Assessment of two techniques for measuring nitrification rates in aerated slurries of seagrass-bed sediments

Two methods were assessed to measure rates of nitrification in aerated slurries of seagras-bed sediments; (1) monitoring ammonium concentrations in samples with and without nitrapyrin ((2-chloro-6-(trichloromethyl) pyridine) treatment and (2) monitoring the accumulation of nitrate plus nitrite. Nitrifying bacteria were inhibited completely after 14 days exposure to nitrapyrin at 5 mg⁻¹, but the effect was poor over shorter periods, probably owing to limited diffusion of the inhibitor. Hence, nitrapyrin was not a satisfactory inhibitor to use in these sediments. Nitrification rates could not be measured by monitoring the concentrations of nitrate plus nitrite, because nitrate reduction occurred simultaneously with nitrification, even though the slurries contained from 3.5 to 8.9 mg O₂ l⁻¹.     

The effects of nitrate:ammonium ratios and nitrapyrin on the nitrogen response ofZea mays L. under field conditions

Five nitrate:ammonium ratios at two N-levels were tested with and without nitrapyrin [2 chloro-6-(trichloromethyl) pyridine] for grain production on a sandy soil. Treatments were applied to field maize as nutrient solutions, in one application, six weeks after planting. Nytrapyrin resulted in an increase in grain yield at a nitrate:ammonium ratio of 1:3 but in a decrease at a 0:1 ratio. The optimum nitrate:ammonium ratio was close to 1:3 with nitrapyrin and close to 3:1 without nitrapyrin. Nitrapyrin had an effect on NH₄ ⁺-N in the topsoil and NO₃ ⁻-N in the subsoil at 70 days after application. Interactions of nitrate:ammonium ratios and N-levels were shown for leaf N concentration, soil mineral N and soil pH.     

Comparative phytotoxicity of nitrapyrin and ATC to several leguminous species

Summary The relative toxicity of nitrapyrin 2-chloro-6-(trichloromethyl) pyridine and ATC (4-amino-1, 2, 4-triazole) on the growth of chick peas (Cicer arietinum L.) cow peas (Vigna sinensis L.), green beans (Phaseolus vulgaris L.), green peas (Pisum sativum L.) and mung beans (Phaseolus aureus Roxb.) and their effectiveness as nitrification inhibitor were studied under greenhouse conditions. ATC produced no toxicity symptoms in green peas, whereas resulted in leaf chlorosis in cow peas, chick peas and green beans. However, nitrapyrin toxicity appeared as leaf chlorosis in cow peas, and interveinal chlorosis in chick peas. Moreover, nitrapyrin-treated green beans and peas developed leaf curling and cupping. Although ATC had no significant effect on growth, a suppression in plant growth was associated with nitrapyrin application. Furthermore, green beans was the most resistant and chick peas the most sensitive to nitrapyrin. Nitrapyrin was more effective nitrification inhibitor than ACT, especially at the lower rates.     

Oxidation of Nitrapyrin to 6-Chloropicolinic Acid by the Ammonia-Oxidizing Bacterium Nitrosomonas europaea

Suspensions of Nitrosomonas europaea catalyzed the oxidation of the commercial nitrification inhibitor nitrapyrin [2-chloro-6-(trichloromethyl)-pyridine]. Rapid oxidation of nitrapyrin (at a concentration of 10 μM) required the concomitant oxidation of ammonia, hydroxylamine, or hydrazine. The turnover rate was highest in the presence of 10 mM ammonia (0.8 nmol of nitrapyrin per min/mg of protein). The product of the reaction was 6-chloropicolinic acid. By the use of ¹⁸O₂, it was shown that one of the oxygens in 6-chloropicolinic acid came from diatomic oxygen and that the other came from water. Approximately 13% of the radioactivity of [2,6-¹⁴C]nitrapyrin was shown to bind to cells. Most (94%) of the latter was bound indiscriminately to membrane proteins. The nitrapyrin bound to membrane proteins may account for the observed inactivation of ammonia oxidation.     

