Exploring potential impact(s) of cerium in mining wastewater on the performance of partial-nitrification process and nitrogen conversion microflora.

Cerium Ce(III) is one of the major pollutants contained in wastewater generated during Ce(III) mining. However, the effect(s) of Ce(III) on the functional genera responsible for removing nitrogen biologically from wastewater has not been studied and reported. In this study, the effects of Ce(III) on aspects of partial-nitritation-(PN) process including ammonia oxidation rate (AOR), process kinetics, and microbial activities were investigated. It was found that the effect of dosing Ce(III) in the PN system correlated strongly with the AOR. Compared to the control, batch assays dosed with 5 mg/L Ce(III) showed elevated PN efficiency of about 121%, an indication that maximum biological response was feasible upon Ce(III) dose. It was also found that, PN performance was not adversely affected, given that Ce(III) dose was ≤20 mg/L. Process kinetics investigated also suggested that the maximum Ce(III) dose without any visible inhibition to the activities of ammonium oxidizing bacteria was 1.37 mg/L, but demonstrated otherwise when Ce(III) dose exceeded 5.63 mg/L. Compared to the control, microbes conducted efficient Ce(III) removal (averaged 98.66%) via biosorption using extracellular polymeric substances (EPS). Notably, significant deposits of Ce(III) was found within the EPS produced as revealed by SEM, EDX, CLSM and FTIR. 2-dimensional correlation infrared-(2DCOS-IR) revealed ester group (uronic acid) as a major organic functional group that promoted Ce(III) removal. Excitation-emission matrix-(EEM) spectrum and 2DCOS-IR suggested the dominance of Fulvic acid, hypothesized to have promoted the performance of the PN process under Ce(III) dosage.

Do soil property variations affect dicyandiamide efficiency in inhibiting nitrification and minimizing carbon dioxide emissions?

Nitrification inhibitors (NIs) are used to retard the nitrification process and reduce nitrogen (N) losses. However, the effects of soil properties on NI efficacy are less clear. Moreover, the direct and indirect effects of soil property variations on NI efficiency in minimizing carbon dioxide (CO2) emissions have not been previously studied. An incubation experiment was conducted for 40 days with two treatments, N (200 mg N-urea kg-1) and N + dicyandiamide (DCD) (20 mg DCD kg-1), and a control group (without the N) to investigate the response of ammonia-oxidizing bacteria (AOB) and archaea (AOA) to DCD application and the consequences for CO2, nitrous oxide (N2O) and ammonia (NH3) emissions from six soils from the Loess Plateau with different properties. The nitrification process completed within 6-18 days for the N treatment and within 30->40 days for the N + DCD treatment. AOB increased significantly with N fertilizer application, while this effect was inhibited in soils when DCD was applied. AOA was not sensitive to N fertilizer and DCD application. The nitrification rate was positively correlated with the clay (p < 0.05) and SOM contents (p < 0.01); DCD was more effective in loam soil with low SOM and high soil pH. Soil pH significantly was decreased with N fertilizer application, while it increased when DCD was applied. Moreover, DCD application decreased CO2 emissions from soils by 22%-172%; CO2 emissions were negatively correlated with the clay and SOM contents. DCD application decreased N2O emissions in each soil by 1.0- to 94-fold compared with those after N fertilizer application. In contrast, DCD application increased NH3 release from soils by 59-278%. NH3 volatilization was negatively correlated with clay (p < 0.05) and SOM (p < 0.01) contents and positively correlated with soil pH (p < 0.01). Therefore, soil texture, SOM and soil pH have significant effects on the DCD performance, nitrification process and gaseous emissions.

Trichloroethylene inhibits nitrogen transformation and microbial community structure in Mollisol.

