FEMS Microbiol Lett, 1986, 35, 55 - 8 The occurrence of denitrification in extremely halophilic bacteria; Mancinelli RL et al.; The ability of Halobacterium vallismortis, Halobacterium mediterranei and Halobacterium marismortui (Ginzburg strain) to grow anaerobically and denitrify was determined . Each organism grew anaerobically only in the presence of nitrate . H . marismortui produced nitrite and dinitrogen from nitrate during exponential growth . However, as the culture entered stationary phase, dinitrogen production ceased and nitrous oxide was detected . H . vallismortis produced nitrous oxide and dinitrogen during exponential growth, with dinitrogen production ceasing at the onset of stationary phase . H . mediterranei produced dinitrogen during exponential growth and did not produce nitrous oxide . These results confirm the occurrence of denitrification in the halobacteria. J Mol Evol, 1985, 22, 46 - 52 Phylogenetic origins of the plant mitochondrion based on a comparative analysis of 5S ribosomal RNA sequences; Villanueva E et al.; The complete nucleotide sequences of 5S ribosomal RNAs from Rhodocyclus gelatinosa, Rhodobacter sphaeroides, and Pseudomonas cepacia were determined . Comparisons of these 5S RNA sequences show that rather than being phylogenetically related to one another, the two photosynthetic bacterial 5S RNA sequences show that rather than being phylogenetically related to one another, the two photosynthetic bacterial 5S RNAs share more sequence and signature homology with the RNAs of two nonphotosynthetic strains . Rhodobacter sphaeroides is specifically related to Paracoccus denitrificans and Rc . gelatinosa is related to Ps . cepacia . These results support earlier 16S ribosomal RNA studies and add two important groups to the 5S RNA data base . Unique 5S RNA structural features previously found in P . denitrificans are present also in the 5S RNA of Rb . sphaeroides; these provide the basis for subdivisional signatures . The immediate consequence of our obtaining these new sequences is that we are able to clarify the phylogenetic origins of the plant mitochondrion . In particular, we find a close phylogenetic relationship between the plant mitochondria and members of the alpha subdivision of the purple photosynthetic bacteria, namely, Rb . sphaeroides, P . denitrificans, and Rhodospirillum rubrum. Int Biodeterior Biodegradation, 1998, 41(1), 25 - 33 The role of microbial biofilms in deterioration of space station candidate materials; Gu JD et al.; Formation of microbial biofilms on surfaces of a wide range of materials being considered as candidates for use on the International Space Station was investigated . The materials included a fibre-reinforced polymeric composite, an adhesive sealant, a polyimide insulation foam, teflon cable insulation, titanium, and an aliphatic polyurethane coating . They were exposed to a natural mixed population of bacteria under controlled conditions of temperature and relative humidity (RH) . Biofilms formed on the surfaces of the materials at a wide range of temperatures and RHs . The biofilm population was dominated by Pseudomonas aeruginosa, Ochrobactrum anthropi, Alcaligenes denitrificans, Xanthomonas maltophila, and Vibrio harveyi . The biocide, diiodomethyl-p-tolyl sulfone, impregnated in the polyurethane coating, was ineffective against microbial colonization and growth . Degradation of the polyurethane coatings was monitored with electrochemical impedance spectroscopy (EIS) . The impedance spectra indicated that microbial degradation of the coating occurred in several stages . The initial decreases in impedance were due to the transport of water and solutes into the polymeric matrices . Further decreases were a result of polymer degradation by microorganisms . Our data showed that these candidate materials for space application are susceptible to biofilm formation and subsequent degradation . Our study suggests that candidate materials for use in space missions need to be carefully evaluated for their susceptibility to microbial biofilm formation and biodegradation. FEMS Microbiol Lett, 1985, 27, 329 - 31 Denitrification by extremely halophilic bacteria; Hochstein LI et al.; Extremely halophilic bacteria were isolated from widely separated sites by anaerobic enrichment in the presence of nitrate . The anaerobic growth of several of these isolates was accompanied by the production of nitrate, nitrous oxide, and dinitrogen . These results are a direct confirmation of the existence of extremely halophilic denitrifying bacteria, and suggest that such bacteria may be common inhabitants of hypersaline environments. J Phycol, 1994 Jun, 30(3), 431 - 8 Metabolic activity of microorganisms in evaporites; Rothschild LJ et al.; Crystalline salt is generally considered so hostile to most forms of life that it has been used for centuries as a preservative . Here, we present evidence that prokaryotes inhabiting a natural evaporite crust of halite and gypsum are metabolically active while inside the evaporite for at least 10 months . In situ measurements demonstrated that some of these "endoevaporitic" microorganisms (probably the cyanobacterium Synechococcus Nageli) fixed carbon and nitrogen . Denitrification was not observed . Our results quantified the slow microbial activity that can occur in salt crystals . Implications of this study include the possibility that microorganisms found in ancient evaporite deposits may have been part of an evaporite community. Life Support Biosph Sci, 1996, 3(1-2), 43 - 6 Mass transfer in the biological fast lane: high CO2 and a shallow root zone; Smart D et al.; Elevated atmospheric CO2, which is common in regenerative systems, increases photosynthesis, plant growth, and root respiration, which increases the O2 demand in the root zone . Closed systems must make efficient use of volume and thus have shallow root zones . The root density and O2 demand in these artificial systems is 10 to 100 times higher than in field environments . Rapid hydroponic flow rates supply O2 to the root zone, but anaerobic microsites occur because of nonuniform flow rates . Our measurements suggest that, probably because of low O2 in such microsites, up to 30% of the nitrogen can volatilize from denitrification . We improved nitrogen recovery to about 85% by increasing the solution flow rate and reducing the nitrate concentration in solution to 100 micromoles. Life Support Biosph Sci, 1996, 3(1-2), 31 - 4 Denitrification: microbiology and ecology; Knowles R; The ability of some aerobic microorganisms to reduce nitrogen oxides, especially nitrate, to gaseous products enables them to grow in oxygen-limiting conditions by using the oxides as electron acceptors . Denitrifying respiration is not as efficient as oxygen respiration so growth is slower . The ability to denitrify is found in a wide variety of unrelated bacteria and even in some fungi, and biochemical mechanisms are now moderately well understood . Major factors regulating denitrification are the availability of the nitrogen oxides, the availability of reductant (mostly organic carbon compounds but inorganic compounds may be used), and decreased oxygen concentration . These three factors are in turn governed by many other factors such as water content, pH, porosity, and the presence of inhibitory compounds, which may act to cause accumulation of ionic (nitrite) or gaseous (nitric oxide, nitrous oxide) intermediates . The prediction of rates of denitrification and release of intermediates such as nitrous oxide in particular environments is proving to be difficult. Adv Space Res, 1992, 12(5), 53 - 6 Catalytic wet-oxidation of human wastes produced in space: the effects of temperature elevation; Takeda N et al.; The filtrate of non-catalytical wet-oxidation sewage sludge was wet-oxidized again at 290 degrees C and 300 degrees C with a Ru-Rh catalyst . At each temperature, repeated batch tests were carried out . Both oxidation and denitrification efficiency of organic matter in the raw material were studied . In the 16 times batch tests at 300 degrees C, high and stable oxidation occurred . 98.0% of organic carbon in the raw material was oxidized and 98.3% of organic nitrogen was denitrified . At 290 degrees C, though high and stable denitrification occurred, oxidation did not occur highly and stably . A catalytic wet-oxidation system studied at 300 degrees C will be useful as a waste management system for a human life support system, where almost all food is resupplied from the earth . This system can prevent organic waste accumulation in the life support system. Appl Environ Microbiol, 1997 Nov, 63(11), 4621 - 4 Evidence that elevated CO2 levels can indirectly increase rhizosphere denitrifier activity; Smart DR et al.; We examined the influence of elevated CO2 concentration on denitrifier enzyme activity in wheat rhizoplanes by using controlled environments and solution culture techniques . Potential denitrification activity was from 3 to 24 times higher on roots that were grown under an elevated CO2 concentration of 1,000 micromoles of CO2 mol-1 than on roots grown under ambient levels of CO2 . Nitrogen loss, as determined by a nitrogen mass balance, increased with elevated CO2 levels in the shoot environment and with a high NO3- concentration in the rooting zone . These results indicated that aerial CO2 concentration can play a role in rhizosphere denitrifier activity. Appl Environ Microbiol, 1997 Jul, 63(7), 2613 - 8 Physiology and enzymology involved in denitrification by Shewanella putrefaciens; Krause B et al.; Nitrate reduction to N2O was investigated in batch cultures of Shewanella putrefaciens MR-1, MR-4, and MR-7 . All three strains reduced nitrate to nitrite to N2O, and this reduction was coupled to growth, whereas ammonium accumulation was very low (0 to 1 micromol liter-1) . All S . putrefaciens isolates were also capable of reducing nitrate aerobically; under anaerobic conditions, nitrite levels were three- to sixfold higher than those found under oxic conditions . Nitrate reductase activities (31 to 60 micromol of nitrite min-1 mg of protein-1) detected in intact cells of S . putrefaciens were equal to or higher than those seen in Escherichia coli LE 392 . Km values for nitrate reduction ranged from 12 mM for MR-1 to 1.3 mM for MR-4 with benzyl viologen as an artifical electron donor . Nitrate and nitrite reductase activities in cell-free preparations were demonstrated in native gels by using reduced benzyl viologen . Detergent treatment of crude and membrane extracts suggested that the nitrate reductases of MR-1 and MR-4 are membrane bound . When the nitrate reductase in MR-1 was partially purified, three subunits (90, 70, and 55 kDa) were detected in denaturing gels . The nitrite reductase of MR-1 is also membrane bound and appeared as a 60-kDa band in sodium dodecyl sulfate-polyacrylamide gels after partial purification. Annu Rev Microbiol, 1988, 42, 231 - 61 The enzymes associated with denitrification; Hochstein LI et al.; NASA: The enzymes involved in the reduction of nitrogenous oxides are thought to be intermediates in denitrification processes . This review examines the roles of nitrate reductase, nitrite reductases, nitric oxide reductase, mechanisms of N-N bond formation, and nitrous oxide reductases . Microbiology, 2001 Sep, 147(Pt 9), 2505 - 15 Characterization of nirV and a gene encoding a novel pseudoazurin in Rhodobacter sphaeroides 2.4.3; Jain R et al.; Sequencing of the region flanking nirK, the gene encoding the copper-containing nitrite reductase in Rhodobacter sphaeroides 2.4.3, has identified two genes whose products could potentially be involved in nitrite reductase expression and activity . One of the genes has been designated nirV . Putative nirV orthologues are found in other denitrifiers, where they are also located downstream of the structural gene for nitrite reductase . The nirV in 2.4.3 is apparently cotranscribed with nirK . Inactivation of nirV had no effect on cell growth, or on nitrite reductase expression or activity . Downstream of nirV and divergently transcribed is a gene, designated ppaZ, encoding a protein with significant similarity to pseudoazurins from other denitrifiers . However, three of the four residues required for binding of the type I copper centre are not conserved in the deduced sequence of the protein in 2.4.3 . ppaZ is expressed only when oxygen becomes limiting . ppaZ expression is dependent on both FnrL and NnrR, and a putative binding site for these proteins has been identified . Expression of ppaZ is also dependent on the two-component PrrB/PrrA system . Inactivation of ppaZ had no significant effect on cell growth or on nitrite reductase expression or activity . Expression of a maltose-binding protein-PpaZ fusion indicated that the protein could not bind copper . Examination of the genome of the related bacterium R . sphaeroides 2.4.1 revealed that it encodes ppaZ but not nirV and evidence is presented suggesting that a common ancestor of 2.4.3 and 2.4.1 had both nitrite and nitric oxide reductase activity but as the strains diverged 2.4.1 lost nirK and nirV, making it incapable of nitrite reduction. Biochemistry, 2001 Sep 11, 40(36), 10774 - 81 Binding of NO and CO to the d(1) Heme of cd(1) nitrite reductase from Pseudomonas aeruginosa; Das TK et al.; The cd(1) nitrite reductase, a key enzyme in bacterial denitrification, catalyzes the one-electron reduction of nitrite to nitric oxide . The enzyme contains two redox centers, a c-type heme and a unique d(1) heme, which is a dioxoisobacteriochlorin . Nitric oxide, generated by this enzymatic pathway, if not removed from the medium, can bind to the ferrous d(1) cofactor with extremely high affinity and inhibit enzyme activity . In this paper, we report the resonance Raman