Toxicity of nitrapyrin to sunflower under different N nutrition regimes

Summary The purpose of this study was to investigate the phytotoxicity of nitrapyrin 2-chloro-6-(trichloromethyl)pyridine to sunflower (Helianthus annuus L.) under different N regimes and to see if N forms affect the phytotoxicity of nitrapyrin. Sunflower was grown in pot culture for 21 days and was fertilized with (NH₄)₂SO₄, NH₄NO₃ and NaNO₃ to provide 0, 100 and 200 ppm N and with nitrapyrin application of 0 and 20 ppm. All N-treated sunflower plants in all N regimes and regardless of titrapyrin treatment produced more root and shoot dry weights and contained a significantly higher N than untreated check. Nitrapyrin toxicity appeared as a curling of leaf margin and a tendril type of stem growth, the visible toxicity symptoms decreased in the order: (NH₄)₂SO₄>NH₄NO₃>NaNO₃. Furthermore nitrapyrin addition suppressed sunflower growth in each N regime, the suppressing effect being greater with (NH₄)₂SO₄ and NH₄NO₃ than as with NaNO₃. Although, shoot growth from plants receiving nitrapyrin was not significantly affected by any N regime, root growth of nitrapyrin-treated plants was somewhat restricted by NH₄ ⁺−N nutrition relative to NO₃ ⁻−N nutrition.     

Contributions of Autotrophic and Heterotrophic Nitrifiers to Soil NO and N(2)O Emissions

Soil emission of gaseous N oxides during nitrification of ammonium represents loss of an available plant nutrient and has an important impact on the chemistry of the atmosphere. We used selective inhibitors and a glucose amendment in a factorial design to determine the relative contributions of autotrophic ammonium oxidizers, autotrophic nitrite oxidizers, and heterotrophic nitrifiers to nitric oxide (NO) and nitrous oxide (N(2)O) emissions from aerobically incubated soil following the addition of 160 mg of N as ammonium sulfate kg. Without added C, peak NO emissions of 4 mug of N kg h were increased to 15 mug of N kg h by the addition of sodium chlorate, a nitrite oxidation inhibitor, but were reduced to 0.01 mug of N kg h in the presence of nitrapyrin [2-chloro-6-(trichloromethyl)-pyridine], an inhibitor of autotrophic ammonium oxidation. Carbon-amended soils had somewhat higher NO emission rates from these three treatments (6, 18, and 0.1 mug of N kg h after treatment with glucose, sodium chlorate, or nitrapyrin, respectively) until the glucose was exhausted but lower rates during the remainder of the incubation. Nitrous oxide emission levels exhibited trends similar to those observed for NO but were about 20 times lower. Periodic soil chemical analyses showed no increase in the nitrate concentration of soil treated with sodium chlorate until after the period of peak NO and N(2)O emissions; the nitrate concentration of soil treated with nitrapyrin remained unchanged throughout the incubation. These results suggest that chemoautotrophic ammonium-oxidizing bacteria are the predominant source of NO and N(2)O produced during nitrification in soil.     

Anticholinesterase effect and toxicity of bis(trichloromethyl) sulfone (N-1386 Biocide) in rats

The oral LD50 for bis(trichloromethyl) sulfone (N-1386 Biocide) in male rats was 691 mg/kg. Deaths occurred 1-5 days after treatment and signs of toxicity suggestive of an anticholinesterase effect were noted. However, neither plasma cholinesterase nor brain acetylcholinesterase was inhibited 2, 4 or 24 hours after a single, oral dose of 500 mg/kg. Atropine (300 mg/kg, s.c.) or scopolamine (670 mg/kg, s.c.) pretreatments did not protect against the acute lethality of bis(trichloromethyl) sulfone although signs of toxicity were alleviated by both pretreatments. Bis(trichloromethyl) sulfone produced in vitro inhibition of rat plasma cholinesterase and brain acetylcholinesterase. The inhibition was competitive in brain. IC50's for these 2 enzymes were 8 microM in plasma and 25 microM in brain. In summary, bis(trichloromethyl) sulfone produced in vitro cholinesterase inhibition not demonstrated in vivo. Doses of anticholinergic compounds that ameliorated many toxic signs did not protect against lethality produced by bis(trichloromethyl) sulfone.     

Kinetic parameters and relative turnovers of some important catabolic reactions in digesting sludge.