Trichloroethylene (TCE) is the most ubiquitous halogenated organic pollutant in the environment, it is one of the 129 priority control pollutants. In order to clarify the influence of TCE on microorganisms and nitrogen transformation in Mollisol is the core purpose of this study. Results showed that 10 mg kg-1 TCE is the concentration limit of ammonification in Mollisol. When the concentration of TCE reached 10 mg kg-1 and the effect lasted for over 7 days, the process of ammonia oxidation to nitric acid in Mollisol will be affected. TCE affected the process of nitrate (NO3-) transformation into nitrite (NO2-) by affecting the activity of nitrate reductase, thereby affected the denitrification process in soil. When the concentration of TCE is more than 10 mg kg-1 it reduced the ability of soil microorganisms to obtain nitrogen, thereby affecting soil nitrogen transformation. RDA (Redundancy analysis) showed that the activity of nitrate reductase and the number of nitrifying bacteria and denitrifying bacteria in soil was negatively correlated with the incubation of TCE. In addition, soil nitrate reductase, nitrite reductase, peroxidase activity, ammonifying bacteria, nitrifying bacteria and denitrifying bacteria were negatively correlated with TCE concentration. Beyond that PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) of functional gene structure depend on KEGG (Kyoto Encyclopedia of Genes and Genomes) showed that 20 mg kg-1 TCE significantly inhibited the metabolism of energy and other substances in Mollisol. Based on the above, it is found that TCE significantly affected nitrification and denitrification in Mollisol, thus the nitrogen transformation in Mollisol was affected by TCE contamination.

Tetracyclines lead to ammonium accumulation during nitrification process.

The effect of tetracyclines used for swine food-production (tetracycline and oxytetracycline) on enriched nitrifying bacteria cultures over time was investigated in this study. Short-term exposure assays were performed in different concentrations of each antibiotic, using ammonia oxidizing bacteria (AOB) culture and nitrifying bacteria. The results pointed out a higher inhibitory effect of tetracycline on both bacterial communities. The AOB was more sensitive to antibiotic exposure when compared to the nitrifying culture. Although high antibiotic concentrations were applied, the half maximal inhibitory concentration (IC50) was achieved only for the AOB culture exposed to tetracycline at a concentration of 273 mg L-1. Nonetheless, the long-term exposure assay demonstrated a reduction of the tetracycline inhibition effect against AOB. The exposure to 100 mg L-1 of tetracycline (TC) did not show relevant influence over ammonium conversion efficiency; however, at 128 mg L-1 of TC, the efficiency decreased from 94% to 72%. Further investigation revealed that TC reduced the final effluent quality due to the development of a resistance mechanism by AOB culture against this antibiotic. This mechanism involves increasing the excretion of extracellular polymeric substances (EPS) and soluble microbial products (SMP), which probably increases BOD, and reduces ammonia consumption by the bacterial culture.

Nitrification inhibitors effectively target N2 O-producing Nitrosospira spp. in tropical soil.

The nitrification inhibitors (NIs) 3,4-dimethylpyrazole (DMPP) and dicyandiamide (DCD) can effectively reduce N2 O emissions; however, which species are targeted and the effect of these NIs on the microbial nitrifier community is still unclear. Here, we identified the ammonia oxidizing bacteria (AOB) species linked to N2 O emissions and evaluated the effects of urea and urea with DCD and DMPP on the nitrifying community in a 258 day field experiment under sugarcane. Using an amoA AOB amplicon sequencing approach and mining a previous dataset of 16S rRNA sequences, we characterized the most likely N2 O-producing AOB as a Nitrosospira spp. and identified Nitrosospira (AOB), Nitrososphaera (archaeal ammonia oxidizer) and Nitrospira (nitrite-oxidizer) as the most abundant, present nitrifiers. The fertilizer treatments had no effect on the alpha and beta diversities of the AOB communities. Interestingly, we found three clusters of co-varying variables with nitrifier operational taxonomic units (OTUs): the N2 O-producing AOB Nitrosospira with N2 O, NO3 - , NH4 + , water-filled pore space (WFPS) and pH; AOA Nitrososphaera with NO3 - , NH4 + and pH; and AOA Nitrososphaera and NOB Nitrospira with NH4 + , which suggests different drivers. These results support the co-occurrence of non-N2 O-producing Nitrososphaera and Nitrospira in the unfertilized soils and the promotion of N2 O-producing Nitrosospira under urea fertilization. Further, we suggest that DMPP is a more effective NI than DCD in tropical soil under sugarcane.

Acute and chronic toxicity of nickel to nitrifiers at different temperatures.

