Water Sci Technol, 2002, 46(8), 119 - 26 Effect of loading rate and intermittent aeration cycle on nitrogen removal in membrane separation activated sludge process; Nagaoka H et al.; The performance of the submerged membrane separation activated sludge process with intermittent aeration was investigated in a laboratory scale experiment by changing organic loading rate and intermittent aeration cycle . A rectangular PVC tank was used as an aeration tank, in which a flat-sheet type Micro-Filtration membrane made of poly-olefin with a pore size of 0.2 microm was submerged . Organic loading rate to the reactor was set at 0.3 and 0.8 g-TOC/L/day . C/N ratio in the feed was set at around 5.0 for every condition . Aeration cycle was changed from 10 min-10 min (aeration-stop) to 120 min-120 min in different organic loading conditions . Flux through the membrane was set at 0.25 m/day . Membrane fouling proceeded rapidly in 0.8 g-TOC/L/day conditions . However, when organic loading rate was 0.3 g-TOC/L/day, bacterial metabolic substances were degraded rapidly compared to the production, thereby decreasing viscosity in mixed liquor . Nitrogen removal rate was between 60% and 80% for 0.8 g-TOC/L/day loading, and between 50% and 65% for 0.3 g-TOC/L/day loading . And the nitrogen removal was highest in 40 min to 60 min aeration cycle conditions . Too short aeration cycle did not result in sufficiently long anoxic periods for denitrification while too long a cycle resulted in unnecessary anaerobic periods after depletion of nitrate . Intermittent aeration was effective also for decreasing viscosity in mixed liquor. Water Sci Technol, 2002, 46(8), 75 - 82 Retrofitting activated sludge systems to intermittent aeration for nitrogen removal; Hanhan O et al.; The paper provides the basis and the conceptual approach of applying process kinetics and modelling to the design of alternating activated sludge systems for retrofitting existing activated sludge plants to intermittent aeration for nitrogen removal . It shows the significant role of the two specific parameters, namely, the aerated fraction and the cycle time ratio on process performance through model simulations and proposes a way to incorporate them into a design procedure using process stoichiometry and mass balance . It illustrates the effect of these parameters, together with the sludge age, in establishing the balance between the denitrification potential and the available nitrogen created in the anoxic/aerobic sequences of system operation. Arch Microbiol, 2002 Dec, 178(6), 506 - 16 Epub 2002 Oct 03. Genes involved in the anaerobic degradation of ethylbenzene in a denitrifying bacterium, strain EbN1; Rabus R et al.; Genes involved in anaerobic degradation of the petroleum hydrocarbon ethylbenzene in the denitrifying Azoarcus-like strain EbN1 were identified on a 56-kb DNA contig obtained from shotgun sequencing . Ethylbenzene is first oxidized via ethylbenzene dehydrogenase to (S)-1-phenylethanol; this is converted by (S)-1-phenylethanol dehydrogenase to acetophenone . Further degradation probably involves acetophenone carboxylase forming benzoylacetate, a ligase forming benzoylacetyl-CoA, and a thiolase forming acetyl-CoA and benzoyl-CoA . Genes of this pathway were identified via N-terminal sequences of proteins isolated from strain EbN1 and by sequence similarities to proteins from other bacteria . Ethylbenzene dehydrogenase is encoded by three genes (ebdABC), in accordance with the heterotrimeric enzyme structure . Binding domains for a molybdenum cofactor (in subunit EbdA) and iron/sulfur-clusters (in subunits EbdA and EbdB) were identified . The previously observed periplasmic location of the enzyme was corroborated by the presence of a twin-arginine leader peptide characteristic of the Tat system for protein export . A fourth gene (ebdD) was identified, the product of which may act as an enzyme-specific chaperone in the maturation of the molybdenum-containing subunit . A distinct gene (ped) coding for (S)-1-phenylethanol dehydrogenase apparently forms an operon with the ebdABCD genes . The ped gene product with its characteristic NAD(P)-binding motif in the N-terminal domain belongs to the short-chain dehydrogenase/reductase (SDR) superfamily . A further operon apparently contains five genes (apc1-5) suggested to code for subunits of acetophenone carboxylase . Four of the five gene products are similar to subunits of acetone carboxylase from Xanthobacter autotrophicus . Upstream of the apc genes, a single gene (bal) was identified which possibly codes for a benzoylacetate CoA-ligase and which is co-transcribed with the apc genes . In addition, an apparent operon containing almost all genes required for beta-oxidation of fatty acids was detected; one of the gene products may be involved in thiolytic cleavage of benzoylacetyl-CoA . The DNA fragment also included genes for regulatory systems; these were two sets of two-component systems, two LysR homologs, and a TetR homolog . Some of these proteins may be involved in ethylbenzene-dependent gene expression. Water Res, 2002 Nov, 36(18), 4616 - 26 Hydrodynamic control of nitrogen and phosphorus turnover in an eutrophicated estuary in the Baltic; Nilsson P et al.; The turnover of phosphorus and nitrogen have been studied in a low salinity estuary in the Bothnian Sea . The estuary, Gardsfjarden, has a high loading of nutrients from a pulp and paper mill which supplies the estuary with three times the annual natural input of phosphorus and nitrogen, respectively . The turnover of nutrients was to a great extent determined by hydrodynamic variations . During periods of intense resuspension there was a net export of particulate bound nutrients from the estuary to the sea but since these periods were short there was an overall net retention of particulate nutrients . The estuary was a source for dissolved phosphorus to the sea . The major source of phosphorus export was release of dissolved P from anoxic sediments . Gardsfjarden was a sink for dissolved nitrogen which most likely escaped the estuary by denitrification . Nitrate was supplied by degradation of particulate organic nitroge'n but the main source was import from the sea. Water Environ Res, 2002 Jul-Aug, 74(4), 338 - 45 Biological denitrification of hydrolysates from octahydro-1,3,5,7 tetranitro-1,3,5,7-tetrazocine; Ogden KL et al.; Alternatives for the destruction of common military explosives, including trinitrotoluene; hexahydro-1,3,5-trinitro-1,3,5-triazine; and octahydro-1,3,5,7 tetranitro-1,3,5,7-tetrazocine (HMX) are being investigated in the post-cold war period . One alternative combines chemical treatment (i.e., base hydrolysis of the explosives) and biological treatment (i.e., denitrification of the hydrolysate) . This paper focuses on results of the biological part of the treatment process, during which Hyphomicrobium sp . bacteria were isolated from a seed obtained from a denitrification facility . The bacteria were enriched and maintained on a surrogate waste with methanol as the carbon source . The resulting culture is capable of anoxic growth in waste solutions containing up to 5000 mg/L of nitrite-nitrogen . The culture efficiently denitrifies both surrogate and actual hydrolysate wastes . A substrate inhibition model was used to accurately predict denitrification rates . Comparisons are made between denitrification rates obtained for surrogate versus actual wastes . Denitrification rates were higher when actual waste streams were used . This work demonstrates the feasibility of using Hyphomicrobiun sp . bacteria to treat HMX hydrolysate and presents a model that can be used to design a large-scale system. Water Environ Res, 2002 Jul-Aug, 74(4), 324 - 37 Long-term effect of total cycle time and aerobic/anoxic phase ratio on nitrogen removal in a sequencing batch reactor; Katsogiannis AN et al.; A laboratory-scale sequencing batch reactor was used to study nitrogen removal from a synthetic wastewater with an ammonium-nitrogen concentration of 50 mg/L . The effect of two key parameters (i.e., the total cycle time and the aerobic/anoxic phase duration ratio) on the performance of the reactor was investigated . Four sets of operational parameters resulting from the combination of 6 and 8 hours of cycle time and 1:1 and 1:3 aerobic/anoxic ratios were studied . Denitrification was based mainly on endogenous carbon sources as long as organic carbon, provided in the form of acetate, was quickly removed from the mixed liquor during the aerobic phase of the reactor operation . In terms of nitrogen removal efficiency, the set of 8 hours of cycle time and 1:3 aerobic/anoxic phase ratio was found to be superior in that it consistently yielded an effluent total nitrogen concentration of less than 15 mg/L (consisting almost exclusively of nitrate-nitrogen) and exhibited a percentage nitrogen removal rate of 77 +/- 2.5% . In terms of wastewater throughput (i.e., wastewater volume treated per day), however, the implementation of 6 hours of cycle time and 1:3 aerobic/anoxic phase ratio was satisfactory for nitrogen removal efficiency (72 +/- 2%), although elevated amounts of ammonium- and nitrite-nitrogen were found in the effluent . It was also demonstrated that under certain operating conditions nitrogen removal via nitrite is observed, as nitratification (i.e., oxidation of nitrite-nitrogen to nitrate) and denitratification (i.e., reduction of nitrate-nitrogen to nitrite) are bypassed. Appl Environ Microbiol, 2002 Nov, 68(11), 5231 - 40 Characterization of the adaptive response to trichloroethylene-mediated stresses in Ralstonia pickettii PKO1; Park J et al.; In Ralstonia pickettii PKO1, a denitrifying toluene oxidizer that carries a toluene-3-monooxygenase (T3MO) pathway, the biodegradation of toluene and trichloroethylene (TCE) by the organism is induced by TCE at high concentrations . In this study, the effect of TCE preexposure was studied in the context of bacterial protective response to TCE-mediated toxicity in this organism . The results of TCE degradation experiments showed that cells induced by TCE at 110 mg/liter were more tolerant to TCE-mediated stress than were those induced by TCE at lower concentrations, indicating an ability of PKO1 to adapt to TCE-mediated stress . To characterize the bacterial protective response to TCE-mediated stress, the effect of TCE itself (solvent stress) was isolated from TCE degradation-dependent stress (toxic intermediate stress) in the subsequent chlorinated ethylene toxicity assays with both nondegradable tetrachloroethylene and degradable TCE . The results of the toxicity assays showed that TCE preexposure led to an increase in tolerance to TCE degradation-dependent stress rather than to solvent stress . The possibility that such tolerance was selected by TCE degradation-dependent stress during TCE preexposure was ruled out because a similar extent of tolerance was observed in cells that were induced by toluene, whose metabolism does not produce any toxic products . These findings suggest that the adaptation of TCE-induced cells to TCE degradation-dependent stress was caused by the combined effects of solvent stress response and T3MO pathway expression. Water Res, 2002 Sep, 36(16), 4088 - 94 Microbial population in a hydrogen-dependent denitrification reactor; Szekeres S et al.; The bacterial population in an H2-dependent denitrification system was studied . The laboratory set-up was designed for the treatment of potable water and consisted of an electrochemical cell, where the water to be treated was enriched with H2 prior to entering a bioreactor . Bioreactors (columns packed with granulated active carbon) were inoculated with denitrifying bacterial strains isolated from a previous reactor, then sampled immediately after inoculation, or after 1 or 3 months of continuous operation . Total number of the bacteria and numbers of each different strain were determined at various levels of the bioreactor . The strains present in the inoculum were identified as Ochrobactrum anthropi, Pseudomonas stutzeri, Paracoccus panthotrophus and Paracoccus denitrificans . Numbers of the latter declined markedly with time with the other three strains being responsible for nitrate removal . A correlation was found between the relative abundance of each strain and its specific denitrification activity. J Bioenerg Biomembr, 2002 Aug, 34(4), 269 - 78 Sulfite inhibits the F1F0-ATP synthase and activates the F1F0-ATPase of Paracoccus denitrificans; Pacheco-Moises F et al.; The F1F0 complex of Paracoccus denitrificans (PdF1F0) is the fastest ATP synthase but the slowest ATPase . Sulfite exerts maximal activation of the PdF1F0-ATPase (Pacheco-Moises, F., Garcia, J . J., Rodriguez-Zavala, J . S., and Moreno-Sanchez, R . (2000) . Eur J . Biochem . 