Silver Nanoparticles Induced Cell Apoptosis, Membrane Damage of Azotobacter vinelandii and Nitrosomonas europaea via Generation of Reactive Oxygen Species.

Silver nanoparticles (AgNPs) is widely used as an antibacterial agent, but the specific antibacterial mechanism is still conflicting. This study aimed to investigate the size dependent inhibition of AgNPs and the relationship between inhibition and reactive oxygen species (ROS). Azotobactervinelandii and Nitrosomonaseuropaea were exposed to AgNPs with different particles size (10 nm and 50 nm). The ROS production was measured and the results showed that the generation of ROS related to the particle size and concentrations of AgNPs. At 10 mg/L of 10 nm Ag particles, the apoptosis rate of A. vinelandii and N. europaea were 20.23% and 1.87% respectively. Additionally, the necrosis rate of A. vinelandii and N. europaea reached to 15.20% and 42.20% respectively. Furthermore, transmission electron microscopy images also indicated that AgNPs caused severely bacterial cell membrane damage. Together these data suggested that the toxicity of AgNPs depends on its particle size and overproduction of ROS.

Regulation of membrane fixation and energy production/conversion for adaptation and recovery of ZnO nanoparticle impacted Nitrosomonas europaea.

The ZnO nanoparticle (NP) effects on typical ammonia-oxidizing bacteria, Nitrosomonas europaea in a chemostat bioreactor, and the cells' toxicity adaptation and recovery potentials were explored. Hardly any inhibition was observed when the NP concentration was high up to 10 mg/L. The cells exposed to 50 mg/L ZnO NPs displayed time-dependent impairment and recovery potentials in terms of cell density, membrane integrity, nitrite production rate, and ammonia monooxygenase activity. The 6-h NP stress impaired cells were nearly completely restored during a 12-h recovery incubation, while the longer exposure time would cause irretrievable cell damage. Microarray analysis further indicated the transcriptional adaptation of N. europaea to NP stress. The regulations of genes encoding for membrane permeability or osmoprotectant, membrane integrity preservation, and inorganic ion transport during NP exposure and cell recovery revealed the importance of membrane fixation and the associated metabolisms for cells' self-protection and the following recovery from NP stress. The oxidative phosphorylation, carbon assimilation, and tricarboxylic acid (TCA) cycling pathways involved in the cells' antitoxicity activities and would promote the energy production/conversion efficiency for cell recovery. The heavy metal resistance, histidine metabolism, toxin-antitoxin defense, glycolysis, and sulfate reduction pathways were also suggested to participate in the cell detoxication and recovery processes. All these findings provided valuable insights into the mechanisms of cell-mediated ZnO NP cytotoxicity and their potential impacts on wastewater nitrogen removal system.

Effects of Simulated Rare Earth Recycling Wastewaters on Biological Nitrification.

Increasing rare earth element (REE) supplies by recycling and expanded ore processing will result in generation of new wastewaters. In some cases, disposal to a sewage treatment plant may be favored, but plant performance must be maintained. To assess the potential effects of such wastewaters on biological treatment, model nitrifying organisms Nitrosomonas europaea and Nitrobacter winogradskyi were exposed to simulated wastewaters containing varying levels of yttrium or europium (10, 50, and 100 ppm), and the extractant tributyl phosphate (TBP, at 0.1 g/L). Y and Eu additions at 50 and 100 ppm inhibited N. europaea, even when virtually all of the REE was insoluble. Provision of TBP with Eu increased N. europaea inhibition, although TBP alone did not substantially alter activity. For N. winogradskyi cultures, Eu or Y additions at all tested levels induced significant inhibition, and nitrification shut down completely with TBP addition. REE solubility was calculated using the previously developed MSE (Mixed-Solvent Electrolyte) thermodynamic model. The model calculations reveal a strong pH dependence of solubility, typically controlled by the precipitation of REE hydroxides but also likely affected by the formation of unknown phosphate phases, which determined aqueous concentrations experienced by the microorganisms.