investigation of the properties of nitric oxide and carbon monoxide binding to the d(1) site of the reduced enzyme . The Fe-ligand (Fe-NO and Fe-CO) stretching vibrational frequencies are unusually high in comparison to those of other ferrous heme complexes . The frequencies of the Fe-NO and N-O stretching modes appear at 585 and 1626 cm(-1), respectively, in the NO complex, while the frequencies of the Fe-CO and C-O stretching modes are at 563 and 1972 cm(-1), respectively, for the CO complex . Also, the widths (fwhm) of the Fe-CO and C-O stretching modes are smaller than those observed in the corresponding complexes of other heme proteins . The unusual spectroscopic characteristics of the d(1) cofactor are discussed in terms of both its unique electronic properties and the strongly polar distal environment around the iron-bound ligand . It is likely that the influence of a highly ruffled structure of heme d(1) on its electronic properties is the major factor causing anomalous Fe-ligand vibrational frequencies. J Theor Biol, 2001 Sep 21, 212(2), 237 - 51 The dynamics of the macromolecular composition of biomass; Hanegraaf PP et al.; The biomass composition of microorganisms depends on the growth conditions . This study explores whether a two-component model can explain how the elemental and macromolecular composition of the biomass of bacteria varies with the specific growth rate . The model describes the rates at which microorganisms assimilate substrates into reserves and utilize reserves for maintenance and growth . Crucial model assumptions are that biomass consists of reserves and structure and that each of these components has an invariant composition . The composition of biomass can vary when the ratio between reserves and structure varies . Literature data on the macromolecular composition of Escherichia coli, cultivated on various substrates, show that the protein, RNA and DNA content of biomass follow a distinctive trend when plotted as a function of the dry-weight-specific growth rate . This observation leads to the proposition that the macromolecular composition of E . coli depends directly on the growth rate, and only indirectly on the carbon- and energy-source used as substrate . We show that the variation of the macromolecular composition of E . coli over its entire range of growth rates can be described with invariant macromolecular compositions of the reserve and structural components of biomass . The model is also applied to our data on a succinate-limited continuous culture of Paracoccus denitrificans . Syst Appl Microbiol, 2001 Jul, 24(2), 303 - 10 Identity and potential functions of heterotrophic bacterial isolates from a continuous-upflow fixed-bed reactor for denitrification of drinking water with bacterial polyester as source of carbon and electron donor; Mergaert J et al.; A collection of 186 heterotrophic bacteria, isolated directly from a continuous-upflow fixed-bed reactor for the denitrification of drinking water, in which poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) granules acted as biofilm carrier, carbon source and electron donor, was studied with regard to taxonomic affiliation and degradation and denitrification characteristics . Two granule samples were taken from a fully operating reactor for enumeration and isolation of heterotrophic bacteria . One sample was drawn from the lower part of the reactor, near the oxic zone, and the other sample from the upper, anoxic part of the fixed bed . Dominant colonies were isolated and the cultures were identified using fatty acid analysis and 16S rDNA sequencing . Their ability to degrade the polymer and 3-hydroxybutyrate and to denitrify in pure culture was assessed . The results show that high numbers of heterotrophic bacteria were present in the biofilms on the polymer granules, with marked differences in taxonomic composition and potential functions between the lower and upper part of the fixed bed . The majority of the isolates were Gram negative bacteria, and most of them were able to reduce nitrate to nitrite or to denitrify, and to utilize 3-hydroxybutyrate as sole source of carbon . Only two groups, one identified as Acidovorax facilis and the other phylogenetically related to Brevundimonas intermedia, could combine denitrification and utilization of poly(3-hydroxybutyrate) (PHB), and were found only in the upper sample . The other groups occurred either in the lower or upper part, or in both samples . They were assigned to Brevundimonas, Pseudomonas, Agrobacterium, Achromobacter, or Phyllobacterium, or were phylogenetically related to Afipia or Stenotrophomonas. FEBS Lett, 2001 Aug 17, 503(2-3), 142 - 6 Zn(2+) binding to the cytoplasmic side of Paracoccus denitrificans cytochrome c oxidase selectively uncouples electron transfer and proton translocation; Kannt A et al.; Using a combination of stopped-flow spectrophotometric proton pumping measurements and time-resolved potential measurements on black lipid membranes, we have investigated the effect of Zn(2+) ions on the proton transfer properties of Paracoccus denitrificans cytochrome c oxidase . When zinc was enclosed in the interior of cytochrome c oxidase containing liposomes, the H/e stoichiometry was found to gradually decrease with increasing Zn(2+) concentration . Half-inhibition of proton pumping was observed at {Zn(2+)}(i)=75 microM corresponding to about 5-6 Zn(2+) ions per oxidase molecule . In addition, there was a significant increase in the respiratory control ratio of the proteoliposomes upon incorporation of Zn(2+) . Time-resolved potential measurements on a black lipid membrane showed that the electrogenic phases slowed down in the presence of Zn(2+) correspond to phases that have been attributed to proton uptake from the cytoplasmic side and to proton pumping . We conclude that Zn(2+) ions bind close to or within the two proton transfer pathways of the bacterial cytochrome c oxidase. Chemosphere, 2001 Aug, 44(5), 1047 - 53 Denitrification with acrylamide by pure culture of bacteria isolated from acrylonitrile-butadiene-styrene resin manufactured wastewater treatment system; Wang CC et al.; Acrylamide is widely used in industrial applications as cement binder and solidification agent . Due to its carcinogenicity and toxicity, discharge of acrylamide to the natural water and soil systems may lead to an adverse environmental impact on water quality and thus endanger public health and welfare . This study attempts to isolate and identify the denitrifying bacteria, which utilize acrylamide as the substrate from the acrylonitrile-butadiene-styrene resin manufactured wastewater treatment system . The performance of the denitrifying bacteria for treating different initial acrylamide concentrations was also investigated under aerobic and anaerobic conditions . The test results indicated that the Pseudomonas stutzeri could remove acrylamide at concentrations below 440 mg/l under aerobic conditions . The acrylic acid and ammonia intermediates were used as carbon and nitrogen sources, respectively . However, P . stutzeri did not show the capability of metabolizing acrylonitrile under aerobic conditions . Furthermore, the P . stutzeri could utilize both acrylamide and acrylic acid in the presence of nitrate (denitrification) and acrylamide could be removed completely from the wastewater. Water Sci Technol, 2001, 44(1), 67 - 76 Contribution of P-bacteria in biological nutrient removal processes to overall effects on the environment; Hao X et al.; P-bacteria can combine denitrification and P-uptake . This category of P-bacteria is abbreviated DPB . Use of DPB in BNR processes, instead of obligate aerobic PAOs, reduces oxygen consumption . Moreover, less COD is needed for the nitrogen removal . Non-required COD can be removed by presettling and used for methanation . This leads to a lower sludge production . As a result, CO2 emissions are reduced owing to less net energy consumption . Simulation for a planned WWTP with the BCFS process indicates that DPB can save 53-59% of required COD . The optimal ratios of COD/N and COD/P for simultaneous N and P removal are determined to be 3.9-4.5 and 32.2-35.2 at 12-20 degrees C . 80-95% of particulate COD can be removed from the influent, thereby CH4 production is increased by 154-271%, and the total volume of reactors can be reduced by about 50% compared to a minimised process design . Less net energy consumption over the whole WWTP contributes to a net reduction of the total CO2 emissions up to 16-21% . The energy production from CH4 is excessive enough to balance the energy consumption from aeration, dewatering and incineration . It is concluded that contribution of P-bacteria to saving COD has overall positive effects on the environment. Water Sci Technol, 2001, 44(1), 49 - 56 Modelling of full-scale wastewater treatment plants with different treatment processes using the Activated Sludge Model no . 3; Wichern M et al.; In 1999 the Activated Sludge Model no . 3 (ASM 3) by the IWA task Group on Mathematical Modeling for Design and Operation of Biological Wastewater Treatment was presented . The model is used for simulation of nitrogen removal . On the basis of a new calibration of the ASM 3 with the easy degradable COD measured by respiration simulation runs of this paper have been done . In 2000 a biological phosphorus removal module by the EAWAG was added to the calibrated version of ASM 3 and is now serving the current requirements for modelling the enhanced biological P-removal . Only little experiences with different load situations of large-scale wastewater treatment plants were made with both new models so far . This article reports the experiences with the simulation and calibration of the biological parameters using ASM 3 and the EAWAG BioP Module . Three different large-scale wastewater treatment plants in Germany with different treatment systems will be discussed (Koblenz: pre-denitrification; Hildesheim: simultaneous denitrification with EBPR; Duderstadt: intermediate denitrification with EBPR) . Informations regarding the choice of kinetic and stoichiometric parameters will be given. Water Sci Technol, 2001, 44(1), 187 - 94 Denitrification potential enhancement by addition of anaerobic fermentation products from the organic fraction of municipal solid waste; Bolzonella D et al.; The aim of this study is to demonstrate the denitrification potential enhancement determined by the addition of the anaerobic fermentation products from the organic fraction of municipal solid waste in BNR wastewater treatment plants . The denitrification potential rose from 6 to 17 mgNO3-N/l thanks to the good performances showed by the fermentation products in the denitrification step: a typical maximum denitrification rate of about 5 mgN/gVSS d was observed . This value is similar to those obtained by using pure organic substrates . The denitrification capacity increase is determined by a significant increase of S8 and Xs availability in the influent due to the anaerobic fermentation products addition which leads to an influent COD increase equal to 68% of the total load: this value is significantly higher than the one obtained by fermentation of primary and/or waste activated sludge (3-5%). Int J Syst Evol Microbiol, 2001 Jul, 51(Pt 4), 1257 - 65 Bordetella petrii sp . nov., isolated from an anaerobic bioreactor, and emended description of the genus Bordetella; von Wintzingerode F et al.; A novel Bordetella species was isolated from an anaerobic, dechlorinating bioreactor culture enriched from river sediment . The only strain, Se-1111R(T) (= DSM 12804T = CCUG 43448T), for which the name Bordetella petrii is proposed, is designated the type strain of the novel species . Strain Se-1111R(T) was isolated from the dechlorinating mixed culture due to its ability to anaerobically reduce selenate to elemental selenium . Comparative 16S rDNA sequence analysis showed a close relationship between Se-1111R(T) and members of the genus Bordetella within the beta-Proteobacteria . This close phylogenetic relatedness was also reflected in several metabolic properties of Se-1111R(T), including its incapacity to utilize carbohydrates, by the high G+C content (63.8 mol%) of its DNA and by the presence of Q-8 as the major isoprenoid quinone . DNA-DNA hybridization experiments with type strains of all species of the genus Bordetella and closely related species Achromobacter xylosoxidans subsp . denitrificans provided further evidence for the assignment of strain Se-1111R(T) as a novel species of the genus Bordetella . This genus currently consists of seven aerobic species, all of which are known to occur in close pathogenic, opportunistic or possibly commensal relationships with various host organisms . B . petrii is the first member of this genus isolated from the environment and capable of anaerobic growth . The proposal of the novel species and an emended description of the genus Bordetella is presented. Water Res, 2001 Sep, 35(13), 3272 - 5 Atrazine degradation under denitrifying conditions in continuous culture of Pseudomonas ADP; Katz I et al.; The simultaneous removal of atrazine and nitrate in continuous culture under denitrifying conditions using Pseudomonas sp . strain ADP was investigated . Under all operational conditions the nitrate removal efficiency was always higher than 90%, while atrazine degradation deteriorated with time due to contamination by foreign denitrifying bacteria, lacking the ability to degrade atrazine . Recovery of atrazine degradation ability was achieved by applying aerobic conditions with atrazine as the sole nitrogen source. Water Res, 2001 Sep, 35(13), 3127 - 36 Integrated biological and physiochemical treatment process for nitrate and fluoride removal; Mekonen A et al.; The feasibility of an integrated biological and