The kinetics of propionate degradation, acetate splitting, and hydrogen consumption in digesting sludge were investigated in a lab-scale digester. At natural steady-state conditions, the acetate-splitting systems in well-digested sludge were about half saturated. Propionate-degrading systems were saturated to only 10 to 15%, and hydrogen removal was less than 1% of the maximum possible rate. It was concluded that acetate splitting rather than "methanogenesis from fatty acids" is the rate-limiting reaction in the anaerobic degradation of dissolved organic matter and that a methoanogenic anaerobic ecosystem is stabilized by its large unused capacity of hydrogen consumption which is "buffering" the partial pressure of dissolved hydrogen in the system at sufficiently low values to permit rapid fatty acid oxidation. A tentative scheme of the substrate flow in sludge digestion is presented. It suggests that acid formation coupled with hydrogen formation via pyridine dinucleotide oxidation yields the immediate substrates, namely acetate and hydrogen, for about 54% of the total methanogenesis.     

Isolation and Characterization of a Thermophilic Strain of Methanosarcina Unable to Use H(2)-CO(2) for Methanogenesis

A thermophilic strain of Methanosarcina, designated Methanosarcina strain TM-1, was isolated from a laboratory-scale 55 degrees C anaerobic sludge digestor by the Hungate roll-tube technique. Penicillin and d-cycloserine, inhibitors of peptidoglycan synthesis, were used as selective agents to eliminate contaminating non-methanogens. Methanosarcina strain TM-1 had a temperature optimum for methanogenesis near 50 degrees C and grew at 55 degrees C but not at 60 degrees C. Substrates used for methanogenesis and growth by Methanosarcina strain TM-1 were acetate (12-h doubling time), methanol (7- to 10-h doubling time), methanol-acetate mixtures (5-h doubling time), methylamine, and trimethylamine. When radioactively labeled acetate was the sole methanogenic substrate added to the growth medium, it was predominantly split to methane and carbon dioxide. When methanol was also present in the medium, the metabolism of acetate shifted to its oxidation and incorporation into cell material. Electrons derived from acetate oxidation apparently were used to reduce methanol. H(2)-CO(2) was not used for growth and methanogenesis by Methanosarcina strain TM-1. When presented with both H(2)-CO(2) and methanol, Methanosarcina strain TM-1 was capable of limited hydrogen metabolism during growth on methanol, but hydrogen metabolism ceased once the methanol was depleted. Methanosarcina strain TM-1 required a growth factor (or growth factors) present in the supernatant of anaerobic digestor sludge. Growth factor requirements and the inability to use H(2)-CO(2) are characteristics not found in other described Methanosarcina strains. The high numbers of Methanosarcina-like clumps in sludges from thermophilic digestors and the fast generation times reported here for Methanosarcina TM-1 indicate that Methanosarcina may play an important role in thermophilic methanogenesis.     

Determination of the phamacophore of penclomedine,a clinically-evaluated antitumor pyridine derivative

The main objective of this investigation was to identify the reactive pharmacophore in penclomedine (PEN, 3,5-dichloro-4,6-dimethoxy-2-(trichloromethyl) pyridine) for in vivo antitumor activity and also to discover related ring structures and sulfur analogues that might exhibit superior antitumor activity in vivo. Several new analogues of PEN and related structural variants have been synthesized and evaluated in vivo against MX-1 human breast tumor xenograft implanted subcutaneously (sc), although none of them demonstrated significant activity.     