267, 993-1000) but its effect on the PdF1F0-ATP synthase activity remains unknown . Therefore, we studied the effect of sulfite on ATP synthesis and 32Pi <--> ATP exchange reactions of inside-out membrane vesicles of P . denitrificans . Sulfite inhibited both reactions under conditions of maximal delta pH and normal sensitivity to dicyclohexylcarbodiimide . Sulfite increased by 10- and 5-fold the K0.5 for Mg2+-ADP and Pi during ATP synthesis, respectively, and by 4-fold the IC50 of Mg2+-ADP for inhibition of the PdF1F0-ATPase activity . Thus, sulfite exerts opposite effects on the forward and reverse functioning of the PdF1F0 complex . These effects are not due to membrane or PdF1F0 uncoupling . Kinetic and structural modifications that could account for these results are discussed. Anal Chem, 2002 Oct 1, 74(19), 4905 - 12 Measurement of the oxygen isotopic composition of nitrate in seawater and freshwater using the denitrifier method; Casciotti KL et al.; We report a novel method for measurement of the oxygen isotopic composition (18O/16O) of nitrate (NO3-) from both seawater and freshwater . The denitrifier method, based on the isotope ratio analysis of nitrous oxide generated from sample nitrate by cultured denitrifying bacteria, has been described elsewhere for its use in nitrogen isotope ratio (15N/14N) analysis of nitrate . (1) Here, we address the additional issues associated with 18O/16O analysis of nitrate by this approach, which include (1) the oxygen isotopic difference between the nitrate sample and the N20 analyte due to isotopic fractionation associated with the loss of oxygen atoms from nitrate and (2) the exchange of oxygen atoms with water during the conversion of nitrate to N2O . Experiments with 18O-labeled water indicate that water exchange contributes less than 10%, and frequently less than 3%, of the oxygen atoms in the N20 product for Pseudomonas aureofaciens . In addition, both oxygen isotope fractionation and oxygen atom exchange are consistent within a given batch of analyses . The analysis of appropriate isotopic reference materials can thus be used to correct the measured 18O/16O ratios of samples for both effects . This is the first method tested for 18O/16O analysis of nitrate in seawater . Benefits of this method, relative to published freshwater methods, include higher sensitivity (tested down to 10 nmol and 1 microM NO3-), lack of interference by other solutes, and ease of sample preparation. Environ Sci Technol, 2002 Oct 1, 36(19), 4087 - 90 TBA biodegradation in surface-water sediments under aerobic and anaerobic conditions; Bradley PM et al.; The potential for {U-14C} TBA biodegradation was examined in laboratory microcosms under a range of terminal electron accepting conditions . TBA mineralization to CO2 was substantial in surface-water sediments under oxic, denitrifying, or Mn(IV)-reducing conditions and statistically significant but low under SO4-reducing conditions . Thus, anaerobic TBA biodegradation may be a significant natural attenuation mechanism for TBA in the environment, and stimulation of in situ TBA bioremediation by addition of suitable terminal electron acceptors may be feasible . No degradation of {U-14C} TBA was observed under methanogenic or Fe(III)-reducing conditions. Biochem J, 2003 Feb 1, 369(Pt 3), 619 - 26 The transition between active and de-activated forms of NADH:ubiquinone oxidoreductase (Complex I) in the mitochondrial membrane of Neurospora crassa; Grivennikova VG et al.; The mammalian mitochondrial NADH:ubiquinone oxidoreductase (Complex I) has been shown to exist in two kinetically and structurally distinct slowly interconvertible forms, active (A) and de-activated (D) {Vinogradov and Grivennikova (2001) IUBMB Life 52, 129-134} . This work was undertaken to investigate the putative Complex I A-D transition in the mitochondrial membrane of the lower eukaryote Neurospora crassa and in plasma membrane of the prokaryote Paracoccus denitrificans, organisms that are eligible for molecular genetic manipulations . The potential interconversion between A and D forms was assessed by examination of the initial and steady-state rates of NADH oxidation catalysed by inside-out submitochondrial ( N . crassa ) and sub-bacterial ( P . denitrificans ) particles and their sensitivities to N -ethylmaleimide and Mg(2+) . All diagnostic tests provide evidence that slow temperature- and turnover-dependent A-D transition is an explicit feature of eukaryotic N . crassa Complex I, whereas the phenomenon is not seen in the membranes of the prokaryote P . denitrificans . Significantly lower activation energy for A-to-D transition characterizes the N . crassa enzyme compared with that determined previously for the mammalian Complex I . Either a lag or a burst in the onset of the NADH oxidase assayed in the presence of Mg(2+) is seen when the reaction is initiated by the thermally de-activated or NADH-activated particles, whereas the delayed final activities of both preparations are the same . We conclude that continuous slow cycling between A and D forms occurs during the steady-state operation of Complex I in N . crassa mitochondria. J Environ Qual, 2002 Sep-Oct, 31(5), 1610 - 22 Relating net nitrogen input in the Mississippi River basin to nitrate flux in the lower Mississippi River: a comparison of approaches; McIsaac GF et al.; A quantitative understanding of the relationship between terrestrial N inputs and riverine N flux can help guide conservation, policy, and adaptive management efforts aimed at preserving or restoring water quality . The objective of this study was to compare recently published approaches for relating terrestrial N inputs to the Mississippi River basin (MRB) with measured nitrate flux in the lower Mississippi River . Nitrogen inputs to and outputs from the MRB (1951 to 1996) were estimated from state-level annual agricultural production statistics and NOy (inorganic oxides of N) deposition estimates for 20 states that comprise 90% of the MRB . A model with water yield and gross N inputs accounted for 85% of the variation in observed annual nitrate flux in the lower Mississippi River, from 1960 to 1998, but tended to underestimate high nitrate flux and overestimate low nitrate flux . A model that used water yield and net anthropogenic nitrogen inputs (NANI) accounted for 95% of the variation in riverine N flux . The NANI approach accounted for N harvested in crops and assumed that crop harvest in excess of the nutritional needs of the humans and livestock in the basin would be exported from the basin . The U.S . White House Committee on Natural Resources and Environment (CENR) developed a more comprehensive N budget that included estimates of ammonia volatilization, denitrification, and exchanges with soil organic matter . The residual N in the CENR budget was weakly and negatively correlated with observed riverine nitrate flux . The CENR estimates of soil N mineralization and immobilization suggested that there were large (2000 kg N ha-1) net losses of soil organic N between 1951 and 1996 . When the CENR N budget was modified by assuming that soil organic N levels have been relatively constant after 1950, and ammonia volatilization losses are redeposited within the basin, the trend of residual N closely matched temporal variation in NANI and was positively correlated with riverine nitrate flux in the lower Mississippi River . Based on results from applying these three modeling approaches, we conclude that although the NANI approach does not address several processes that influence the N cycle, it appears to focus on the terms that can be estimated with reasonable certainty and that are correlated with riverine N flux. Huan Jing Ke Xue, 2002 Jul, 23(4), 37 - 41 {Oxidative degradation of chlorinated hydrocarbons under anaerobic conditions}; Lu X et al.; Based on column experiments, the oxidative degradations of some chlorinated hydrocarbons under three less-reduced redox conditions were investigated . The results showed that in the presence of nitrate and manganese oxide, 1,2-dechloroethane (1,2-DCA) and vinyl chloride (VC) could be oxidized . The transformation rates of 1,2-DCA under denitrification and manganese reduction were 1.18/h and 0.54/h, respectively, while those of VC were 0.29/h and 0.15/h, respectively . In the presence of iron, degradation of VC was not clear . In addition, the degradation of 1,2-DCA was inhibited . For other chlorinated hydrocarbons, such as 1,1,1-trichloroethane, trichloroethene, cis-dichloroethene and trans-dichloroethene, no degradation occurred under the three studied redox conditions . Monochlorobenzene exhibited relative high removal in the columns, however, due to its high soil adsorption potential, it was not known yet whether microbial activities were involved. Bioresour Technol, 2002 Dec, 85(3), 243 - 8 Effect of hydraulic retention time on inorganic nutrient recovery and biodegradable organics removal in a biofilm reactor treating plant biomass leachate; Krumins V et al.; A fixed-film (biofilm) reactor was designed and its performance was determined at various retention times . The goal was to find the optimal retention time for recycling plant nutrients in an advanced life support system, to minimize the size, mass, and volume (hold-up) of a production model . The prototype reactor was tested with aqueous leachate from wheat crop residue at 24, 12, 6, and 3 h hydraulic retention times (HRTs) . Biochemical oxygen demand (BOD), nitrates and other plant nutrients, carbohydrates, total phenolics, and microbial counts were monitored to characterize reactor performance . BOD removal decreased significantly from 92% at the 24 h HRT to 73% at 3 h . Removal of phenolics was 62% at the 24 h retention time, but 37% at 3 h . Dissolved oxygen concentrations, nitric acid consumption, and calcium and magnesium removals were also affected by HRT . Carbohydrate removals, carbon dioxide (CO2) productions, denitrification, potassium concentrations, and microbial counts were not affected by different retention times . A 6 h HRT will be used in future studies to determine the suitability of the bioreactor effluent for hydroponic plant production. Acta Microbiol Pol, 2002, 51(2), 171 - 82 Microflora of soils under pine forests area affected by gradation of leaf-eating insects; Streminska MA et al.; Soils of pine forests in the Bytnica Forestry District, Poland, are poor in nutrients readily accessible to plants . The excessively acidic reaction of the soils, typical for soils under pine forests, unfavourably affects the growth of microorganisms whose numbers are lower than in soils under deciduous and mixed forests . In the pine forests of the studied forestry there were outbreaks of a defoliating insect - pine beauty moth (Panolis flammea L.), which resulted in over 60% defoliation of the trees . The studies were carried out on the area of tree stands subjected to gradation by leaf-eating insects (sprayed and not sprayed) and healthy stand of the same age class (age 60 to 70 years) . The studies revealed increased number of soil microorganisms in samples taken from the area affected by pine beauty moth gradation in the case of both unsprayed areas and those sprayed with the pesticide . The occurrence in these soils of larger numbers of ammonifying and denitrifying bacteria points to the presence of conditions favouring the growth of heterotrophic organisms . Changes in the number of actinomycetes and fungi in soils under tree stands subjected to gradation by insects, compared to healthy stands, can be a consequence of a change of environmental conditions (e.g . % content of organic carbon) . Soils under defoliated tree stands show higher biochemical activity related to nitrogen cycling in the pine forest ecosystem . This leads to higher availability of organic nitrogen for conversion to inorganic forms of nitrogen, which are utilised by trees . Further changes occurring in soils under forest stands affected by gradation by leaf-eating insects would allow to gain knowledge on the ecological consequences of the use of insecticides in the protection of pine stands against harmful insects, with particular stress on those situations in which pine stands not threatened by complete defoliation are sprayed. Environ Technol, 2002 Sep, 23(9), 1017 - 26 Screening and characterization of facultative psychrophilic denitrifiers for treatment of nitrate contaminated groundwater using starch-based biodegradable carriers; Kim YS et al.; Potential starch degrading denitrifying microorganisms that can grow at 4 degrees C were isolated from lake sediments to remove nitrate from groundwater . Initial screening using soluble starch as the sole carbon source confirmed that two out of twenty-five isolates (strain no . 