physiochemical water treatment process for nitrate and fluoride removal has been evaluated . It consisted of two sequencing batch reactors (SBRs) in series . Performance of the process in the treatment of 24 synthetic water samples having nitrate concentrations of 40, 80, 120, 160, 200, and 250 mg/l (as N) and fluoride concentrations of 6, 10, 15, and 20 mg/l at different combinations was studied . Denitrification followed by defluoridation proved to be the best sequence of treatment . In all cases nitrate could be reduced to an acceptable level of less than 10 mg/l (as N) at 3, 5, and 7 h hydraulic retention times (HRTs) depending on its initial concentration . Fluoride concentrations up to 15 mg/l associated with nitrate concentrations up to 80 mg/l (as N) could be reduced acceptable 1.5 mg/l by alum-PAC slurry using alum doses up 850 mg/l {as Al2(SO4)3 x 16H2O} along with 100 mg/l of powdered activated carbon (PAC) . Additional alkalinity produced during denitrification was used up during defluoridation for maintenance of pH avoiding the need for lime addition . On the other hand, residual organics, turbidity, and sulfide present in the denitrified water are removed by alum and PAC at the defluoridation stage along with fluoride, eliminating the need for an additional post-treatment step . At higher nitrate concentrations (> or = 120 mg/l as N), the alkalinity produced at the denitrification stage was in the range of 715-1175 mg/l as CaCO3 . This excessive alkalinity inhibited reduction of fluoride to the level of 1.5 mg/l at the defluoridation stage, using alum doses up to 900 mg/l along with 100 mg/l of PAC . In all cases a fluoride concentration of 20 mg/l in water could not be reduced to the acceptable level of 1.5 mg/l. Biotechnol Prog, 2001 Jul-Aug, 17(4), 768 - 74 Analysis of a microbial community oxidizing inorganic sulfide and mercaptans; Duncan KE et al.; Successful treatment of refinery spent-sulfidic caustic (which results from the addition of sodium hydroxide solutions to petroleum refinery waste streams) was achieved in a bioreactor containing an enrichment culture immobilized in organic polymer beads with embedded powdered activated carbon (Bio-Sep) . The aerobic enrichment culture had previously been selected using a gas mixture of hydrogen sulfide and methyl mercaptan (MeSH) as the sole carbon and energy sources . The starting cultures for the enrichment consisted of several different Thiobacilli spp . (T . thioparus, T . denitrificans, T . thiooxidans, and T . neopolitanus), as well as activated sludge from a refinery aerobic wastewater treatment system and sludge from an industrial anaerobic digester . Microscopic examination (light and SEM) of the beads and of microbial growth on the walls of the bioreactor revealed a great diversity of microorganisms . Further characterization was undertaken starting with culturable aerobic heterotrophic microorganisms (sequencing of PCR-amplified DNA coding for 16S rRNA, Gram staining) and by PCR amplification of DNA coding for 16S rRNA extracted directly from the cell mass, followed by the separation of the PCR products by DGGE (denaturing gradient gel electrophoresis) . Eight prominent bands from the DGGE gel were sequenced and found to be closest to sequences of uncultured Cytophagales (3 bands), Gram-positive cocci (Micrococcineae), alpha proteobacteria (3 bands), and an unidentified beta proteobacterium . Culturable microbes included several genera of fungi as well as various Gram-positive and Gram-negative heterotrophic bacteria not seen in techniques using direct DNA extraction. Bioresour Technol, 2001 Sep, 79(2), 165 - 70 Nitrogen removal from wastewaters at low C/N ratios with ammonium and acetate as electron donors; Cervantes FJ et al.; A denitrifying upflow anaerobic sludge blanket (UASB) reactor was operated at different nitrate loading rates at a C/N ratio of 1.2, with acetate as an electron donor . This resulted in an increase in the accumulation of nitrite . After this, the UASB reactor was supplemented with 100 mg NH4+-Nl(-1) d(-1), while acetate was gradually limited in the medium . This prevented nitrite accumulation at a C/N ratio of 0.6 due to an enhanced nitrite reduction rate achieved in the reactor . An increasing amount of ammonium was consumed when the C/N ratio was lowered in the medium . This suggested that ammonium was used as an alternative electron donor during denitrification, which is supported by nitrogen balances . Nitrite was shown to be toxic for the nitrogen removal process at 200-400 mg NO2--N(l(-1) when the C/N ratio was decreased to 0.4 leading to formation of ammonium . The present study showed that addition of ammonium as an alternative electron donor for denitrification achieved a nitrogen removal process with negligible accumulation of undesirable intermediates. Arch Microbiol, 2001 Jul, 176(1-2), 136 - 42 The nir, nor, and nos denitrification genes are dispersed over the Bradyrhizobium japonicum chromosome; Mesa S et al.; Cleavage of genomic DNA from Bradyrhizobium japonicum strain 3I1b110 by the restriction enzymes PmeI, PacI, and SwaI has been used together with pulsed-field gel electrophoresis and Southern hybridization to locate the nirK, norCBQD, and nosRZDFYLX denitrification genes on the chromosomal map of B . japonicum strain 110spc4 . Mutant strains GRK13, GRC131, and GRZ25 were obtained by insertion of plasmid pUC4-KIXX-aphII-PSP, which carries recognition sites for the enzymes PacI, PmeI and SwaI, into the B . japonicum 3I1b110 nirK, norC and nosZ genes, respectively . Restriction of strain 3I1b110 genomic DNA with PacI, PmeI and SwaI yielded three, five and nine fragments, respectively . Pulsed-field gel electrophoresis of restricted mutant DNAs resulted in an altered fragment pattern that allowed determination of the position of the selected genes . Complementary mapping data were obtained by hybridization using digoxigenin-labeled B . japonicum 3I1b110 nirK, norBQD and nosZD as gene probes . The nirK, norCBQD and nosRZDFYLX genes were located close to the groEL(2), cycH and cycVWX genes, respectively, on the strain 110spc4 genetic map . In contrast to other denitrifiers, B . japonicum 3I1b110 denitrification genes were dispersed over the entire chromosome. Arch Microbiol, 2001 Jul, 176(1-2), 129 - 35 (S)-1-phenylethanol dehydrogenase of Azoarcus sp . strain EbN1, an enzyme of anaerobic ethylbenzene catabolism; Kniemeyer O et al.; The initial steps in the anaerobic oxidation of the aromatic hydrocarbon ethylbenzene by denitrifying bacteria are two sequential dehydrogenation reactions of ethylbenzene to (S)-1-phenylethanol and further to acetophenone . The enzyme catalysing the second oxidation step, (S)-1-phenylethanol dehydrogenase, was analysed in the denitrifying bacterium Azoarcus sp . strain EbN1 . An NAD+-dependent 1-phenylethanol dehydrogenase for each of the enantiomers of 1-phenylethanol was identified in this bacterium; the two enzymes were induced under different growth conditions . (S)-1-phenylethanol dehydrogenase from ethylbenzene-grown cells was purified and biochemically characterised . The enzyme is a typical secondary alcohol dehydrogenase and consists of two subunits of 25.5 kDa . The enantioselective enzyme catalyses the oxidation of (S)-1-phenylethanol or the reduction of acetophenone and is inhibited by high concentrations of (R)-1-phenylethanol . The enzyme exhibits low apparent K(m) values for (S)-1-phenylethanol and acetophenone and is rather substrate-specific, using only a few chemically similar secondary alcohols, such as 1-phenylpropanol and isopropanol. Plant Cell Physiol, 2001 Jul, 42(7), 703 - 9 Mutational analysis of regulatory cis-acting elements for the transcriptional activation of the dmsCBA operon in Rhodobacter sphaeroides f . sp . denitrificans; Yamamoto I et al.; Four direct repeats of a 10-nt sequence, called dms boxes, are located upstream of the dmsCBA operon encoding dimethyl sulfoxide (DMSO) reductase in Rhodobacter sphaeroides f . sp . denitrificans IL106 . Two dms boxes 1 and 2 have been shown to be binding sites of DmsR protein, a response regulator of a two-component system involved in the anaerobic induction by DMSO of DMSO reductase synthesis . In this study, functions of four dms boxes in the transcriptional regulation of the dmsCBA operon were investigated . The transcription start site of the dmsCBA genes was identified at the distance of 23 nt downstream of the closest dms box 4 . Expression of the dmsC-lacZ gene fusion which included the dmsCBA promoter region containing the dms boxes was examined and its anaerobic induction by DMSO and DmsR-dependency were demonstrated in the phototroph . The examination with nucleotide substitutions in the four respective dms boxes showed that the set of four dms boxes is required for the dmsCBA operon activation . Moreover, the importance of the nucleotide sequence of TTCAC in dms box 4 and of A at the center in dms box 1 was significantly shown . These facts suggest that the pentad nucleotides TTCAC and TTAAC in the dms boxes serve as cis-acting elements in the transcriptional activation of the dmsCBA operon. J Environ Qual, 2001 Jul-Aug, 30(4), 1411 - 20 Denitrification at a long-term forested land treatment system in the Piedmont of Georgia; Meding SM et al.; Spray irrigation of forested land can provide an effective system for nutrient removal and treatment of municipal wastewater . Evolution of N2 + N2O from denitrifying activity is an important renovation pathway for N applied to forested land treatment systems . Federal and state guidance documents for design of forested land treatment systems indicate the expected range for denitrification to be up to 25% of applied N, and most forest land treatment systems are designed using values from 15 to 20% of applied N . However, few measurements of denitrification following long-term wastewater applications at forested land treatment sites exist . In this study, soil N2 + N2O-N evolution was directly measured at four different landscape positions (hilltop, midslope, toe-slope, and riparian zone) in a forested land treatment facility in the Georgia Piedmont that has been operating for more than 13 yr . Denitrification rates within effluent-irrigated areas were significantly greater than rates in adjacent nonirrigated buffer zones . Rates of N2 + N2O-N evolved from soil in irrigated forests ranged from 5 to 10 kg ha(-1) yr(-1) N on the three upland landscape positions and averaged 38 kg ha(-1) yr(-1) N within the riparian zone . The relationship between measured riparian zone denitrification rates and soil physical and chemical properties was poor . The best relationship was with soil temperature, with an r2 of 0.18 . Overall, on a landscape position weighted basis, only 2.4% of the wastewater-applied N was lost through denitrification. J Environ Qual, 2001 Jul-Aug, 30(4), 1163 - 75 Tracing nitrate transport and environmental impact from intensive swine farming using delta nitrogen-15; Karr JD et al.; Natural-abundance delta15N showed that nitrate generated from commercial land application of swine (Sus scrofa domesticus) waste within a North Carolina Coastal Plain catchment was being discharged to surface waters by ground water passing beneath the sprayfields and adjacent riparian buffers . This was significant because intensive swine farms in North Carolina are considered non-discharge operations, and riparian buffers with minimum widths of 7.6 m (25 ft) are the primary regulatory control on ground water export of nitrate from these operations . This study shows that such buffers are not always adequate to prevent discharge of concentrated nitrate in ground water from commercial swine farms in the Mid-Atlantic Coastal Plain, and that additional measures are required to ensure non-discharge conditions . The median delta15N-total N of liquids in site swine waste lagoons was +15.4 +/- 0.2% vs . atmospheric nitrogen . The median delta15N-NO3 values of shallow ground water beneath and adjacent to site sprayfields, a stream draining sprayfields, and waters up to 1.5 km downstream were + 15.3 +/- 0.2 to + 15.4 +/- 0.2% . Seasonal and spatial isotopic variations in lagoons and well waters were greatly homogenized during ground water transport and discharge to streams . Neither denitrification nor losses of ammonia during spraying significantly altered the bulk ground water delta15N signal being delivered to streams . The lagoons were sources of chloride and potassium enrichment, and shallow ground water showed strong correlation between nitrate N, potassium, and chloride . The 15N-enriched nitrate in ground water beneath swine waste sprayfields can thus be successfully traced during transport and discharge into nearby surface waters. Appl Environ Microbiol, 2001 Aug, 67(8), 3481 - 7 Presence of nitrate-accumulating sulfur bacteria and their influence on nitrogen cycling in a shallow coastal marine sediment; Sayama M; Nitrate flux between sediment and water, nitrate concentration profile at the sediment-water interface, and in situ sediment denitrification activity were measured seasonally at the innermost part of Tokyo Bay, Japan . For the determination of sediment nitrate concentration, undisturbed sediment cores were sectioned into 5-mm depth intervals and each segment was stored frozen at -30 degrees C . The nitrate concentration was determined for the supernatants after centrifuging the frozen and thawed sediments . Nitrate in the uppermost sediment showed a remarkable seasonal change, and its seasonal maximum of up to 400 microM was found in October . The directions of the diffusive nitrate fluxes predicted from the interfacial concentration gradients were out of the sediment throughout the year . In contrast, the directions of the total nitrate fluxes measured by