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1.
One host ( Rana catesbiana)-associated and two free-living mesophilic strains of bacteria with violet pigmentation and biochemical characteristics of Chromobacterium violaceum were isolated from freshwater habitats. Cells of each freshly isolated strain and of strain ATCC 12472 (the neotype strain) grew anaerobically with glucose as the sole carbon and energy source. The major fermentation products of cells grown in Trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.) supplemented with glucose included acetate, small amounts of propionate, lactate, and pyruvate. The final cell yield and culture growth rate of each strain cultured anaerobically in this medium increased approximately twofold with the addition of 2 mM NaNO 3. Final growth yields increased in direct proportion to the quantity of added NaNO 3 over the range of 0.5 to 5 mM. Each strain reduced NO 3−, producing NO 2−, NO, and N 2O. NO 2− accumulated transiently. With 2 mM NaNO 3 in the medium, N 2O made up 85 to 98% of the N product recovered with each strain. N-oxides were recovered in the same quantity and distribution whether 0.01 atm (ca. 1 kPa) of C 2H 2 (added to block N 2O reduction) was present or not. Neither N 2 production nor gas accumulation was detected during NO 3− reduction by growing cells. Cell growth in media containing 0.5 to 5 mM NaNO 2 in lieu of NaNO 3 was delayed, and although N 2O was produced by the end of growth, NO 2− -containing media did not support growth to an extent greater than did medium lacking NO 3− or NO 2−. The data indicate that C. violaceum cells ferment glucose or denitrify, terminating denitrification with the production of N 2O, and that NO 2− reduction to N 2O is not coupled to growth but may serve as a detoxification mechanism. No strain detectably fixed N 2 (reduced C 2H 2). 相似文献
2.
A modification of the Adler capillary assay was used to evaluate the chemotactic responses of several denitrifiers to nitrate and nitrite. Strong positive chemotaxis was observed to NO 3− and NO 2− by soil isolates of Pseudomonas aeruginosa, Pseudomonas fluorescens, and Pseudomonas stutzeri, with the peak response occurring at 10 −3 M for both attractants. In addition, a strong chemoattraction to serine (peak response at 10 −2 M), tryptone, and a soil extract, but not to NH 4+, was observed for all denitrifiers tested. Chemotaxis was not dependent on a previous growth on NO 3−, NO 2−, or a soil extract, and the chemoattraction to NO 3− occurred when the bacteria were grown aerobically or anaerobically. However, the best response to NO 3− was usually observed when the cells were grown aerobically with 10 mM NO 3− in the growth medium. Capillary tubes containing 10 −3 M NO 3− submerged into soil-water mixtures elicited a significant chemotactic response to NO 3− by the indigenous soil microflora, the majority of which were Pseudomonas spp. A chemotactic strain of P. fluorescens also was shown to survive significantly better in aerobic and anaerobic soils than was a nonmotile strain of the same species. Both strains had equal growth rates in liquid cultures. Thus, chemotaxis may be one mechanism by which denitrifiers successfully compete for available NO 3− and NO 2−, and which may facilitate the survival of naturally occurring populations of some denitrifiers. 相似文献
3.
Two fast-growing strains of cowpea rhizobia (A26 and A28) were found to grow anaerobically at the expense of NO 3−, NO 2−, and N 2O as terminal electron acceptors. The two major differences between aerobic and denitrifying growth were lower yield coefficients ( Y) and higher saturation constants ( Ks) with nitrogenous oxides as electron acceptors. When grown aerobically, A26 and A28 adhered to Monod kinetics, respectively, as follows: Ks, 3.4 and 3.8 μM; Y, 16.0 and 14.0 g · cells eq −1; μ max, 0.41 and 0.33 h −1. Yield coefficients for denitrifying growth ranged from 40 to 70% of those for aerobic growth. Only A26 adhered to Monod kinetics with respect to growth on all three nitrogenous oxides. The apparent Ks values were 41, 270, and 460 μM for nitrous oxide, nitrate, and nitrite, respectively; the Ks for A28 grown on nitrate was 250 μM. The results are kinetically and thermodynamically consistent in explaining why O 2 is the preferred electron acceptor. Although no definitive conclusions could be drawn regarding preferential utilization of nitrogenous oxides, nitrite was inhibitory to both strains and effected slower growth. However, growth rates were identical (μ max, 0.41 h −1) when A26 was grown with either O 2 or NO 3− as an electron acceptor and were only slightly reduced when A28 was grown with NO 3− (0.25 h −1) as opposed to O 2 (0.33 h −1). 相似文献
4.
