Cobalt-dependent inhibition of nitrite oxidation in Nitrobacter winogradskyi.

Nitrobacter winogradskyi is an abundant, intensively studied autotrophic nitrite-oxidizing bacterium, which is frequently used as a model strain in the two-step nitrification of ammonia (NH3) to nitrate (NO3-) via nitrite (NO2-), either in activated sludge, agricultural field studies or more recently in artificial microbial consortia for organic hydroponics. We observed a hitherto unknown cobalt ion-dependent inhibition of cell growth and NO2- oxidation activity of N. winogradskyi in a mineral medium, which strongly depended on accompanying Ca2+ and Mg2+ concentrations. This inhibition was bacteriostatic, but susceptible to natural chelators. l-Histidine effectively restored cell growth and NO2- oxidation activity of N. winogradskyi in mineral media containing Co2+ with >90% recovery. Our results suggest that Co2+ competed with alkaline earth metals during uptake and that its toxicity was significantly reduced by complexation.

Toxicity of TiO2 nanoparticles on soil nitrification at environmentally relevant concentrations: Lack of classical dose-response relationships.

Titanium-dioxide nanoparticles (TiO2-NPs) are increasingly released in agricultural soils through, e.g. biosolids, irrigation or nanoagrochemicals. Soils are submitted to a wide range of concentrations of TiO2-NPs depending on the type of exposure. However, most studies have assessed the effects of unrealistically high concentrations, and the dose-response relationships are not well characterized for soil microbial communities. Here, using soil microcosms, we assessed the impact of TiO2-NPs at concentrations ranging from 0.05 to 500 mg kg-1 dry-soil, on the activity and abundance of ammonia-oxidizing archaea (AOA) and bacteria (AOB), and nitrite-oxidizing bacteria (Nitrobacter and Nitrospira). In addition, aggregation and oxidative potential of TiO2-NPs were measured in the spiking suspensions, as they can be important drivers of TiO2-NPs toxicity. After 90 days of exposure, non-classical dose-response relationships were observed for nitrifier abundance or activity, making threshold concentrations impossible to compute. Indeed, AOA abundance was reduced by 40% by TiO2-NPs whatever the concentration, while Nitrospira was never affected. Moreover, AOB and Nitrobacter abundances were decreased mainly at intermediate concentrations nitrification was reduced by 25% at the lowest (0.05 mg kg-1) and the highest (100 and 500 mg kg-1) TiO2-NPs concentrations. Path analyses indicated that TiO2-NPs affected nitrification through an effect on the specific activity of nitrifiers, in addition to indirect effects on nitrifier abundances. Altogether these results point out the need to include very low concentrations of NPs in soil toxicological studies, and the lack of relevance of classical dose-response tests and ecotoxicological dose metrics (EC50, IC50…) for TiO2-NPs impact on soil microorganisms.

[Effect of Low-concentration Ciprofloxacin on the Nitrification and Nitrifying Microorganisms of Biofilms in Biological Aerated Filter].

Effect of low-concentration ciprofloxacin (CIP) on nitrification and nitrifying microorganisms of biofilms was studied in biological aerated filters (BAF). Quantitative PCR (qPCR) was used to determine the abundance variance of four ciprofloxacin resistance genes (CIP-ARGs) during nitrification in biofilms. The correlations between the abundances of CIP-ARGs and nitrifying microorganisms were also discussed. The results showed that CIP had little influence on the ammonium oxidation process of biofilm microorganisms, whereas inhibition of the nitrite oxidation process was found. The quantitative results of ammonium-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) including Nitrobacter and Nitrospira indicated that the inhibition on the transformation of nitrite was resulted from the inhibition on Nitrobacter and Nitrospira. In addition, little influence of CIP on the relative abundance of aac and qepA in biofilms was found, but the influence on parC and oqxB was great. The abundance of Nitrotacter exhibited significant positive correlation with the abundance of parC. Similar significant correlation was also found between the abundances of Nitrospira and oqxB. It could be speculated that the genetic elements of different nitrifying microorganisms in biofilms possibly carried CIP-ARGs.