the whole-core incubation were into the sediment at all seasons . This contradiction between directions indicates that a large part of the nitrate pool extracted from the frozen surface sediments is not a pore water constituent, and preliminary examinations demonstrated that the nitrate was contained in the intracellular vacuoles of filamentous sulfur bacteria dwelling on or in the surface sediment . Based on the comparison between in situ sediment denitrification activity and total nitrate flux, it is suggested that intracellular nitrate cannot be directly utilized by sediment denitrification, and the probable fate of the intracellular nitrate is hypothesized to be dissimilatory reduction to ammonium . The presence of nitrate-accumulating sulfur bacteria therefore may lower nature's self-purification capacity (denitrification) and exacerbate eutrophication in shallow coastal marine environments. Water Res, 2001 Aug, 35(12), 2995 - 3003 Feasibility of a multi-component additive for efficient control of activated sludge filamentous bulking; Seka AM et al.; Instantaneous improvement of the settling of bulking filamentous activated sludge can be achieved by the addition of a polymer or a large amount (up to 100% of the MLSS concentration) of talc powder to the sludge . Long-term improvement relies on repeated additions, as these additives have no adverse effects on the causative filaments . A multi-component additive was compared to the traditional additives in lab-scale activated sludge units using three highly filamentous sludges from different industrial treatment plants . The study demonstrated that the multi-component additive was superior to the traditional remedies . It was shown that, in the case of severe filamentous bulking, a single addition of the new additive immediately improved sludge settling and exerted a destructive effect on the causative filamentous bacteria . Thus, the latter additive also ensured a long-term sludge sedimentation improvement . The traditional additives exhibited an immediate and short-term effect . The novel additive also retarded sludge rising due to denitrification and it improved sludge dewaterability . The study revealed Nostocoido limicola II, with slightly hydrophobic cell wall, to be somewhat resistant to the quaternary ammonium salt present as biocide in the additive. Water Res, 2001 Aug, 35(12), 2851 - 60 Model-based evaluation of two BNR processes--UCT and A2N; Hao X et al.; The activity of denitrifying P-accumulating bacteria (DPB) has been verified to exist in most WWTPs with biological nutrient removal (BNR) . The modified UCT process has a high content of DPB . A new BNR process with a two-sludge system named A2N was especially developed to exploit denitrifying dephosphatation . With the identical inflow and effluent standards, an existing full-scale UCT-type WWTP and a designed A2N process were evaluated by simulation . The used model is based on the Delft metabolical model for bio-P removal and ASM2d model for COD and N removal . Both processes accommodate denitrifying dephosphatation, but the A2N process has a more stable performance in N removal . Although excess sludge is increased by 6%, the A2N process leads to savings of 35, 85 and 30% in aeration energy, mixed liquor internal recirculation and land occupation respectively, as compared to the UCT process . Low temperature has a negative effect on growth of poly-P bacteria, which becomes to especially appear in the A2N process. FEMS Microbiol Lett, 2001 Jul 24, 201(2), 213 - 9 A physical map of the megaplasmid pHG1, one of three genomic replicons in Ralstonia eutropha H16; Schwartz E et al.; We have used pulsed field gel electrophoresis and megabase DNA techniques to investigate the basic genomic organization of Ralstonia eutropha H16, and to construct a physical map of its indigenous megaplasmid pHG1 . This Gram-negative, soil-dwelling bacterium is a facultative chemolithoautotroph and a denitrifier . In the absence of organic substrates it can grow on H2 as its sole energy source and CO2 as its sole source of carbon . Under anaerobic conditions it can utilize nitrate as a terminal electron acceptor, whereby dinitrogen is released . Essential genetic determinants of the enzyme systems responsible for these metabolic processes are linked to the 0.44-Mb conjugative megaplasmid pHG1 . Aside from pHG1, the genome of R . eutropha H16 is comprised of two circular chromosomes measuring 4.1 and 2.9 Mb, adding up to a total genome size of 7.1 Mb . An estimated five copies of rDNA are distributed on the two chromosomes . A macrorestriction map of pHG1 was derived for the endonucleases DraI and XbaI . Hybridization studies showed that genes for anaerobic metabolism are located on all three genomic replicons. Rapid Commun Mass Spectrom, 2001, 15(15), 1274 - 8 Development of delta(15)N stratification of NO(3)(-) in soil profiles; Handley LL et al.; New evidence, obtained using a robust method for measuring the delta(15)N of NO(3)(-)-N in soil, is consistent with denitrification being the major determinant in the vertical distribution of NO(3)(-)-delta(15)N in soil profiles . These data also suggest that varying moisture regimes result in different effects of soil NO(3)(-)-N leaching on residual whole soil delta(15)N . J Bioenerg Biomembr, 2001 Apr, 33(2), 99 - 105 The carboxin-binding site on Paracoccus denitrificans succinate:quinone reductase identified by mutations; Matsson M et al.; Succinate:quinone reductase catalyzes electron transfer from succinate to quinone in aerobic respiration . Carboxin is a specific inhibitor of this enzyme from several different organisms . We have isolated mutant strains of the bacterium Paracoccus denitrificans that are resistant to carboxin due to mutations in the succinate:quinone reductase . The mutations identify two amino acid residues, His228 in SdhB and Asp89 in SdhD, that most likely constitute part of a carboxin-binding site . This site is in the same region of the enzyme as the proposed active site for ubiquinone reduction . From the combined mutant data and structural information derived from Escherichia coli and Wolinella succinogenes quinol:fumarate reductase, we suggest that carboxin acts by blocking binding of ubiquinone to the active site . The block would be either by direct exclusion of ubiquinone from the active site or by occlusion of a pore that leads to the active site. Water Res, 2001 Aug, 35(11), 2794 - 8 Effect of nitrate exposure history on the oxygen/nitrate diauxic growth of Pseudomonas denitrificans; Gouw M et al.; Pseudomonas denitrificans pre-cultured (revived from agar plates) under varying conditions (nitrate absent or present; dissolved oxygen absent or present) was subsequently grown aerobically with or without nitrate present, and finally exposed to anoxic conditions (i.e., aeration stopped and dissolved oxygen stripped from solution) . The occurrence and length of diauxic lags following transition from aerobic to anoxic conditions were affected strongly by the nitrate and oxygen exposure history of the biomass. Water Res, 2001 Aug, 35(11), 2749 - 57 Study of dissolved silicon and nitrate dynamics in a freshwater stream; House WA et al.; Dissolved reactive silicon and nitrate were measured at weekly intervals over a 3 year period (1991-94) on a 1.2km reach of a gauged Dorset Mill Stream . In addition, dissolved nitrite was measured over a 8 month period from the spring to late autumn in 1992 . Two intensive studies with sampling at 2h intervals were also completed in low and high riverflow conditions . The results were analysed using a mass-balance approach with the loss and gains in nutrients dissolved in the water expressed in terms of areal rates . Losses of both nutrients occurred during periods of low streamflow in spring and summer . Losses of silicon are attributed to growth of epilithic diatoms whereas nitrate losses are consistent with a number of processes including the growth of aquatic plants, the development of epilithic biofilms and nitrogen transformations, such as denitrification, in bed-sediments . Stream water gained dissolved nitrite during its passage through the section . Silicon losses from the stream amounted to between 52 and 63 mmol m(-2) d(-1) (expressed per area of bed-sediment) for the spring periods in 1992-94 . Nitrate losses were more variable with overall rates between 24 and 89 mmol m(-2) d(-1) for the summer periods in 1991-93. Mikrobiologiia, 2001 May-Jun, 70(3), 329 - 36 {Microbial decomposition of 3,4-dichloroaniline, adsorbed by activated charcoal}; Bakhaeva LP et al.; The accessibility of 3,4-dichloroaniline (DCA) sorbed by activated carbon to degradative microorganisms was studied . A Paracoccus denitrificans strain capable of growing on medium with DCA as the only source of energy, carbon, and nitrogen was used in the experiment . The high sorption capacity of all the carbons studied (powdered RS and SKT-6A and granular AG-3) in relation to DCA (350 to 360, 480 to 520, and 540 to 580 mg/g, respectively) was demonstrated . The sorption capacity correlated positively with the specific surface area and the total volume of the sorbent micropores . The bulk of the DCA was reversibly sorbed and amenable to microbial decomposition; however, the decomposition rates significantly differed . When RS, SKT, and Agrosorb preliminarily saturated with DCA were incubated in a culture of P . denitrificans, the bulk of the reversibly sorbed DCA was decomposed (in the absence of the other carbon sources) in 2, 5, and 10 weeks, respectively, after which the process slowed down . At the end of the experiment (29 weeks), 81 to 87% of the DCA underwent full mineralization, which was accompanied by the release of chlorine ions; a small fraction of the xenobiotic (0.8 to 1.9%) remained a reversibly sorbed fraction (extractable with acetone), and 12 to 17% of the initial DCA seemed to have been chemically transformed and bound by carbon . The studied carbons may be used in biological decontamination of chloroaniline-polluted soils to decrease the toxic effect of chloroanilines on microorganisms. J Med Chem, 2001 Jul 19, 44(15), 2469 - 78 Significance of asymmetric sites in choosing siderophores as deferration agents; Bergeron RJ et al.; The syntheses of the microbial iron chelators L-fluviabactin, its unnatural enantiomer, D-fluviabactin, L-homofluviabactin, and L-agrobactin, are described . The key steps involve the selective bis-acylation of the terminal nitrogens of norspermidine, spermidine, or homospermidine with 2,3-bis(benzyloxy)benzoic acid in the presence of 1,1-carbonyldiimidazole, followed by coupling of the N-hydroxysuccinimide ester of CBZ-protected L- or D-threonine with the central nitrogen . The effectiveness of each of these ligands in supporting the growth of Paracoccus denitrificans in a low-iron environment and the ability of these compounds to promote iron uptake are evaluated . The stereochemical configuration of the oxazoline ring is shown to be the major structural factor controlling both microbial growth stimulation and iron uptake . L-Fluviabactin, L-homofluviabactin, and L-agrobactin all promoted growth and iron uptake; D-fluviabactin was only marginally active . As with the microorganism's native siderophore, L-parabactin, all three ligands in the L-configuration investigated exhibited biphasic, i.e., both high-affinity and low-affinity, kinetics . The high-affinity system (iron concentration < 1 microM) yielded K(m) values between 0.11 and 0.23 microM and V(max) values from 157 to 129 pg-atoms Fe min(-1) (mg of protein)(-1), whereas the low-affinity scheme (iron concentration > 1 microM) gave K(m) values from 0.53 to 3.5 microM and V(max) values between 96 and 413 pg-atoms Fe min(-1) (mg of protein)(-1) . Both L- and D-fluviabactin are very effective at clearing iron from the bile duct-cannulated rodent; when given subcutaneously at a dose of 150 micromol/kg, both ligands had iron clearing efficiencies of >13%, which is much greater than that of desferrioxamine in this model . Thus, by altering the stereochemistry of certain microbial siderophores, it is possible to generate deferration agents that are still effective at clearing iron from animals, yet do not promote microbial growth. Chemosphere, 2001 Jul, 44(2), 231 - 6 Degradation of herbicides in two sandy aquifers under different redox conditions; Larsen L et al.; We examined the potential for complete degradation (mineralisation) of the four {ring-U-14C}herbicides mecoprop, isoproturon, atrazine, and metsulphuron-methyl in two sandy aquifers representing aerobic, denitrifying, sulphate-reducing, and methanogenic conditions . Slurries with sediment and groundwater were set-up aerobically or anaerobically in the presence of the electron-acceptor prevailing at the sampling site, amended with 25 microg l(-1) herbicide, and incubated at 10 degrees C . Considerable mineralisation was only observed in sediment from the plough layer incubated aerobically . Here, 30% of 14C-mecoprop was recovered as 14CO2 after 15 days and 15% of isoproturon was recovered as 14CO2 after 267 days . Only 7% of mecoprop was recovered as 14CO2 after 313 days in sediment from the aquifer below sampled at 1.95-3.00 mbs (m below the surface) . In denitrifying and methanogenic slurries, 3% of 14C added as mecoprop was recovered as 14CO2 . Isoproturon was not mineralised except in the aerobic plough layer, and atrazine and metsulphuron-methyl were not mineralised under any of the conditions applied. Water Sci Technol, 2001, 43(11), 99 - 108 Influence of pre-treatments and recycled flows on the COD fractions of a wastewater: a case study; Anderson E et al.; Physical-chemical and biological fractionation methods were used at different parts of the Evry WWTP in order to assess the influence