Competition of Fe(III) reduction and methanogenesis in an acidic fen

Peatlands are sources of relevant greenhouse gases such as CH4, but the temporal presence of Fe(III) may inhibit methanogenesis. Because excess of carbon during the vegetation period might allow concomitant electron-accepting processes, Fe(III) reduction and methanogenesis were studied during an annual season in an acidic fen. The upper peat layer displayed the highest Fe(II)- and CH4-forming activities. The rates of Fe(II) formation did not change during the year and methanogenesis started mostly when Fe(II) formation reached a plateau. Most of the Fe(III) pool seemed to be bioavailable, and addition of nitrilotriacetic acid stimulated only light Fe(II) formation, whereas EDTA and anthraquinone-2,6-disulfonate had no effect. In the presence of an inhibitor for methanogenesis (sodium 2-bromoethanesulfonate), Fe(II) formation was inhibited to 45%. Addition of Fe(III) during ongoing methanogenesis led only to a partial inhibition of CH4 formation. The proportion of acetoclastic methanogenesis varied between 42% and 90%, but no trend with time was observed. The number of acetate-, ethanol- or lactate-utilizing Fe(III) reducers approximated 10(5)-10(6) cells g (fresh wt peat)(-1). Fermentative glucose-utilizing Fe(III)-reducers were most abundant. Our results suggest that (1) methanogens used Fe(III) as an electron acceptor and (2) fermenting bacteria, which do not compete with methanogens for common electron donors, dominated the reduction of Fe(III) in this fen.     

Effects of Nitrapyrin [2-Chloro-6-(Trichloromethyl) Pyridine] on the Obligate Methanotroph Methylosinus trichosporium OB3b

Nitrapyrin inhibited growth, CH₄ oxidation, and NH₄⁺ oxidation, but not the oxidation of CH₃OH, HCHO, or HCOONa, by Methylosinus trichosporium OB3b, suggesting that nitrapyrin acts against the methane monooxygenase enzyme system. The inhibition of CH₄ oxidation could be reversed by repeated washing of nitrapyrin-inhibited cells, indicating that its effect is bacteriostatic. The addition of Cu²⁺ did not release the inhibition. Methane oxidation was also inhibited by 6-chloro-2-picoline. These data suggest that the mode of action of nitrapyrin on M. trichosporium is different from that on chemoautotrophic NH₄⁺ oxidizers or methanogens.     

Effects of N-serve (2-chloro-6-(trichloromethyl)pyridine) formulations on nitrification and on loss of nitrate in sand culture experiments

Summary N-serve (2-chloro-6-(trichloromethyl)pyridine) was tested as an inhibitor of nitrification of ammonium or urea in sand cultures. Nitrification was reduced but not prevented by N-Serve present at between 5 and 20 ppm in solution or by weight of sand. In the presence of root debris and acetone, used in some experiments at 2–4 ml/l of nutrient to convey N-Serve, denitrification was stimulated under the same conditions and resulted in loss of a large proportion of nitrate, probably mainly as gaseous products and some nitrite. These losses were greater when N-serve was also present. There was also conversion of nitrate to an insoluble form in the sand. A smaller proportional loss of nitrate occurred in other treatments in the presence of root debris when N-Serve was added without acetone, either as the commercial formulation 24E or as a solid. Thus, using N-Serve to inhibit nitrification may encourage denitrifying organisms especially in the presence of carbon sources including root debris or acetone. Large decreases of nitrate reductase activity in plants produced by using N-Serve in the presence of ammonium or urea were caused as much by losses of nitrate in the presence of acetone as by prevention of nitrate formation. Other N-Serve treatments (solid or 24E) decreased enzyme induction by between 50 and 90 per cent as a result mainly of reduced nitrification.     

Contributions of Autotrophic and Heterotrophic Nitrifiers to Soil NO and N2O Emissions

Soil emission of gaseous N oxides during nitrification of ammonium represents loss of an available plant nutrient and has an important impact on the chemistry of the atmosphere. We used selective inhibitors and a glucose amendment in a factorial design to determine the relative contributions of autotrophic ammonium oxidizers, autotrophic nitrite oxidizers, and heterotrophic nitrifiers to nitric oxide (NO) and nitrous oxide (N₂O) emissions from aerobically incubated soil following the addition of 160 mg of N as ammonium sulfate kg⁻¹. Without added C, peak NO emissions of 4 μg of N kg⁻¹ h⁻¹ were increased to 15 μg of N kg⁻¹ h⁻¹ by the addition of sodium chlorate, a nitrite oxidation inhibitor, but were reduced to 0.01 μg of N kg⁻¹ h⁻¹ in the presence of nitrapyrin [2-chloro-6-(trichloromethyl)-pyridine], an inhibitor of autotrophic ammonium oxidation. Carbon-amended soils had somewhat higher NO emission rates from these three treatments (6, 18, and 0.1 μg of N kg⁻¹ h⁻¹ after treatment with glucose, sodium chlorate, or nitrapyrin, respectively) until the glucose was exhausted but lower rates during the remainder of the incubation. Nitrous oxide emission levels exhibited trends similar to those observed for NO but were about 20 times lower. Periodic soil chemical analyses showed no increase in the nitrate concentration of soil treated with sodium chlorate until after the period of peak NO and N₂O emissions; the nitrate concentration of soil treated with nitrapyrin remained unchanged throughout the incubation. These results suggest that chemoautotrophic ammonium-oxidizing bacteria are the predominant source of NO and N₂O produced during nitrification in soil.     