Disinfectant-induced ammonia oxidation disruption in microbial N-cycling process in aquatic ecosystem after the COVID-19 outbreak.

Anthropogenic activities significantly impact the elemental cycles in aquatic ecosystems, with the N-cycling playing a critical role in potential nutrient turnover and substance cycling. We hypothesized that measures to prevent COVID-19 transmission profoundly altered the nitrogen cycle in riverine ecosystems. To investigate this, we re-analyzed metagenomic data and identified 60 N-cycling genes and 21 host metagenomes from four urban reaches (one upstream city, Wuhan, and two downstream cities) along the Yangtze River. Our analyses revealed a marked decrease in the abundance of bacterial ammonia monooxygenase genes, as well as in the host, ammonia-oxidizing autotrophic Nitrosomonas, followed by a substantial recovery post-pandemic. We posited that discharge of sodium hypochlorite (NaOCl) disinfectant may be a primary factor in the reduction of N-cycling process. To test this hypothesis, we exposed pure cultures of Nitrosomonas europaea to NaOCl to explore the microbial stress response. Results indicated that NaOCl exposure rapidly compromised the cell structure and inhibited ammonia oxidation of N. europaea, likely due to oxidative stress damage and reduced expression of nitrogen metabolism-related ammonia monooxygenase. Using the functional tagging technique, we determined that NaOCl directly destroyed the ammonia monooxygenase protein and DNA structure. This study highlights the negative impacts of chlorine disinfectants on the function of aquatic ecosystems and elucidates potential mechanisms of action.

Influence of bovine serum albumin and alginate on silver nanoparticle dissolution and toxicity to Nitrosomonas europaea.

Bovine serum albumin (BSA), a model protein, reduced the toxicity of 20 nm citrate silver nanoparticles (AgNP) toward Nitrosomonas europaea, a model ammonia oxidizing bacteria, through a dual-mode protection mechanism. BSA reduced AgNP toxicity by chelating the silver ions (Ag(+)) released from the AgNPs. BSA further reduced AgNP toxicity by binding to the AgNP surface thus preventing NH3-dependent dissolution from occurring. Due to BSA's affinity toward Ag(+) chemisorbed on the AgNP surface, increased concentrations of BSA lead to increased AgNP dissolution rates. This, however, did not increase AgNP toxicity as the dissolved Ag(+) were adsorbed onto the BSA molecules. Alginate, a model extracellular polysaccharide (EPS), lacks strong Ag(+) ligands and was unable to protect N. europaea from Ag(+) toxicity. However, at high concentrations, alginate reduced AgNP toxicity by binding to the AgNP surface and reducing AgNP dissolution rates. Unlike BSA, alginate only weakly interacted with the AgNP surface and was unable to completely prevent NH3-dependent AgNP dissolution from occurring. Based on these results, AgNP toxicity in high protein environments (e.g., wastewater) is expected to be muted while the EPS layers of wastewater biofilms may provide additional protection from AgNPs, but not from Ag(+) that have already been released.

Impacts of silver nanoparticle coating on the nitrification potential of Nitrosomonas europaea.

Silver nanoparticles (AgNPs) are increasingly used as bacteriostatic agents to prevent microbial growth. AgNPs are manufactured with a variety of coatings, and their potential impacts on wastewater treatment in general are poorly understood. In the present study, Nitrosomonas europaea, a model ammonia oxidizing bacterium, was exposed to AgNPs with citrate, gum arabic (GA), and polyvinylpyrrolidone (PVP). GA and citrate AgNPs inhibited nitrification most strongly (67.9 ± 3.6% and 91.4 ± 0.2%, respectively at 2 ppm). Our data indicate that Ag(+) dissolution and colloid stability of AgNPs were the main factors in AgNP toxicity. In general, low amounts of dissolved Ag initially caused a post-transcriptional interruption of membrane-bound nitrifying enzyme function, reducing nitrification by 10% or more. A further increase in dissolved Ag resulted in heavy metal stress response (e.g., merA up-regulation) and ultimately led to membrane disruption. The highest effect on membrane disruption was observed for citrate AgNPs (64 ± 11% membranes compromised at 2 ppm), which had high colloidal stability. This study demonstrates that coating plays a very important role in determining Ag dissolution and ultimately toxicity to nitrifiers. More research is needed to characterize these parameters in complex growth media such as wastewater.

