stutzeri enzyme was purified in a form with tightly bound Ca(2+) . The affinity for Ca(2+) in the mixed valence enzyme is so high that Ca(2+) returns to it from the EGTA which was added to empty the site in the oxidized enzyme . Molecular mass determination by ultracentrifugation and behavior on gel filtration chromatography have revealed that this CCP is isolated as an active dimer, in contrast to the Pa . denitrificans CCP which requires added Ca(2+) for formation of the dimer and also for activation of the enzyme . This is consistent with the proposal that Ca(2+) in the bacterial peroxidases influences the monomer/dimer equilibrium and the transition to the active form of the enzyme . Additional Ca(2+)does affect both the kinetics of oxidation of horse heart cytochrome c (but not cytochrome c(551)) and higher aggregation states of the enzyme . This suggests the presence of a superficial Ca(2+)binding site of low affinity.

J Biol Chem, 2003 Feb 14, 278(7), 4404 - 9 Epub 2002 Nov 27.
Cytochrome c maturation . The in vitro reactions of horse heart apocytochrome c and Paracoccus dentrificans apocytochrome c550 with heme; Daltrop O et al.; C-type cytochromes are characterized by having the heme moiety covalently attached via thioether bonds between the heme vinyl groups and the thiols of conserved cysteine residues of the polypeptide chain . Previously, we have shown the in vitro formation of Hydrogenobacter thermophilus cytochrome c(552) (Daltrop, O., Allen, J . W . A., Willis, A . C., and Ferguson, S . J . (2002) Proc . Natl . Acad . Sci . U . S . A . 99, 7872-7876) . In this work we report that thioether bonds can form spontaneously in vitro between heme and the apocytochromes c from horse heart and Paracoccus denitrificans via b-type cytochrome intermediates . Both apocytochromes, but not the holo forms, bind 8-anilino-1-naphthalenesulfonate, indicating that the apoproteins each have an affinity for a hydrophobic ligand . Furthermore, for both apocytochromes c an intramolecular disulfide can form between the cysteines of the CXXCH motif that is characteristic of c-type cytochromes . In vitro reaction of these apocytochromes c with heme to yield holocytochromes c, and the tendency to form a disulfide, have implications for the different systems responsible for cytochrome c maturation in vivo in various organisms.

J Struct Biol, 2002 Sep, 139(3), 171 - 80
Three-dimensional structure of manganese superoxide dismutase from Bacillus halodenitrificans, a component of the so-called "green protein"; Liao J et al.; A so-called "green protein" has been purified from a moderate halophilic eubacterium, Bacillus halodenitrificans (ATCC 49067), under anaerobic conditions . The protein, which might play an important role in denitrification, dissociates mainly into two components after exposure to air: a manganese superoxide dismutase (GP-MnSOD) and a nucleoside diphosphate kinase . As a first step in elucidating the overall structure of the green protein and the role of each component, the 2.8-A resolution crystal structure of GP-MnSOD was determined . Compared with other manganese dismutases, GP-MnSOD shows two significant characteristics . The first is that the entrance to its substrate channel has an additional basic residue-Lys38 . The second is that its surface is decorated with an excess of acidic over basic residues . All these structural features may be related to GP-MnSOD's high catalytic activity and its endurance against the special cytoplasm of B . halodenitrificans . The structure of GP-MnSOD provides the basis for recognizing its possible role and assembly state in the green protein.

Prikl Biokhim Mikrobiol, 2002 Nov-Dec, 38(6), 649 - 52
{Reduction of nitrates by cultures of Azotobacter indicum and Azotobacter chroococcum }; Furina EK et al.; The capacity for denitrification was studied in Azotobacter bacteria, which are free-living nitrogen-fixing obligatory aerobes . Data on the nitrate reduction to nitrites and nitric oxide by A . indicum under anaerobic conditions were obtained for the first time for genus Azotobacter.

Mikrobiologiia, 2002 Sep-Oct, 71(5), 604 - 10
{Thiodiglycol metabolism in Alcaligenes xylosoxydans subsp . denitrificans}; Ermakova IT et al.; The investigation of the degradation of thiodiglycol (the major product of mustard gas hydrolysis) by Alcaligenes xylosoxydans subsp . denitrificans strain TD2 showed that thiodiglycol is metabolized through the oxidation of its primary alcohol groups and the subsequent cleavage of C-S bonds in the intermediate products, thiodiglycolic and thioglycolic acids . The end products of these reactions are SO4(2-) ions and acetate, the latter being involved in the central metabolism of strain TD2 . The oxidation of the sulfur atom gives rise to diglycolsulfoxide, which is recalcitrant to further microbial degradation . Based on the data obtained, a metabolic pathway of thiodiglycol transformation by A . xylosoxydans subsp . denitrificans strain TD2 is proposed.