Reductive dehalogenation of the trichloromethyl group of nitrapyrin by the ammonia-oxidizing bacterium Nitrosomonas europaea.

Suspensions of Nitrosomonas europaea catalyzed the reductive dehalogenation of the commercial nitrification inhibitor nitrapyrin (2-chloro-6-trichloromethylpyridine). The product of the reaction was identified as 2-chloro-6-dichloromethylpyridine by its mass fragmentation and nuclear magnetic resonance spectra. A small amount of 2-chloro-6-dichloromethylpyridine accumulated during the conversion of nitrapyrin to 6-chloropicolinic acid in an aerated solution in the presence of ammonia (T. Vannelli and A.B. Hooper, Appl. Environ. Microbiol. 58:2321-2325, 1992). Nearly stoichiometric conversion of nitrapyrin to 2-chloro-6-dichloromethylpyridine occurred at very low oxygen concentrations and in the presence of hydrazine as a source of electrons. Under these conditions the turnover rate was 0.37 nmol of nitrapyrin per min per mg of protein. Two specific inhibitors of ammonia oxidation, acetylene and allylthiourea, inhibited the rate of the dehalogenation reaction by 80 and 84%, respectively. In the presence of D2O, all 2-chloro-6-dichloromethylpyridine produced in the reaction was deuterated at the methyl position. In an oxygenated solution and in the presence of ammonia or hydrazine, cells did not catalyze the oxidation of exogenously added 2-chloro-6-dichloromethylpyridine to 6-chloropicolinic acid. Thus, 2-chloro-6-dichloromethylpyridine is apparently not an intermediate in the aerobic production of 6-chloropicolinic acid from nitrapyrin.     

Polycyclic Aromatic Hydrocarbon (PAH) Degradation Coupled to Methanogenesis

Baltimore Harbor (Baltimore, MD) sediments were utilized to initiate anaerobic enrichment cultures with polycyclic aromatic hydrocarbons (PAHs) in the absence of supplementary electron acceptors. Cultures amended with naphthalene and phenanthrene exhibited sustained, transferable degradation of the PAHs. Bromoethanesulfonic acid, a selective inhibitor of methanogenesis, inhibited the degradation of 200 μm naphthalene and phenanthrene; molecular characterization based on 16S rRNA sequences confirmed that methanogenic Archaea were eliminated, thus providing evidence that methanogenesis is involved in the degradation pathway. Revisions requested 16 November 2005; Revisions received 14 December 2005     

Inhibition of Ammonium Oxidation by Nitrapyrin in Soil and Liquid Culture

Inhibition of growth of axenic cultures of Nitrosomonas europaea by nitrapyrin was investigated in liquid culture and in soil. In liquid culture, exponentially growing cells were more sensitive than stationary-phase cells, possibly due to a requirement for uptake of nitrapyrin, metabolism of nitrapyrin, or both before inhibition. Differences in sensitivity were observed between the parent strain and two strains, sp1 and sp2, that were selected through repeated subculturing. These differences were reflected in the length of the lag period induced by nitrapyrin and in the specific growth rate and were due to different bactericidal and bacteriostatic effects. Soil provided significant protection from inhibition, with concentrations of nitrapyrin approximately one order of magnitude greater than those required for equivalent inhibition in liquid culture. The data show that strain differences alone do not explain differences in sensitivity between nitrification in soil and in liquid culture and suggest that the inhibitor may be more effective against actively nitrifying soils.     