Determination of the effects of medium composition on the monochloramine disinfection kinetics of Nitrosomonas europaea by the propidium monoazide quantitative PCR and Live/Dead BacLight methods.

Various medium compositions (phosphate, 1 to 50 mM; ionic strength, 2.8 to 150 meq/liter) significantly affected Nitrosomonas europaea monochloramine disinfection kinetics, as determined by the Live/Dead BacLight (LD) and propidium monoazide quantitative PCR (PMA-qPCR) methods (lag coefficient, 37 to 490 [LD] and 91 to 490 [PMA-qPCR] mg·min/liter; Chick-Watson rate constant, 4.0 × 10(-3) to 9.3 × 10(-3) [LD] and 1.6 × 10(-3) to 9.6 × 10(-3) [PMA-qPCR] liter/mg·min). Two competing effects may account for the variation in disinfection kinetic parameters: (i) increasing kinetics (disinfection rate constant [k] increased, lag coefficient [b] decreased) with increasing phosphate concentration and (ii) decreasing kinetics (k decreased, b increased) with increasing ionic strength. The results support development of a standard medium for evaluating disinfection kinetics in drinking water.

Copper toxicity to bioluminescent Nitrosomonas europaea in soil is explained by the free metal ion activity in pore water.

The terrestrial biotic ligand model (BLM) for metal toxicity in soil postulates that metal toxicity depends on the free metal ion activity in solution and on ions competing for metal sorption to the biotic ligand. Unequivocal evidence for the BLM assumptions is most difficult to obtain for native soil microorganisms because the abiotic and biotic compartments cannot be experimentally separated. Here, we report copper (Cu) toxicity to a bioluminescent Nitrosomonas europaea reporter strain that was used in a solid phase-contact assay and in corresponding soil extracts and artificial soil solutions. The Cu(2+) ion activities that halve bioluminescence (EC50) in artificial solutions ranged 10(-5) to 10(-7) M and increased with increasing activities of H(+), Ca(2+) and Mg(2+) according to the BLM concept. The solution based Cu(2+) EC50 values of N. europaea in six contaminated soils ranged 2 × 10(-6) to 2 × 10(-9) M and these thresholds for both solid phase or soil extract based assays were well predicted by the ion competition model fitted to artificial solution data. In addition, solution based Cu(2+) EC50 of the solid phase-contact assay were never smaller than corresponding values in soil extracts suggesting no additional solid phase toxic route. By restricting the analysis to the same added species, we show that the Cu(2+) in solution represents the toxic species to this bacterium.

Membrane-aerated biofilm proton and oxygen flux during chemical toxin exposure.

Bioreactors containing sessile bacteria (biofilms) grown on hollow fiber membranes have been used for treatment of many wastestreams. Real time operational control of bioreactor performance requires detailed knowledge of the relationship between bulk liquid water quality and physiological transport at the biofilm-liquid interface. Although large data sets exist describing membrane-aerated bioreactor effluent quality, very little real time data is available characterizing boundary layer transport under physiological conditions. A noninvasive, microsensor technique was used to quantify real time (≈1.5 s) changes in oxygen and proton flux for mature Nitrosomonas europaea and Pseudomonas aeruginosa biofilms in membrane-aerated bioreactors following exposure to environmental toxins. Stress response was characterized during exposure to toxins with known mode of action (chlorocarbonyl cyanide phenyl-hydrazone and potassium cyanide), and four environmental toxins (rotenone, 2,4-dinitrophenol, cadmium chloride, and pentachlorophenol). Exposure to sublethal concentrations of all environmental toxins caused significant increases in O(2) and/or H(+) flux (depending on the mode of action). These real time microscale signatures (i.e., fingerprints) of O(2) and H(+) flux can be coupled with bulk liquid analysis to improve our understanding of physiology in counter-diffusion biofilms found within membrane aerated bioreactors; leading to enhanced monitoring/modeling strategies for bioreactor control.