This study investigated the acute nickel toxicity on nitrification of low ammonia synthetic wastewater at 10, 23, and 35°C. The nickel inhibition half-velocity constants (KI,Ni) for ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) based on Ni/MLSS ratio at 10, 23, and 35°C were 5.4 and 5.6 mg Ni/g MLSS, 4.6 and 3.5 mg Ni/g MLSS, and 9.1 and 2.7 mg Ni/g MLSS, respectively. In addition, chronic toxicity of nickel to nitrification of low ammonia synthetic wastewater was investigated at 10°C in two sequencing batch reactors (SBRs). Long-term SBRs operation and short-term batch tests were comparable with respect to the extent of inhibition and corresponding Ni/MLSS ratio. The µmax, b, and Ko of AOB were 0.16 day-1, 0.098 day-1 and 2.08 mg O2/L after long-term acclimatization to nickel of 1 mg/L at 10°C, high dissolved oxygen (DO) (7 mg/L) and long solids retention time (SRT) of 63-70 days. Acute nickel toxicity of nitrifying bacteria was completely reversible.

Simultaneous nitrification and denitrification with different mixed nitrogen loads by a hypothermia aerobic bacterium.

Microorganism with simultaneous nitrification and denitrification ability plays a significant role in nitrogen removal process, especially in the eutrophic waters with excessive nitrogen loads. The nitrogen removal capacity of microorganism may suffer from low temperature or nitrite nitrogen source. In this study, a hypothermia aerobic nitrite-denitrifying bacterium, Pseudomonas tolaasii strain Y-11, was selected to determine the simultaneous nitrification and denitrification ability with mixed nitrogen source at 15 °C. The sole nitrogen removal efficiencies of strain Y-11 in simulated wastewater were obtained. After 24 h of incubation at 15 °C, the ammonium nitrogen fell below the detection limit from an initial value of 10.99 mg/L. Approximately 88.0 ± 0.33% of nitrate nitrogen was removed with the initial concentration of 11.78 mg/L and the nitrite nitrogen was not detected with the initial concentration of 10.75 mg/L after 48 h of incubation at 15 °C. Additionally, the simultaneous nitrification and denitrification nitrogen removal ability of P. tolaasii strain Y-11 was evaluated using low concentration of mixed NH4+-N and NO3--N/NO2--N (about 5 mg/L-N each) and high concentration of mixed NH4+-N and NO3--N/NO2--N (about 100 mg/L-N each). There was no nitrite nitrogen accumulation at the time of evaluation. The results demonstrated that P. tolaasii strain Y-11 had higher simultaneous nitrification and denitrification capacity with low concentration of mixed inorganic nitrogen sources and may be applied in low temperature wastewater treatment.

Roxarsone exposure jeopardizes nitrogen removal and regulates bacterial community in biological sequential batch reactors.

Roxarsone is widely present in wastewaters of many animal farms in China. However, little is known about how long-term roxarsone exposure influences the nitrogen removal of biological wastewater treatment in agricultural settings. Here we investigated the nitrogen removal performance of a biological sequential batch reactor (SBR) and the changes of bacterial community, upon long-term roxarsone exposure. The long-term roxarsone dosing decreased the SBR nitrogen removal by 52.4%, with an immediate inhibition on denitrification and a delayed inhibition on nitrification. The analyses of bacterial enzymatic activities and 16 S rRNA sequencing revealed that bacterial activities generally decreased, and the nitrogen-cycling bacterial community was changed, particularly by the decrease (Acinetobacter and Methylophilaceae), persistence (Flavobacterium and Methylotenera), and emergence (Aeromonas) of certain bacterial genera. Overall, chronic roxarsone exposure could suppress nitrification and denitrification, which may even have broad implications on the use efficiency and cycling of nitrogen in agroecosystems.

Effect of the nitrification inhibitor (3, 4-dimethylpyrazole phosphate) on the activities and abundances of ammonia-oxidizers and denitrifiers in a phenanthrene polluted and waterlogged soil.