Biodegradation, 2002, 13(2), 149 - 54
Biochemical and genetic evidence of benzylsuccinate synthase in toluene-degrading, ferric iron-reducing Geobacter metallireducens; Kane SR et al.; In vitro assays demonstrated that toluene-grown cells of Geobacter metallireducens catalyzed the addition of toluene to fumarate to form benzylsuccinate under anaerobic conditions . The specific in vitro rate of benzylsuccinate formation was ca . 45% of the specific in vivo rate of toluene consumption . In addition, bssA and bssB, which code for the alpha and beta subunits of benzylsuccinate synthase (BSS), respectively, were found to have sequences in G . metallireducens similar to the only sequences heretofore available (for three denitrifying strains) . This is the first report of the presence of BSS in a ferric iron-reducing bacterium; BSS activity has previously been reported in denitrifying, sulfate-reducing, and anoxygenic phototrophic toluene degraders, as well as in a highly enriched methanogenic, toluene-degrading culture.

Water Res, 2002 Nov, 36(19), 4801 - 10
High-rate denitrification and SS rejection by biofilm-electrode reactor (BER) combined with microfiltration; Prosnansky M et al.; In this study, a multi-cathode biofilm-electrode reactor (BER) combined with microfiltration (MF) was investigated using a laboratory-scale experimental apparatus for treatment of nitrate-contaminated water . The multi-cathode electrodes were composed of multiple-granular activated carbons (GACs) . GACs attached to each cathode to enlarge surface area of electrodes and to attach bacteria quickly and firmly . In BER, H2 gas is produced by applying electric current, which serves as an electron donor in biological reduction of nitrate to N2 gas . Since some suspended solids were escaping from BER, MF membrane with plate modules and a pore size of 0.2 microm was placed after BER . Experimental results demonstrated that it was possible to operate the multi-cathode BER with high denitrification rates and hydraulic retention time (HRT) as low as HRT = 20 min . The denitrification rate was enhanced by 3-60 times in comparison with former studies . MF membrane successfully rejected the bacteria escaping from BER, so that the effluent concentration of SS was kept below 1 mg SS/l throughout the experiment . It was also possible to operate MF membrane at flux 2-9 times higher and pressure 2.5-31 times smaller than in former studies . This higher performance was mainly brought about by using biofilm and H2 gas as an electron donor . Also, an economic evaluation of BER/MF was included, showing the feasibility of this process . The present BER/MF process is considered advantageous for the enhanced treatment of nitrate-polluted groundwater.

Water Res, 2002 Nov, 36(19), 4683 - 90
Hydrogenotrophic denitrification in a microporous membrane bioreactor; Mansell BO et al.; Hydrogenotrophic denitrification of nitrate contaminated groundwater in a bench-scale microporous membrane bioreactor has been investigated . To prevent microbial contamination of the effluent from the reactor the nitrate-laden water treated was separated from the denitrifying culture with a 0.02 microm pore diameter membrane . Equal pressure was maintained across the membrane and nitrate was removed by molecular diffusion through the membrane and into the denitrifying culture . The system was operated with a hydrogenotrophic denitrification culture to circumvent the addition of an organic substrate to the water . Removal efficiencies ranging from 96% to 92% were achieved at influent concentrations ranging from 20 to 40 mg/L NO3(-)-N . The flux values achieved in this study were 2.7-5.3 g NO3-N m 2d(-1) . The microporous membrane served as an effective barrier for preventing microbial contamination of the product water as evidenced by the effluent heterotrophic plate count of 9 (+/- 3.5) CFU/mL . The hydrogenotrophic culture was analyzed using available 16S and 23S rRNA-targeted oligonucleotide probes . It was determined that the enrichment process selected for organisms belonging to the beta subclass of Proteobacteria . Further analysis of the hydrogenotrophic culture indicated that the organisms may belong to the beta-3 subgroup of Proteobacteria and have yet to be identified as hydrogenotrophic denitrifiers.

Water Sci Technol, 2002, 46(9), 237 - 46
Assessment of the denitrification potential for biological nutrient removal processes using OUR/NUR measurements; Sozen S et al.; The denitrification potential, a key parameter in nutrient removal activated sludge systems, is mathematically described in terms of mass balance expressions for different carbon sources, namely, easily biodegradable substrate, slowly biodegradable substrate and biomass . Mass balance was derived both for single-anoxic (pre-denitrification) and dual anoxic (Bardenpho) systems . Correction factors for anoxic growth were experimentally determined using respirometry for domestic sewage and meat processing wastewater . The denitrification potential expressions were evaluated for different process configurations such as pre-denitrification, Bardenpho process and University of Cape Town (UCT) process.

