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In this study, an expanded granular sludge blanket reactor (EGSB) was proposed to achieve stable mainstream deammonification process by adding hydrazine (N 2 H 4 ). Two N 2 H 4 addition methods consisted of constant concentration (strategy A) and variable concentration (strategy B) both can inhibit nitrite oxidizing bacteria. A efficient performance was achieved with higher total nitrogen removal efficiency (82 ± 6%) and nitrogen removal rate (0.32 ± 0.02 kg N/(m 3 ·d)) under strategy B. For strategy A, anaerobic ammonia oxidizing bacteria (AnAOB) in-situ activity was decreased from 2.76 to 0.68 mg N/(g VSS·h) at 42 mg/L NH 4 + -N. Candidatus Brocadia abundance increase from 14.62% to 20.07% under the strategy may indicated the self-regulate mechanism of AnAOB. Aerobic ammonia oxidizing bacteria (AOB, mainly Nitrosomonas) and AnAOB (mainly Candidatus Brocadia) were always dominated under two strategies. Strategy B provided better environment for most microorganisms (mainly Chloroflexri, Planctomycetes, Proteobacteria and Chlorobi)., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

A few previous studies showed that the low soil moisture could interact with the toxic effect of the polycyclic aromatic hydrocarbons (PAHs) towards animals (mostly invertebrates). In the present research the impact of the soil moisture in the wide range (from the drought to high moisture conditions) in three different soil materials on toxic effect of the PAH (phenanthrene) towards soil microorganisms (nitrifying bacteria activity) was evaluated. The three dry soil materials were artificially contaminated with phenanthrene (0, 1, 10, 100 and 1000 mg kg -1 dry mass of soil) and moistened to the varied levels of the soil moisture (30% WHC (dry), 55% WHC (optimal) and 80% WHC (highly wet conditions)). After 7 days incubation, the nitrification potential was measured. The results of the proposed ANCOVA multiple regression model (adjusted R 2  = 0.91), showed that the increase of soil moisture enhanced the toxicity of the phenanthrene towards nitrification potential and this combined moisture-phenanthrene effect was soil dependent. Therefore, the effect of the soil moisture in combination with the soil diversity should not be missed in the ecotoxicological risk assessment of the PAHs., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Keratosis pilaris (KP) is a common skin finding that presents as follicular hyperkeratotic papules on the proximal extremities in patients with a propensity for atopy. Although often asymptomatic, the stippled appearance is cosmetically disturbing to patients and difficult to treat as current therapies are limited in availability and efficacy. Nitric oxide (NO) has been found to be essential in basic systemic and cutaneous physiologic function, specifically in terms of its anti-microbial and anti-inflammatory properties, which evolutionarily was maintained by ammonia-oxidizing bacteria (AOB). As modern hygiene practices have improved, there has been a gradual loss of cutaneous AOB and, therefore, the availability of an important source of human physiologic NO. We propose that restoring this dermal microflora with a purified strain of AOB, Nitrosomonas eutropha (D23), may reduce the overall cutaneous inflammatory state and, thus, be a potential therapeutic option for improving the cosmetic appearance of a skin condition such as KP which is often found in association with xerosis and atopic dermatitis. Clinical trial registry number: NCT03243617

J Drugs Dermatol. 2018;17(3):285-288.

The proper use of selective ammonia-oxidizing archaea (AOA) and/or ammonia-oxidizing bacteria (AOB) inhibitors is critical to distinguish AOA and AOB contribution. In this research, three inhibitors including ampicillin, dicyandiamide (DCD), and allylthiourea (ATU) were examined mainly focusing on inhibiting dosage, adaptability, and effects. The results showed that the optimized inhibitory dosage of ampicillin, DCD, and ATU was separately 1.5 g L -1 , 1 mM, and 25 μM. Among the three inhibitors, ATU exhibited the strongest and persistent inhibition effects and resulted in up to 90% inhibition in the AOB-enriched culture. The seemingly weakening inhibiting effects of ATU in the simulated river systems can be attributed to the involved role of AOA, the uneven spatial distribution of ATU, and protection by sediment structure in complex malodorous rivers. The high-throughput pyrosequencing analysis showed the AOB-related genus Nitrosomonas and Nitrosococcus were mostly affected by ATU in the enrichments and the river systems, respectively. The inhibition of ATU was realized mainly by reducing the abundance and activity of AOB. The decrease of the ratio of AOB/AOA amoA gene copy numbers after addition of ATU further confirmed the inhibiting effectiveness of ATU in complex microbial community of malodorous rivers.