Through a 60-day microcosm incubation, the effect of 3, 4-dimethylpyrazole phosphate (DMPP) on the activities and abundances of ammonia-oxidizers and denitrifiers in phenanthrene-polluted soil was investigated. Five treatments were conducted for clean soil (CK), phenanthrene added (P), phenanthrene and DMPP added (PD), phenanthrene and urea added (PU), and phenanthrene, urea, and DMPP added (PUD) soils. The results indicate that the potential nitrification rate (PNR) in the P treatment was significantly higher than that in the PD treatment only on day 7, whereas the PNR in the PU treatment was significantly higher than that in the PUD treatment on each sampling day. The abundance of soil ammonia-oxidizing bacteria (AOB) in the PU treatment was significantly higher than that in the PUD treatment on each sampling day. Moreover, the abundance of AOB but rather than the ammonia-oxidizing archaea (AOA) had significantly positive correlation with soil PNR (P < 0.05). DMPP showed no obvious effect on the soil denitrification enzyme activity (DEA), which could have inhibited the abundances of denitrification-related narG, nirS, and nirK genes. The results of this study should provide a deeper understanding of the interaction between soil polycyclic aromatic hydrocarbons (PAH) contamination, ammonia oxidization, and denitrification, and offer valuable information for assessing the potential contribution of denitrification for soil PAH elimination.

Investigation of copper inhibition of nitrifying moving bed biofilm (MBBR) reactors during long term operations.

Copper, a prevalent heavy metal in industrial mining wastewaters, has been shown to inhibit nitrification in wastewater treatment systems. Biofilm treatment systems have an inherent potential to reduce inhibition. This study investigated the effects of copper concentration on nitrifying biofilms in moving bed biofilm reactor (MBBR) systems across long term operation using influent ammonia concentrations representative of gold mining wastewater. Conventional isotherm models did not adequately model the attachment of copper to the biofilm. Long term nitritation was shown to be uninhibited at influent copper concentrations between 0.13 and 0.61 mg Cu/L. Nitratation was inhibited with influent copper concentrations of 0.28-0.61 mg Cu/L. There was no statistical difference in biofilm characteristics, including biofilm thickness, mass and density, across all copper concentrations tested, however, changes in biofilm morphology were observed. The demonstrated resistance of the nitrifying biofilm to copper inhibition makes the MBBR system a promising technology for treating ammonia in mining wastewaters.

Acute Impact of Chlortetracycline on Nitrifying and Denitrifying Processes.

In the current study, sequential nitrification and anoxic experiments in synthetic municipal wastewater were exposed to 0.5 to 100 mg/L of chlortetracycline for 24 h to evaluate acute impact on the nitrification, and denitrification processes of biological treatment. Both processes were significantly (p < 0.05) inhibited at >50 mg/L of chlortetracycline, and the results revealed that nitrification was adversely affected by chlortetracycline compared with the anoxic process. In nitrification, chemical oxygen removal (COD) and ammonia oxidation kinetics were 50% inhibited at 10 mg chlortetracycline/L, and nitrite oxidation kinetics at 0.5 mg chlortetracycline/L. Likewise, in the anoxic process, 14 and 10 mg/L of chlortetracycline inhibited 50% of COD removal and nitrate reduction kinetics, respectively. In nitrification and denitrification, 90% of chlortetracycline was removed by adsorbing onto sludge suspended solids. In addition, a higher chlortetracycline concentration in anoxic effluent, compared with aerobic effluents, indicated a dissimilarity in the composition of sludge solids, pH, and biomass production for both processes.

Effects of metal oxide nanoparticles on nitrification in wastewater treatment systems: A systematic review.

While the variety of engineered nanoparticles used in consumer products continues to grow, the use of metal oxide nanoparticles in electronics, textiles, cosmetics and food packaging industry has grown exponentially in recent years, which will inevitably result in their release into wastewater streams in turn impacting the important biological processes in wastewater treatment plants. Among these processes, nitrification play a critical role in nitrogen removal during wastewater treatment, however, it is sensitive to a wide range of inhibitory substances including metal oxide nanoparticles. Therefore, it is essential to systematically asses the effects of metal oxide nanoparticles on nitrification in biological wastewater treatment systems. In this review we discuss the present scenario of metal oxide nanoparticles and their impact on biological wastewater treatment processes, specifically nitrogen removal through nitrification. We also summarize the various methods used to measure nitrification inhibition by metal oxide nanoparticles and highlight corresponding results obtained using those methods. Finally, the key research gaps that need to be addressed in future are discussed.

Effects of 1-octyl-3-methylimidazolium nitrate on the microbes in brown soil.