Inhibition of biofilm populations of Nitrosomonas europaea

The influence of surface attachment and growth on inhibition of the ammonia oxidizing bacterium, Nitrosomonas europaea, by nitrapyrin was investigated in liquid culture in the presence and absence of glass slides. Significant attachment to glass slides occurred in the absence of ammonia, but the extent of attachment was not affected by nitrapyrin, nor by previous culture of cells in medium containing nitrapyrin. The presence of glass slides affected neither the specific growth rate of N. europaea, measured by changes in nitrite concentration, nor inhibition by nitrapyrin. Inhibitory effects of nitrapyrin on increases in nitrite concentration and in free cell concentration were similar, but greater effects were observed on changes in attached cell concentration. Established biofilms on glass slides grew at a lower specific growth rate than freely suspended cells. Both biofilm cells, and those detached from the biofilm, were protected from inhibition. A mechanism for protection of biofilm populations is proposed involving reduced sensitivity of slowly growing cells producing extracellular polymeric material. Offprint requests to.: J. I. Prosser.     

Heme peptide as a model substance for halogenomethane activation and heme modification.

Octa-heme peptide (CHP) obtained from Candida krusei cytochrome c was tested for suicidal activation of halogenomethanes. Under anaerobic conditions, CHP was kept in the reduced state in the presence of NADPH and NADPH-cytochrome P-450 reductase. Addition of CBrCl3 to the reduced CHP caused spectral changes such as rapid disappearance of alpha and beta bands and gradual decrease in the gamma-peak height, accompanied by oxidation of NADPH. Heme content of the reaction mixture, determined as pyridine hemochrome, also decreased NADPH dependently. CCl4 was less effective than CBrCl3, while CHCl3 had almost no effect. N-tert-butyl-alpha-phenylnitrone (PBN) suppressed the CBrCl3-induced heme damage, and resulted in the formation of radical adduct .PBN-CCl3 as evidenced by ESR spectroscopy. Radical formation was also observed with CCl4. The CHP damage induced by CBrCl3 was also accompanied by the release of Br- about 11-12-times molar excess of CHP, whereas the release of CHCl3 was about 20% that of Br-.FD-MS assay of the product of CHP reaction suggested that 10 trichloromethyl radicals bonded with CHP. Thus, CBrCl3 undergoes single-electron reduction in the presence of reduced CHP to trichloromethyl radicals, which covalently bind to CHP molecules. Heme peptide may be a useful tool in the study of mechanisms involved in the destruction of cytochrome P-450 by halogenomethanes.     

Endogenous methanogenesis stimulates oxidation of atmospheric CH(4) in alpine tundra soil

Experiments were done to test the hypothesis that atmospheric CH(4) oxidizers in a well-drained alpine tundra soil are supported by CH(4) production from anaerobic microsites in the soil. Soil was subjected to 22 days of anaerobic conditions with elevated H(2) and CO(2) in order to stimulate methanogenesis. This treatment stimulated subsequent atmospheric CH(4) consumption, probably by increasing soil methanogenesis. After removal from anaerobic conditions, soils emitted CH(4) for up to 6 h, then oxidized atmospheric CH(4) at 111 (+/- 5.7) pmol (g dry weight)(-1) h(-1), which was more than 3 times the rate of control soils. Further supporting our hypothesis, additions of lumazine, a highly specific inhibitor of methanogenesis, prevented the stimulation of atmospheric CH(4) oxidation by the anaerobic treatment. The method used to create anaerobic conditions with elevated H(2) and CO(2) also elevated headspace CH(4) concentrations. However, elevated CH(4) concentrations under aerobic conditions did not stimulate CH(4) oxidation as much as preexposure to H(2) and CO(2) under anaerobic conditions. Anaerobic conditions created by N(2) flushing did not stimulate atmospheric CH4 oxidation, probably because N2 flushing inhibited methanogenesis by removing necessary precursors for methane production. We conclude that anaerobic conditions with elevated H(2) and CO(2) stimulate atmospheric CH(4) oxidation in this dry alpine tundra soil by increasing endogenous CH(4) production. This effect was prevented by inhibiting methanogenesis, indicating the importance of endogenous CH(4) production in a CH(4-) consuming soil.