2 and 47) significantly reduced nitrate in the medium and liberated nitrogen gas during culture . In a second screening, several commercially available starch based materials and different kinds of starch were tested . Strain 47 was found to have the best denitrification performance compared with strain 2 . Using starch based carrier C (a commercial packing material) as carbon source, strain 47 could completely reduce the nitrate nitrogen in the medium after one week of batch culture even at 10 degrees C . Strain 47 could remove nitrate even without trace element supplementation, and it could perform optimally at 1X (10ml l(-1) of trace element solution) level of trace element supplement . The best temperature for denitrification for strain 47 was 15 degrees C and 20 degrees C, but it could also remove nitrate nitrogen at 10 degrees C and 30 degrees C, although at a slower rate . Reactor studies in a simulated treatment well (a cylindrical reciprocating basket reactor) in a repeated fed batch mode showed a good stable denitrification performance as long as substrate limitation is avoided by adequate supply of starch based carrier . Although the similarity score obtained was not enough for phylogenic identification, the results of 16SrRNA sequences analysis for the strain 47 showed a dose relation to Janthinobacterium lividum or Pseudomonas (Janth) mephitica (95.77%). Water Sci Technol, 2002, 46(4-5), 421 - 5 Effects of ozone treatment on the biodegradability of sludge from municipal wastewater treatment plants; Yeom IT et al.; The effects of ozone pretreatment on the biodegradability of municipal wastewater sludge were determined . Three types of experiments were conducted: anaerobic digestion, aerobic biodegradation, and denitrification using ozone-treated sludge as a carbon source . For 5 days, ozonated sludge at 0.1 gO3/g-SS showed about 2-3 times greater biodegradation compared to the raw sludge in both aerobic and anaerobic conditions . In anaerobic experiments, biodegradation increased with ozone dosage up to 0.2 gO3/g-SS . Further increase of ozone dosage did not improve the biodegradation . In aerobic condition, about 77% of the ozonated sludge at 0.1 gO3/g-SS could be biodegraded after 15 days and is compared with 36% degradation of the untreated sludge . Most of the biodegradation of the ozonated sludge occurred within 5 days while the raw sludge was biodegraded steadily throughout the experimental period . The biodegradation enhancement of ozonated sludge was confirmed in batch denitrification experiments. Water Sci Technol, 2002, 46(4-5), 193 - 200 Combined denitrification and excess biological phosphorus removal in discontinuous operated biofilm systems; Brandt D et al.; The sorption-denitrification-P-removal (S-DN-P) process combines biological excess P-removal (BEPR) and denitrification using immobilized biomass . The accumulation of denitrifying polyP organisms is achieved by sequencing anaerobic/anoxic conditions . The immobilized biomass is in alternating contact with primary treated wastewater (anaerobic sorption-phase) and nitrified wastewater (denitrification phase) . In the sorption phase, P-release takes place and readily biodegradable organic substrate, e.g . volatile fatty acid, is taken up and stored by polyP accumulating organisms (PAO) . In addition to this, other organic matter is physically/chemically adsorbed in the biofilm structures . In the denitrification phase, the biomass denitrifies the stored and adsorbed organic substrate and, at the same time, P-uptake and polyP formation occurs . This paper presents results of investigations at laboratory and half-technical scale . At laboratory scale different types of carriers were tested regarding their suitability for the S-DN-P-process . In half-technical scale a biofilter and a moving bed reactor (MBR) were tested . In the biofilter a stable removal of nitrate and phosphate was achieved . However, it was not possible to achieve similar results in the MBR process . Especially the release and uptake of phosphate showed no clear tendency although the uptake of acetate was good . Reasons for this could be the accumulation of glycogen accumulating organisms which impair the metabolism of PAO. Water Sci Technol, 2002, 46(4-5), 131 - 7 Use pH and ORP as fuzzy control parameters of denitrification in SBR process; Peng YZ et al.; In order to achieve fuzzy control of denitrification in a Sequencing Batch Reactor (SBR) brewery wastewater was used as the substrate . The effects of brewery wastewater, sodium acetate, methanol and andogenous carbon source on the relationships between pH, ORP and denitrification were investigated . Also different quantities of brewery wastewater were examined . All the results indicated that the nitrate apex and nitrate knee occurred in the pH and ORP profiles at the end of denitrification . And when carbon was the limiting factor, through comparing the different increasing rate of pH whether the carbon was enough or not could be known, and when the carbon should be added again could be decided . On the basis of this, the fuzzy controller for denitrification in SBR was constructed, and the on-line fuzzy control experiments comparing three methods of carbon addition were carried out . The results showed that continuous carbon addition at a low rate might be the best method, it could not only give higher denitrification rate but also reduce the re-aeration time as much as possible . It appears promising to use pH and ORP as fuzzy control parameters to control the denitrification time and the addition of carbon. Biochim Biophys Acta, 2002 Sep 27, 1577(3), 355 - 76 Denitrifying genes in bacterial and Archaeal genomes; Philippot L; Denitrification, the reduction of nitrate or nitrite to nitrous oxide or dinitrogen, is the major mechanism by which fixed nitrogen returns to the atmosphere from soil and water . Although the denitrifying ability has been found in microorganisms belonging to numerous groups of bacteria and Archaea, the genes encoding the denitrifying reductases have been studied in only few species . Recent investigations have led to the identification of new classes of denitrifying reductases, indicating a more complex genetic basis of this process than previously recognized . The increasing number of genome sequencing projects has opened a new way to study the genetics of the denitrifying process in bacteria and Archaea . In this review, we summarized the current knowledge on denitrifying genes and compared their genetic organizations by using new sequences resulting from the analysis of finished and unfinished microbial genomes with a special attention paid to the clustering of genes encoding different classes of reductases . In addition, some evolutionary relationships between the structural genes are presented. Biochem J, 2003 Jan 1, 369(Pt 1), 77 - 88 Crystal structure of nitrous oxide reductase from Paracoccus denitrificans at 1.6 A resolution; Haltia T et al.; N2O is generated by denitrifying bacteria as a product of NO reduction . In denitrification, N2O is metabolized further by the enzyme N2O reductase (N2OR), a multicopper protein which converts N2O into dinitrogen and water . The structure of N2OR remained unknown until the recent elucidation of the structure of the enzyme isolated from Pseudomonas nautica . In the present paper, we report the crystal structure of a blue form of the enzyme that was purified under aerobic conditions from Paracoccus denitrificans . N2OR is a head-to-tail homodimer stabilized by a multitude of interactions including two calcium sites located at the intermonomeric surface . Each monomer is composed of two domains: a C-terminal cupredoxin domain that carries the dinuclear electron entry site known as Cu(A), and an N-terminal seven-bladed beta-propeller domain which hosts the active-site centre Cu(Z) . The electrons are transferred from Cu(A) to Cu(Z) across the subunit interface . Cu(Z) is a tetranuclear copper cluster in which the four copper ions (Cu1 to Cu4) are ligated by seven histidine imidazoles, a hydroxyl or water oxygen and a bridging inorganic sulphide . A bound chloride ion near the Cu(Z) active site shares one of the ligand imidazoles of Cu1 . This arrangement probably influences the redox potential of Cu1 so that this copper is stabilized in the cupric state . The treatment of N2OR with H2O2 or cyanide causes the disappearance of the optical band at 640 nm, attributed to the Cu(Z) centre . The crystal structure of the enzyme soaked with H2O2 or cyanide suggests that an average of one copper of the Cu(Z) cluster has been lost . The lowest occupancy is observed for Cu3 and Cu4 . A docking experiment suggests that N(2)O binds between Cu1 and Cu4 so that the oxygen of N2O replaces the oxygen ligand of Cu4 . Certain ligand imidazoles of Cu1 and Cu2, as well as of Cu4, are located at the dimer interface . Particularly those of Cu2 and Cu4 are parts of a bonding network which couples these coppers to the Cu(A) centre in the neighbouring monomer . This structure may provide an efficient electron transfer path for reduction of the bound N2O. Mol Microbiol, 2002 Sep, 45(6), 1741 - 50 The multicopper oxidase of Pseudomonas aeruginosa is a ferroxidase with a central role in iron acquisition; Huston WM et al.; Recently it has been observed that multicopper oxidases are present in a number of microbial genomes, raising the question of their function in prokaryotes . Here we describe the analysis of an mco mutant from the opportunistic pathogen Pseudomonas aeruginosa . Unlike wild-type Pseudomonas aeruginosa, the mco mutant was unable to grow aerobically on minimal media with Fe(II) as sole iron source . In contrast, both the wild-type and mutant strain were able to grow either anaerobically via denitrification with Fe(II) or aerobically with Fe(III) . Analysis of iron uptake showed that the mco mutant was impaired in Fe(II) uptake but unaffected in Fe(III) uptake . Purification and analysis of the MCO protein confirmed ferroxidase activity . Taken together, these data show that the mco gene encodes a multicopper oxidase that is involved in the oxidation of Fe(II) to Fe(III) subsequent to its acquisition by the cell . In view of the widespread distribution of the mco gene in bacteria, it is suggested that an iron acquisition mechanism involving multicopper oxidases may be an important and hitherto unrecognized feature of bacterial pathogenicity. Appl Environ Microbiol, 2002 Oct, 68(10), 5017 - 25 Detection and diversity of expressed denitrification genes in estuarine sediments after reverse transcription-PCR amplification from mRNA; Nogales B et al.; The expression of five denitrification genes coding for two nitrate reductases (narG and napA), two nitrite reductases (nirS and nirK), and nitrous oxide reductase (nosZ) was analyzed by reverse transcription (RT)-PCR of mRNA extracted from two sediment samples obtained in the River Colne estuary (United Kingdom), which receives high nitrogen inputs and for which high denitrification rates have been observed . The presence of all five genes in both sediment samples was confirmed by PCR amplification from extracted DNA prior to analysis of gene expression . Only nirS and nosZ mRNAs were detected; nirS was detected directly as an RT-PCR amplification product, and nosZ was detected following Southern blot hybridization . This indicated that active expression of at least the nirS and nosZ genes was occurring in the sediments at the time of sampling . Amplified nirS RT-PCR products were cloned and analyzed by sequencing, and they were compared with amplified nirS gene sequences from isolates obtained from the same sediments . A high diversity of nirS sequences was observed . Most of the cloned nirS sequences retrieved were specific to one site or the other, which underlines differences in the compositions of the bacterial communities involved in denitrifrification in the two sediments analyzed. J Biotechnol, 2002 Oct 23, 99(2), 161 - 71 Use of limestone for pH control in autotrophic denitrification: continuous flow experiments in pilot-scale packed bed reactors; Koenig A et al.; The sulfur-utilizing autotrophic denitrification process consumes about 4 g alkalinity (as CaCO(3)) per g NO(3)-N reduced resulting in a decrease of pH . Using limestone as an alkalinity source to control the pH, autotrophic denitrification of synthetic wastewater with varying alkalinity to NO(3)-N ratios was evaluated in pilot-scale packed bed reactors operating in the upflow mode, which contained limestone and sulfur granules in different volumetric ratios . The results demonstrated that limestone supplies effective buffering capacity, if the initial alkalinity of the wastewater is insufficient for complete denitrification . The alkalinity supplied by limestone is a function of hydraulic retention time and the pH, which in turn depends on the extent of biological denitrification and the initial alkalinity to NO(3)-N ratio in the wastewater . The dissolution rate of limestone is inversely proportional to pH for pH values lower than 7.1 . It was found that the ratio of influent alkalinity to theoretically required alkalinity in the wastewater should not be lower than 0.5 in order to prevent a decrease in nitrate removal performance . Based on the established chemical-biological interactive relationships, a multilayer approach was proposed to determine the optimum sulfur:limestone ratio for nitrate removal under steady state conditions, taking into account the characteristics of the influent wastewater . Environ Sci Technol, 2002 Sep 15, 36(18), 3977 - 84 A real-time polymerase chain reaction method for monitoring anaerobic, hydrocarbon-degrading bacteria based on a catabolic gene; Beller HR et al.; We have developed a real-time polymerase chain reaction (PCR) method that can quantify hydrocarbon-degrading bacteria in sediment samples based on a catabolic gene associated with the first step of anaerobic toluene and xylene degradation . The target gene, bssA, codes for the alpha-subunit of benzylsuccinate synthase . The primer-probe set for real-time PCR was based on consensus regions of bssA from four denitrifying bacterial strains; bssA sequences for two of these strains were determined during this study . The method proved to be sensitive (detection limit ca . 