Development and Quality Control of Nanohexaconazole as an Effective Fungicide and Its Biosafety Studies on Soil Nitifiers.

The study was aimed to develop a nano form of an existing fungicide for improving plant protection and reducing crop losses caused by fungal pathogens. The protocol for the preparation and estimation of nanohexaconazole was developed. Technically pure hexaconazole was converted into its nanoform using polyethyleneglycol-400 (PEG) as the surface stabilizing agent. Nanohexaconazole was characterized using Scanning Electron Microscopy (SEM) and Dynamic Light Scattering (DLS) studies. The average particle size of nanohexaconazole was about 100 nm. An analytical method was also developed for quality control of the nanofungicide by GLC fitted with flame ionization detector. Its limit of detection was 2.5 ppm. Fungicidal potential of nanohexaconazole was better in comparison to that of conventional hexaconazole. Hydrolytic and thermal stability studies confirmed its stability at par with the conventional formulation of fungicide. Impact of nanohexaconazole on soil nitrifiers was tested in vitro and there were no significant adverse effect in their numbers observed as compared to conventional registered formulation, proving the safety of the nanofungicide.

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.

Effect of penicillin on nitrite-oxidizing bacteria in activated sludge.

The effects of penicillin G on the number and activity of nitrite-oxidizing bacteria were investigated in laboratory-scale reactors and batch tests. At a concentration of 100 mg L(-1), addition of penicillin G for short periods did not significantly affect nitrite oxidation, while addition for more than 2 months suppressed nitrite oxidation. Fluorescence in situ hybridization with 16S ribosomal RNA-targeted probes revealed a slight decrease in the abundance of Nitrospira, while Nitrobacter was not affected by addition of penicillin G for more than 39 days. The resistance of nitrite-oxidizing bacteria to penicillin appeared to be positively affected by intermittent aeration only when accompanied by denitrification; otherwise, the aeration mode (continuous or intermittent aeration) did not significantly affect the abundance of Nitrobacter and Nitrospira.

Limited impact of free ammonia on Nitrobacter spp. inhibition assessed by chemical and molecular techniques.

Free ammonia has long been identified as a nitrite oxidation inhibitor. However, past attempts to use this compound to eliminate nitrite oxidation and thereby promote more efficient nitrogen removal strategies during biological wastewater treatment have often failed. Additionally, contradictory results exist in the literature where direct measurements of free ammonia inhibition of nitrite oxidation have been reported. In this study, suspended biomass samples (nitrifier enriched activated sludge) were collected from a bench scale nitrification reactor with Nitrobacter spp. as the dominant nitrite oxidizer and subjected to batch respirometric experiments designed to quantify free ammonia inhibition of nitrite oxidization. A variety of data including ammonia, nitrite, and nitrate conversion rates, oxygen consumption rates, and Nitrobacter ribosomal RNA transcript abundance, a molecular indicator of growth activity, were used to assess nitrite oxidation and growth activity. Both the traditional and molecular activity assessments indicated that free ammonia had a limited inhibitory effect on Nitrobacter spp. In fact, the pH changes necessary to induce high free ammonia concentrations (>10mg-N/L) had a demonstrably more important inhibiting effect on nitrite oxidation than free ammonia. In contrast, during high ammonia oxidizing activity (5.3mg-N/L/h), low nitrite oxidation rates (0.2mg-N/L/h) and severely impaired Nitrobacter spp. growth activity, indicated by a low abundance of the Nitrobacter spp. ribosomal gene transcript relative to the ribosomal gene (0.08), were measured. The findings suggest that pH changes and ammonia oxidizing bacteria activity are more important factors limiting Nitrobacter spp. mediated nitrite oxidation, rather than the free ammonia concentration.