Autotrophic nitrification in biological nitrogen removal systems has been shown to be sensitive to the presence of heavy metals in wastewater treatment plants. Using transcriptase-quantitative polymerase chain reaction (RT-qPCR) data, we examined the effect of copper on the relative expression of functional genes (i.e., amoA, hao, nirK, and norB) involved in redox nitrogen transformation in batch enrichment cultures obtained from a nitrifying bioreactor operated as a continuous reactor (24-h hydraulic retention time). 16S ribosomal RNA (rRNA) gene next-generation sequencing showed that Nitrosomonas-like populations represented 60-70% of the bacterial community, while other nitrifiers represented <5%. We observed a strong correspondence between the relative expression of amoA and hao and ammonia removal in the bioreactor. There were no considerable changes in the transcript levels of amoA, hao, nirK, and norB for nitrifying samples exposed to copper dosages ranging from 0.01 to 10 mg/L for a period of 12 h. Similar results were obtained when ammonia oxidation activity was measured via specific oxygen uptake rate (sOUR). The lack of nitrification inhibition by copper at doses lower than 10 mg/L may be attributed to the role of copper as cofactor for ammonia monooxygenase or to the sub-inhibitory concentrations of copper used in this study. Overall, these results demonstrate the use of molecular methods combined with conventional respirometry assays to better understand the response of wastewater nitrifying systems to the presence of copper.

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.

In this study, the effect of the organic micropollutant and known inhibitor of nitrification, p-cresol, was investigated on the metabolism of the ammonia oxidizing bacteria (AOB) Nitrosomonas eutropha C91 using MS-based quantitative proteomics. Several studies have demonstrated that AOB are capable of biotransforming a wide variety of aromatic compounds making them suitable candidates for bioremediation, yet the underlying molecular mechanisms are poorly described. The effect of two different concentrations of the aromatic micropollutant p-cresol (1 and 10 mg L(-1)) on the metabolism of N. eutropha C91, relative to a p-cresol absent control, was investigated. Though the rate of nitrification in N. eutropha C91 appeared essentially unaffected at both concentrations of p-cresol relative to the control, the expressional pattern of the proteins of N. eutropha C91 changed significantly. The presence of p-cresol resulted in the repressed expression of several key proteins related to N-metabolism, seemingly impairing energy production in N. eutropha C91, contradicting the observed unaltered rates of nitrification. However, the expression of proteins of the TCA cycle and proteins related to xenobiotic degradation, including a p-cresol dehydrogenase, was found to be stimulated by the presence of p-cresol. This indicates that N. eutropha C91 is capable of degrading p-cresol and that it assimilates degradation intermediates into the TCA cycle. The results reveal a pathway for p-cresol degradation and subsequent entry point in the TCA cycle in N. eutropha C91. The obtained data indicate that mixotrophy, rather than cometabolism, is the major mechanism behind p-cresol degradation in N. eutropha C91.

A flow biosensor for the detection of toxicity in water using the ammonia-oxidizing bacterium (AOB) Nitrosomonas europaea as a bioreceptor and a polymeric membrane ammonium-selective electrode as a transducer is described. The system is based on the inhibition effects of toxicants on the activity of AOB, which can be evaluated by measuring the ammonium consumption rates with the ammonium-selective membrane electrode. The AOB cells are immobilized on polyethersulfone membranes packed in a holder, while the membrane electrode is placed downstream in the flow cell. Two specific inhibitors of the ammonia oxidation-allylthiourea and thioacetamide-have been tested. The IC50 values defined as the concentration of an inhibitor causing a 50% reduction in the ammonia oxidation activity have been measured as 0.17 μM and 0.46 μM for allylthiourea and thioacetamide, respectively. The proposed sensor offers advantages of simplicity, speed and high sensitivity for measuring toxicity in water.