Monochloramine disinfection kinetics of Nitrosomonas europaea by propidium monoazide quantitative PCR and Live/dead BacLight methods.

Monochloramine disinfection kinetics were determined for the pure-culture ammonia-oxidizing bacterium Nitrosomonas europaea (ATCC 19718) by two culture-independent methods, namely, Live/Dead BacLight (LD) and propidium monoazide quantitative PCR (PMA-qPCR). Both methods were first verified with mixtures of heat-killed (nonviable) and non-heat-killed (viable) cells before a series of batch disinfection experiments with stationary-phase cultures (batch grown for 7 days) at pH 8.0, 25 degrees C, and 5, 10, and 20 mg Cl(2)/liter monochloramine. Two data sets were generated based on the viability method used, either (i) LD or (ii) PMA-qPCR. These two data sets were used to estimate kinetic parameters for the delayed Chick-Watson disinfection model through a Bayesian analysis implemented in WinBUGS. This analysis provided parameter estimates of 490 mg Cl(2)-min/liter for the lag coefficient (b) and 1.6 x 10(-3) to 4.0 x 10(-3) liter/mg Cl(2)-min for the Chick-Watson disinfection rate constant (k). While estimates of b were similar for both data sets, the LD data set resulted in a greater k estimate than that obtained with the PMA-qPCR data set, implying that the PMA-qPCR viability measure was more conservative than LD. For N. europaea, the lag phase was not previously reported for culture-independent methods and may have implications for nitrification in drinking water distribution systems. This is the first published application of a PMA-qPCR method for disinfection kinetic model parameter estimation as well as its application to N. europaea or monochloramine. Ultimately, this PMA-qPCR method will allow evaluation of monochloramine disinfection kinetics for mixed-culture bacteria in drinking water distribution systems.

Expression of merA, trxA, amoA, and hao in continuously cultured Nitrosomonas europaea cells exposed to cadmium sulfate additions.

The effects of CdSO(4) additions on the gene expressions of a mercury reductase, merA, an oxidative stress protein, trxA, the ammonia-monooxygenase enzyme (AMO), amoA, and the hydroxylamine oxidoreductase enzyme (HAO), hao, were examined in continuously cultured N. europaea cells. The reactor was fed 50 mM NH(4)+ and was operated for 78 days with a 6.9 days hydraulic retention time. Over this period, six successive batch additions of CdSO(4) were made with increasing maximum concentrations ranging from 1 to 60 microM Cd(2+). The expression of merA was highly correlated with the level of Cd(2+) within the reactor (Rs = 0.90) with significant up-regulation measured at non-inhibitory Cd(2+) concentrations. Cd(2+) appears to target AMO specifically at lower concentrations and caused oxidative stress at higher concentrations, as indicated by the SOURs (specific oxygen uptake rates) and the up-regulation of trxA. Since Cd(2+) inhibition is irreversible and amoA was up-regulated in response to Cd(2+) inhibition, it is hypothesized that de novo synthesis of the AMO enzyme occurred and was responsible for the observed recovery in activity. Continuously cultured N. europaea cells were more resistant to Cd(2+) inhibition than previously examined batch cultured cells due to the presence of Mg(2+) and Ca(2+) in the growth media, suggesting that Cd(2+) enters the cell through Mg(2+) and Ca(2+) import channels. The up-regulation of merA during exposure to non-inhibitory Cd(2+) levels indicates that merA is an excellent early warning signal for Cd(2+) inhibition.

Expression of merA, amoA and hao in continuously cultured Nitrosomonas europaea cells exposed to zinc chloride additions.