Expression of a putative nitrite reductase and the reversible inhibition of nitrite-dependent respiration by nitric oxide in Nitrobacter winogradskyi Nb-255.

The nitrite oxidizing Alphaproteobacterium, Nitrobacter winogradskyi, primarily conserves energy from the oxidation of nitrite (NO(2)(-))to nitrate (NO(3)(-)) through aerobic respiration. Almost 20 years ago, NO-dependent NADH formation was reported to occur in both aerobic and anaerobic cell suspensions of N. winogradskyi strain 'agilis', suggesting that NO oxidation might contribute to energy conservation by Nitrobacter. Recently, the N. winogradskyi Nb-255 genome was found to contain a gene (Nwin_2648) that encodes a putative copper-containing nitrite reductase (NirK), which may reduce NO(2)(-) to NO. In this study, the putative nirK was found to be maximally transcribed under low O(2) (between zero and 4% O(2)) in the presence of NO(2)(-). Transcription of nirK was not detected under anaerobic conditions in the absence of NO(2)(-) or in the presence of NO(3)(-) and pyruvate. Although net production of NO could not be detected from either aerobically grown or anaerobically incubated cells, exogenous NO was consumed by viable cells and concomitantly inhibited NO(2)(-)-dependent O(2) uptake in a reversible, concentration dependent manner. Both NO(2(-)-dependent O(2) uptake and NO consumption were inhibited by 1 mM cyanide suggesting involvement of cytochrome oxidase with NO consumption. Abiotic consumption of NO was measured, yet, both the rates and kinetics of NO transformation in buffer alone, or by heat killed, or cyanide-treated cells differed from those of viable cells. In light of this new information, a modified model is proposed to explain how NirK and NO manage electron flux in Nitrobacter.

Response of Nitrobacter spp. ribosomal gene and transcript abundance following nitrite starvation and exposure to mechanistically distinct inhibitors.

The Nitrobacter spp. rRNA gene (rDNA) and relative rRNA transcript abundance (rRNAt/rDNA ratio) were evaluated in response to sudden changes in the nitrite oxidation rate. The rDNA abundance poorly indicated sudden transitions in the rate, whereas the relative rRNAt abundance usually varied quickly and significantly. In response to changes in nitrite concentration, 8 h were required for the rRNAt/rDNA ratio to transition from a minimum value at nitrite starvation (approximately 0.07) to a maximum value with excess nitrite present (approximately 4), and 5 h were required for this metric to return to the minimum value after nitrite starvation re-ensued. Generally, the relative rRNAt abundance dropped significantly after 4.5 h of exposure to three different inhibitors. A sharp decline in the rRNAt/rDNA ratio occurred during exposure to 3,5-DCP (from 4 down to 0.2) even as the fractional inhibition level remained low (< 0.10); the minimum ratio value was observed when nitrite oxidation was completely inhibited. The ratio decreased significantly during exposure to azide (from 4 to 0.5) and H+ (from 2 to 0.2), but only when the fractional inhibition levels were high (> 0.8). Interestingly, when the pH was suddenly changed to 4.5, inhibiting nitrite oxidation completely, the rRNAt/rDNA metric did not decline suggesting that rRNAt processing was inhibited. This effect was not observed during severe inhibition with 3,5-DCP and azide. Overall, the findings indicate the relative rRNAt abundance can be used to closely track in situ Nitrobacter spp. activity and in most instances will reveal inhibition events with the potential to impact treatment performance in reactors where Nitrobacter spp. are dominant.

Free ammonia and free nitrous acid inhibition on the anabolic and catabolic processes of Nitrosomonas and Nitrobacter.