of effluents recycled from the primary settling tanks and sludge treatment processes on the characteristics of the wastewater . The RBCOD doubled between the WWTP influent and the biological reactor inlet, thus doubling the denitrifying potential of the plant . The primary sludge gravity thickener effluent, subject to a great variability, appeared to contribute up to 38% of the RBCOD contained in the biological reactor influent, whereas only 30% seemed to come from the WWTP inlet . No notable increase of RBCOD was observed at the primary settling tanks' outlet for the longer residence time . The significant variations of RBCOD across the plant were not caused by fluctuations of the physical-chemical COD fractions. Water Sci Technol, 2001, 43(11), 337 - 44 Moving-bed biological treatment (MBBT) of municipal wastewater: denitrification; Maurer M et al.; Denitrification in a full-scale installation and a pilot plant for moving-bed biological treatment (MBBT) was subject to detailed investigation . Two different types of carriers were used in conventional activated sludge reactors: foam cubes and plastic tubes (Kaldnes) . Both investigated carriers showed the same behavior with regard to denitrification capacity, temperature dependency and maximum COD and nitrate turnover . In contrast to the plastic tubes (Kaldnes), the sponge cubes stored remarkable amounts of substrate . The maximum denitrification rate with acetate as a substrate was 420 gNm-3d-1 at 10 degrees C and 730 gNm-3d-1 at 20 degrees C . An average denitrification rate of 240 gNm-3d-1 (10 degrees C) was achieved with wastewater . A maximum of 37% of the COD in the influent was denitrified with a volumetric loading rate in the anoxic zone of 2.2 kgCODm-3d-1. Water Sci Technol, 2001, 43(11), 25 - 33 Nitrate and pesticide removal by a combined bioelectrochemical/adsorption process; Feleke Z et al.; Biological denitrification and trace pesticide removal in a combined biofilm-electrode reactor/adsorption process has been investigated . In long-term (more than 260 days) continuous experiments, influent and effluent concentrations of nitrate, nitrite, isoprothiolane and gas composition were measured at different electric current and pesticide loading conditions . Experimental results showed that complete and stable denitrification was achieved in BER without accumulation of nitrite and nitrous oxide . Isoprothiolane (IPT) was removed by adsorption onto either granular activated carbon or silicone resin . Removal efficiency of IPT exceeding 97% was achieved and effluent concentration was below the guideline value (40 micrograms/l) . Theoretically predicted effluent concentrations were in good agreement with the observed results . From these results, it is concluded that the combined process is applicable to treat nitrate and pesticide contaminated drinking water . Moreover, from comparison with former studies, different possible options to further enhance the decomposition of pesticide were suggested. Water Sci Technol, 2001, 43(11), 161 - 71 A modification to the Activated Sludge Model No . 2 based on the competition between phosphorus-accumulating organisms and glycogen-accumulating organisms; Manga J et al.; A modification to the ASM2 is proposed which permits representation of the competition between phosphorus accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs) in a nutrient removal activated sludge system . Some important aspects, which are not considered in ASM2, are discussed . The proposed modification includes denitrification by PAOs, PAO glycogen storage capability and GAO metabolism model . It is shown that the proposed modification is capable of describing pilot plant data using a single set of stoichiometric and kinetic parameters over three different sludge ages (16, 14 and 12 days) . The modified ASM2 may be applicable to a wide range of situations where PAOs and GAOs can compete . This modification may well provide a better understanding about GAO behaviour. Water Sci Technol, 2001, 43(11), 109 - 17 Impact of cold and dilute sewage on pre-fermentation--a case study; Bixio D et al.; The municipal sewage treatment plant (STP) of the city of Ghent (Belgium) has to be retrofitted to a 43%-increase in the nitrogen treatment capacity and to phosphorus removal . Cold weather, dilute sewage and a critical COD over N ratio make the retrofit a challenge for full biological nutrient removal . The potential for fermentation of primary sludge to alter those critical feed sewage characteristics was experimentally evaluated . The idea was that the pinpoint introduction of fermentate could optimise the available reactors by achieving high-rate denitrification and enhanced biological phosphorus removal . The fermentation process was evaluated with a bench scale apparatus . At 20 degrees C (heated process), the hydrolysis yield--expressed in terms of soluble COD--varied from 11% to 24% of the total sludge COD . The fermentation yield expressed in VFA COD varied from 8% to 13% of the total sludge COD . The efficiency of heated fermentation of primary sludge was lower during cold and wet weather, due to the different sewage characteristics, as a result of extended dilution periods and low temperature . The raw sewage, the primary effluent and the fermentate were fractionated according to the requirements for the IAWQ Activated Sludge Model No . 2d . The results clearly show that fermentation in the sewer played an important role and temperature was the driving parameter for the characteristics of the dissolved COD . Instead, the weather flow conditions were the driving parameter for the characteristics of the suspended COD . The results of the detailed fractionation were used as background for process evaluation . The final scenario choice for the retrofit depends on a cost-efficiency calculation. J Bacteriol, 2001 Aug, 183(15), 4536 - 42 Isolation and characterization of anaerobic ethylbenzene dehydrogenase, a novel Mo-Fe-S enzyme; Johnson HA et al.; The first step in anaerobic ethylbenzene mineralization in denitrifying Azoarcus sp . strain EB1 is the oxidation of ethylbenzene to (S)-(-)-1-phenylethanol . Ethylbenzene dehydrogenase, which catalyzes this reaction, is a unique enzyme in that it mediates the stereoselective hydroxylation of an aromatic hydrocarbon in the absence of molecular oxygen . We purified ethylbenzene dehydrogenase to apparent homogeneity and showed that the enzyme is a heterotrimer (alphabetagamma) with subunit masses of 100 kDa (alpha), 35 kDa (beta), and 25 kDa (gamma) . Purified ethylbenzene dehydrogenase contains approximately 0.5 mol of molybdenum, 16 mol of iron, and 15 mol of acid-labile sulfur per mol of holoenzyme, as well as a molydopterin cofactor . In addition to ethylbenzene, purified ethylbenzene dehydrogenase was found to oxidize 4-fluoro-ethylbenzene and the nonaromatic hydrocarbons 3-methyl-2-pentene and ethylidenecyclohexane . Sequencing of the encoding genes revealed that ebdA encodes the alpha subunit, a 974-amino-acid polypeptide containing a molybdopterin-binding domain . The ebdB gene encodes the beta subunit, a 352-amino-acid polypeptide with several 4Fe-4S binding domains . The ebdC gene encodes the gamma subunit, a 214-amino-acid polypeptide that is a potential membrane anchor subunit . Sequence analysis and biochemical data suggest that ethylbenzene dehydrogenase is a novel member of the dimethyl sulfoxide reductase family of molybdopterin-containing enzymes. Biodegradation, 2000, 11(2-3), 85 - 105 Metabolism of alkylbenzenes, alkanes, and other hydrocarbons in anaerobic bacteria; Spormann AM et al.; Aromatic and aliphatic hydrocarbons are the main constituents of petroleum and its refined products . Whereas degradation of hydrocarbons by oxygen-respiring microorganisms has been known for about a century, utilization of hydrocarbons under anoxic conditions has been investigated only during the past decade . Diverse strains of anaerobic bacteria have been isolated that degrade toluene anaerobically, using nitrate, iron(III), or sulfate as electron acceptors . Also, other alkylbenzenes such as m-xylene or ethylbenzene are utilized by a number of strains . The capacity for anaerobic utilization of alkylbenzenes has been observed in members of the alpha-, beta-, gamma- and delta-subclasses of the Proteobacteria . Furthermore, denitrifying bacteria and sulfate-reducing bacteria with the capacity for anaerobic alkane degradation have been isolated, which are members of the beta- and delta-subclass, respectively . The mechanism of the activation of hydrocarbons as apolar molecules in the absence of oxygen is of particular interest . The biochemistry of anaerobic toluene degradation has been studied in detail . Toluene is activated by addition to fumarate to yield benzylsuccinate, which is then further metabolized via benzoyl-CoA . The toluene-activating enzyme presents a novel type of glycine radical protein . Another principle of anaerobic alkylbenzene activation has been observed in the anaerobic degradation of ethylbenzene . Ethylbenzene in denitrifying bacteria is dehydrogenated to 1-phenylethanol and further to acetophenone; the latter is also metabolized to benzoyl-CoA . Naphthalene is presumably activated under anoxic conditions by a carboxylation reaction . Investigations into the pathway of anaerobic alkane degradation are only at the beginning . The saturated hydrocarbons are most likely activated by addition of a carbon compound rather than by desaturation and hydration, as speculated about in some early studies . An anaerobic oxidation of methane with sulfate as electron acceptor has been documented in aquatic sediments . The process is assumed to involve a reversal of methanogenesis catalyzed by Archaea, and scavenge of an electron-carrying metabolite by sulfate-reducing bacteria . Among unsaturated non-aromatic hydrocarbons, anaerobic bacterial degradation has been demonstrated and investigated with n-alkenes, alkenoic terpenes and the alkyne, acetylene. Biodegradation, 2000, 11(2-3), 159 - 70 Enhanced anaerobic bioremediation of groundwater contaminated by fuel hydrocarbons at Seal Beach, California; Cunningham JA et al.; Enhanced anaerobic biodegradation of groundwater contaminated by fuel hydrocarbons has been evaluated at a field experiment conducted at the Naval Weapons Station, Seal Beach, California . This experiment included the establishment of three different remediation zones in situ: one zone was augmented with sulfate, one was augmented with sulfate and nitrate, and the third was unaugmented . This enables a comparison of hydrocarbon biodegradation under sulfate-reducing, sequential denitrifying/sulfate-reducing, and methanogenic conditions, respectively . In general, the results from the field experiment are: (1) Certain fuel hydrocarbons were removed preferentially over others, but the order of preference is dependent upon the geochemical conditions; and (2) In the zones that were augmented with sulfate and/or nitrate, the added electron acceptors were consumed quickly, indicating that enhancement via electron acceptor injection accelerates the biodegradation process . More specifically, in the sulfate-reducing zone, sulfate was utilized with an apparent first-order rate coefficient of approximately 0.1 day(-1) . In the combined denitrifying/sulfate-reducing zone, nitrate was utilized preferentially over sulfate, with an apparent first-order rate coefficient of 0.1-0.6 day(-1) . However, the data suggest that slow sulfate utilization does occur in the presence of nitrate, i.e., the two processes are not strictly sequential . With regard to the aromatic BTEX hydrocarbons, toluene was preferentially removed under intrinsic conditions; biodegradation of benzene was slow if it occurred at all; augmentation with sulfate preferentially stimulated biodegradation of o-xylene; and ethylbenzene appeared recalcitrant under sulfate-reducing conditions but readily degradable under denitrifying conditions. Biosci Biotechnol Biochem, 2001 May, 65(5), 1206 - 10 A novel C1-using denitrifier alcaligenes sp . STC1 and its genes for copper-containing nitrite reductase and azurin; Ozeki S et al.; A novel denitrifier Alcaligenes sp . STC1 was identified . The strain efficiently denitrifies under an atmosphere of 10% oxygen (O2) where Paracoccus denitrificans, one of the most studied aerobic denitrifiers, had less denitrifying activity, indicating that the strain has an O2-torelant denitrifying system . It denitrified by using C1-carbon sources such as formate and methanol as well as glucose, glycerol, and succinate . The genes for the copper-containing nitrite reductase and azurin of this C1-using denitrifier were cloned . Their predicted products of them were similar to those of their counterparts and the maximum similarities were 90% and 92%, respectively. J Biol Chem, 2001 Sep 7, 276(36), 34142 - 7 Epub 2001 Jun 27. Protein dynamics enhance electronic coupling in electron transfer complexes; Chohan KK et al.; Electron-transferring flavoproteins (ETFs) from human and Paracoccus denitrificans have been analyzed by small angle x-ray scattering, showing that neither molecule exists in a rigid conformation in solution . Both ETFs sample a range of conformations corresponding to a large rotation of domain II with respect to domains I and III . A model of the human ETF.medium chain acyl-CoA dehydrogenase complex, consistent with x-ray scattering data, indicates that optimal electron transfer requires domain II of ETF to rotate by approximately 30 to 50 degrees toward domain I relative to its position in the x-ray structure . Domain motion establishes a new "robust engineering principle" for electron transfer complexes, tolerating multiple configurations of the complex while retaining efficient electron transfer. Biochemistry, 2001 Jul 3, 40(26), 7806 - 11 Dynamics of nitric oxide in the active site of reduced cytochrome c oxidase aa3; Vos MH et al.; Nitric oxide (NO) is involved in the regulation of respiration by acting as a competitive ligand for molecular oxygen at the binuclear active site of cytochrome c oxidase . The dynamics of NO in and near this site are not well understood . We performed flash photolysis studies of NO from heme a3 in cytochrome c oxidase from Paracoccus denitrificans, using femtosecond transient absorption spectroscopy . The formation of the product state--the unliganded heme a3 ground state--occurs in a similar stepwise manner (period approximately 700 fs) as previously observed for carbon monoxide photolysis from this enzyme and interpreted in terms of ballistic ligand motions in the active site on the subpicosecond time scale {Liebl, U., Lipowski, G., Negrerie, M., Lambry, J.