5 gene copies) and had a linear range of >7 orders of magnitude . We used the method to investigate how gasohol releases from leaking underground storage tanks could affect indigenous toluene-degrading bacteria . Microcosms inoculated with aquifer sediments from four different sites were incubated anaerobically with BTEX (benzene, toluene, ethylbenzene, and xylenes) and nitrate in the presence and absence of ethanol . Overall, population trends were consistent with observed toluene degradation activity: the microcosms with the most rapid toluene degradation also had the largest numbers of bssA copies . In the microcosms with the most rapid toluene degradation, numbers of bssA copies increased 100-to 1000-fold over the first 4 days of incubation, during which time most of the toluene had been consumed . These results were supported by slot blot analyses with unamplified DNA and by cloning and sequencing of putative bssA amplicons, which confirmed the real-time PCR method's specificity for bssA . Use of a companion real-time PCR method for estimating total eubacterial populations (based on 16S rDNA) indicated that, in some cases, ethanol disproportionately supported the growth of bacteria that did not contain bssA . The real-time PCR method for bssA could be a powerful tool for monitored natural attenuation of BTEX in fuel-contaminated groundwater . To our knowledge, this is the first reported molecular method that targets anaerobic, hydrocarbon-degrading bacteria based on a catabolic gene. Biomed Environ Sci, 2002 Jun, 15(2), 130 - 44 Microbial aspects of anaerobic BTEX degradation; Lin B et al.; Combined with conventional methods, developments in both geochemical (delineation of redox processes) and molecular microbial methods (analysis of 16S rDNA genes and functional genes) have allowed us to study in details microorganisms and genes involved in the anaerobic degradation of benzene, toluene, ethylbenzene and xylene (BTEX) under specific redox conditions . This review summarizes recent research in this field . The potential for anaerobic BTEX degradation is widely spread . Specific groups of microorganisms appear to be involved in degradation under different redox conditions . Members of the Azoarcus/Thauera cluster perform BTEX degradation under denitrifying conditions, Geobacteraceae under Fe (III) reducing conditions and Desulfobacteriaceae under sulfate reducing conditions . The information so far obtained on biochemistry and molecular genetics of BTEX degradation indicates that each BTEX compound is funneled into the central benzyol-CoA pathway by a different peripheral pathway . The peripheral pathways of per BTEX compound show similarities among different physiological groups of microorganisms . We also describe how knowledge obtained on the microbial aspects of BTEX degradation can be used to enhance and monitor anaerobic BTEX degradation. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1996, 28(5), 471 - 478 Cloning and Expression of the Cytochrome P-450nor2 cDNA from Cylindrocarpon tonkinense; Liu DL et al.; Cytochrome P-450nor is involved in the fungal denitrification and acts as a nitric oxide reductase . The cDNA library from Cylindrocarpon tonkinense was constructed with lambdagtll, and screened with antibodies . From the positive clones, the P-450nor2 cDNA fragments were recovered, and subcloned into the expression vector pYES2, then expressed in the yeast system . Western blot analysis showed that the expressed protein was hybridized with the antibody . Enzyme assay indicated that the expressed protein had the activities of P-450nor2, which reduced NO to form N(2)O, employing NADH or NADPH as the sole electron donor. Water Res, 2002 Aug, 36(14), 3497 - 506 A novel in situ technology for the treatment of nitrate contaminated groundwater; Haugen KS et al.; A novel in situ membrane technology was developed to remove nitrate (NO3-) from groundwater . Membrane-fed hydrogen gas (H2) was used as an electron donor to stimulate denitrification . A flow-through reactor fit with six hollow-fiber membranes (surface area = 93 cm2) was designed to simulate groundwater flowing through an aquifer with a velocity of 0.3 m/day . This membrane technology supported excellent NO3- and nitrite (NO2-) removal once H2 and carbon limitations were corrected . The membrane module achieved a maximum H2 flux of 1.79 x 10(-2) mg H2/m2 s, which was sufficient to completely remove 16.4 mg/L NO3(-)-N from a synthetic groundwater with no NO2- accumulation . In addition, this model in situ treatment process produced a high quality water containing <0.5 mg/L total organic carbon. Bioresour Technol, 2002 Nov, 85(2), 147 - 54 Nitrogen transformations during aerobic/anoxic sludge digestion; Al-Ghusain I et al.; Laboratory experiments were conducted to study and compare nitrogen transformations occurring under both aerobic digestion and aerobic/anoxic (A/A) digestion . The process performance was examined at different sludge residence times (SRTs), temperatures and anoxic cycles . Both modes of operation gave comparable solids reduction results . However, introduction of anoxic periods to aerobic sludge digestion appears to be a promising alternative to control pH during digestion through endogenous nitrate respiration (ENR) . Operating an aerobic digester with an anoxic phase to achieve complete denitrification would also improve supernatant quality over that achieved solely by aerobic digestion . Alternating A/A operation can conserve most of the influent alkalinity and maintain near neutral pH condition over prolonged periods . The A/A digestion of mixed primary/waste-activated sludge achieved up to 43.7% reductions in volatile suspended solids, 33.7% removal of total nitrogen, and a specific ENR rate of 5.75 x 10(-2) mg NO3-N/mg VSSd . Optimum results were obtained at 10 d SRT, 30 degrees C temperature, and 50% anoxic cycle length. Biochim Biophys Acta, 2002 Sep 10, 1555(1-3), 154 - 9 Respiratory chain supercomplexes of mitochondria and bacteria; Schagger H; Respiratory chain complexes are fragments of larger structural and functional units, the respiratory chain supercomplexes or "respirasomes", which exist in bacterial and mitochondrial membranes . Supercomplexes of mitochondria and bacteria contain complexes III, IV, and complex I, with the notable exception of Saccharomyces cerevisiae, which does not possess complex I . These supercomplexes often are stable to sonication but sensitive to most detergents except digitonin . In S . cerevisiae, a major component linking complexes III and IV together is cardiolipin.In Paracoccus denitrificans, complex I itself is rather detergent-sensitive and thus could not be obtained in detergent-solubilized form so far . However, it can be isolated as part of a supercomplex . Stabilization of complex I by binding to complex III was also found in human mitochondria . Further functional roles of the organization in a supercomplex are catalytic enhancement by reducing diffusion distances of substrates or, depending on the organism, channelling of the substrates quinone and cytochrome c . This makes redox reactions less dependent of midpoint potentials of substrates, and permits electron flow at low degree of substrate reduction.A dimeric state of ATP synthase seems to be specific for mitochondria . Exclusively, monomeric ATP synthase was found in Acetobacterium woodii, in P . denitrificans, and in spinach chloroplasts. Biochim Biophys Acta, 2002 Sep 10, 1555(1-3), 75 - 82 A turbo engine with automatic transmission? How to marry chemicomotion to the subtleties and robustness of life; Koefoed S et al.; Most genomes are much more complex than required for the minimum chemistry of life . Evolution has selected sophistication more than life itself . Could this also apply to bioenergetics? We first examine mechanisms through which bioenergetics could deliver sophistication . We illustrate possible benefits of the turbo-charging of catabolic pathways, of loose coupling, low-gear catabolism, automatic transmission in energy coupling, and of homeostasis . Mechanisms for such phenomena may reside at the level of individual proton pumps, or consist of rerouting of electrons over parallel pathways . The mechanisms may be confined to preexisting components, or involve the plasticity of gene expression that is so characteristic of most living organisms . These possible benefits lead us to the conjecture that also bioenergetics has evolved more for sophistication than for necessity.We next discuss a hitherto unresolved enigma, i.e . that bioenergetics does not seem to be critical for the physiological state . To decide on how critical bioenergetics is, we quantified the control exerted by catabolism on important physiological functions such as growth rate and growth yield . We also determined whether a growth inhibition mostly affected bioenergetics (catabolism) or anabolism; if ATP increases with growth rate, then growth should be considered energy (catabolism) limited . The experimental results for Escherichia coli pinpoint the enigma: its energy metabolism (catabolism) is not critical for growth rate.These results might suggest that because it has no direct control over cell function, bioenergetics is unimportant . Paradoxically however, in biology, highly important mechanisms tend to have little control on cell function, precisely because of that importance . Sophistication in terms of homeostatic mechanisms has evolved to guarantee robustness of the most important functions: The most important mechanisms are redundant in biology . Bioenergetics may be an excellent example of this paradox, in line with the above conjecture . It may be highly important and sophisticated.We then discuss work that has begun to focus on the sophistication of bioenergetics . Homeostasis of the energetics of DNA structure in E . coli is extensive . It relies both on preexisting components and on responsive gene expression . The vastly parallel electron-transfer network of Paracoccus denitrificans engages in sophisticated dynamic and hierarchical regulation . The growth yield of the organism can depend on which terminal oxidases are active . Effective proton translocation may vary due to rerouting of electrons . We conclude that much sophistication of bioenergetics will be discovered in this era of functional genomics. Water Sci Technol, 2002, 46(1-2), 55 - 60 Biological iron oxidation-reduction and the effects on sulfur oxidation-reduction, denitrification and poly-P accumulation in an anaerobic-oxic activated sludge; Yamamoto-Ikemoto R et al.; Iron oxidation and reduction were examined using the activated sludge from a municipal plant . Iron contents of the activated sludge were 1-2% . Iron oxidation rates were correlated with the initial iron concentrations . Iron reducing rates could be described by the Monod equation . The effects of iron reducing bacteria on sulfate reduction, denitrification and poly-P accumulation were examined . Iron reduction suppressed sulfate reduction by competing with hydrogen produced from protein . Denitrification was outcompeted with iron reduction and sulfate reduction . These phenomena could be explained thermodynamically . Poly-P accumulation was also suppressed by denitrification . The activity of iron reduction was relatively high. Water Sci Technol, 2002, 46(1-2), 389 - 95 Quantification of functional groups in activated sludge by microautoradiography; Nielsen JL et al.; Different functional groups of bacteria in activated sludge from a Danish municipal wastewater treatment plant were investigated and quantified on the basis of their ability to take up acetate under different electron acceptor conditions . The number of bacteria in the different functional groups was quantified by microautoradiography or by fluorescence in situ hybridization (FISH) . Uptake of radiolabeled acetate was tested under conditions where oxygen, nitrate, ferric iron, and sulfate served as electron acceptors and under methanogenic conditions . Ammonia-oxidizing bacteria and nitrite-oxidizing bacteria were enumerated by applying oligonucleotide probes (FISH) . 80% of the total DAPI count hybridised with a mixture of bacterial probes . Most baceria were able to take up acetate with oxygen or nitrate as electron acceptors (74 and 71% of all DAPI-stained bacteria, respectively) . The very similar numbers indicate that the alternating aerobic and anoxic conditions in the wastewater treatment plant investigated favoured the presence of facultative aerobic and denitrifying heterotrophic bacteria . The number of bacteria able to take up acetate under anaerobic conditions was around 8% of the total DAPI count . About half of these were able to take up acetate and store it and were thus probably phosphate-accumulating organisms . The remaining 4% of anaerobic acetate-consuming bacteria were mainly iron reducers . By incubating the sludge samples with specific inhibitors against sulfate reduction and methanogenic activity, it was found that sulfate reducers and methanogenic bacteria constituted approximately 1% and less than 0.5% of the total DAPI count, respectively. Water Sci Technol, 2002, 46(1-2), 333 - 6 PCR-DGGE analysis of denitrifying bacteria in a metallurgic wastewater treatment process; Noda N et al.; The wastewater generated from the processes of recovering precious metals from industrial wastes contains high concentrations of acids such as nitric acid and of salts . Biological nitrogen removal from this wastewater was attempted by using a circulating bioreactor system equipped with an anoxic packed bed or an anoxic fluidized bed and an aerobic three-phase fluidized bed . The system was found to effectively remove nitrogen from the diluted wastewater (T-N; 1,000-4,000 mg litre(-1)) . The microbial population structure of activated sludge in an anoxic reactor was analyzed by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA (rDNA) fragments . DGGE analysis under different operating conditions demonstrated the presence of some distinguishable bands in the separation pattern, which were most likely derived from many different species constituting the microbial communities . Furthermore, the population diversity varied in accordance with the nitrate-loading rate, water temperature and reactor condition . Some major DGGE bands were excised, reamplified and directly sequenced . It was revealed that the dominant population in the anoxic reactor were affiliated with the beta subclass of the class Proteobacteria. Water Sci Technol, 2002, 46(1-2), 323 - 6 Modelling population dynamics of denitrifying phosphorus accumulating organisms in activated sludge; Spagni A et al.; In this paper a kinetic model for simulating population dynamics is developed . The aim was to simulate anoxic and aerobic phosphorus accumulating organisms dynamics in a lab-scale sequencing batch reactor (SBR) operating under different conditions . Denitrifying phosphorus accumulating organisms (DNPAOs) successfully competed in almost all the simulated operational conditions tested . Using this model, the anoxic energy disadvantage of DNPAOs was not as strong as using metabolic models . The model was