Nitrosomonas eutropha is an ammonia-oxidizing betaproteobacterium found in environments with high ammonium levels, such as wastewater treatment plants. The effects of NO(2) on gene and protein expression under oxic and anoxic conditions were determined by maintaining N. eutropha strain C91 in a chemostat fed with ammonium under oxic, oxic-plus-NO(2), and anoxic-plus-NO(2) culture conditions. Cells remained viable but ceased growing under anoxia; hence, the chemostat was switched from continuous to batch cultivation to retain biomass. After several weeks under each condition, biomass was harvested for total mRNA and protein isolation. Exposure of N. eutropha C91 to NO(2) under either oxic or anoxic conditions led to a decrease in proteins involved in N and C assimilation and storage and an increase in proteins involved in energy conservation, including ammonia monooxygenase (AmoCAB). Exposure to anoxia plus NO(2) resulted in increased representation of proteins and transcripts reflective of an energy-deprived state. Several proteins implicated in N-oxide metabolism were expressed and remained unchanged throughout the experiment, except for NorCB nitric oxide reductase, which was not detected in the proteome. Rather, NorY nitric oxide reductase was expressed under oxic-plus-NO(2) and anoxic-plus-NO(2) conditions. The results indicate that exposure to NO(2) results in an energy-deprived state of N. eutropha C91 and that anaerobic growth could not be supported with NO(2) as an oxidant.

Metasystox-R is a systemic soluble liquid insecticide for the control of aphids on brassica vegetable crops, cotton and lupins and it is possible enter to the marine environment and may be have a hazard effects for the marine organisms and nitrification processes. Effect of Metasystox-R on ammonia oxidizing activity by marine Nitrosomonas sp. was investigated by determining nitrification inhibitor assay in the cell suspension. Results showed that 8 microg mL(-1) of Metasystox-R with PI50 = 4.48 significantly inhibited nitrite production by marine Nitrosomonas sp. These results suggested marine Nitrosomonas sp. may be one of the target bacteria which was inhibitor and decreasing nitrification in the marine environment.

The kinetics of the NO2-dependent ammonia oxidation was developed for ammonia oxidizer mixed culture when there was no molecular oxygen in the batch tests. The kinetics parameters were determined, where the half saturate coefficient of NO2 was 0.821 micromol x L(-1), inhibition coefficient of NO2 concentration was 1.721 micromol x L(-1), and the maximum ammonia oxidation rate were 0.144 mg x (mg x h)(-1). After adding the volume fraction of O2 was 2% to trace NO2, the ammonia oxidation rates increased obviously. The maximum ammonia oxidation rate, 0.198 mg x (mg x h)(-1) occurred under the condition of the mixed gasses containing the volume fraction of O2 was 2% and 50 x 10(-6) NO2. Under the condition of mixed gasses containing the volume fraction of O2 was 21% to trace NO2, the ammonia oxidation rates further increased greatly. The maximum ammonia oxidation rate, 0.477 mg x (mg x h)(-1) occurred when the volume fraction of O2 was 21% and 100 x 10(-6) NO2 in the mixed gas, which is 3 times higher than the general aerobic ammonia oxidation rate. The function for NO2 apparently to enhance ammonia oxidation was suggested. The kinetics model of ammonia oxidation under the conditions of O2 and trace NO2 mixed gasses was developed. The model was validated by the results of ammonia oxidation experiments under the conditions of the mixed gasses containing 2% O2 and trace NO2. The mechanism for NO2 to enhance ammonia oxidation under the conditions of O2 and trace NO2 mixed gasses was discussed.

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.

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.

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.

We found in the environmental strain Nitrosomonas europaea a chromosomal integron-like structure with an integrase gene, intI(Neu). We have tested the capacity of the IntINeu integrase to excise and integrate several resistance gene cassettes. The results allow us to consider IntINeu a new functional integron integrase.

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.

The effect of acetylene ((14)C(2)H(2)) on aerobic and anaerobic ammonia oxidation by Nitrosomonas eutropha was investigated. Ammonia monooxygenase (AMO) was inhibited and a 27 kDa polypeptide (AmoA) was labelled during aerobic ammonia oxidation. In contrast, anaerobic, NO(2)-dependent ammonia oxidation (NO(2)/N(2)O(4) as oxidant) was not affected by acetylene. Further studies gave evidence that the inhibition as well as the labelling reaction were O(2)-dependent. Cells pretreated with acetylene under oxic conditions were unable to oxidize ammonia with O(2) as oxidant. After these cell suspensions were supplemented with gaseous NO(2), ammonia oxidation activity of about 140 micromol NH(4)(+) (g protein)(-1) h(-1) was detectable under both oxic and anoxic conditions. A significantly reduced acetylene inhibition of the ammonia oxidation activity was observed for cells incubated in the presence of NO. This suggests that NO and acetylene compete for the same binding site on AMO. On the basis of these results a new hypothetical model of ammonia oxidation by N. eutropha was developed.