-C., Martin, J.-L., and Vos, M . H . (1999) Nature 401, 181-184} . A fraction (approximately 35% at very low NO concentrations) of the dissociated NO recombines with heme a3 in 200-300 ps . The presence of this recombination phase indicates that a transient bond to the second ligand-binding site, a copper atom (CuB), has a short lifetime or may not be formed . Increasing the NO concentration increases the recombination yield on the hundreds of picoseconds time scale . This effect, unprecedented for heme proteins, implies that, apart from the one NO molecule bound to heme a3, a second NO molecule can be accommodated in the active site, even at relatively low (submicromolar) concentrations . Models for NO accommodation in the active site, based on molecular dynamics energy minimizations are presented . Pathways for NO motion and their relevance for the regulation of respiration are discussed. Environ Technol, 2001 May, 22(5), 553 - 9 Nitrogen removal from sewage by continuous flow SBR system with intermittent aeration; Chen AC et al.; A continuous-flow sequential batch reactor system with intermittent aeration known as the intermittent cycle extended aeration system was used to investigate the efficiencies of nitrogen removal for sewage . In the conventional intermittent cycle extend aeration system with continuous aeration process to treat the synthetic sewage wastewater under 3 cycles per day (cycle time = 8 hours), high COD removal efficiencies were achieved, but high levels of nitrate (22.3-23.6 mg l-1) and decreased pH values were also found in the effluent . In the intermittent aeration, the dissolved oxygen in the reactor was reduced from the range of 3.5-5.0 mg l-1 to 0.5-1.2 mg l-1, the nitrogen in the influent was partially oxidized to nitrite-nitrogen, although nitrate-nitrogen was also present in the effluent . In detailed cyclic study of the reactor with relatively low D.O . (0.3-1.0 mg l-1) and high MLSS (3200 mg l-1 at half full water level), the experimental results of intermittent aeration processes showed higher oxidation rates of ammonia to nitrite (1.44-3.55 mg N g-1 MLSS hr-1) than the conversion rates of nitrite to nitrate (0.00-1.22 mg N g-1 MLSS hr-1) . It was thus, concluded that denitrification rates were not affected by the limited supplement of soluble organic carbon in the system . In addition, NOx(-)-N could be removed with a total nitrogen removal rate up to 94.9% when the MLSS was controlled in concentrations of 3200 mg l-1 at half full water level with low D.O . The process of intermittent aeration of ICEAS could provide an economic and effective means for nitrogen removal in treating sewage. J Ind Microbiol Biotechnol, 1999 Oct, 23(4-5), 341 - 346 Degradation of azo dyes containing aminonaphthol by Sphingomonas sp strain 1CX; Coughlin MF et al.; Sphingomonas sp strain 1CX was isolated from a wastewater treatment plant and is capable of aerobically degrading a suite of azo dyes, using them as a sole source of carbon and nitrogen . All azo dyes known to be decolorized by strain 1CX (Orange II, Acid Orange 8, Acid Orange 10, Acid Red 4, and Acid Red 88) have in their structure either 1-amino-2-naphthol or 2-amino-1-naphthol . In addition, an analysis of the structures of the dyes degraded suggests that there are certain positions and types of substituents on the azo dye which determine if degradation will occur . Growth and dye decolorization occurs only aerobically and does not occur under fermentative or denitrification conditions . The mechanism by which 1CX decolorizes azo dyes appears to be through reductive cleavage of the azo bond . In the case of Orange II, the initial degradation products were sulfanilic acid and 1-amino-2-naphthol . Sulfanilic acid, however, was not used by 1CX as a growth substrate . The addition of glucose or inorganic nitrogen inhibited growth and decoloration of azo dyes by 1CX . Attempts to grow the organism on chemically defined media containing several different amino acids and sugars as sources of nitrogen and carbon were not successful . Phylogenetic analysis of Sphingomonas sp strain 1CX shows it to be related to, but distinct from, other azo dye-decolorizing Sphingomonas spp strains isolated previously from the same wastewater treatment facility. J Bacteriol, 2001 Jul, 183(14), 4288 - 95 Succinyl-CoA:(R)-benzylsuccinate CoA-transferase: an enzyme of the anaerobic toluene catabolic pathway in denitrifying bacteria; Leutwein C et al.; Anaerobic microbial toluene catabolism is initiated by addition of fumarate to the methyl group of toluene, yielding (R)-benzylsuccinate as first intermediate, which is further metabolized via beta-oxidation to benzoyl-coenzyme A (CoA) and succinyl-CoA . A specific succinyl-CoA:(R)-benzylsuccinate CoA-transferase activating (R)-benzylsuccinate to the CoA-thioester was purified and characterized from Thauera aromatica . The enzyme is fully reversible and forms exclusively the 2-(R)-benzylsuccinyl-CoA isomer . Only some close chemical analogs of the substrates are accepted by the enzyme: succinate was partially replaced by maleate or methylsuccinate, and (R)-benzylsuccinate was replaced by methylsuccinate, benzylmalonate, or phenylsuccinate . In contrast to all other known CoA-transferases, the enzyme consists of two subunits of similar amino acid sequences and similar sizes (44 and 45 kDa) in an alpha(2)beta(2) conformation . Identity of the subunits with the products of the previously identified toluene-induced bbsEF genes was confirmed by determination of the exact masses via electrospray-mass spectrometry . The deduced amino acid sequences resemble those of only two other characterized CoA-transferases, oxalyl-CoA:formate CoA-transferase and (E)-cinnamoyl-CoA:(R)-phenyllactate CoA-transferase, which represent a new family of CoA-transferases . As suggested by kinetic analysis, the reaction mechanism of enzymes of this family apparently involves formation of a ternary complex between the enzyme and the two substrates. J Bacteriol, 2001 Jul, 183(14), 4149 - 56 Purification, characterization, and genetic analysis of Cu-containing dissimilatory nitrite reductase from a denitrifying halophilic archaeon, Haloarcula marismortui; Ichiki H et al.; Cu-containing dissimilatory nitrite reductase (CuNiR) was purified from denitrifying cells of a halophilic archaeon, Haloarcula marismortui . The purified CuNiR appeared blue in the oxidized state, possessing absorption peaks at 600 and 465 nm in the visible region . Electron paramagnetic resonance spectroscopy suggested the presence of type 1 Cu (g(II) = 2.232; A(II) = 4.4 mT) and type 2 Cu centers (g(II) = 2.304; A(II) = 13.3 mT) in the enzyme . The enzyme contained two subunits, whose apparent molecular masses were 46 and 42 kDa, according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis . N-terminal amino acid sequence analysis indicated that the two subunits were identical, except that the 46-kDa subunit was 16 amino acid residues longer than the 42-kDa subunit in the N-terminal region . A nirK gene encoding the CuNiR was cloned and sequenced, and the deduced amino acid sequence with a residual length of 361 amino acids was homologous (30 to 41%) with bacterial counterparts . Cu-liganding residues His-133, Cys-174, His-182, and Met-187 (for type 1 Cu) and His-138, His-173, and His-332 (for type 2 Cu) were conserved in the enzyme . As generally observed in the halobacterial enzymes, the enzymatic activity of the purified CuNiR was enhanced during increasing salt concentration and reached its maximum in the presence of 2 M NaCl with the value of 960 microM NO(2)(-) x min(-1) x mg(-1). Arch Microbiol, 2001 May, 175(5), 344 - 52 Identification and characterization of a periplasmic nitrate reductase in Azospirillum brasilense Sp245; Steenhoudt O et al.; The Azospirillum brasilense Sp245 napABC genes, encoding nitrate reductase activity, were isolated and sequenced . The derived protein sequences are very similar throughout the whole Nap segment to the NapABC protein sequences of Escherichia coli, Pseudomonas sp . G-179, Ralstonia eutropha, Rhodobacter sphaeroides, and Paracoccus denitrificans . Based on whole-cell nitrate reductase assays with the artificial electron donors benzyl viologen and methyl viologen, and assays with periplasmic cell-free extracts, it was concluded that the napABC-encoded enzyme activity in Azospirillum brasilense Sp245 corresponds to a periplasmic dissimilatory nitrate reductase, which was expressed under anoxic conditions and oxic conditions . A kanamycin-resistant Azospirillum brasilense Sp245 napA insertion mutant was constructed . The mutant still expressed assimilatory nitrate reductase activity, but was devoid of its periplasmic dissimilatory nitrate reductase activity. Int J Syst Evol Microbiol, 2001 May, 51(Pt 3), 999 - 1006 Comamonas denitrificans sp . nov., an efficient denitrifying bacterium isolated from activated sludge; Gumaelius L et al.; To find a biomarker for denitrification in activated sludge, five denitrifying strains isolated from three wastewater treatment plants were studied . These strains were selected from among 1,500 isolates for their excellent denitrifying properties . They denitrify quickly and have no lag phase when switching from aerobic to anoxic conditions . All strains have the cd1-type of nitrite reductase . The strains are Gram-negative rods and they all grow as filamentous chains when cultivated in liquid solution . The strains differ in colony morphology when grown on nutrient agar . Almost full-length 16S rDNA sequences were determined and phylogenetic analysis revealed that these strains are positioned among members of the genus Comamonas in the beta-subclass of the Proteobacteria . Signature nucleotides and bootstrap percentages were also analysed to verify this position . Strains 110, 123T, 2.99g, 5.38g and P17 were < or = 96.7% similar to known strains, but > or = 99.7% similar to each other, as judged from their 16S rDNA sequences, and grouped tightly together in the phylogenetic tree . Sequence motifs in the 16S rRNA gene were also found, suggesting the monophyletic origin of these strains . Nevertheless, some strains differed from the others, for example strain 110 branches early from the other strains and 5.38g is phenotypically more inert . Therefore, it is proposed that strains 110, 123T, 2.99g and P17 are classified into a new species, Comamonas denitrificans sp . nov., while the taxonomic status of strain 5.38g will have to await the outcome of further studies . The type strain of Comamonas denitrificans is 123T (ATCC 700936T). Int J Syst Evol Microbiol, 2001 May, 51(Pt 3), 977 - 83 Comamonas nitrativorans sp . nov., a novel denitrifier isolated from a denitrifying reactor treating landfill leachate; Etchebehere C et al.; A group of Gram-negative denitrifying bacteria has been isolated from a denitrifying reactor treating landfill leachate . The new isolates produced both oxidase and catalase and showed growth on acetate, butyrate, n-caproate, i-butyrate, i-valerate, propionate, n-valerate, lactate, alanine, benzoate, phenylalanine and ethanol . No growth was observed on sugars . The bacteria could perform anoxic reduction of nitrate, nitrite and nitrous oxide to nitrogen, coupled to the oxidation of the same substrates as those used under aerobic conditions, except for aromatic compounds . They were very efficient denitrifiers, as estimated from the specific rate of N2 gas production . All the strains showed the same 16S rDNA restriction profile and one of them, designated 23310T, was selected for phylogenetic analysis . The organism clustered within the family Comamonadaceae, being related to Comamonas terrigena (95.8% sequence similarity) . On the basis of the phylogenetic analysis, physiological characterization and the ability to efficiently reduce nitrate to N2, it is proposed that the bacterium be assigned to a new species, Comamonas nitrativorans . The type strain is 23310T (= DSM 13191T = NCCB 100007T = CCT 7062T). FEMS Microbiol Lett, 2001 Jun 12, 200(1), 9 - 15 PhaR, a protein of unknown function conserved among short-chain-length polyhydroxyalkanoic acids producing bacteria, is a DNA-binding protein and represses Paracoccus denitrificans phaP expression in vitro; Maehara A et al.; A putative regulatory protein, PhaR, which was identified in the polyhydroxyalkanoic acid synthetic locus (phaZCPR) in Paracoccus denitrificans, was investigated . The PhaR protein purified from a recombinant Escherichia coli was estimated to be 22 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, being consistent with the mass calculated from the nucleotide sequence . The molecular mass was determined to be 93 kDa by size-exclusion chromatography, suggesting that the protein formed a tetramer . A gel mobility shift assay showed that PhaR specifically bound to the intergenic region of phaC--phaP . In a cell-free protein synthesis system using E . coli S30 extract, the expression of the phaP gene was repressed by the addition of purified PhaR . These results suggest that PhaR is a DNA-binding protein and may play a role in the regulation of phaP gene expression. Curr Opin Biotechnol, 2001 Jun, 12(3), 283 - 8 Microbiology and application of the anaerobic ammonium oxidation ('anammox') process; Jetten MS et al.; Ten years ago, an anaerobic ammonium oxidation ('anammox') process was discovered in a denitrifying pilot plant reactor . From this system, a highly enriched microbial community was obtained, dominated by a single deep-branching planctomycete, Candidatus Brocadia anammoxidans . Phylogenetic inventories of different wastewater treatment plants with anammox activity have suggested that at least two genera in Planctomycetales can catalyse the anammox process . Electron microscopy of the ultrastructure of B . anammoxidans has shown that several membrane-bounded compartments are present inside the cytoplasm . Hydroxylamine oxidoreductase, a key anammox enzyme, is found exclusively inside one of these compartments, tentatively named the 'anammoxosome'. Biochemistry, 2001 Jun 19, 40(24), 7077 - 83 Charge translocation coupled to electron injection into oxidized cytochrome c oxidase from Paracoccus denitrificans; Verkhovsky MI et al.; Electrons were discretely injected into oxidized cytochrome c oxidase in liposomes by laser flash excitation of bound ruthenium {II} bispyridyl, and the membrane potential was recorded by time-resolved electrometry . Membrane potential is generated in a fast phase when an electron is transferred from the excited dye, via the CuA center, to heme a at a relative dielectric depth d inside the membrane {Zaslavsky, D., Kaulen, A . D., Smirnova, I . A., Vygodina, T., and Konstantinov, A . A . (1993) FEBS Lett . 