The effects of ZnCl2 additions on a mercuric reductase, merA, ammonia monooxygenase, amoA, and hydroxylamine (NH2OH) oxidoreductase, hao, gene expression were examined in continuously cultured Nitrosomonas europaea cells. The reactor was operated for 85 days with a 6.9 d hydraulic retention time and with four successive additions of ZnCl2 achieving maximum concentrations from 3 to 90 microM Zn2+. Continuously cultured N. europaea cells were more resistant to Zn2+ inhibition than previously examined batch cultured cells due to the presence of Mg2+ in the growth media, suggesting that Zn2+ enters the cell through Mg2+ import channels. The maximum merA up-regulation was 45-fold and expression increased with increases in Zn2+ concentration and decreased as Zn2+ concentrations decreased. Although Zn2+ irreversibly inactivated ammonia oxidation in N. europaea, the addition of either 600 microM CuSO4 or 2250 microM MgSO4 protected N. europaea from ZnCl2 inhibition, indicating a competition between Zn2+ and Cu2+/Mg2+ for uptake and/or AMO active sites. Since ZnCl2 inhibition is irreversible and amoA was up-regulated at 30 and 90 microM additions, it is hypothesized that de novo synthesis of the AMO enzyme is needed to overcome inhibition. The up-regulation of merA during exposure to non-inhibitory Zn2+ levels indicates that merA is an excellent early warning signal for Zn2+ inhibition.

Whole-genome transcriptional and physiological responses of Nitrosomonas europaea to cyanide: identification of cyanide stress response genes.

Nitrosomonas europaea (ATCC 19718) is one of several nitrifying species that participate in the biological removal of nitrogen from wastewater by oxidizing ammonia to nitrite, the first step in nitrification. Because nitrification is quite sensitive to cyanide, a compound often encountered in wastewater treatment plants, we characterized the physiological and transcriptional responses of N. europaea cells to cyanide. The cells were extremely sensitive to low concentrations of cyanide, with NO-(2)production and ammonia-dependent oxygen uptake rates decreasing by 50% within 30 min of exposure to 1 microM NaCN. Whole-genome transcriptional responses of cells exposed to 1 microM NaCN were examined using Affymetrix microarrays to identify stress-induced genes. The transcript levels of 35 genes increased more than 2-fold while transcript levels of 29 genes decreased more than 20-fold. A gene cluster that included moeZ (NE2353), encoding a rhodanese homologue and thought to be involved in detoxification of cyanide, showed the highest up-regulation (7-fold). The down-regulated genes included genes encoding proteins involved in the sulfate reduction pathway, signal transduction mechanisms, carbohydrate transport, energy production, coenzyme metabolism, and amino acid transport.

Effect of analgesics and their derivatives on antibiotic resistance of environmental microbes.

This research is a preliminary study conducted to determine the effects of aspirin (acetyl-salicylic acid) and salicylic acid (analgesics and their derivatives) on the antibiotic resistance of ammonia oxidizing bacterium (AOB) (a non-pathogenic environmental microbe) cultured from the Texas Tech University-Water Recovery System that treats a space related wastewater for NASA. The effect of salicylic acid was investigated by obtaining the minimal inhibition concentration (MIC) of antibiotics (amoxicillin, ciprofloxacin, and nalidixic acid) in the presence of aspirin and salicylic acid. The possibility of transfer of resistance genes between unrelated species was investigated by analyzing the similarity of the AcrA protein (a multi-drug efflux protein) in Nitrosomonas europaea, Escherichia coli and Salmonella enterica. The protein alignment analysis was done using ExPASy, a proteomics tool. The results of this preliminary study indicated that the antibiotic resistance of AOBs increased in the presence of aspirin and salicylic acid and similarities in the AcrA protein of different species indicated the likelihood of possible resistance transfer between the species. This paper high lights the importance of research and further investigation on antibiotic resistance and resistance transfer, highlighting the number of parameters that should be considered while assessing antibiotic resistance in environmental samples.

Effects of Exogenous N-Acyl-Homoserine Lactone as Signal Molecule on Nitrosomonas Europaea under ZnO Nanoparticle Stress.