Strong inhibitory effects of the anionic surfactant linear alkylbenzene sulfonate (LAS) on four strains of autotrophic ammonia-oxidizing bacteria (AOB) are reported. Two Nitrosospira strains were considerably more sensitive to LAS than two Nitrosomonas strains were. Interestingly, the two Nitrosospira strains showed a weak capacity to remove LAS from the medium. This could not be attributed to adsorption or any other known physical or chemical process, suggesting that biodegradation of LAS took place. In each strain, the metabolic activity (50% effective concentration [EC(50)], 6 to 38 mg liter(-1)) was affected much less by LAS than the growth rate and viability (EC(50), 3 to 14 mg liter(-1)) were. However, at LAS levels that inhibited growth, metabolic activity took place only for 1 to 5 days, after which metabolic activity also ceased. The potential for adaptation to LAS exposure was investigated with Nitrosomonas europaea grown at a sublethal LAS level (10 mg liter(-1)); compared to control cells, preexposed cells showed severely affected cell functions (cessation of growth, loss of viability, and reduced NH(4)(+) oxidation activity), demonstrating that long-term incubation at sublethal LAS levels was also detrimental. Our data strongly suggest that AOB are more sensitive to LAS than most heterotrophic bacteria are, and we hypothesize that thermodynamic constraints make AOB more susceptible to surfactant-induced stress than heterotrophic bacteria are. We further suggest that AOB may comprise a sensitive indicator group which can be used to determine the impact of LAS on microbial communities.

Cells of Nitrosomonas eutropha grown under anoxic conditions with hydrogen as electron donor and nitrite as electron acceptor were initially unable to oxidize ammonia (ammonium) and hydroxylamine when transferred to oxic conditions. Recovery of ammonia and hydroxylamine oxidation activity was dependent on the presence of NO2. Under oxic conditions, without addition of NO2, ammonia consumption started after 8 - 9 days, and small amounts of NO and NO2 were detectable in the gas atmosphere. Removing these nitrogen oxides by intensive aeration, ammonia oxidation activity decreased and broke off after 15 days. Addition of gaseous NO2 (25 ppm1) led to a fast recovery of ammonia oxidation (3 days). Simultaneously, the arrangement of intracytoplasmic membranes (ICM) changed from circular to flattened vesicles, the protein pattern revealed an increase in the concentration of a 27 and a 30 kDa polypeptide, and the cytochrome c content increased significantly.

An expression vector for the luxAB genes, derived from Vibrio harveyi, was introduced into Nitrosomonas europaea. Although the recombinant strain produced bioluminescence due to the expression of the luxAB genes under normal growing conditions, the intensity of the light emission decreased immediately, in a time-and dose-dependent manner, with the addition of ammonia monooxygenase inhibitors, such as allylthiourea, phenol, and nitrapyrin. When whole cells were challenged with several nitrification inhibitors and toxic compounds, a close relationship was found between the change in the intensity of the light emission and the level of ammonia-oxidizing activity. The response of bioluminescence to the addition of allylthiourea was considerably faster than the change in the ammonia-oxidizing rate, measured as both the O2 uptake and NO2- production rates. The bioluminescence of cells inactivated by ammonia monooxygenase inhibitor was recovered rapidly by the addition of certain substrates for hydroxylamine oxidoreductase. These results suggested that the inhibition of bioluminescence was caused by the immediate decrease of reducing power in the cell due to the inactivation of ammonia monooxygenase, as well as by the destruction of other cellular metabolic pathways. We conclude that the assay system using luminous Nitrosomonas can be applied as a rapid and sensitive detection test for nitrification inhibitors, and it will be used to monitor the nitrification process in wastewater treatment plants.