The inhibitory effects of free ammonia (FA) and free nitrous acid (FNA) on the catabolic and anabolic processes of Nitrosomonas and Nitrobacter were investigated using a method that allows decoupling the growth and energy generation processes. Lab-scale sequencing batch reactors (SBRs) were operated for the enrichment of Nitrosomonas and Nitrobacter. Fluorescent In-Situ Hybridization (FISH) analysis showed that the reactors were 82% and 73% enriched with Nitrosomonas and Nitrobacter, respectively. Batch tests were carried out to measure the oxygen uptake rate (OUR) by the enriched cultures at various FA and FNA levels, in the presence (OUR with CO2 ) or absence (OUR without CO2) of inorganic carbon (CO2, HCO*3 and CO 2*3). FA up to 16.0 mgNH3-N.L(-1) was not found to have any inhibitory effect on either the catabolic or anabolic processes of the Nitrosomonas culture, but both these processes were inhibited by FNA. While an FNA level of 0.40-0.63 mgHNO2-N.L(-1) inhibited the energy production capability of Nitrosomonas by 50%, the growth process of the culture was completely inhibited by FNA at a concentration of 0.40 mgHNO2-N.L(-1). Both FA and FNA were found to have strong inhibition on the anabolic processes of Nitrobacter, but with limited inhibitory effects on the catabolism of this culture. The biosynthesis of Nitrobacter was totally inhibited at an FA level of 6.0 mgNH3-N.L(-1) (or above) or an FNA level of 0.02 mgHNO2-N.L(-1) (or above). At the same level of FA, the energy production capability of Nitrobacter was only inhibited by 12%, whereas an FNA level of up to 0.024 mgHNO2-N.L(-1) did not show any inhibition on the energy production of Nitrobacter. Further, these inhibitory effects appears to be much stronger on Nitrobacter than on Nitrosomonas, supporting that FA and FNA inhibition may play a major role in the elimination of nitrite oxidizing bacteria in processes treating wastewater containing a high level of nitrogen.

Kinetic characterisation of an enriched Nitrospira culture with comparison to Nitrobacter.

Nitrospira and Nitrobacter are nitrite-oxidising bacteria commonly identified in nitrogen removal wastewater treatment plants. Little is known about the growth parameters of Nitrospira or the effects of environmental conditions or inhibitory compounds on Nitrospira activity. These bacterial properties were investigated using an enriched Nitrospira culture and an enriched Nitrobacter culture or Nitrobacter literature values. Compared to Nitrobacter, Nitrospira was found to have a comparable optimal pH range (8.0-8.3); similar normalised activity-temperature relationship (0.44e(0.055(T-15))) for temperatures between 15 and 30 degrees C and a similar oxygen half-saturation constant, K(O) (0.54+/-0.14 mgL(-1)). The major differences identified were that Nitrospira had a lower nitrite half-saturation constant, K(S) (0.9+/-0.07 mgNO(2)-NL(-1)); lower inhibition threshold concentrations for free ammonia (between 0.04 and 0.08 mg NH(3)-NL(-1)) and free nitrous acid (less than 0.03 mg HNO(2)-NL(-1)) and a higher yield (0.15+/-0.04 g VSS g N(-1)). Therefore, Nitrospira is more likely to dominate nitrite oxidation under conditions with low ammonium and nitrite concentrations, which would provide an advantage to them due to their lower K(S) value while avoiding any free ammonia or free nitrous acid inhibition.

The inhibitory effects of free nitrous acid on the energy generation and growth processes of an enriched nitrobacter culture.