A more sensitive analytical method for NO 3− was developed based on the conversion of NO 3− to N 2O by a denitrifier that could not reduce N 2O further. The improved detectability resulted from the high sensitivity of the 63Ni electron capture gas chromatographic detector for N 2O and the purification of the nitrogen afforded by the transformation of the N to a gaseous product with a low atmospheric background. The selected denitrifier quantitatively converted NO 3− to N 2O within 10 min. The optimum measurement range was from 0.5 to 50 ppb (50 μg/liter) of NO 3− N, and the detection limit was 0.2 ppb of N. The values measured by the denitrifier method compared well with those measured by the high-pressure liquid chromatographic UV method above 2 ppb of N, which is the detection limit of the latter method. It should be possible to analyze all types of samples for nitrate, except those with inhibiting substances, by this method. To illustrate the use of the denitrifier method, NO 3− concentrations of <2 ppb of NO 3− N were measured in distilled and deionized purified water samples and in anaerobic lake water samples, but were not detected at the surface of the sediment. The denitrifier method was also used to measure the atom% of 15N in NO 3−. This method avoids the incomplete reduction and contamination of the NO 3− -N by the NH 4+ and N 2 pools which can occur by the conventional method of 15NO 3− analysis. N 2O-producing denitrifier strains were also used to measure the apparent Km values for NO 3− use by these organisms. Analysis of N 2O production by use of a progress curve yielded Km values of 1.7 and 1.8 μM NO 3− for the two denitrifier strains studied. 相似文献
5.
Heterotrophic bacteria, yeasts, fungi, plants, and animal breath were investigated as possible sources of N 2O. Microbes found to produce N 2O from NO 3− but not consume it were: (i) all of the nitrate-respiring bacteria examined, including strains of Escherichia, Serratia, Klebsiella, Enterobacter, Erwinia, and Bacillus; (ii) one of the assimilatory nitrate-reducing bacteria examined, Azotobacter vinelandii, but not Azotobacter macrocytogenes or Acinetobacter sp.; and (iii) some but not all of the assimilatory nitrate-reducing yeasts and fungi, including strains of Hansenula, Rhodotorula, Aspergillus, Alternaria, and Fusarium. The NO 3−-reducing obligate anaerobe Clostridium KDHS2 did not produce N 2O. Production of N 2O occurred only in stationary phase. The nitrate-respiring bacteria produced much more N 2O than the other organisms, with yields of N 2O ranging from 3 to 36% of 3.5 mM NO 3−. Production of N 2O was apparently not regulated by ammonium and was not restricted to aerobic or anaerobic conditions. Plants do not appear to produce N 2O, although N 2O was found to arise from some damaged plant tops, probably due to microbial growth. Concentrations of N 2O above the ambient level in the atmosphere were found in human breath and appeared to increase after a meal of high-nitrate food. 相似文献
6.
The capacity for dissimilatory reduction of NO 3− to N 2 (N 2O) and NH 4+ was measured in 15NO 3−-amended marine sediment. Incubation with acetylene (7 × 10 −3 atmospheres [normal]) caused accumulation of N 2O in the sediment. The rate of N 2O production equaled the rate of N 2 production in samples without acetylene. Complete inhibition of the reduction of N 2O to N 2 suggests that the “acetylene blockage technique” is applicable to assays for denitrification in marine sediments. The capacity for reduction of NO 3− by denitrification decreased rapidly with depth in the sediment, whereas the capacity for reduction of NO 3− to NH 4+ was significant also in deeper layers. The data suggested that the latter process may be equally as significant as denitrification in the turnover of NO 3− in marine sediments. 相似文献
7.
Microbial Fe reduction in acetate- and succinate-containing enrichment cultures initiated with an estuarine sediment inoculum was studied. Fe reduction was unaffected when SO 42− reduction was inhibited by MoO 42−, indicating that both processes could occur independently. Bacterially produced sulfide precipitated as FeS but was not completely responsible for Fe reduction. The separation of oxidized Fe particles from bacteria by dialysis tubing demonstrated that direct bacterial contact was necessary for Fe reduction. Fe reduction in cultures amended with NO 3− was delayed until NO 3− and NO 2− were removed. However, bacterial attachment to oxidized Fe particles in NO 3−-amended cultures occurred early during growth in a manner similar to NO 3−-free cultures. During late stages of growth, bacteria not attached to Fe particles became pale and swollen, while attached cells remained bright blue when examined by 4′,6-diamidine-2-phenylindole epifluo-rescence microscopy. The presence of added oxidized Mn had no effect on Fe reduction. The results suggested that enzymatic Fe reduction was responsible for reducing Fe in these cultures even in the presence of sulfide and that cells incapable of Fe reduction became unhealthy when Fe(III) was the only available electron acceptor. 相似文献
8.