able to predict reasonably well N, P and acetate profiles in the SBR with only a minor modification of the ASM2d default values of the parameters. Water Sci Technol, 2002, 46(1-2), 119 - 22 First results from a screening of filamentous organisms present in Buenos Aires's activated sludge plants; Di Marzio WD; Activated sludge samples from municipal and industry plants were evaluated with the aim to recognise the mainly filamentous bacteria found . The routine tests recommended were performed: Gram, Neisser, PHB-Nilo blue epifluorescence reaction and S . The morphologic characteristics were determined . Correlation between environmental conditions and abundance of dominant bacteria were made . All plants were completely mixed configurations with anoxic reactor for denitrification ahead of main aerobic reactor . Also all systems were working at low F/M values or high sludge age (> ten days) and in some cases with low oxygen concentration . The dominant species were Type 021 N, Thiothrix I, Sphaerotilus natans, Microthrix parvicella, Nocardia sp, Type 1701, Type 1863, Type 0041 and Type 0092. Environ Sci Technol, 2002 Aug 15, 36(16), 3635 - 44 Development, operation, and long-term performance of a full-scale biocurtain utilizing bioaugmentation; Dybas MJ et al.; A full-scale field evaluation of bioaugmentation was conducted in a carbon tetrachloride (CT)- and nitrate-impacted aquifer at Schoolcraft, MI . The added organism was Pseudomonas stutzeri KC (strain KC), a denitrifying bacterium that cometabolically degrades CT without producing chloroform (CF) . To introduce and maintain strain KC in the aquifer, a row of closely spaced (1-m) injection/extraction wells were installed normal to the direction of groundwater flow near the leading edge of the CT plume . The resulting system of wells was used to establish and maintain a "biocurtain" for CT degradation through the intermittent addition of base to create favorable pH conditions; inoculation; and weekly addition of acetate (electron donor), alkali, and phosphorus . Although half of the test zone was inoculated twice, the long-term performance of both sections was indistinguishable: both had high CT removal efficiencies (median of 98-99.9%) and similar levels of strain KC colonization (>10(5) strain KC/g) . Sustained and efficient (98%) removal of CT has now been observed over 4 yr . Transient low levels of CF (<20 ppb) and H2S (<2 ppm) were observed, but both disappeared when the concentration of acetate in the weekly feed was reduced . Nitrate removal efficiencies ranged from 60% at low acetate concentrations to nearly 100% at high acetate concentrations . We conclude that closely spaced wells and intermittent substrate addition are effective means of delivering organisms and substrates to subsurface environments . At the Schoolcraft site, we achieved uniform removal efficiencies over a significant vertical depth (15 m), despite significant variability in hydraulic conductivity . This was accomplished by pumping 65% (v/v) of the natural gradient flow passing through the biocurtain during a given week in a single 6-h pumping event . Approximately 18,600 m3 of contaminated groundwater was treated during the project. J Environ Sci (China), 2002 Jul, 14(3), 296 - 302 Continuous treatment of azo acid dyes by photo-dependent denitrifying sludge; Hong JL et al.; Simultaneous removals of dye and nitrate by photo-dependent denitryfying sludge(PDDS) have been demonstrated in a continuous-flow bench-scale reactor . The best C/N for the degradation of azo dyes by PDDS was 1.5 . The specific removal rate of azo dye AB92 decreased with a decrease in hydraulic retention time and increased with a decrease in solids retention time . The degradation rate of TOC decreased with a decrease in hydraulic retention time . AB92, which has nitro and hydroxyl substitutions in non-para positions, was uniquely degraded . During continuous flow treatment experiments using PDDS, complete degradation of azo dyes AB92 and AO20 at influent concentrations of 40 mg/L and 30 mg/L, respectively, was achieved with an HRT of 16. J Basic Microbiol, 2002, 42(4), 268 - 76 The effects of NaCl and some heavy metals on the denitrification activity of Ochrobactrum anthropi; Kesseru P et al.; Ochrobactrum anthropi is a well-known Gram-negative bacterium, with the ability to degrade atrazine, urea-formaldehyde and chlorophenols . Investigation were made of the nitrate and nitrite reduction capacities of the strain in succinate and glucose media, and the tolerance of its denitrification to NaCl and some heavy metals . Succinate proved to be a better carbon source to drive denitrification by O . anthropi . Batch fermentation studies in anaerobic succinate medium indicated reduction capacities of 85.4 +/- 9.1 and 48.6 +/- 5.2 mgh(-1)g(-1) dry cell for NO(3) (-) and NO(2) (-), respectively . The nitrite accumulation of the cells revealed that O . anthropi is a group C denitrifying bacterium . Its growth in DSM 1 broth containing NaCl up to 40 g l(-1) demonstrates that O . anthropi belongs in the group of moderately halophilic bacteria . Despite the fact that 42.5 g NaCl l(-1) caused 50% growth inhibition in DSM 1 broth, the cells in the stationary phase readily tolerated NaCl concentrations up to 100 g l(-1) . Complete denitrification was achieved in test media containing 30 g NaCl l(-1) after 1 week and the nitrate reductase retained its activity up to 100 g NaCl l(-1) . The cells were tolerant to Hg, Zn, Pb, Cu and Ni, and N(2) was producted at tolerated concentrations of the metal in the cases of Hg and Pb. Arch Microbiol, 2002 Oct, 178(4), 279 - 87 Epub 2002 Jul 12. Aerobic and anaerobic metabolism of squalene by a denitrifying bacterium isolated from marine sediment; Rontani JF et al.; The aerobic and anaerobic metabolism of the isoprenoid alkene squalene was investigated in a new type of marine denitrifying bacterium, strain 2sq31, isolated from marine sediment . Strain 2sq31 was identified as a species of Marinobacter . Under denitrifying conditions, the strain efficiently degraded squalene; of 0.7 mmol added per liter of medium, 77% was degraded within 120 days under anoxic conditions with nitrate as electron acceptor . Tertiary diols and methyl ketones were identified as metabolites, and an anaerobic pathway was suggested to explain the formation of such compounds . The first step in anaerobic degradation of squalene by strain 2sq31 involves hydration of double bonds to tertiary alcohols . Under oxic conditions, the degradation of squalene by strain 2sq31 was rapid and involved oxidative splitting of the C-10/C-11 or C-14/C-15 double bonds, in addition to the pathways observed under denitrifying conditions. J Clin Microbiol, 2002 Sep, 40(9), 3538 - 9 Disseminated infection due to Blastobacter denitrificans following routine appendectomy in an adolescent; Trotha R et al.; Until now, Blastobacter denitrificans has not been mentioned in the context of human infections . A case of severe complication caused by B . denitrificans after routine appendectomy in a young girl is described and confirms this organism to be an opportunistic human pathogen. Water Sci Technol, 2002, 45(12), 53 - 61 Sequential variation of groundwater quality in an agricultural area with greenhouses near the coast; Fujiwara T et al.; The quality of coastal groundwater utilized for greenhouse-farming is liable to be affected by not only fertilization practices but also seawater intrusion into the aquifer . In this study, the sequential variations of groundwater quality and the mechanisms of its pollution processes were analyzed through successive field measurements from June 2000 to May 2001 in such a coastal agricultural area located in Kochi prefecture, Japan . The analysis revealed that the ions for NO3-, SO4(2-), Mg2+ and Ca2+ in the groundwater originated primarily from the ground surface due to the seasonal flooding activities . It was also estimated that denitrification occurred at three well points, and the rate constants were 0.010, 0.019 and 0.089 d-1, respectively . Na+, Mg2+ and Ca2+ concentrations in the groundwater governed by seawater intrusion phenomena showed strong correlations with the concentration of Cl- by excluding the influence of fertilizer . Cation exchange phenomena between Ca2+ and Na+ and as well as the mixing with seawater had a considerable effect on the groundwater quality. Water Sci Technol, 2002, 45(12), 13 - 21 Present state of food and feed cycle and accompanying issues around Japan; Ukita M et al.; Nitrogen (N) and phosphorus (P) cycles of food and feed, and the regulation systems for industrial wastewater are studied . The rate of domestic supply of food in Japan is 41% in 1970, 32% in 1990, and 29% in 1998 for N and 33%, 29%, and 28% for P, excluding grass feed . Among 3 countries, Japan, USA and Thailand, Japan is in the most difficult state to recycle food wastes to farmland . Comparing the estimated load of wastewater from food processing industries with the estimated load according to the budget study for N and P cycles, the tentative criteria of wastewater of food processing for P seem to be loose, and denitrification might be considered in every stage of N cycles . New regulation of industrial wastewater recently proposed in Japan may induce the chance for environmental business to remove N and P. Appl Environ Microbiol, 2002 Sep, 68(9), 4539 - 45 The bacterivorous soil flagellate Heteromita globosa reduces bacterial clogging under denitrifying conditions in sand-filled aquifer columns; Mattison RG et al.; An exopolymer (slime)-producing soil bacterium Pseudomonas sp . (strain PS+) rapidly clogged sand-filled columns supplied with air-saturated artificial groundwater containing glucose (500 mg liter(-1)) as a sole carbon source and nitrate (300 mg liter(-1)) as an alternative electron acceptor . After 80 days of operation under denitrifying conditions, the effective porosity and saturated hydraulic conductivity (permeability) of sand in these columns had fallen by 2.5- and 26-fold, respectively . Bacterial biofilms appeared to induce clogging by occluding pore spaces with secreted exopolymer, although there may also have been a contribution from biogas generated during denitrification . The bacterivorous soil flagellate Heteromita globosa minimized reductions in effective porosity (1.6-fold) and permeability (13-fold), presumably due to grazing control of biofilms . Grazing may have limited growth of bacterial biomass and hence the rate of exopolymer and biogas secretion into pore spaces . Evidence for reduction in biogas production is suggested by increased nitrite efflux from columns containing flagellates, without a concomitant increase in nitrate consumption . There was no evidence that flagellates could improve flow conditions if added once clogging had occurred (60 days) . Presumably, bacterial biofilms and their secretions were well established at that time . Nevertheless, this study provides evidence that bacterivorous flagellates may play a positive role in maintaining permeability in aquifers undergoing remediation treatments. BMC Microbiol . 2002 Aug 28;2(1):23. Diversity and abundance of bacteria in an underground oil-storage cavity; Watanabe K et al.; BACKGROUND: Microorganisms inhabiting subterranean oil fields have recently attracted much attention . Since intact groundwater can easily be obtained from the bottom of underground oil-storage cavities without contamination by surface water, studies on such oil-storage cavities are expected to provide valuable information to understand microbial ecology of subterranean oil fields . RESULTS: DNA was extracted from the groundwater obtained from an oil-storage cavity situated at Kuji in Iwate, Japan, and 16S rRNA gene (16S rDNA) fragments were amplified by PCR using combinations of universal and Bacteria-specific primers . The sequence analysis of 154 clones produced 31 different bacterial sequence types (a unique clone or group of clones with sequence similarity of > 98) . Major sequence types were related to Desulfotomaculum, Acetobacterium, Desulfovibrio, Desulfobacula, Zoogloea and Thiomicrospira denitrificans . The abundance in the groundwater of bacterial populations represented by these major sequence types was assessed by quantitative competitive PCR using specific primers, showing that five rDNA types except for that related to Desulfobacula shared significant proportions (more than 1%) of the total bacterial rDNA . CONCLUSIONS: Bacteria inhabiting the oil-storage cavity were unexpectedly diverse . A phylogenetic affiliation of cloned 16S rDNA sequences suggests that bacteria exhibiting different types of energy metabolism coexist in the cavity. Biochem Soc Trans, 2002 Aug, 30(4), 662 - 7 Comparison between the nitric oxide reductase family and its aerobic relatives, the cytochrome oxidases; de Vries S et al.; The denitrification pathway has been studied in the hyperthermophilic archaeon Pyrobaculum aerophilum . In contrast with Gram-negative bacteria, all four denitrification enzymes are membrane-bound . P . aerophilum is also the only denitrifyer identified so far in which menaquinol is the electron donor to all four denitrification reductases . The NO reductase (NOR) of P . aerophilum belongs to the superfamily of haem-copper oxidases and is of the qNOR (quinol-dependent) type . Three types of NOR have been purified so far: cNOR (cytochrome c/pseudoazurin-dependent), qNOR and qCu(A)NOR (qNOR that contains Cu(A) at the electron entry site) . It is proposed that the NORs and the various cytochrome oxidases have evolved by modular evolution, in view of the structure of their electron donor sites . qNOR is further proposed to be the ancestor of all NORs and cytochrome oxidases belonging to