Water Sci Technol, 2002, 46(9), 211 - 8
Denitrifying activity measurements using an anoxic titration (pHstat) bioassay; Foxon KM et al.; An anoxic titrimetric test was investigated for measuring denitrifying activity in an activated sludge system . The method measures the amount of acid that is required to maintain the pH set-point value in a batch denitrification experiment . An iterative algorithm was implemented to extract nitrate uptake rate (NUR) data from titration data, since the accumulation and depletion (stripping) of reaction by-products HCO3- and CO2 affects the direct calculation of denitrifying activity from titration data . This method was performed using an automatic pH-stat acid dosing system, and the data were analysed using the simulation software package, AQUASIM.

Microb Ecol, 2003 Jan, 45(1), 39 - 52 Epub 2002 Nov 27.
Maintenance and impacts of an inoculated mer/luc-tagged Pseudomonas fluorescens on microbial communities in birch rhizospheres developed on humus and peat; Bjorklof K et al.; Antagonistic bacteria represent promising biocontrol agents for improving forest production in seedling nurseries or forest soils . The fate of an introduced mer/luc-tagged antagonistic Pseudomonas fluorescens 31K3 was monitored in the rhizosphere of silver birch (Betula pendula) seedlings grown in microcosms containing forest humus or nursery peat . The inoculated strain (10(8) cfu g(-1) soil) was unable to establish in significant numbers in either soil type and turned nonculturable in humus . Detection in both soils was possible only via luminescence of enrichment cultures 80 days post-inoculation . Despite low P . fluorescens survival, inoculation had a positive effect on seedling growth . Limited impact of inoculation on the indigenous microbial communities was identified following analyses of respiration and denitrification potential, community-level physiological profiles and molecular fingerprinting of fungi and eubacteria, and Pseudomonas community structures . The minor changes observed in the indigenous microbial communities, including mycorrhiza development, were not consistent between humus and peat growth substrates . It was concluded that the rhizosphere-related microbial communities developed in both of these highly organic soil systems are highly buffered against introduction of foreign bacteria.

FEMS Microbiol Lett, 2002 Nov 19, 217(1), 37 - 42
Construction and characterization of insertion/deletion mutations of the tutF, tutD, and tutG genes of Thauera aromatica strain T1; Coschigano PW; Thauera aromatica T1 was isolated for its ability to use toluene as a sole carbon source under denitrifying conditions . A genetic approach was used to examine the roles of the tutF, tutD, and tutG gene products (part of a single operon) in the metabolism of toluene . The genes were individually deleted from the chromosome and each resulting mutant strain was unable to metabolize toluene . Plasmids carrying individual in-frame gene deletions failed to complement the corresponding chromosomal deletions but did complement chromosomal deletions downstream of the in-frame deletion . Hence, the tutF, tutD, and tutG genes are each essential for toluene metabolism in T . aromatica T1.

Curr Microbiol, 2003 Jan, 46(1), 65 - 9
Metabolism of DDT {1,1,1-Trichloro-2,2-bis(4-chlorophenyl)ethane} by Alcaligenes denitrificans ITRC-4 under aerobic and anaerobic conditions; Ahuja R et al.; An isolated bacterium, Alcaligenes denitrificans ITRC-4, metabolizes 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) under both aerobic and anaerobic conditions . The aerobic metabolism is inhibited by 38% and 47% in the presence of 1.0 g L(-1) of sodium acetate and sodium succinate, respectively, but remains uninhibited in the presence of 1.0 g L(-1) of glucose . Also, the metabolism is inhibited completely in the presence of biphenyl vapors, as well as 0.8 g L(-1) of 2,2'-bipyridyl . Under anaerobic conditions, DDT is metabolized into 1,1-dichloro-2,2-bis(4-chlorophenyl)ethane (DDD), which is further enhanced by 50% in the presence of 1.0 g L(-1) of glucose . Besides, the bacterium also metabolizes 4-chlorobenzoate, which is accompanied by the release of chloride ions.