The inhibitory effects of nitrite (NO2-)/free nitrous acid (HNO2-FNA) on the metabolism of Nitrobacter were investigated using a method allowing the decoupling of the growth and energy generation processes. A lab-scale sequencing batch reactor was operated forthe enrichment of a Nitrobacter culture. Fluorescent in situ hybridization (FISH) analysis showed that 73% of the bacterial population was Nitrobacter. Batch tests were carried out to assess the oxygen and nitrite consumption rates of the enriched culture at low and high nitrite levels, in the presence or absence of inorganic carbon. It was observed that in the absence of CO2, the Nitrobacter culture was able to oxidize nitrite at a rate that is 76% of that in the presence of CO2, with an oxygen consumption rate that is 85% of that measured in the presence of CO2. This enabled the impacts of nitrite/FNA on the catabolic and anabolic processes of Nitrobacter to be assessed separately. FNA rather than nitrite was likely the actual inhibitor to the Nitrobacter metabolism. It was revealed that FNA of up to 0.05 mg HNO2-N x L(-1) (3.4 microM), which was the highest FNA concentration used in this study, did not have any inhibitory effect on the catabolic processes of Nitrobacter. However, FNA initiated its inhibition to the anabolic processes of Nitrobacterat approximately 0.011 mg HNO2-N x L(-1) (0.8 microM), and completely stopped biomass synthesis at a concentration of approximately 0.023 mg HNO2-N x L(-1) (1.6 microM). The inhibitory effect could be described by an empirical inhibitory model proposed in this paper, but the underlying mechanisms remain to be revealed.

Selective enrichment and molecular characterization of a previously uncultured Nitrospira-like bacterium from activated sludge.

Previously uncultured nitrite-oxidizing bacteria affiliated to the genus Nitrospira have for the first time been successfully enriched from activated sludge from a municipal wastewater treatment plant. During the enrichment procedure, the abundance of the Nitrospira-like bacteria increased to approximately 86% of the total bacterial population. This high degree of purification was achieved by a novel enrichment protocol, which exploits physiological features of Nitrospira-like bacteria and includes the selective repression of coexisting Nitrobacter cells and heterotrophic contaminants by application of ampicillin in a final concentration of 50 microg ml(-1). The enrichment process was monitored by electron microscopy, fluorescence in situ hybridization (FISH) with rRNA-targeted probes and fatty acid profiling. Phylogenetic analysis of 16S rRNA gene sequences revealed that the enriched bacteria represent a novel Nitrospira species closely related to uncultured Nitrospira-like bacteria previously found in wastewater treatment plants and nitrifying bioreactors. The enriched strain is provisionally classified as 'Candidatus Nitrospira defluvii'.

Pilot-scale studies on biological treatment of hypersaline wastewater at low temperature.

In order to investigate the feasibility of biological treatment of hypersaline wastewater produced from toilet flushing with seawater at low temperature, pilot-scale studies were established with plug-flow activated sludge process at low temperature (5-9 degrees C) based on bench-scale experiments. The critical salinity concentration of 30 g/L, which resulted from the cooperation results of the non-halophilic bacteria and the halophilic bacteria, was drawn in bench-scale experiments. Pilot-scale studies showed that high COD removal efficiency, higher than 80%, was obtained at low temperature when 30 percent seawater was introduced. The salinity improved the settleability of activated sludge, and average sludge value dropped down from 38% to 22.5% after adding seawater. Seawater salinity had a strong negative effect on notronomonas and nitrobacter growth, but much more on the nitrobacter. The nitrification action was mainly accomplished by nitrosomonas. Bench-scale experiments using two SBRs were carried out for further investigation under different conditions of salinities, ammonia loadings and temperatures. Biological nitrogen removal via nitrite pathway from wastewater containing 30 percent seawater was achieved, but the ammonia removal efficiency was strongly related not only to the influent ammonia loading at different salinities but also to temperature. When the ratio of seawater to wastewater was 30 percent, and the ammonia loading was below the critical value of 0.15 kgNH4+-N/(kgMLSS.d), the ammonia removal efficiency via nitrite pathway was above 90%. The critical level of ammonia loading was 0.15, 0.08 and 0.03 kgNH4+-N/(kgMLSS.d) respectively at the different temperature 30 degrees C, 25 degrees C and 20 degrees C when the influent ammonia concentration was 60-80 mg/L and pH was 7.5-8.0.

Effects of seawater salinity on nitrite accumulation in short-range nitrification to nitrite as end product.