Nitrate (NO 3−) and nitrite (NO 2−) are the physiological sources of nitric oxide (NO), a key biological messenger molecule. NO 3−/NO 2− exerts a beneficial impact on NO homeostasis and its related cardiovascular functions. To visualize the physiological dynamics of NO 3−/NO 2− for assessing the precise roles of these anions, we developed a genetically encoded intermolecular fluorescence resonance energy transfer (FRET)-based indicator, named sNOOOpy (sensor for NO 3−/NO 2− in physiology), by employing NO 3−/NO 2−-induced dissociation of NasST involved in the denitrification system of rhizobia. The in vitro use of sNOOOpy shows high specificity for NO 3− and NO 2−, and its FRET signal is changed in response to NO 3−/NO 2− in the micromolar range. Furthermore, both an increase and decrease in cellular NO 3− concentration can be detected. sNOOOpy is very simple and potentially applicable to a wide variety of living cells and is expected to provide insights into NO 3−/NO 2− dynamics in various organisms, including plants and animals. 相似文献
9.
We adapted a method for the rapid screening of colonies of free-living Rhizobium japonicum for hydrogenase activity to determine the hydrogenase status of individual soybean nodules. Crude bacteroid suspensions from nodules containing strains known to be hydrogen uptake positive (Hup +) caused a localized decolorization of filter paper disks, whereas suspensions from nodules arising from inoculation with hydrogen uptake-negative (Hup −) mutants or strains did not decolorize the disks. The reliability of the method was demonstrated by its successful application to 29 slow-growing rhizobia. The Hup phenotype on methylene blue filters agreed with that determined amperometrically with either methylene blue or oxygen as the electron acceptor. 相似文献
10.
Until recently, denitrification was thought to be the only significant pathway for N 2 formation and, in turn, the removal of nitrogen in aquatic sediments. The discovery of anaerobic ammonium oxidation in the laboratory suggested that alternative metabolisms might be present in the environment. By using a combination of 15N-labeled NH 4+, NO 3−, and NO 2− (and 14N analogues), production of 29N 2 and 30N 2 was measured in anaerobic sediment slurries from six sites along the Thames estuary. The production of 29N 2 in the presence of 15NH 4+ and either 14NO 3− or 14NO 2− confirmed the presence of anaerobic ammonium oxidation, with the stoichiometry of the reaction indicating that the oxidation was coupled to the reduction of NO 2−. Anaerobic ammonium oxidation proceeded at equal rates via either the direct reduction of NO 2− or indirect reduction, following the initial reduction of NO 3−. Whether NO 2− was directly present at 800 μM or it accumulated at 3 to 20 μM (from the reduction of NO 3−), the rate of 29N 2 formation was not affected, which suggested that anaerobic ammonium oxidation was saturated at low concentrations of NO 2−. We observed a shift in the significance of anaerobic ammonium oxidation to N 2 formation relative to denitrification, from 8% near the head of the estuary to less than 1% at the coast. The relative importance of anaerobic ammonium oxidation was positively correlated ( P < 0.05) with sediment organic content. This report of anaerobic ammonium oxidation in organically enriched estuarine sediments, though in contrast to a recent report on continental shelf sediments, confirms the presence of this novel metabolism in another aquatic sediment system. 相似文献
11.
The acetylene block technique was employed to study denitrification in intertidal estuarine sediments. Addition of nitrate to sediment slurries stimulated denitrification. During the dry season, sediment-slurry denitrification rates displayed Michaelis-Menten kinetics, and ambient NO 3− + NO 2− concentrations (≤26 μM) were below the apparent Km (50 μM) for nitrate. During the rainy season, when ambient NO 3− + NO 2− concentrations were higher (37 to 89 μM), an accurate estimate of the Km could not be obtained. Endogenous denitrification activity was confined to the upper 3 cm of the sediment column. However, the addition of nitrate to deeper sediments demonstrated immediate N 2O production, and potential activity existed at all depths sampled (the deepest was 15 cm). Loss of N 2O in the presence of C 2H 2 was sometimes observed during these short-term sediment incubations. Experiments with sediment slurries and washed cell suspensions of a marine pseudomonad confirmed that this N 2O loss was caused by incomplete blockage of N 2O reductase by C 2H 2 at low nitrate concentrations. Areal estimates of denitrification (in the absence of added nitrate) ranged from 0.8 to 1.2 μmol of N 2 m −2 h −1 (for undisturbed sediments) to 17 to 280 μmol of N 2 m −2 h −1 (for shaken sediment slurries). 相似文献
13.