Microbiology, 2002 Nov, 148(Pt 11), 3553 - 60
Characterization of the norCBQD genes, encoding nitric oxide reductase, in the nitrogen fixing bacterium Bradyrhizobium japonicum; Mesa S et al.; The genes norCBQD that encode the bc-type nitric oxide reductase from Bradyrhizobium japonicum USDA110 have been isolated and characterized . norC and norB encode the cytochrome c-containing subunit II and cytochrome b-containing subunit I of nitric oxide reductase, respectively . norQ encodes a protein with an ATP/GTP-binding motif, and the predicted norD gene product shows similarity with NorD from other denitrifiers . Mutational analysis indicates that the two structural norC and norB genes are required for microaerobic growth under nitrate-respiring conditions . A mutant strain lacking a functional norC gene also lacked the 16 kDa c-type cytochrome that is normally detectable by haem-staining of proteins from membranes of microaerobically grown wild-type cells . Expression of a transcriptional fusion of the nor promoter region to the reporter gene lacZ (P(norC)-lacZ) was not detected in aerobically grown cells of USDA110, but the fusion was induced threefold when the cells were cultured under microaerobic conditions (1% O(2)) with either nitrite or nitric oxide, and about 18-fold when nitrate was the N oxide present in the medium . The P(norC)-lacZ fusion was not expressed in the B . japonicum fixK(2) mutant strain 9043, but complementation of the mutant with the fixK(2) gene restored beta-galactosidase activity to levels similar to those found in the parental strain . The promoter region of the norCBQD genes has been characterized by primer extension . A major transcript initiates 45.5 bp downstream of the centre of a putative binding site for the transcription factor FixK(2).

Biochemistry, 2002 Nov 19, 41(46), 13736 - 43
Spectroelectrochemical evaluation of redox potentials of cysteine tryptophylquinone and two hemes c in quinohemoprotein amine dehydrogenase from Paracoccus denitrificans; Fujieda N et al.; Quinohemoprotein amine dehydrogenase (QH-AmDH) from Paracoccus denitrificans has a novel cofactor cysteine tryptophylquinone (CTQ) in the smallest gamma subunit and two hemes c in the largest alpha subunit {Datta, S., Mori, Y., Takagi, K., Kawaguchi, K., Chen, Z., Okajima, T., Kuroda, S., Ikeda, T., Kano, K., Tanizawa, K., and Mathews, F . S . (2001) Proc . Natl . Acad . Sci . U.S.A . 98, 14268-14273} . The spectral change of QH-AmDH was assigned to the redox reaction of the hemes c alone . The redox potentials of the two hemes c with His and Met as the second axial ligands, respectively, were determined to be 0.149 and 0.235 V versus SHE at pH 7.0 by a mediator-assisted continuous-flow column electrolytic spectroelectrochemistry (MCES) . The monomeric gamma subunit of QH-AmDH was isolated from urea-treated QH-AmDH . The fully oxidized and reduced forms of the gamma subunit exhibited a unique absorption band centered at 380 nm and a shoulder band around 315 nm, respectively, at neutral pH . The two-electron redox potential of CTQ in the isolated gamma subunit was evaluated to be 65 mV at pH 7.0 by MCES . The redox reaction was linked to the two-proton transfer at pH <8.6 and to a single-proton transfer at pH >8.6 . The pK(a) value (K(a) being the acid dissociation constant) of 8.6 was assigned to one of the phenolic OH groups of the quinol form . Upon deprotonation, the red shift of the shoulder band was observed . The gamma subunit adsorbed on a glassy carbon electrode, and gave a direct but quasi-reversible electrochemical signal . Intra- and interprotein electron transfers of QH-AmDH are discussed from thermodynamic and structural points of view.

J Bacteriol, 2002 Dec, 184(23), 6515 - 21
Long-chain acyl-homoserine lactone quorum-sensing regulation of Rhodobacter capsulatus gene transfer agent production; Schaefer AL et al.; Many proteobacteria use acyl-homoserine lactones as quorum-sensing signals . Traditionally, biological detection systems have been used to identify bacteria that produce acyl-homoserine lactones, although the specificities of these detection systems can limit discovery . We used a sensitive approach that did not require a bioassay to detect production of long-acyl-chain homoserine lactone production by Rhodobacter capsulatus and Paracoccus denitrificans . These long-chain acyl-homoserine lactones are not readily detected by standard bioassays . The most abundant acyl-homoserine lactone was N-hexadecanoyl-homoserine lactone . The long-chain acyl-homoserine lactones were concentrated in cells but were also found in the culture fluid . An R . capsulatus gene responsible for long-chain acyl-homoserine lactone synthesis was identified . A mutation in this gene, which we named gtaI, resulted in decreased production of the R . capsulatus gene transfer agent, and gene transfer agent production was restored by exogenous addition of N-hexadecanoyl-homoserine lactone . Thus, long-chain acyl-homoserine lactones serve as quorum-sensing signals to enhance genetic exchange in R . capsulatus.

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