Despite the adverse effects of emerging ZnO nanoparticles (nano-ZnO) on wastewater biological nitrogen removal (BNR) systems being widely documented, strategies for mitigating nanoparticle (NP) toxicity impacts on nitrogen removal have not been adequately addressed. Herein, N-acyl-homoserine lactone (AHL)-based quorum sensing (QS) was investigated for its effects against nano-ZnO toxicity to a model nitrifier, Nitrosomonas europaea. The results indicated that AHL-attenuated nano-ZnO toxicity, which was inversely correlated with the increasing dosage of AHL from 0.01 to 1 µM. At 0.01 µM, AHL notably enhanced the tolerance of N. europaea cells to nano-ZnO stress, and the inhibited cell proliferation, membrane integrity, ammonia oxidation rate, ammonia monooxygenase activity and amoA gene expression significantly increased by 18.2 ± 2.1, 2.4 ± 0.9, 58.7 ± 7.1, 32.3 ± 1.7, and 7.3 ± 5.9%, respectively, after 6 h of incubation. However, increasing the AHL dosage compromised the QS-mediated effects and even aggravated the NPs' toxicity effects. Moreover, AHLs, at all tested concentrations, significantly increased superoxide dismutase activity, indicating the potential of QS regulations to enhance cellular anti-oxidative stress capacities when facing NP invasion. These results provide novel insights into the development of QS regulation strategies to reduce the impact of nanotoxicity on BNR systems.

Physiological state, growth mode, and oxidative stress play a role in Cd(II)-mediated inhibition of Nitrosomonas europaea 19718.

The goal of this study was to determine the impact of physiological growth states (batch exponential and batch stationary growth) and growth modes (substrate-limited chemostat, substrate-sufficient exponential batch, and substrate-depleted stationary batch growth) on several measures of growth and responses to Cd(II)-mediated inhibition of Nitrosomonas europaea strain 19718. The specific oxygen uptake rate (sOUR) was the most sensitive indicator of inhibition among the different responses analyzed, including total cell abundance, membrane integrity, intracellular 16S rRNA/DNA ratio, and amoA expression. This observation remained true irrespective of the physiological state, the growth mode, or the mode of Cd(II) exposure. Based on the sOUR, a strong time-dependent exacerbation of inhibition (in terms of an inhibition coefficient [K(i)]) in exponential batch cultures was observed. Long-term inhibition levels (based on K(i) estimates) in metabolically active chemostat and exponential batch cultures were also especially severe and comparable. In contrast, the inhibition level in stationary-phase cultures was 10-fold lower and invariable with exposure time. Different strategies for surviving substrate limitation (a 10-fold increase in amoA expression) and starvation (the retention of 16S rRNA levels) in N. europaea cultures were observed. amoA expression was most negatively impacted by Cd(II) exposure in the chemostat cultures, was less impacted in exponential batch cultures, and was least impacted in stationary batch cultures. Although the amoA response was consistent with that of the sOUR, the amoA response was not as strong. The intracellular 16S rRNA/DNA ratio, as determined by fluorescence in situ hybridization, also did not uniformly correlate with the sOUR under conditions of inhibition or no inhibition. Finally, Cd(II)-mediated inhibition of N. europaea was attributed partially to oxidative stress.

Candidate stress genes of Nitrosomonas europaea for monitoring inhibition of nitrification by heavy metals.

Heavy metals have been shown to be strong inhibitors of nitrification in wastewater treatment plants. In this research, the effects of cadmium, copper, and mercury on Nitrosomonas europaea were studied in quasi-steady-state batch reactors. When cells were exposed to 1 microM CdCl2, 6 microM HgCl2, or 8 microM CuCl2, ammonia oxidation rates were decreased by about 90%. Whole-genome transcriptional and proteomic responses of N. europaea to cadmium were used to identify heavy metal stress response genes. When cells were exposed to 1 microM CdCl2 for 1 h, 66 genes (of the total of 2,460 genes) were upregulated, and 50 genes were downregulated more than twofold. Of these, the mercury resistance genes (merTPCADE) averaged 277-fold upregulation under 1 microM CdCl2, with merA (mercuric reductase) showing 297-fold upregulation. In N. europaea cells exposed to 6 microM HgCl2 or to 8 microM CuCl2, merA showed 250-fold and 1.7-fold upregulation, respectively. Cells showed the ability to recover quickly from Hg2+-related toxic effects, apparently associated with upregulation of the mercury resistance genes and amoA, but no such recovery was evident in Cd2+-exposed cells even though merTPCADE were highly upregulated. We suggest that the upregulation of merA in response to CdCl2 and HgCl2 exposure may provide a means to develop an early-warning indicator for inhibition of nitrification by these metals.