A recent study (D. C. Cooper, F. W. Picardal, A. Schimmelmann, and A. J. Coby, Appl. Environ. Microbiol. 69:3517-3525, 2003) has shown that NO 3− and NO 2− (NO x−) reduction by Shewanella putrefaciens 200 is inhibited in the presence of goethite. The hypothetical mechanism offered to explain this finding involved the formation of a Fe(III) (hydr)oxide coating on the cell via the surface-catalyzed, abiotic reaction between Fe 2+ and NO 2−. This coating could then inhibit reduction of NO x− by physically blocking transport into the cell. Although the data in the previous study were consistent with such an explanation, the hypothesis was largely speculative. In the current work, this hypothesis was tested and its environmental significance explored through a number of experiments. The inhibition of ~3 mM NO 3− reduction was observed during reduction of a variety of Fe(III) (hydr)oxides, including goethite, hematite, and an iron-bearing, natural sediment. Inhibition of oxygen and fumarate reduction was observed following treatment of cells with Fe 2+ and NO 2−, demonstrating that utilization of other soluble electron acceptors could also be inhibited. Previous adsorption of Fe 2+ onto Paracoccus denitrificans inhibited NO x− reduction, showing that Fe(II) can reduce rates of soluble electron acceptor utilization by non-iron-reducing bacteria. NO 2− was chemically reduced to N 2O by goethite or cell-sorbed Fe 2+, but not at appreciable rates by aqueous Fe 2+. Transmission and scanning electron microscopy showed an electron-dense, Fe-enriched coating on cells treated with Fe 2+ and NO 2−. The formation and effects of such coatings underscore the complexity of the biogeochemical reactions that occur in the subsurface. 相似文献
14.
Aquaspirillum magnetotacticum MS-1 grew microaerobically but not anaerobically with NO 3− or NH 4+ as the sole nitrogen source. Nevertheless, cell yields varied directly with NO 3− concentration under microaerobic conditions. Products of NO 3− reduction included NH 4+, N 2O, NO, and N 2. NO 2− and NH 2OH, each toxic to cells at 0.2 mM, were not detected as products of cells growing on NO 3−. NO 3− reduction to NH 4+ was completely repressed by the addition of 2 mM NH 4+ to the growth medium, whereas NO 3− reduction to N 2O or to N 2 was not. C 2H 2 completely inhibited N 2O reduction to N 2 by growing cells. These results indicate that A. magnetotacticum is a microaerophilic denitrifier that is versatile in its nitrogen metabolism, concomitantly reducing NO 3− by assimilatory and dissimilatory means. This bacterium appears to be the first described denitrifier with an absolute requirement for O 2. The process of NO 3− reduction appears well adapted for avoiding accumulation of several nitrogenous intermediates that are toxic to cells. 相似文献
15.
Biological N 2 fixation is the dominant supply of new nitrogen (N) to the oceans, but is often inhibited in the presence of fixed N sources such as nitrate (NO 3
−). Anthropogenic fixed N inputs to the ocean are increasing, but their effect on marine N 2 fixation is uncertain. Thus, global estimates of new oceanic N depend on a fundamental understanding of factors that modulate N source preferences by N 2-fixing cyanobacteria. We examined the unicellular diazotroph Crocosphaera watsonii (strain WH0003) to determine how the light-limited growth rate influences the inhibitory effects of fixed N on N 2 fixation. When growth ( µ) was limited by low light ( µ = 0.23 d −1), short-term experiments indicated that 0.4 µM NH 4
+ reduced N 2-fixation by ∼90% relative to controls without added NH 4
+. In fast-growing, high-light-acclimated cultures ( µ = 0.68 d −1), 2.0 µM NH 4
+ was needed to achieve the same effect. In long-term exposures to NO 3
−, inhibition of N 2 fixation also varied with growth rate. In high-light-acclimated, fast-growing cultures, NO 3
− did not inhibit N 2-fixation rates in comparison with cultures growing on N 2 alone. Instead NO 3
− supported even faster growth, indicating that the cellular assimilation rate of N 2 alone (i.e. dinitrogen reduction) could not support the light-specific maximum growth rate of Crocosphaera. When growth was severely light-limited, NO 3
− did not support faster growth rates but instead inhibited N 2-fixation rates by 55% relative to controls. These data rest on the basic tenet that light energy is the driver of photoautotrophic growth while various nutrient substrates serve as supports. Our findings provide a novel conceptual framework to examine interactions between N source preferences and predict degrees of inhibition of N 2 fixation by fixed N sources based on the growth rate as controlled by light. 相似文献
16.