A pure culture of the obligately lithoautotrophic ammonia-oxidizer Nitrosomonas eutropha was grown in a laboratory-scale bioreactor with complete biomass retention. The air supply was supplemented with nitrogen dioxide (NO2; 25 or 50 ppm) or nitric oxide (NO; 25 or 50 ppm). Compared to cultures grown without these nitrogenous oxides, the addition of NO2 or NO to the culture resulted in a significant increase of the nitrification rate, specific activity of ammonia oxidation, growth rate, and maximum cell densities. In contrast, the growth yield slightly decreased in the presence of NO or NO2. Maximum cell densities of about 2 x 10(10) cells ml-1 and a maximum nitrification rate of about 221 mmol NH4+ l-1 day-1 were obtained after 3 weeks in the presence of 50 ppm NO2. Furthermore, in the stationary phase about 50% of the nitrite produced was aerobically denitrified to dinitrogen (N2) and traces of nitrous oxide (N2O). When cells were supplemented with NO, a high rate of aerobic denitrification occurred only during the first days of the exponential growth phase.

In Nitrosomonas europaea, ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO) catalyse the oxidation of ammonia (NH3) to nitrite (NO2-). A transcript of 3500 bases hybridizes to probes for amoA and amoB (genes that code for AMO proteins). A transcript of 2100 bases hybridizes to probes for hao (the gene that codes for HAO). Induction of the mRNAs detected by amo and hao probes required the presence of ammonium (NH4+). To correlate new levels of mRNA with de novo activity, existent mRNA pools and AMO activity were depleted prior to induction by NH4+. The mRNAs of AMO and HAO were depleted by depriving the cells of energy for at least 8 h; AMO activity was inactivated with acetylene (C2H2) after mRNA depletion. In cells treated this way, levels of new AMO mRNA and de novo AMO enzyme activity were correlated with increased NH4+ concentrations up to 1 mM after 3 h of incubation. HAO mRNA also increased in the NH4(+)-treated cells. Other proteins and RNAs induced by NH4+ were detected in 14CO2-labelling experiments. The AMO and HAO mRNAs were preferentially synthesized during energy-limiting conditions.

Nitrate production by Nitrosomonas europaea in inorganic liquid medium containing ammonium was limited by reduction in pH. In the presence of montmorillonite and vermiculite, expanding clays with high cation-exchange-capacity (CEC), nitrite yield was increased, ammonia oxidation continued at pH values below those which inhibited growth in the absence of clays and growth was biphasic. The first phase was similar to that in the absence of clays, while the second was characterized by a lower rate of nitrite production. Illite, a non-expanding clay with low CEC, had no significant effect on ammonia oxidation, while oxidation of ammonia-treated vermiculite (ATV) occurred with no significant change in the pH of the medium. ATV, montmorillonite and vermiculite, but not illite, protected cells from inhibition by nitrapyrin at concentrations inhibitory to cells growing in suspended culture. This protection was maintained in ATV homo-ionic to Al3+, but montmorillonite made homo-ionic to Al3+ did not provide protection from inhibition. Attachment of cells to clays with high CEC is therefore advantageous in providing exchange at the clay surface of NH+4 and H+ produced by ammonia oxidation, in reducing pH toxicity, and in protecting cells from inhibition.

Cytochrome P-460 of Nitrosomonas europaea [Erickson, R.H. and Hooper, A.B. (1972) Biochim. Biophys. Acta 275, 231-244] was further purified to an electrophoretically homogeneous state. The cytochrome molecule was composed of three molecules of subunits with Mr of 17,300-18,500, and contained three atoms of iron, which seemed to be heme iron, and six cysteine residues, but did not contain nonheme iron or inorganic sulfide. The cytochrome showed absorption peaks at 460 and 688 nm with a broad shoulder at 635 nm in the reduced form. The ESR spectrum of ferricytochrome P-460 showed signals at g = 5.91, 5.63, and 1.99, indicating that the protein was a high spin hemoprotein. The heme of the cytochrome was not cleaved by the methods which were available for cleavage of heme c. The pyridine ferrohemochrome of the hemoprotein did not show the distinct alpha and beta peaks which are shown by the ferrohemochromes of many other cytochromes so far known. The N-terminal amino acid sequence of cytochrome P-460 differed from that of hydroxylamine oxidoreductase. Therefore, cytochrome P-460 did not seem to be the solubilized P-460 moiety of hydroxylamine oxidoreductase, in agreement with the finding by D.J. Miller et al. [J. Gen. Microbiol. 130, 3049-3054 (1984)]. However, cytochrome P-460 had several enzymatic activities which hydroxylamine oxidoreductase showed. Although most of the activities of the cytochrome were lower than the corresponding activities of the oxidoreductase, the hydroxylamine-cytochrome c-552 reductase activity of the cytochrome was about 5-times as high as that of the oxidoreductase.