The toxicity of ionic liquids (ILs) on soil organisms has aroused wide attention due to their high-solubility. The present investigation focused on the toxicity of 1-octyl-3-methylimidazolium nitrate ([C8mim]NO3) on the microbial populations (bacteria, fungi, and actinomycetes), soil enzyme (urease, dehydrogenase, acid phosphatase, and ß-glucosidase) activities, microbial community diversity using terminal restriction fragment length polymorphism (T-RFLP), and abundance of the ammonia monooxygenase (amoA) genes of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) using quantitative real-time polymerase chain reaction (q-PCR) in brown soil at each trial with doses of 0, 1.0, 5.0, and 10.0mg/kg on days 10, 20, 30, and 40. The contents of [C8mim]NO3 in soil were measured using high performance liquid chromatography with recoveries of 84.3% to 85.2%, and changed less than 10% during the experimental period. A significant decrease was observed from the bacteria, fungi and actinomycetes populations at 10.0mg/kg, at which the urease activity was inhibited and the ß-glucosidase activity was stimulated on days 20, 30, and 40. In addition, [C8mim]NO3 inhibited the dehydrogenase activity at 10mg/kg on days 30 and 40 and the acid phosphatase activity on day 20. The diversity of the soil microbial community and the gene abundance of AOA- and AOB- amoA were also inhibited. Furthermore, the present investigation provided more scientific information for the toxicity evaluation of ILs in soil.

Nanosilver inhibits nitrification and reduces ammonia-oxidising bacterial but not archaeal amoA gene abundance in estuarine sediments.

Silver nanoparticles (AgNPs) enter estuaries via wastewater treatment effluents, where they can inhibit microorganisms, because of their antimicrobial properties. Ammonia-oxidising bacteria (AOB) and archaea (AOA) are involved in the first step of nitrification and are important to ecosystem function, especially where effluent discharge results in high nitrogen inputs. Here, we investigated the effect of a pulse addition of AgNPs on AOB and AOA ammonia monooxygenase (amoA) gene abundances and benthic nitrification potential rates (NPR) in low-salinity and mesohaline estuarine sediments. Whilst exposure to 0.5 mg L-1 AgNPs had no significant effect on amoA gene abundances or NPR, 50 mg L-1 AgNPs significantly decreased AOB amoA gene abundance (up to 76% over 14 days), and significantly decreased NPR by 20-fold in low-salinity sediments and by twofold in mesohaline sediments, after one day. AgNP behaviour differed between sites, whereby greater aggregation occurred in mesohaline waters (possibly due to higher salinity), which may have reduced toxicity. In conclusion, AgNPs have the potential to reduce ammonia oxidation in estuarine sediments, particularly where AgNPs accumulate over time and reach high concentrations. This could lead to long-term risks to nitrification, especially in polyhaline estuaries where ammonia-oxidation is largely driven by AOB.

Nitrifier-induced denitrification is an important source of soil nitrous oxide and can be inhibited by a nitrification inhibitor 3,4-dimethylpyrazole phosphate.

Soil ecosystem represents the largest contributor to global nitrous oxide (N2 O) production, which is regulated by a wide variety of microbial communities in multiple biological pathways. A mechanistic understanding of these N2 O production biological pathways in complex soil environment is essential for improving model performance and developing innovative mitigation strategies. Here, combined approaches of the 15 N-18 O labelling technique, transcriptome analysis, and Illumina MiSeq sequencing were used to identify the relative contributions of four N2 O pathways including nitrification, nitrifier-induced denitrification (nitrifier denitrification and nitrification-coupled denitrification) and heterotrophic denitrification in six soils (alkaline vs. acid soils). In alkaline soils, nitrification and nitrifier-induced denitrification were the dominant pathways of N2 O production, and application of the nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) significantly reduced the N2 O production from these pathways; this is probably due to the observed reduction in the expression of the amoA gene in ammonia-oxidizing bacteria (AOB) in the DMPP-amended treatments. In acid soils, however, heterotrophic denitrification was the main source for N2 O production, and was not impacted by the application of DMPP. Our results provide robust evidence that the nitrification inhibitor DMPP can inhibit the N2 O production from nitrifier-induced denitrification, a potential significant source of N2 O production in agricultural soils.

Microbial N Transformations and N2O Emission after Simulated Grassland Cultivation: Effects of the Nitrification Inhibitor 3,4-Dimethylpyrazole Phosphate (DMPP).