The effect of seawater salinity on nitrite accumulation in short-range nitrification to nitrite as the end product was studied by using a SBR. Experimental results indicated that the growth of nitrobacteria was inhibited and very high levels of nitrite accumulation at different salinities were achieved under the conditions of 25-28 degrees C, pH 7.5-8.0, and the influent ammonia nitrogen of 40-70 mg/L when seawater flow used to flush toilet was less than 35% (salinity 12393 mg/L, Cl- 6778 mg/L) of total domestic wastewater flow, which is mainly ascribed to much high chlorine concentration of seawater. Results showed that high seawater salinity is available for short-range nitrification to nitrite as the end product. When the seawater flow used to flush toilet accounting for above 70% of the total domestic wastewater flow, the removal efficiency of ammonia was still above 80% despite the removal of organics declined obviously (less than 60%). It was found that the effect of seawater salinity on the removal of organics was negative rather than positive one as shown for ammonia removal.

Influence of long-term diesel fuel pollution on nitrite-oxidising activity and population size of nitrobacter spp in soil.

Previous investigations have shown that ammonia oxidation is not inhibited by diesel fuel in a soil with a long history of contamination contrary to a non-contaminated soil. As a consequence, ammonia oxidation does not constitute a Limited step in nitrification process (Appl. Environ. Microbiol. 65 (1999) 4008). Moreover, this type of soil also has had the opportunity to develop an abundant microbial population able to metabolise the diesel hydrocarbons. Whether the properties of soil with a long history of diesel fuel contamination may affect the activity of nitrite-oxidising bacteria was investigated. It was observed that re-exposure of soil to diesel fuel apparently stimulated the proliferation of nitrite-oxidising bacteria, as determined by most probable number (MPN) culture technique and MPN-polymerase chain reaction technique. The potential of nitrite-oxidising activity in soil treated with diesel fuel was about 4 times higher than in the control without addition. In the presence of diesel fuel and ammonium, the potential nitrite-oxidising activity was 40% higher than in presence of ammonium only. However, in the presence of hydrocarbon only, low proliferation of Nitrobacter was observed, probably because the heterotrophic bacteria were strongly limited by lack of nitrogen and did not produce sufficient organic metabolites that could be used by the Nitrobacter cells.

Susceptibility of ammonia-oxidizing bacteria to nitrification inhibitors.

Activity of nitrification inhibitors to several typical ammonia-oxidizing bacteria isolated recently, i. e. Nitrosococcus, Nitrosolobus, Nitrosomonas, Nitrosospira and Nitrosovibrio species was assayed using 2-amino-4-methyl-trichloromethyl-1,3,5-triazine (MAST), 2-amino-4-tribromomethyl-6-trichloromethyl-1,3,5-triazine (Br-MAST), 2-chloro-6-trichloromethylpyridine (nitrapyrin) and others, and compared to confirm the adequate control of ammonia-oxidizing bacteria by the inhibitors. The order of activity of the inhibitors to 13 species of ammonia-oxidizing bacteria examined was approximately summarized as Br-MAST > or = nitrapyrin > or = MAST > other inhibitors. Two Nitrosomonas strains, N. europaea ATCC25978 and N. sp. B2, were extremely susceptible to Br-MAST, exhibiting a pI50 > or = 6.40. These values are the position logarithms of the molar half-inhibition concentration. The 16S rRNA gene sequence similarity for the highly susceptible 4 strains of genus Nitrosomonas was 94% to 100% of Nitrosomonas europaea, although those of the less susceptible 3 strains of ammonia-oxidizing bacteria, Nitrosococcus oceanus C-107 ATCC19707, Nitrosolobus sp. PJA1 and Nitrosolobus multiformis ATCC25196, were 77.85, 91.53 and 90.29, respectively. However, no clear correlation has been found yet between pI50-values and percent similarity of 16S rRNA gene sequence among ammonia-oxidizing bacteria.