Acetylene brings about a progressive inactivation of ammonia mono-oxygenase, the ammonia-oxidizing enzyme in Nitrosomonas europaea. High NH4+ ion concentrations were protective. The inactivation followed first-order kinetics, with a rate constant of 1.5 min-1 at saturating concentrations of acetylene. If acetylene was added in the absence of O2, the cells remained active until O2 was re-introduced. A protective effect was also demonstrated with thiourea, a reversible non-competitive inhibitor of ammonia oxidation. Incubation of cells with [14C]acetylene was found to cause labelling of a single membrane polypeptide. This ran on dodecyl sulphate/polyacrylamide-gel electrophoresis with an Mr value of 28 000. It is concluded that acetylene is a suicide substrate for the mono-oxygenase. The labelling experiment provides the first identification of a constituent polypeptide of ammonia mono-oxygenase.

Studies on the effect of herbicides on nitrification in field and laboratory conditions have shown that steady use of these substances in the same field does not exert a significant effect on this process in the soil. The herbicides caused only shortlasting disturbances. Nitrification usually returned to a relative equilibrium in several days after the herbicide application.

Nitrification by intertidal sediments was measured by using a tide simulator that approximated the cycle of seawater on tidal flats. Sediments were chosen from sites affected by industrial and municipal effluents and pastoral seepage and runoff. The ability of sediments from different sites to nitrify endogenous nitrogen varied markedly. All sites exhibited an initial lag before activity commenced. The duration of this lag and the rate of nitrate production were different at each site. The sediments were also capable of oxidizing NH3-N supplied to them in seawater. This "nitrification potential" was highest at sites receiving nitrogenous effluents (slaughterhouse and sewage), but was also substantial in sediments affected by bark extract effluent and pasture runoff. The lowest potential and the longest lag were exhibited by sediments in an apple cannery effluent area. Enrichment cultures of nitrifying microorganisms were obtained from all sites using NH4+ as a source of energy, but enrichments for nitrite oxidizers were unsuccessful. Concentrated pine bark tannins, similar in origin to those in effluents at the well-nitrifying chipmill site, were tested for toxicity to pure cultures of nitrifying bacteria. Two Nitrobacter strains and one Nitrosomonas strain were unaffected by tannins even at 5 mg/ml. A Nitrosolobus and a Nitrosospira strain were inhibited partially at 5 mg/ml and only slightly or not at all at 1 mg/ml.

The effects of polychlorinated biphenyls (PCBs) on nitrification were examined for pure cultures and natural reservoir samples. PCBs at concentrations greater than 10 microgram liter-1 inhibited nitrification, principally ammonium oxidation, in one of two natural reservoir environments. However, this inhibition could not be reproduced in pure high-cell-density cultures or in previously contaminated reservoir waters. A PCB environmental biotransformation product, p-chlorophenylglyoxylic acid, and p-chloromandelic acid had no effect on nitrification.

Growth responses of Nitrosomonas europaea to individual amino acids or vitamins was observed in log-phase cultures, as was the incorporation of carbon-14 labeled amino acids. Nitrite formation and protein synthesis were increased by l-glutamic acid, l-aspartic acid, l-serine, and l-glutamine. l-Lysine, l-histidine, l-threonine, l-valine, l-methionine, and l-arginine were inhibitory. The other amino acids had no effect on growth. All of the uniformly labeled amino acids added at low concentrations were taken up by growing cells and distributed into cell fractions. From 1 to 12% of the added radioactivity was present in cells analyzed in late log phase, depending on the amino acid; glycine and l-serine caused accumulation of the label to the greatest extent, whereas l-aspartic and l-glutamic acids were among those incorporated to the least extent. Aspartic acid increased both cell protein and nitrite values, but did not alter the ratio of protein to nitrite from that found in controls.