the superfamily of haem-copper oxidases. Biochemistry, 2002 Sep 3, 41(35), 10858 - 65 Properties of a soluble domain of subunit C of a bacterial nitric oxide reductase; Oubrie A et al.; Bacterial nitric oxide reductases are integral membrane proteins that catalyze the reduction of two molecules of nitric oxide to nitrous oxide and water . They are diverged members of the superfamily of heme/copper oxidases . The enzyme from Paracoccus denitrificans (NorBC) contains two subunits; NorB comprises the membrane-integrated active site, which harbors a heme iron/non-heme iron dinuclear center . NorC is a membrane-anchored c-type cytochrome and presumably the site of electron uptake . A DNA construct encoding the water-soluble domain of NorC (NorC(sol)) was coexpressed with the cytochrome c maturation genes in Escherichia coli . Using redox potentiometry, electronic absorption, circular dichroism (CD), magnetic CD (MCD), nuclear magnetic resonance, and electron paramagnetic resonance (EPR) spectroscopy the following observations were made: (i) NorC(sol) was folded into a alpha-helical structure . (ii) The low-spin heme iron was coordinated by histidine and methionine in both redox states . (iii) The midpoint redox potential of the NorC(sol) heme was 183 mV, much lower than the corresponding value of 275 mV in the NorBC complex . This points to an increased solvent exposure of the NorC(sol) heme compared to in the native NorBC complex and shows that the electronic properties of NorC are modulated by NorB in the complex . (iv) The EPR and MCD spectra of NorC(sol) were considered alongside the spectra of NorBC, which has helped to resolve the contribution that different redox centers make in the holo-enzyme complex. Environ Monit Assess, 2002 Aug, 77(3), 293 - 309 Microbiological characteristics in a zero-valent iron reactive barrier; Gu B et al.; Zero-valent iron (Fe0)-based permeable reactive barrier treatment has been generating great interest for passive groundwater remediation, yet few studies have paid particular attention to the microbial activity and characteristics within and in the vicinity of the Fe0-barrier matrix . The present study was undertaken to evaluate the microbial population and community composition in the reducing zone of influence by Fe0 corrosion in the barrier at the Oak Ridge Y-12 Plant site . Both phospholipid fatty acids and DNA analyses were used to determine the total microbial population and microbial functional groups, including sulfate-reducing bacteria, denitrifying bacteria, and methanogens, in groundwater and soil/iron core samples . A diverse microbial community was identified in the strongly reducing Fe0 environment despite a relatively high pH condition within the Fe0 barrier (up to pH approximately 10) . In comparison with those found in the background soil/groundwater samples, the enhanced microbial population ranged from approximately 1 to 3 orders of magnitude and appeared to increase from upgradient of the barrier to downgradient soil . In addition, microbial community composition appeared to change over time, and the bacterial types of microorganisms increased consistently as the barrier aged . DNA analysis indicated the presence of sulfate-reducing and denitrifying bacteria in the barrier and its surrounding soil . However, the activity of methanogens was found to be relatively low, presumably as a result of the competition by sulfate/metal-reducing bacteria and denitrifying bacteria because of the unlimited availability of sulfate and nitrate in the site groundwater . Results of this study provide evidence of a diverse microbial population within and in the vicinity of the iron barrier, although the important roles of microbial activity, either beneficially or detrimentally, on the longevity and enduring efficiency of the Fe0 barriers are yet to be evaluated. Water Sci Technol, 2002, 45(10), 365 - 70 Sludge bed development in denitrifying reactors using different inocula-performance and microbiological aspects; Etchebehere C et al.; Aerobic and methanogenic consortia were evaluated as inocula for laboratory scale denitrifying reactors, fed with a synthetic wastewater with acetate as the main electron donor . The denitrifying microflora of inocula and reactors was evaluated by specific denitrifying activity, enumeration and isolation of denitrifiers, which were screened by amplified ribosomal DNA restriction analysis . Reactor performance was monitored by COD and nitrate removal efficiencies and granule size . The aerobic sludge failed to form granules, probably due to the development of a filamentous, nitrate-reducing organism which was characterised by 16SrDNA sequencing as Bacillus cereus . The methanogenic sludge showed denitrifying activity and adapted very rapidly to denitrifying conditions in the two reactors seeded with granules of different sizes . Denitrifiers grew around the granules, increasing the specific denitrifying activity of the sludge over 10-fold . Exopolymer-forming organisms, belonging to the same species, were isolated from both reactors . Granule size increased during operation, but flotation of the aggregates, related to gas retention was observed. Water Sci Technol, 2002, 45(10), 335 - 40 Adaptation of the methanogenic granules to denitrification in anaerobic-anoxic USSB reactor; Jenicek P et al.; Continuous laboratory experiments have confirmed that the construction of the USSB reactor was suitable for the combination of several biological wastewater treatment processes and that both anaerobic and denitrifying anoxic granules could be cultivated inside a compact reactor . The anoxic granules were transformed from the anaerobic granules after the shift from anaerobic to anoxic conditions in the upper compartments of the USSB reactor . The denitrification in such a system can be very intensive and efficient because of the high concentration of the sludge in anoxic compartments, and the high activity of the sludge due to a high and stable temperature . The denitrification rate of 62 mg N/l.h (at 35 degrees C) was achieved in spite of COD-limited conditions in the anoxic compartment of the USSB reactor . Maximum specific activity of anoxic granules 47 mg N/g VSS.h (at 35 degrees C, with glucose as substrate) was found in batch tests . The anoxic granules originating from the anaerobic granules have still demonstrated a significant methanogenic activity comparable with the anaerobic granules. Water Sci Technol, 2002, 45(10), 281 - 6 Co-digestion of domestic kitchen waste and night soil sludge in a full-scale sludge treatment plant; Yoneyama Y et al.; A study was made on the domestic kitchen waste and night soil treatment performance of a full-scale sludge treatment plant . The sludge treatment at this plant was by thermophilic methane fermentation . The initial treatment, mesophilic to thermophilic fermentation, was able to be started up within a short time by adjusting the amount of influent waste . Thermophilic methane fermentation was carried out for five months (May-October) and the performance under a mean residual time of 22 days indicated a VTS decomposition of 42%, gas generation of 54-1,610 m3/day (average: 755 m3/day), and a mean methane concentration of 60% . The methane gas was used to generate power in the plant and the amount of power generated by methane gas was highest in October (average of 1,200 kWh/day) . This was equivalent to about 7% of the power consumed at the entire sludge treatment plant . The BOD/NH4-N of the activated sludge influent water was lower, compared to a case where there is no recycle flow, due to the recycle flow from the methane fermentation process . There was, therefore, a tendency for an increase in the amount of methanol charged into the secondary denitrification tank . However, the quality of the effluent was satisfactory (BOD< 10 mg/L, SS< 5 mg/L, and T-N< 25 mg/L) . Study results indicated that it was possible to implement a full-scale plant for recovering organic waste. Water Res, 2002 Jul, 36(13), 3330 - 40 Improved brine recycling during nitrate removal using ion exchange; Bae BU et al.; Ion exchange technology is currently the best for removing nitrate from drinking water . However, problems related to the disposal of spent brine from regeneration of exhausted resins must be overcome so that ion exchange can be applied more widely and economically, especially in small communities . For this purpose, a novel spent brine recycling system using combined biological denitrification and sulfate reduction processes was developed for more efficient reuse of brine . A granular activated carbon (GAC) adsorption column was introduced as an additional step to prevent contamination of resins by bio-polymers and dissolved organics present in the bio-reactor effluent . Two upflow sludge blanket reactors (USBRs) were operated in series for 166 days to provide denitrification and sulfate reduction . The denitrification reactor provided a nitrate removal efficiency of 96% at a nitrate-N loading rate of 5.4 g NO3(-)-N/l d . The sulfate reduction efficiency of the sulfate reduction reactor remained approximately 62% at a sulfate loading rate of 1.8 g SO4(2-)/l d . Five ion exchange columns containing A520E resins were repeatedly operated in up to 25 cycles of service and regeneration using five kinds of brine: one virgin 3% NaCl and four differently recycled spent brines . Throughput decreased remarkably when the biologically recycled brine was not treated with the GAC column, probably due to the presence of bio-polymers and dissolved organic compounds . The sulfate reduction reactor placed after the denitrification step increased the bicarbonate concentration, which could be used as a co-regenerant with chloride . The inclusion of the sulfate reduction reactor into the conventional brine recycling system allowed more efficient reuse of brine, resulting in both reduced salt consumption and brine discharge. J Biochem Mol Biol Biophys, 2002 Jun, 6(3), 221 - 4 Gene cluster of nitrous oxide reduction in the deep sea of mariana trench; Tamegai H et al.; The gene of nitrous oxide reductase (nosZ) and entire region of nos gene cluster from Pseudomonas sp . strain MT-1, which is isolated from the sediment of Mariana Trench, was identified . Gene organization in nos gene cluster is similar to that of other bacteria, and nosZ was highly homologous to nosZ of other Pseudomonads . The remarkable property was the existence of a gap from Arg-302 to Ser-309 (numbered by NosZ of P . aeruginosa), which might be a feature of marine bacterial NosZ . It is interesting that nosZ of the strain MT-1 is not related so closely to P . stutzeri, although 16S rDNA of the strain MT-1 is highly homologous to that of P . stutzeri . The existence of nosZ gene in the strain MT-1 indicated the presence of a complete denitrification (nitrate to N(2)) in the Mariana Trench . This is the first report on genetic analysis of denitrification in the deep sea. Biochem J, 2002 Dec 1, 368(Pt 2), 425 - 32 Identification of two domains and distal histidine ligands to the four haems in the bacterial c-type cytochrome NapC; the prototype connector between quinol/quinone and periplasmic oxido-reductases; Cartron ML et al.; NapC is a tetra-haem member of a family of bacterial membrane-anchored multi-haem c -type cytochromes implicated in electron transfer between membrane quinols and periplasmic enzymes . The water-soluble tetra-haem fragment of Paracoccus pantotrophus NapC has been expressed as a periplasmic protein (NapC(sol)) in Paracoccus denitrificans, P . pantotrophus and Escherichia coli . Site-specific mutagenesis of NapC(sol), combined with spectroscopic studies, suggests that each haem iron centre has bis -histidinyl co-ordination . Four proximal ligands arise from each of four Cys-Xaa-Xaa-Cys-His haem-binding motifs; candidates for the four distal ligands are His(81), His(99), His(174) and His(194) . NapC(H81A), NapC(H99A), NapC(H174A) and NapC(H194A) mutants (with alanine substituted for each of the four candidate residues) have all been purified from E . coli . In each case, one of the haems has become high-spin, as judged by the presence of a broad absorption band between 620 nm and 650 nm for the oxidized cytochrome; this feature is absent for wild-type protein and presumably arises because of the absence of the distal histidine ligand from one of the haems . NapC(H81A) and NapC(H174A) are less well expressed in E . coli than NapC(H99A) and NapC(H194A) and cannot be detected when expressed in P . denitrificans or P . pantotrophus . In vitro and in vivo complementation studies demonstrate that the soluble periplasmic NapC can mediate electron transfer from quinols to the periplasmic nitrate reductase . This capacity was retained in vitro with the NapC(H99A) and NapC(H194A) mutants but was lost in vivo . A model for the structural organization of NapC(sol) into two domains, each containing a di-haem pair, is proposed . In this model, each haem pair obtains one distal haem ligand from its own domain and a second from the other domain . The suggestion of two domains is supported by observations that the 24 kDa NapC(sol) cleaves to yield a 12 kDa haem-staining band . Determination of the cleavage site showed it was between two equally sized di-haem domains predicted from sequence analysis. Biochemistry, 2002 Aug 27, 41(34), 10629 - 34 Specificity of the interaction between the Paracoccus denitrificans oxidase and its substrate cytochrome c: comparing the mitochondrial to the homologous bacterial cytochrome c(552), and its truncated and site-directed mutants; Drosou V et al.; Under in vitro conditions, bacterial cytochrome c oxidases may accept several nonhomologous c-type electron donors, including the evolutionarily related