DNA microarray mediated transcriptional profiling of Nitrosomonas europaea in response to linear alkylbenzene sulfonates.

Linear alkylbenzene sulfonates (LAS) constitute, quantitatively, the most important group of synthetic surfactants used today. We studied the gene expression of Nitrosomonas europaea in response to LAS using a DNA microarray because ammonia-oxidizers are thought to be more sensitive to LAS than other microorganisms. Our objective was to elucidate which genes are expressed for N. europaea in response to LAS exposure. Microarray analysis and real-time PCR assay revealed that c. 30 genes were significantly expressed after LAS exposure, in particular genes associated with energy production and conversion. Our findings demonstrate that physical disruption of membrane structures, which contain enzymes associated with energy production and conversion, might be an important explanation for the high sensitivity of N. europaea to LAS exposure.

Inactivation of Nitrosomonas europaea and pathogenic Escherichia coli by chlorine and monochloramine.

The purpose of this study was to measure the chlorine and monochloramine inactivation kinetics of Nitrosomonas europaea at 21 degrees C in the presence and absence of particles. The inactivation kinetics rates were compared with those obtained with Escherichia coli O157:H7. The results show that, in pure water, the use of free chlorine produced 4 log10 of N. europaea inactivation at a CT value of 0.8 mg.min l(-1), whereas monochloramine yielded 4 log10 of inactivation at CT values of approximately 9.9-16.4mg.min l(-1). With E. coli, chlorine produced approximately 4 1og10 of inactivation at a CT of 0.13 mg.min l(-1), whereas monochloramine resulted in 4 logo10 of inactivation at a CT of approximately 9.2 mg.min l(-1). These results suggest that N. europaea is more resistant to monochloramine and chlorine than E. coli. Corrosion debris, soil material and wastewater had no statistically significant (p < 0.05) impact on the inactivation of N. europaea by either chlorine or monochloramine. It seems likely that the CT values present in distribution systems would be sufficient to control suspended cells of these two organisms, especially under conditions of breakpoint chlorination, which could be used to control nitrification. Adequate disinfection should prevent the growth of these organisms in a distribution system.

Functional and physiological evidence for a rhesus-type ammonia transporter in Nitrosomonas europaea.

Ammonium transporters form a conserved family of transport proteins and are widely distributed among all domains of life. The genome of Nitrosomonas europaea codes for a single gene (rh1) that belongs to the family of the AMT/Rh ammonium transporters. For the first time, this study provides functional and physiological evidence for a rhesus-type ammonia transporter in bacteria (N. europaea). The methylammonium (MA) transport activity of N. europaea correlated with the Rh1 expression. The K(m) value for the MA uptake of N. europaea was 1.8+/-0.2 mM (pH 7.25), and the uptake was competitively inhibited by ammonium [K(i)(NH(4) (+)) 0.3+/-0.1 mM at pH 7.25]. The MA uptake rate was pH dependent, indicating that the uncharged form of MA is transported by Rh1. An effect of the glutamine synthetase on the MA uptake was not observed. When expressed in Saccharomyces cerevisiae, the function of Rh1 from N. europaea as an ammonia/MA transporter was confirmed. The results suggest that Rh1 equilibrates the uncharged substrate species. A low pH value in the periplasmic space during ammonia oxidation seems to be responsible for the ammonium accumulation functioning as an acid NH(4) (+) trap.