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