mitochondrial cytochrome c . Several lines of evidence suggest that in intact membranes the heme aa(3) oxidase from Paracoccus denitrificans receives its electrons from the membrane-bound cytochrome c(552) . Both the structures of the oxidase and of a heterologously expressed, soluble fragment of the c(552) have been determined recently, but no direct structural information about a static cocomplex is available . Here, we analyze the kinetic properties of the isolated oxidase with the full-size c(552), with two truncated soluble forms, and with a set of site-specific mutants within the presumed docking site of the cytochrome, all heterologously expressed in Escherichia coli . Our data indicate that all three forms, the wild type and both truncations, are fully competent kinetically and exhibit biphasic kinetic behavior, however, under widely different ionic strength conditions . When mutations in lysine residues clustered around the interaction domain were introduced into the smallest fragment of c(552), both kinetic parameters, K(M) and k(cat), were drastically influenced . On the other hand, when the nonmutated truncated form was used to donate electrons to a set of oxidase mutants with replacements clustered along the docking site on subunit II, we observe distinct differences when comparing the kinetic properties of the widely used horse heart cytochrome c with those of the bacterial c(552) . We conclude that the specific docking sites for the two types of cytochromes differ to some extent. Acta Microbiol Pol, 2002, 51(1), 47 - 56 Effect of nitrates on biotransformation of phosphogypsum and phenol uptake in cultures of autochthonous sludge microflora from petroleum refining wastewaters; Kowalski W et al.; The effect of nitrates on the biotransformation of phosphogypsum at 30 degrees C in stationary cultures of anaerobic, heterogeneous microflora growing in medium with phenol (250-1,000 mg/L) as sole carbon source was studied . The microorganisms used in this study were isolated from sludge in biological petroleum-refining wastewater treatment plant . Phosphogypsum (a waste product in the chemical industry that contains approximately 95% CaSO4) was added in amount of 5 g/L, the source of nitrates was KNO3 in concentration equivalent to that of phenol (250-1,000 mg N-NO3/L) . The presence of nitrates in heterogeneous cultures has an inhibitory effect on the process of phosphogypsum biotransformation and stimulates the uptake of phenol . We have found that in cultures in medium containing phenol, phosphogypsum and nitrates at least three physiological groups of microorganisms were present . These were phenol-biodegrading microorganisms not requiring an external electron acceptor, sulfate-reducing bacteria biodegrading phenol or intermediate products of its breakdown and denitrifying bacteria not utilising phenol as a carbon source . On solid medium these bacteria together formed heterogeneous single colonies . In spite of repeated attempts we were unable to isolate pure strains and the only result of these measures was loss of denitrification ability in medium with phenol. Acta Microbiol Pol, 2002, 51(1), 39 - 45 Phenol removal in packed bed reactor under denitrifying conditions; Blaszczyk M et al.; Detailed studies on the efficiency of phenol degradation by a biofilm in an anaerobic packed bed reactor were carried out . The efficiency of phenol degradation depended on both the concentration of phenol in the medium and the phenol load in anaerobic packed bed reactor . Increasing phenol concentrations from 200 to 1,250 mg l(-1) and retention time (Tr)= 12 h were paralleled by increasing efficiency of the process, which reached a maximum value of 1,390 mg l(-1) day(-1) at 700 mg phenol l(-1) . The highest concentration of phenol used inhibited growth by approximately 95% . When the phenol load in medium containing 200, 300, 400 and 500 mg l(-1) was increased through a shortening of the retention time (Tr from 24 to 2 h) a maximum efficiency of phenol degradation of 2,200 mg l(-1) day(-1) was obtained at Tr=4 h and phenol concentrations in the medium of 200 mg l(-1) . Phenol in concentrations from 300 to 500 mg l(-1) was fully degraded at Tr>9 h and phenol load reaching 530-1330 mg l(-1) day(-1) for the individual concentrations . The post-denitrification effluent leaving packed bed reactor in spite of the absence or even trace amounts of phenol in it requires further purification. Eur J Biochem, 2002 Aug, 269(16), 4020 - 4 NADH oxidation and NAD+ reduction catalysed by tightly coupled inside-out vesicles from Paracoccus denitrificans; Kotlyar AB et al.; Tightly coupled inside-out vesicles were prepared from Paracoccus denitrificans cells (SPP, sub-Paracoccus particles) and characterized kinetically . The rate of NADH oxidation, catalysed by SPP, increases 6-8 times on addition of gramicidin . The vesicles are capable of catalysing Delta micro H+-dependent reverse electron transfer from quinol to NAD+ . The kinetic parameters of the NADH-oxidase and the reverse electron transfer carried out by membrane-bound P . denitrificans complex I were estimated and compared with those of the mitochondrial enzyme . The data demonstrate that catalytic properties of the dinucleotide-binding site of the bacterial and mitochondrial complex I are almost identical, pointing out similar organization of the site in mammals and P . denitrificans . Inhibition of the bacterial complex I by a specific inhibitor of Q reduction, rotenone, is very different from that of the mitochondrial enzyme . The inhibitor is capable of suppressing the NADH oxidation reaction only at micromolar concentrations, while the activity of mitochondrial enzyme is suppressed by nanomolar concentrations of rotenone . In contrast to the mitochondrial enzyme, rotenone, even at concentrations as high as 10 micro m, does not inhibit the reverse, Delta micro H+-dependent NAD+-reductase reaction on SPP. Sci Total Environ, 2002 Jul 22, 294(1-3), 95 - 110 Hydrological controls on nutrient concentrations and fluxes in agricultural catchments; Petry J et al.; Like many streams draining intensively farmed parts of lowland Scotland, water quality in the Newmills burn, Aberdeenshire, is characterized by relatively high nutrient levels; mean concentrations of NO3-N and NH3-N are 6.09 mg l(-1) and 0.28 mg l(-1), respectively, whilst average PO4-P concentrations reach 0.06 mg l(-1) . Nutrient concentrations vary spatially and temporally with levels being highest under arable farming during the autumn and winter . Annual fluxes from the 14.5 km2 catchment are estimated at 25.67 and 1.26 kg ha(-1) a(-1) for NO3-N and NH3-N, respectively, and 0.26 kg ha(-1) a(-1) for PO4-P . Hydrological controls exert a strong influence on both nutrient concentrations and fluxes . Over short timescales nutrient concentrations and fluxes are greatest during storm events when P04-P and NH3-N are mobilized by overland flow in riparian areas, particularly where the soils have been compacted by livestock or farm machinery . Delivery of deeper soil water in subsurface storm flow, facilitated by agricultural under-drainage, provide large contributions of NO3-N on the recession limb of hydrological events . In contrast, groundwater inputs generally have lower NO3 concentrations implying that denitrification may be a pathway of N loss in the saturated zone . Approximately 75% of the N loss for the catchment occurs during the autumn and early winter when high flows dominate the hydrological regime . The close coupling of hydrological pathways and biogeochemical processes has major implications for catchment management strategies such as Nitrate Vulnerable Zones (NVZs) as it is likely that significant groundwater stores with long residence times will continue to cause N losses before water quality improvements become apparent. FEMS Microbiol Lett, 2002 Aug 6, 213(2), 183 - 8 Characterization of bacterial consortia capable of degrading 4-chlorobenzoate and 4-bromobenzoate under denitrifying conditions; Song B et al.; 4-Chlorobenzoate and 4-bromobenzoate were readily degraded in denitrifying enrichment cultures established with river sediment, estuarine sediment or agricultural soil as inoculum . Stable denitrifying consortia were obtained and maintained by serial dilution and repeated feeding of substrates . Microbial community analyses were performed to characterize the 4-chlorobenzoate and 4-bromobenzoate degrading consortia with terminal restriction fragment length polymorphism (T-RFLP) and cloning of 16S rRNA genes from the cultures . Interestingly, two major terminal restriction fragments (T-RFs) in the 4-chlorobenzoate degrading consortia and one T-RF in the 4-bromobenzoate utilizing consortium were observed from T-RFLP analysis regardless of their geographical and ecological origins . The two T-RFs (clones 4CB1 and 4CB2) in 4-chlorobenzoate degrading consortia were identified as members of the beta-subunit of the Proteobacteria on the basis of 16S rRNA sequencing analysis . Phylogenetic analysis of 16S rRNA genes showed that clone 4CB1 was closely related to Thauera aromatica while clone 4CB2 was distantly related to the genera Limnobacter and Ralstonia . The 4-bromobenzoate utilizing consortium mainly consisted of one T-RF, which was identical to clone 4CB2 in spite of different enrichment substrate . This suggests that degradation of 4-chlorobenzoate and 4-bromobenzoate under denitrifying conditions was mediated by bacteria belonging to the beta-subunit of the Proteobacteria. Environ Pollut, 2002, 119(3), 413 - 20 Effects of macrophytes and external carbon sources on nitrate removal from groundwater in constructed wetlands; Lin YF et al.; Several microcosm wetlands unplanted and planted with five macrophytes (Phragmites australis, Commelina communis, Penniserum purpureum, Ipomoea aquatica, and Pistia stratiotes) were employed to remove nitrate from groundwater at a concentration of 21-47 mg NO3-N/l . In the absence of external carbon, nitrate removal rates ranged from 0.63 to 1.26 g NO3-N/m2/day for planted wetlands . Planted wetlands exhibited significantly greater nitrate removal than unplanted wetlands (P<0.01), indicating that macrophytes are essential to efficient nitrate removal . Additionally, a wetland planted with Penniserum showed consistently higher nitrate removal than those planted with the other four macrophytes, suggesting that macrophytes present species-specific nitrate removal efficiency possibly depending on their ability to produce carbon for denitrification . Although adding external carbon to the influent improved nitrate removal, a significant fraction of the added carbon was lost via microbial oxidation in the wetlands . Planting a wetland with macrophytes with high productivity may be an economic way for removing nitrate from groundwater . According to the harvest result, 4-11% of nitrogen removed by the planted wetland was due to vegetation uptake, and 89-96% was due to denitrification. Chemosphere, 2002 Jul, 48(4), 445 - 51 A comparison of organic and inorganic carbon controls over biological denitrification in aquaria; Vidal S et al.; In aquaria and rearing tanks, nitrate accumulation as a result of organic matter degradation is inevitable and has two major negative side effects: direct toxicity to organisms, specially invertebrates, and the introduction of a reducing environment by oxygen consumption . The aim of this study was to compare two alternate methods of removing nitrogen compounds from closed systems, autotrophic columnar denitrification (ACD) and heterotrophic columnar denitrification (HCD) by following end product concentrations as reaction progressed . A pilot plant consisting of two series of 50 dm3 recirculating flow systems (each in triplicate) was used to test both methods . Absence of pH control was also useful in autotrophic denitrification systems in order to follow effects over reaction rates and pathways . Concentrations of NO(3-), NO2- and NH(4+) were followed throughout the experiment, as well as pH, temperature and salinity . Under different flow conditions results show that higher nitrate reduction rates were possible in the autotrophic systems (35.1+/-4.7 microM/day without pH control until reversal of the process and 20.6+/-7.3 microM/day after reestablishment of pH control) in comparison with heterotrophic (9.9+/-1.3 microM/day) . However, pH control through calcium bicarbonate addition was found to be crucial in maintaining constant levels of total denitrification in ACD systems, just as it was necessary to closely maintain organic carbon addition to HCD systems. Appl Environ Microbiol, 2002 Aug, 68(8), 3818 - 29 Biodiversity of denitrifying and dinitrogen-fixing bacteria in an acid forest soil; Rosch C et al.; Isolated soil DNA from an oak-hornbeam forest close to Cologne, Germany, was suitable for PCR amplification of gene segments coding for the 16S rRNA and nitrogenase reductase (NifH), nitrous oxide reductase (NosZ), cytochrome cd(1)-containing nitrite reductase (NirS), and Cu-containing nitrite reductase (NirK) of denitrification . For each gene segment, diverse PCR products were characterized by cloning and sequencing . None of the 16S rRNA gene sequences was identical to any deposited in the data banks, and therefore each of them belonged to a noncharacterized bacterium . In contrast, the analyzed clones of nifH gave only a few different sequences, which occurred many times, indicating a low level of species richness in the N2-fixing bacterial population in this soil . Identical nifH sequences were also detected in PCR amplification products of DNA of a soil approximately 600 km distant from the Cologne area . Whereas biodiversity was high in the case of nosZ, only a few different sequences