Grassland cultivation can mobilize large pools of N in the soil, with the potential for N leaching and N2O emissions. Spraying with the nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) before cultivation was simulated by use of soil columns in which the residue distribution corresponded to plowing or rotovation to study the effects of soil-residue contact on N transformations. DMPP was sprayed on aboveground parts of ryegrass and white clover plants before incorporation. During a 42-day incubation, soil mineral N dynamics, potential ammonia oxidation (PAO), denitrifying enzyme activity (DEA), nitrifier and denitrifier populations, and N2O emissions were investigated. The soil NO3- pool was enriched with 15N to trace sources of N2O. Ammonium was rapidly released from decomposing residues, and PAO was stimulated in soil near residues. DMPP effectively reduced NH4+ transformation irrespective of residue distribution. Ammonia-oxidizing archaea (AOA) and bacteria (AOB) were both present, but only the AOB amoA transcript abundance correlated with PAO. DMPP inhibited the transcription of AOB amoA genes. Denitrifier genes and transcripts (nirK, nirS, and clades I and II of nosZ) were recovered, and a correlation was found between nirS mRNA and DEA. DMPP showed no adverse effects on the abundance or activity of denitrifiers. The 15N enrichment of N2O showed that denitrification was responsible for 80 to 90% of emissions. With support from a control experiment without NO3- amendment, it was concluded that DMPP will generally reduce the potential for leaching of residue-derived N, whereas the effect of DMPP on N2O emissions will be significant only when soil NO3- availability is limiting. IMPORTANCE: Residue incorporation following grassland cultivation can lead to mobilization of large pools of N and potentially to significant N losses via leaching and N2O emissions. This study proposed a mitigation strategy of applying 3,4-dimethylpyrazole phosphate (DMPP) prior to grassland cultivation and investigated its efficacy in a laboratory incubation study. DMPP inhibited the growth and activity of ammonia-oxidizing bacteria but had no adverse effects on ammonia-oxidizing archaea and denitrifiers. DMPP can effectively reduce the potential for leaching of NO3- derived from residue decomposition, while the effect on reducing N2O emissions will be significant only when soil NO3- availability is limiting. Our findings provide insight into how DMPP affects soil nitrifier and denitrifier populations and have direct implications for improving N use efficiency and reducing environmental impacts during grassland cultivation.

Inhibitory effect of thiourea on biological nitrification process and its eliminating method.

Thiourea is a typical nitrification inhibitor that shows a strong inhibitory effect against the biological nitrification process. The 50% inhibitory concentration (IC50) of thiourea on nitrification was determined to be 0.088 mg g VSS-1, and nitrifiers recovered from the thiourea inhibition after it was completely degraded. The thiourea-degrading ability of the sludge system was improved to 3.06 mg gVSS-1 h-1 through cultivation of thiourea-degrading bacteria by stepwise increasing the influent thiourea concentration. The dominant thiourea-degrading bacteria strain that used thiourea as the sole carbon and nitrogen source in the sludge system was identified as Pseudomonas sp. NCIMB. The results of this study will facilitate further research of the biodegradation characteristics of thiourea and similar pollutants.

Optimized inhibition assays reveal different inhibitory responses of hydroxylamine oxidoreductases from beta- and gamma-proteobacterial ammonium-oxidizing bacteria.

Ammonia-oxidizing bacteria (AOB), ubiquitous chemoautotrophic bacteria, convert ammonia (NH3) to nitrite (NO2(-)) via hydroxylamine as energy source. Excessive growth of AOB, enhanced by applying large amounts of ammonium-fertilizer to the farmland, leads to nitrogen leaching and nitrous oxide gas emission. To suppress these unfavorable phenomena, nitrification inhibitors, AOB specific bactericides, are widely used in fertilized farmland. However, new nitrification inhibitors are desired because of toxicity and weak-effects of currently used inhibitors. Toward development of novel nitrification inhibitors that target hydroxylamine oxidoreductase (HAO), a key enzyme of nitrification in AOB, we established inhibitor evaluation systems that include simplified HAO purification procedure and high-throughput HAO activity assays for the purified enzymes and for the live AOB cells. The new assay systems allowed us to observe distinct inhibitory responses of HAOs from beta-proteobacterial AOB (ßAOB) Nitrosomonas europaea (NeHAO) and gamma-proteobacterial AOB (γAOB) Nitrosococcus oceani (NoHAO) against phenylhydrazine, a well-known suicide inhibitor for NeHAO. Consistently, the live cells of N. europaea, Nitrosomonas sp. JPCCT2 and Nitrosospira multiformis of ßAOB displayed higher responses to phenylhydrazine than those of γAOB N. oceani. Our homology modeling studies suggest that different inhibitory responses of ßAOB and γAOB are originated from different local environments around the substrate-binding sites of HAOs in these two classes of bacteria due to substitutions of two residues. The results reported herein strongly recommend inhibitor screenings against both NeHAO of ßAOB and NoHAO of γAOB to develop HAO-targeting nitrification inhibitors with wide anti-AOB spectra.