336, 389-393} . Subsequently, membrane potential may develop further in a slower event, which is due to proton transfer into the enzyme from the opposite side of the membrane {Ruitenberg, M., Kannt, A., Bamberg, E., Ludwig, B., Michel, H., and Fendler, K . (2000) Proc . Natl . Acad . Sci . U.S.A . 97, 4632-4636} . Here, we confirm that injection of the first electron into the fully oxidized cytochrome c oxidase from Paracoccus denitrificans is associated with a fast electrogenic 11 micros phase, but there is no further electrogenic phase up to 100 milliseconds when special care is taken to ensure that only fully oxidized enzyme is present initially . A slower electrogenic 135 micros phase only becomes apparent and grows in amplitude upon increasing the number of light flashes . This occurs in parallel with a decrease in amplitude of the 11 micros phase and correlates with the number of enzyme molecules that are already reduced by one electron before the flash . The electrogenic 135 micros phase does not appear with increasing flash number in the K354M mutant enzyme, where electron and proton transfer into the binuclear center is delayed . We conclude that the 135 micros phase, and its associated proton uptake, take place on electron injection into enzyme molecules where the binuclear heme a3-CuB site is already reduced by one electron, and that it is accompanied by oxidation of heme a with a similar time constant . Reduction of heme a is not associated with electrogenic proton uptake into the enzyme, neither in the fully oxidized nor in the one-electron-reduced enzyme . The extent of the electrogenic 135 micrcos phase also rules out the possibility that reduction of the binuclear center by the second electron would be coupled to proton translocation in addition to the electrogenic uptake of a proton. J Environ Qual, 2001 May-Jun, 30(3), 967 - 81 Precipitation and river water chemistry of the Piracicaba River basin, southeast Brazil; Williams MR et al.; Annual precipitation and river water volumes and chemistry were measured from 1995 to 1998 in a mesoscale agricultural area of southeast Brazil . Precipitation was mildly acidic and solute concentrations were higher in the west than in the east of the basin . Combustion products from biomass burning, automobile exhaust, and industry typically accumulate in the atmosphere from March until October and are responsible for seasonal differences observed in precipitation chemistry . In river waters, the volume-weighted mean (VWM) concentrations of major solutes at 10 sites across the basin were generally lower at upriver than at downriver sampling sites for most solutes . Mass balances for major solutes indicate that, as a regional entity, the Piracicaba River basin was a net sink of H+, PO4(3-), and NH4+, and a net source of other solutes . The main stem of the Piracicaba River had a general increase in solute concentrations from upriver to downriver sampling sites . In contrast, NO3- and NH4+ concentrations increased in the mid-reach sampling sites and decreased due to immobilization or utilization in the mid-reach reservoir, and there was denitrification immediately downriver of this reservoir . Compared with tributaries of the Chesapeake Bay estuary, the Piracicaba River is affected more by point-source inputs of raw sewage and industrial wastes than nonpoint agricultural runoff high in N and P . Inputs of N and C are responsible for a degradation of water quality at downriver sampling sites of the Piracicaba River drainage, and water quality could be considerably improved by augmenting sewage treatment. J Environ Qual, 2001 May-Jun, 30(3), 1051 - 8 Denitrification losses from outdoor piglet production: spatial and temporal variability; Petersen SO et al.; Animal welfare considerations have stimulated the development of outdoor piglet (Sus scrofa) production systems, but the high levels of nutrients excreted suggest that nutrient losses from this system may be high . This study first described the spatial distribution of denitrification activity in a 5- x 5-m grid within and outside a paddock immediately after the sows (32 sows ha(-1) for 6 mo) were removed in October 1997, and again the following March . Denitrification rates averaged 0.01 kg N ha(-1) d(-1) outside, and 0.5 kg N ha(-1) d(-1) inside the paddock in October, while the corresponding figures in March were 0.01 and 0.1 kg N ha(-1) d(-1) . The highest denitrification rates were observed around the feeder, and this was also the case for concentrations of dissolved organic C and inorganic N in the soil . A statistical model that included both soil parameters and distance to feeder and huts gave the best description of the variability, but there was no significant autocorrelation between sampling points . In a second phase, seasonal variation of denitrification activity within a paddock (12 sows ha(-1) yr(-1)) was quantified; 10 soil cores were sampled along a transect 11 times between March 1998 and February 1999 . There was a significant positive effect of dissolved organic carbon (DOC) on denitrification at <25% gravimetric soil moisture (i.e., to November in this study) . Both climate and management (position of huts and feeder) appeared to influence denitrification, which was estimated to be 69 kg N ha(-1) yr(-1), or 11% of the N surplus of this production system. Eur J Biochem, 2001 Jun, 268(11), 3198 - 204 Purification and cDNA cloning of nitric oxide reductase cytochrome P450nor (CYP55A4) from Trichosporon cutaneum; Zhang L et al.; Cytochrome P450nor is involved in fungal denitrification as nitric oxide (NO) reductase . Although the heme protein has been known to occur in restricted species of fungi that belong to ascomycotina, we have previously suggested that it would also occur in the yeast Trichosporon cutaneum, which is phylogenetically far from those P450nor-producing ascomycetous fungi . Here we isolated and characterized the heme protein from the basidiomycetous yeast T . cutaneum . P450nor of the yeast (TcP450nor) exhibited properties in terms of catalysis, absorption spectrum and molecular mass that are almost identical to those of its counterparts in ascomycetous fungi . We also isolated and sequenced its cDNA . The predicted primary structure of TcP450nor showed high sequence identities (around 65%) to those of other P450nors, indicating that they belong to the same family . TcP450nor protein cofractionated with cytochrome c oxidase by subcellular fractionation and its predicted primary structure contained an extension on its amino terminus that is characteristic of a mitochondrial-targeting signal, indicating that it is a mitochondrial protein like some of the isoforms of other fungi . On the other hand, TcP450nor was unique in that inducers such as nitrate, nitrite, or NO were not required for its production in the cells . The occurrence of P450nor across the subdivisions of eumycota suggests that P450nor and denitrification are distributed more universally among fungi than was previously thought. Biochemistry, 2001 Jun 12, 40(23), 6707 - 12 Enthalpic and entropic contributions to the mutational changes in the reduction potential of azurin; Battistuzzi G et al.; The changes in the reduction potential of Pseudomonas aeruginosa and Alcaligenes denitrificans azurins following point mutations and residue ionizations were factorized into the enthalpic and entropic contributions through variable temperature direct electrochemistry experiments . The effects on the reduction enthalpy due to changes in the first coordination sphere of the copper ion, as in the Met121Gln and Met121His variants of A . denitrificans azurin, insertion of a net charge and alteration in the solvation properties and electrostatic potential in proximity of the metal site, as in the Met44Lys and His35Leu variants of P . aeruginosa azurin, respectively, and proton uptake/release in wild-type and mutated species could invariably be accounted for on the basis of simple coordination chemistry and/or electrostatic considerations . The concomitant changes in reduction entropy were found in general to contribute to the E degrees ' variation to a lesser extent as compared to the enthalpy changes . However, their effects were by no means negligible and in some instances were found to heavily contribute to (or even become the main determinant of) the observed change in reduction potential . Several lines of evidence indicate that the entropic effects are notably influenced by reduction-induced solvent reorganization effects . In particular, protein reduction tends to be favored on entropic grounds with increasing exposure of the copper site to the solvent . Moreover, enthalpy-entropy compensation phenomena are invariably observed when residue mutation or pH-induced conformational changes modify the solvent accessibility of the metal site or alter the H-bonding network in the hydration shell of the molecule . Therefore, in these cases, caution must be used in making predictions of E degrees ' changes simply based on Coulombic or coordination chemistry arguments. Biotechnol Prog, 2001 May-Jun, 17(3), 439 - 46 Characterization of a novel biocatalyst system for sulfide oxidation; McComas C et al.; It has been demonstrated that an enrichment culture dominated by Thiomicrospira sp . CVO may be cultured on H2S(g) as an energy source under sulfide-limiting conditions in suspended culture with nitrate as the electron acceptor . Hydrogen sulfide (10,000 ppmv) was completely removed from the feed gas and oxidized to sulfate in <3 s of gas-liquid contacting time . Maximum loading of the biomass for sulfide oxidation was observed to be 5.8 mmol H2S/h-g biomass protein, comparable to that reported previously for Thiobacillus denitrificans under similar conditions . However, the enrichment culture was shown to be more tolerant of extremes in pH and elevated temperature than T . denitrificans . Coupled with a reported tolerance of CVO for up to 10% NaCl, these observations suggest that a CVO-based culture is potentially a more robust biocatalyst system for sulfide oxidation than cultures based on Thiobacilli. Water Sci Technol, 2001, 43(7), 287 - 94 Development of a risk assessment based technique for design/retrofitting of WWTPs; Rousseau D et al.; Up to now, within the design/retrofit of wastewater treatment plants (WWTPs), deterministic models were used to evaluate different scenarios on their merits in terms of effluent compliance . This paper describes an approach in which a Monte Carlo engine is coupled to a deterministic treatment plant model, followed by risk interpretation in the form of concentration-duration-frequency (cdf) curves of norm exceedance . The combination of probabilistic modelling techniques with the currently available deterministic models allows to determine the probability of exceeding the effluent limits of a WWTP . This percentage of exceedance is accompanied by confidence intervals resulting from the inherent uncertainty of influent characteristics and model parameters . The approach is illustrated for a hypothetical case study, consisting of a denitrifying plant model inspired by the benchmark model described by Spanjers et al. Water Sci Technol, 2001, 43(7), 215 - 22 Model system for the management of nitrogen leaching at the scale of river basins and regions; Wendland F et al.; In the framework of the EU-project RANR (Regional analysis of subsurface nitrogen retention and its impact on the nitrogen export from land to sea) a model system was developed to estimate groundwaterborne nitrogen inputs into river systems . The core of this model system is composed of a soil nitrogen leaching model (SOIL-N) and a groundwater residence time/denitrification model (WEKU) . The application of the model system was carried out for the study catchment areas of the Uecker basin (ca . 2400 km2, Germany) and the Gjern basin (ca . 