were obtained with nirK . With respect to nirS, cloning and sequencing of the PCR products revealed that many false gene segments had been amplified with DNA from soil but not from cultured bacteria . With the 16S rRNA gene data, many sequences of uncultured bacteria belonging to the Acidobacterium phylum and actinomycetes showed up in the PCR products when isolated DNA was used as the template, whereas sequences obtained for nifH and for the denitrification genes were closely related to those of the proteobacteria . Although in such an experimental approach one has to cope with the enormous biodiversity in soils and only a few PCR products can be selected at random, the data suggest that denitrification and N2 fixation are not genetic traits of most of the uncultured bacteria. Appl Environ Microbiol, 2002 Aug, 68(8), 3802 - 8 Factors controlling anaerobic ammonium oxidation with nitrite in marine sediments; Dalsgaard T et al.; Factors controlling the anaerobic oxidation of ammonium with nitrate and nitrite were explored in a marine sediment from the Skagerrak in the Baltic-North Sea transition . In anoxic incubations with the addition of nitrite, approximately 65% of the nitrogen gas formation was due to anaerobic ammonium oxidation with nitrite, with the remainder being produced by denitrification . Anaerobic ammonium oxidation with nitrite exhibited a biological temperature response, with a rate optimum at 15 degrees C and a maximum temperature of 37 degrees C . The biological nature of the process and a 1:1 stoichiometry for the reaction between nitrite and ammonium indicated that the transformations might be attributed to the anammox process . Attempts to find other anaerobic ammonium-oxidizing processes in this sediment failed . The apparent K(m) of nitrite consumption was less than 3 microM, and the relative importance of ammonium oxidation with nitrite and denitrification for the production of nitrogen gas was independent of nitrite concentration . Thus, the quantitative importance of ammonium oxidation with nitrite in the jar incubations at elevated nitrite concentrations probably represents the in situ situation . With the addition of nitrate, the production of nitrite from nitrate was four times faster than its consumption and therefore did not limit the rate of ammonium oxidation . Accordingly, the rate of this process was the same whether nitrate or nitrite was added as electron acceptor . The addition of organic matter did not stimulate denitrification, possibly because it was outcompeted by manganese reduction or because transport limitation was removed due to homogenization of the sediment. Water Res, 2002 Jun, 36(11), 2721 - 32 Methanol-induced biological nutrient removal kinetics in a full-scale sequencing batch reactor; Louzeiro NR et al.; The primary goal of this research was to determine the potential for denitrification and phosphorus removal of a full-scale sequencing batch reactor (SBR), with and without the use of methanol as an external carbon source . The control SBR, without methanol addition, achieved negligible denitrification . Two denitrification rates were observed in the experimental SBR, with methanol addition, an initial fast rate and a slower second rate . The denitrification rate during the first rate period increased with increasing methanol concentration, until a maximum denitrification rate of approximately 19 mg NOx-N/g MLVSS/day was attained . Following the depletion of the methanol, denitrification reactions probably continued by using the available natural carbon in the influent, resulting in a slower, second denitrification rate . Biological phosphorus uptake and release was significant only in the SBR with methanol addition . Methanol was probably not utilized as the carbon source for the enhanced biological phosphorus removal (EBPR) process . However, methanol addition was critical, since it depleted the available nitrates and thus allowed EBPR to take place. Chemosphere, 2002 Jul, 48(3), 329 - 34 Anaerobic degradation of pyrrolidine and piperidine coupled with nitrate reduction; Bae HS et al.; Biodegradability of secondary amines (pyrrolidine, piperidine, piperazine, morpholine, and thiomorpholine) under anaerobic conditions was examined in microbial consortia from six different environmental sites . The consortia degraded pyrrolidine and piperidine under denitrifying conditions . Enrichment cultures were established by repeatedly sub-culturing the consortia on pyrrolidine or piperidine in the presence of nitrate . The enrichments strictly required nitrate for the anaerobic degradation and utilized pyrrolidine or piperidine as a carbon, nitrogen, and energy source for their anaerobic growths . The anaerobic degradation of pyrrolidine and piperidine reduced nitrate to nitrogen gas, indicating that these anaerobic degradations were coupled with a respiratory nitrate reduction. J Contam Hydrol, 2002 Jul, 57(1-2), 41 - 59 Natural attenuation of chlorinated solvents at Area 6, Dover Air Force Base: characterization of microbial community structure; Davis JW et al.; A polyphasic approach based on cultivation and direct recovery of 16S rRNA gene sequences was utilized for microbial characterization of an aquifer contaminated with chlorinated ethenes . This work was conducted in order to support the evaluation of natural attenuation of chlorinated ethenes in groundwater at Area 6 at Dover Air Force Base (Dover, DE) . Results from these studies demonstrated the aquifer contained relatively low biomass (e.g . direct microscopic counts of < 10(7) bacteria/g of sediment) comprised of a physiologically diverse group of microorganisms including iron reducers, acetogens, sulfate reducers, denitrifiers, aerobic and anaerobic heterotrophs . Laboratory microcosms prepared with authentic sediment and groundwater provided direct microbiological evidence that the mineralization of vinyl chloride and cis-dichloroethene as well as each step in the complete reductive dechlorination of tetracloroethene to ethene can occur in the Area 6 aquifer . Enrichment cultures capable of the oxidative degradation of cis-1,2-dichloroethene (cis-DCE) and vinyl chloride (VC) were obtained from groundwater across the aquifer demonstrating the possible importance of direct, non-cometabolic oxidation of cis-DCE and VC in natural attenuation . Culture-independent analyses based upon recovery of 16S rRNA gene sequences revealed the presence of anaerobic organisms distributed primarily between two major bacterial divisions: the delta subdivision of the Proteobacteria and low-G + C gram positive . Recovery of sequences affiliated with phylogenetic groups containing known anaerobic-halorespiring organisms such as Desulfitobacterium, Dehalobacter, and certain groups of iron reducers provided qualitative support for a role of reductive dechlorination processes in the aquifer . This molecular data is suggestive of a functional linkage between the microbiology of the site and the apparent natural attenuation process . The presence and distribution of microorganisms were found to be consistent with a microbially driven attenuation of chlorinated ethenes within the aquifer and in accord with a conceptual model of aquifer geochemistry which suggest that both reductive and oxidative mechanisms are involved in heterogeneous, spatially distributed processes across the aquifer. Arch Biochem Biophys, 2002 Aug 1, 404(1), 158 - 62 Redox properties of an engineered purple Cu(A) azurin; Sun D et al.; Purple Cu(A) centers are a class of binuclear, mixed-valence copper complexes found in cytochrome c oxidase and nitrous oxide reductase . An engineered Cu(A) protein was formed by replacing a portion of the amino acid sequence that contains three of the ligands to the native type I copper center of Pseudomonas aeruginosa azurin with the corresponding portion of sequence from the Cu(A) center of cytochrome c oxidase from Paracoccus denitrificans {Proc . Natl . Acad . Sci . USA 93 (1996) 461} . Oxidation-reduction midpoint potential (E(m)) values of the Cu(A) azurin of +399+/-10 and +380+/-2mV, respectively, were determined by cyclic voltammetry and spectrochemical titration . An n value of one was obtained, indicating that the redox reaction is cycling between the mixed valence and the fully reduced states . Whereas the E(m) value of native azurin is pH dependent, the E(m) value of Cu(A) azurin is not, as expected for the Cu(A) center . Similarities and differences in the redox properties are discussed in terms of the known crystal structures of Cu(A) centers in cytochrome c oxidase and Cu(A) azurin. J Inorg Biochem, 2002 Jul 25, 91(1), 132 - 8 Spectroscopic and functional characterization of Cu-containing nitrite reductase from Hyphomicrobium denitrificans A3151; Deligeer et al.; The Cu-containing nitrite reductase from Hyphomicrobium denitrificans (HydNIR) has been spectroscopically and functionally characterized . The visible absorption spectrum implies that the enzyme has two type 1 Cu ions in one subunit (ca . 50 kDa) . The electron paramagnetic resonance (EPR) spectrum of HydNIR is simulated assuming the sum of three distinct S = 1/2 systems: two type 1 Cu signals (axial and rhombic symmetries) and one type 2 Cu signal . The intramolecular electron transfer reaction from the type 1 Cu to the type 2 Cu at pH 6.0 does not occur in the absence of nitrite, but a very slow electron transfer reaction is observed in the presence of nitrite . The apparent first-order rate constants for the intramolecular electron transfer reactions (k(ET(intra))) in the presence of nitrite and also the apparent catalytic rate constants (k(cat)) of HydNIR decrease gradually with increasing pH in the range of pH 4.5-7.5 . These pH profiles are substantially similar to each other, suggesting that the intramolecular electron transfer process is linked to the subsequent nitrite reduction process. Biochemistry, 2002 Jul 23, 41(29), 9116 - 25 Vibrational modes of tyrosines in cytochrome c oxidase from Paracoccus denitrificans: FTIR and electrochemical studies on Tyr-D4-labeled and on Tyr280His and Tyr35Phe mutant enzymes; Hellwig P et al.; A combined electrochemical and FTIR spectroscopic approach was used to identify the vibrational modes of tyrosines in cytochrome c oxidase from Paracoccus denitrificans which change upon electron transfer and coupled proton transfer . Electrochemically induced FTIR difference spectra of the Tyr-D4-labeled cytochrome c oxidase reveal that only small contributions arise from the tyrosines . Contributions between 1600 and 1560 cm(-1) are attributed to nu8a/8b(CC) ring modes . The nu19(CC) ring mode for the protonated form of tyrosines is proposed to absorb with an uncommonly small signal at 1525-1518 cm(-1) and for the deprotonated form at 1496-1486 cm(-1), accompanied by the increase of the nu19(CC) ring mode of the Tyr-D(4)-labeled oxidase at approximately 1434 cm(-1) . A signal at 1270 cm(-1) can be tentatively attributed to the nu7'a(CO) and delta(COH) mode of a protonated tyrosine . Uncommon absorptions, like the mode at 1524 cm(-1), indicate the involvement of Tyr280 in the spectra . Tyr280 is a crucial residue close to the binuclear center and is covalently bonded to His276 . The possible changes of the spectral properties are discussed together with the absorbance spectra of tyrosine bound to histidine . The vibrational modes of Tyr280 are further analyzed in combination with the mutation to histidine, which is assumed to abolish the covalent bonding . The electrochemically induced FTIR difference spectra of the Tyr280His mutant point to a change in protonation state in the environment of the binuclear center . Together with an observed decrease of a signal at 1736 cm(-1), previously assigned to Glu278, a possible functional coupling is reflected . In direct comparison to the FTIR difference spectra of the D4-labeled compound and comparing the spectra at pH 7 and 4.8, the protonation state of Tyr280 is discussed . Furthermore, a detailed analysis of the mutant is presented, the FTIR spectra of the CO adduct revealing a partial loss of Cu(B) . Electrochemical redox titrations reflect a downshift of the heme a3 midpoint potential by 95 +/- 10 mV . Another tyrosine identified to show redox dependent changes upon electron transfer is Tyr35, a residue in the proposed D-pathway of the cytochrome c oxidase. Biochemistry, 2002 Jul 16, 41(28), 8886 - 98 Ca(2+)-binding site in Rhodobacter sphaeroides cytochrome C oxidase; Lee A et al.; Cytochrome c oxidase (COX) from R . sphaeroides contains one Ca(2+) ion per enzyme that is not removed by dialysis versus EGTA . This is similar to COX from Paracoccus denitrificans {Pfitzner, U., Kirichenko, A., Konstantinov, A . A., Mertens, M., Wittershagen, A., Kolbesen, B . O., Steffens, G . C . M., Harrenga, A., Michel, H., and Ludwig, B . (1999) FEBS Lett . 456, 365-369} and is in contrast to the bovine oxidase, which binds Ca(2+) reversibly . A series of R . sphaeroides mutants with replacements of the E54, Q61, and D485 residues, which form the Ca(2+) coordination sphere in subunit I, has been generated . The substitutions for the E54 residue do not assemble normally . Mutants with the Q61 replacements are active and retain the tightly bound Ca(2+); their spectra are not perturbed by added Ca(2+) or EGTA . The D485A mutant is active, binds to Ca(2+) reversibly, like the mitochondrial oxidase, and exhibits the red shift in the heme a absorption spectrum upon Ca(2+) binding for both reduced and oxidized states of heme a . The K(d) value of 6 nM determined by equilibrium titrations is much lower than that reported for the homologous D477A mutant of Paracoccus denitrificans or for bovine COX (K(d) = 1-3 microM) . The rate of Ca(2+) binding with the D4