Effects of the Nitrification Inhibitor 3,4-Dimethylpyrazole Phosphate on Nitrification and Nitrifiers in Two Contrasting Agricultural Soils.

UNLABELLED: The nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) is a powerful tool that can be used to promote nitrogen (N) use efficiency and reduce N losses from agricultural systems by slowing nitrification. Mounting evidence has confirmed the functional importance of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in nitrification and N2O production; however, their responses to DMPP amendment and the microbial mechanisms underlying the variable efficiencies of DMPP across different soils remain largely unknown. Here we compared the impacts of DMPP on nitrification and the dynamics of ammonia oxidizers between an acidic pasture soil and an alkaline vegetable soil using a (15)N tracing and (13)CO2-DNA-stable-isotope probing (SIP) technique. The results showed that DMPP significantly inhibited nitrification and N2O production in the vegetable soil only, and the transient inhibition was coupled with a significant decrease in AOB abundance. No significant effects on the community structure of ammonia oxidizers or the abundances of total bacteria and denitrifiers were observed in either soil. The (15)N tracing experiment revealed that autotrophic nitrification was the predominant form of nitrification in both soils. The (13)CO2-DNA-SIP results indicated the involvement of AOB in active nitrification in both soils, but DMPP inhibited the assimilation of (13)CO2 into AOB only in the vegetable soil. Our findings provide evidence that DMPP could effectively inhibit nitrification through impeding the abundance and metabolic activity of AOB in the alkaline vegetable soil but not in the acidic pasture soil, possibly due to the low AOB abundance or the adsorption of DMPP by organic matter. IMPORTANCE: The combination of the (15)N tracing model and (13)CO2-DNA-SIP technique provides important evidence that the nitrification inhibitor DMPP could effectively inhibit nitrification and nitrous oxide emission in an alkaline soil through influencing the abundance and metabolic activity of AOB. In contrast, DMPP amendment has no significant effect on nitrification or nitrifiers in an acidic soil, potentially owing to the low abundance of AOB and the possible adsorption of DMPP by organic matter. Our findings have direct implications for improved agricultural practices through utilizing the nitrification inhibitor DMPP in appropriate situations, and they emphasize the importance of microbial communities to the efficacy of DMPP.

Biological nitrification inhibition by rice root exudates and its relationship with nitrogen-use efficiency.

Microbial nitrification in soils is a major contributor to nitrogen (N) loss in agricultural systems. Some plants can secrete organic substances that act as biological nitrification inhibitors (BNIs), and a small number of BNIs have been identified and characterized. However, virtually no research has focused on the important food crop, rice (Oryza sativa). Here, 19 rice varieties were explored for BNI potential on the key nitrifying bacterium Nitrosomonas europaea. Exudates from both indica and japonica genotypes were found to possess strong BNI potential. Older seedlings had higher BNI abilities than younger ones; Zhongjiu25 (ZJ25) and Wuyunjing7 (WYJ7) were the most effective genotypes among indica and japonica varieties, respectively. A new nitrification inhibitor, 1,9-decanediol, was identified, shown to block the ammonia monooxygenase (AMO) pathway of ammonia oxidation and to possess an 80% effective dose (ED80 ) of 90 ng µl-1 . Plant N-use efficiency (NUE) was determined using a 15 N-labeling method. Correlation analyses indicated that both BNI abilities and 1,9-decanediol amounts of root exudates were positively correlated with plant ammonium-use efficiency and ammonium preference. These findings provide important new insights into the plant-bacterial interactions involved in the soil N cycle, and improve our understanding of the BNI capacity of rice in the context of NUE.