200 km2, Denmark) . For both catchment areas, the modelled average nitrogen loads leached into the groundwater were about 40 kg N/ha a, while the remaining groundwaterborne nitrogen intake to rivers was quantified to an average of about 2 kg/ha a . The comparision with observed groundwaterborne riverine nitrogen loads showed a very good agreement, proofing the key role nitrogen retention in groundwater plays in the two catchment areas . With regard to the generalisation and transfer of the SOIL-N/WEKU model concept we assume that the model can be applicated in catchment areas in the European Pleistocene Lowland, which ranges from the Netherlands in the west to the Baltic States and the Ukraine in the east. Arch Microbiol, 2001 Apr, 175(4), 270 - 81 Use of isotopic and molecular techniques to link toluene degradation in denitrifying aquifer microcosms to specific microbial populations; Pelz O et al.; Microcosms were inoculated with sediments from both a petroleum-hydrocarbon (PHC)-contaminated aquifer and from a nearby pristine aquifer and incubated under anoxic denitrifying conditions with {methyl-13C}toluene . These microcosms served as a laboratory model system to evaluate the combination of isotope (13C-labeling of polar-lipid-derived fatty acids) and molecular techniques (16S rRNA-targeting gene probes) to identify the toluene-metabolizing population . After total depletion of toluene, the following bacterial phospholipid fatty acids (PLFA) were 13C-enriched: 16:1omega7c, 16:1omega7t, 16:0, cy17:0, and 18:1omega7c . Pure culture experiments demonstrated that these compounds were also found in PLFA profiles of PHC-degrading Azoarcus spp . (beta-Proteobacteria) and related species . The origin of the CO2 evolved in the microcosms was determined by measurements of stable carbon isotope ratios . Toluene represented 11% of the total pool of mineralized substrates in the contaminated sediment and 54% in the pristine sediment . The microbial community in the microcosm incubations was characterized by using DAPI staining and whole-cell hybridization with specific fluorescently labeled 16S rRNA-targeted oligonucleotide probes . Results revealed that 6% of the DAPI-stained cells in the contaminated sediment and 32% in the pristine sediment were PHC-degrading Azoarcus spp . In biotic control microcosms (incubated under denitrifying conditions, no toluene added), Azoarcus spp . cells remained at less than 1% of the DAPI-stained cells . The results show that isotope analysis in combination with whole-cell hybridization is a promising approach to identify and to quantify denitrifying toluene degraders within microbial communities. Water Sci Technol, 2000, 41(3), 231 - 8 Kinetics of accelerated solid-state fermentation of organic-rich municipal solid waste; Vieitez ER et al.; Biotransformation of landfill solid wastes is a slow process requiring decades for completion . Accelerated anaerobic fermentation in modulated landfill environments may alleviate or eliminate pollution of land, water and air . This research was undertaken to demonstrate the application of biphasic fermentation to a simulated laboratory-scale landfill to effect rapid biomethanation of biodegradable solids . The biphasic process consisted of solid-state, acidogenic fermentation of the organic fraction of MSW followed by biomethanation of acidic hydrolysates in a separate methane fermenter . Solid-state fermentation of the MSW with effluent recirculation resulted in rapid hydrolysis, acidification and denitrification, with soluble COD and VFA concentrations accumulating to inhibitory levels of 60,000 mg/l and 13,000 mg/l, respectively, at a pH of 4.5 . The landfill gas methane concentration reached a maximum of 55 mol.% . By comparison, the methanogenic reactor produced high methane-content (70-85 mol.%) gases . The biphasic process effected carbohydrate, lipid, and protein conversion efficiencies of 90%, 49%, and 37%, respectively . Development of a Monod-type product-formation model was undertaken to predict methane formation and to determine kinetic parameters for the methanogenic processes in the simulated landfill and separate methane reactors . A first-order solids hydrolysis rate constant of 0.017 day-1 was evaluated to show that landfill solids hydrolysis was slower than the inhibited methanogenesis rate. Water Sci Technol, 2001, 43(3), 93 - 100 On-line control of a SBR system for nitrogen removal from industrial wastewater; Andreottola G et al.; In this study, laboratory-scale experiments were conducted applying the SBR activated sludge process to a wastewater from a wood factory operating on plywood production . Due to the fact that the wastewater is characterised with a high concentration of ammonia, the aim was to evaluate the nitrogen removal process with SBR system operating with a on-line control of ORP, pH and DO . The complete ammonia removal corresponded exactly to the "Ammonia Valley" in the pH versus time graphic . The ammonia removal efficiency in SBR was 99% at 20 degrees C . The denitrification reaction was completed when in the ORP versus time graphic appeared the "Nitrate Knee" . From the experimental results the optimisation of the SBR cycle allowed the doubling of the applied load with respect to a not optimised cycle (performed in the same volume reactor), without a decrease in the removal efficiency . Moreover, the possibility of stopping the aeration just after the appearance of the Ammonia Valley allows an energy saving . The easy operation and the low cost make the SBR system an interesting option for the biological pre-treatment of plywood industry to be discharged into surface water. Water Sci Technol, 2001, 43(3), 355 - 62 Full-scale demonstration of biological nutrient removal in a single tank SBR process; Keller J et al.; Complete biological nutrient removal (BNR) in a single tank, sequencing batch reactor (SBR) process, is demonstrated here at full-scale on a typical domestic wastewater . The unique feature of the UniFed process is the introduction of the influent into the settled sludge blanket during the settling and decant periods of the SBR operation . This achieves suitable conditions for denitrification and anaerobic phosphate release which is critical to successful biological phosphorus removal . It also achieves a "selector" effect, which helps in generating a compact, well settling biomass in the reactor . The results of this demonstration show that it is possible to achieve well over 90% removal of COD, nitrogen and phosphorus in such a process . Effluent quality achieved over a six-month operating period directly after commissioning was: 29 mg/l COD, 0.5 mg/l NH4-N 1.5 mg/l NOx-N and 1.5 mg/l PO4-P (50%-iles of daily samples) . During an 8-day, intensive sampling period, the effluent BOD5 was < 2 mg/l in all samples and the total phosphorus averaged 0.17 mg/l in the effluent . Detailed sampling and analysis during one cycle and at various depths clearly showed the deliberate stratification achieved in the tank during the settling and decant period, allowing biochemical reactions to occur during this normally "non-productive" period . The simplicity and flexibility of the UniFed system allows it to be used in numerous applications, particularly for industrial situations where a high degree of uncertainty of the wastewater composition during the design stage or where changing requirements based on changes on the production side are present . The single tank operation without any recycle also reduces the capital costs for a full BNR system compared to the comparatively complex continuous flow processes. Water Sci Technol, 2001, 43(3), 275 - 82 Creating anoxic and microaerobic conditions in sequencing batch reactors treating volatile BTX compounds; Ma G et al.; An experimental strategy is introduced for studying the biodegradation of wastewaters containing volatile contaminants using an alternating anoxic/microaerobic sequencing batch reactor (SBR) . Benzene, toluene, and the xylene isomers (BTX) served as model volatile contaminants for this study . The reactor was configured to overcome stripping the volatile BTX compounds into the atmosphere to provide opportunities for BTX biodegradation . Oxygen-free anoxic and microaerobic (< 0.2 mg/L dissolved oxygen) conditions were established using a novel laboratory reactor configuration . ORP was successfully used to monitor different electron acceptor conditions in the SBR . Toluene and m-xylene were amenable to anoxic (denitrifying) metabolism while benzene, o-, and p-xylene were biodegradable under microaerobic conditions . The results demonstrate that establishing microaerobic conditions in full-scale bioreactors may be an appropriate way to encourage the biodegradation of aerobically biodegradable volatile contaminants . Additionally, the laboratory reactor configuration introduced in this paper may be useful in subsequent studies involving microaerobic metabolism. Water Sci Technol, 2001, 43(3), 265 - 74 Characterization and treatment of the liquid effluents from the anaerobic digestion of biogenic solid waste; Graja S et al.; The process wastewater generated by the fermentation of biogenic fractions of solid waste (biowaste) is characterized by very specific composition features, which sets it apart from the well known categories of wastewater . Up to now, there is a definite lack of practical experience and know-how in the treatment of those effluents . In view of the increasingly wide acceptance awarded to the fermentation process, it appears necessary, however, to include the treatment of the generated wastewater in the overall process and to grant it the same priority as the fermentation step . In this study, pilot-scale experiments were conducted over 200 days in a sequencing batch reactor (SBR), with the wastewater from a full-scale Bavarian biowaste digestion plant . Characterization of the influent implementing molecular size distribution studies showed that 97% of the components were either in particular form or < 1 kD . The main classes of compounds present in these fractions have been identified . The treatment objectives of the SBR plant were carbon and nitrogen removal . Soluble COD removal efficiencies were comprised between 40 and 60%, yielding a residual, refractory COD concentration of about 2000 mg/L in the effluent . Ammonium removal efficiencies averaged 96% and denitrification occurred with the addition of acetic acid or pretreated biowaste as a carbon source . Pretreated biowaste also showed synergetic effects as a co-substrate for COD removal. Water Sci Technol, 2001, 43(3), 191 - 4 Biological denitrifying phosphorus removal in SBR: effect of added nitrate concentration and sludge retention time; Merzouki M et al.; Optimizing anoxic biological phosphorus removal in the anaerobic-anoxic sequencing batch reactor (A2 SBR) was observed to depend on two parameters: the amount of added nitrate and the sludge retention time (SRT) . The concentration of 120 mg N-NO3.l-1 in the anoxic medium and the SRT of 15 days were determined as optimal for a complete phosphorus removal in the A2 SBR . The reactor was supplied with synthetic wastewater containing 800 mg COD.l-1 acetic acid, 240 mg N-NH4.l-1 and 30 mg P-PO4.l-1 . This study was completed by microscopic observations which revealed three morphological types of phosphate-accumulating bacteria (PAB). Water Sci Technol, 2001, 43(3), 175 - 82 Modeling effect of remaining nitrate on phosphorus removal in SBR; Kazmi AA et al.; Nitrate shock loading experiments were conducted in a bench scale SBR to investigate the effect of nitrate on phosphorus removal . After achieving satisfactory phosphorus removal under steady state operation, initial NO3-N concentration amounting to 10 and 20 mg/L was fed at the beginning of the cycle . It was observed that, 10 mg/L of NO3-N suppressed phosphorus release during the feed and mix phases . Organic consumption for denitrification lead to limited PHA storage by phosphorus removing bacteria, resulting in less PO4-P removal . For 20 mg/L, influent organic substrate was not sufficient even for complete denitrification, thus leading to the presence of higher NO3-N and PO4-P in effluent . To explain the dynamics of the nutrient removal system under the transient loading, a SBR model based on IAWQ ASM2 was implemented . After adjusting PHA contents, model simulations well predicted dynamic changes of nitrate and phosphate concentrations during a cycle . Based on the model simulations, competition of COD substrate among denitrification, fermentation and oxygen respiration were investigated by calculating their consumption rates during mixing phase . In addition, a nitrate disappearance model was proposed and implemented in conjunction with a settling model to predict remaining and effluent nitrate in a cycle of SBR . Furthermore, integrated model simulations highlighted the effect of remaining nitrate on phosphorus release considering different options of reactions in settling phase. Water Sci Technol, 2001, 43(3), 159 - 65 Phosphorus removal from SBR with controlled denitrification for weak sewage; Choi E et al.; Most SBRs operating for weak sewage have a tendency of poor removal of phosphorus due to NO3N inhibition . To minimize this inhibition, NO3N storage was utilized in this study . With influent BOD 100 mg/L and VFA 30 mg/L, a total of the maximum removable nitrogen and phosphorus was about 25 mg/L with 8 hr cycle . With this storage, VFA utilized for phosphorus removal could be maximized . The effluent nitrogen and phosphorus levels were generally 11 and 1 mg/L, respectively. Water Sci Technol, 2001, 43(3), 151 - 8 Role of storage phenomena on removal of different substrates during pre-denitrification; Majone M et al.; Removal mechanisms of different substrates during the pre-denitrification step of an anoxic/aerobic sequencing process are studied . Biom