Although previous research has demonstrated that NO 3− inhibits microbial Fe(III) reduction in laboratory cultures and natural sediments, the mechanisms of this inhibition have not been fully studied in an environmentally relevant medium that utilizes solid-phase, iron oxide minerals as a Fe(III) source. To study the dynamics of Fe and NO 3− biogeochemistry when ferric (hydr)oxides are used as the Fe(III) source, Shewanella putrefaciens 200 was incubated under anoxic conditions in a low-ionic-strength, artificial groundwater medium with various amounts of NO 3− and synthetic, high-surface-area goethite. Results showed that the presence of NO 3− inhibited microbial goethite reduction more severely than it inhibited microbial reduction of the aqueous or microcrystalline sources of Fe(III) used in other studies. More interestingly, the presence of goethite also resulted in a twofold decrease in the rate of NO 3− reduction, a 10-fold decrease in the rate of NO 2− reduction, and a 20-fold increase in the amounts of N 2O produced. Nitrogen stable isotope experiments that utilized δ 15N values of N 2O to distinguish between chemical and biological reduction of NO 2− revealed that the N 2O produced during NO 2− or NO 3− reduction in the presence of goethite was primarily of abiotic origin. These results indicate that concomitant microbial Fe(III) and NO 3− reduction produces NO 2− and Fe(II), which then abiotically react to reduce NO 2− to N 2O with the subsequent oxidation of Fe(II) to Fe(III). 相似文献
17.
We examined nitrate-dependent Fe 2+ oxidation mediated by anaerobic ammonium oxidation (anammox) bacteria. Enrichment cultures of “ Candidatus Brocadia sinica” anaerobically oxidized Fe 2+ and reduced NO 3− to nitrogen gas at rates of 3.7 ± 0.2 and 1.3 ± 0.1 (mean ± standard deviation [SD]) nmol mg protein −1 min −1, respectively (37°C and pH 7.3). This nitrate reduction rate is an order of magnitude lower than the anammox activity of “ Ca. Brocadia sinica” (10 to 75 nmol NH 4+ mg protein −1 min −1). A 15N tracer experiment demonstrated that coupling of nitrate-dependent Fe 2+ oxidation and the anammox reaction was responsible for producing nitrogen gas from NO 3− by “ Ca. Brocadia sinica.” The activities of nitrate-dependent Fe 2+ oxidation were dependent on temperature and pH, and the highest activities were seen at temperatures of 30 to 45°C and pHs ranging from 5.9 to 9.8. The mean half-saturation constant for NO 3− ± SD of “ Ca. Brocadia sinica” was determined to be 51 ± 21 μM. Nitrate-dependent Fe 2+ oxidation was further demonstrated by another anammox bacterium, “ Candidatus Scalindua sp.,” whose rates of Fe 2+ oxidation and NO 3− reduction were 4.7 ± 0.59 and 1.45 ± 0.05 nmol mg protein −1 min −1, respectively (20°C and pH 7.3). Co-occurrence of nitrate-dependent Fe 2+ oxidation and the anammox reaction decreased the molar ratios of consumed NO 2− to consumed NH 4+ (ΔNO 2−/ΔNH 4+) and produced NO 3− to consumed NH 4+ (ΔNO 3−/ΔNH 4+). These reactions are preferable to the application of anammox processes for wastewater treatment. 相似文献
18.
The effects of several photosynthetic inhibitors and uncouplers of oxidative phosphorylation on NO 3− and NO 2− assimilation were studied using detached barley ( Hordeum vulgare L. cv Numar) leaves in which only endogenous NO 3− or NO 2− were available for reduction. Uncouplers of oxidative phosphorylation greatly increased NO 3− reduction in both light and darkness, while photosynthetic inhibitors did not. The NO2− concentration in the control leaves was very low in both light and darkness; 98% or more of the NO2− formed from NO3− was further assimilated in control leaves. More NO2− accumulated in the leaves in light and darkness in the presence of photosynthetic inhibitors. Of this NO2−, 94% or more was further assimilated. It appears that metabolites, either external or internal to the chloroplast, capable of reducing NADP (which, in turn, could reduce ferredoxin via NADP reductase) might support NO2− reduction in darkness and light when photosynthetic electron flow is inhibited by photosynthetic inhibitors. Nitrite assimilation was much more sensitive to uncouplers in darkness than in light: in darkness, 74% or more of NO2− formed from NO3− was further assimilated, whereas in light, 95% or more of the NO2− was further assimilated. 相似文献
19.
Dark-grown, detopped corn seedlings (cv. Pioneer 3369A) were exposed to treatment solutions containing Ca(NO 3) 2, NaNO 3, or KNO 3; KNO 3 plus 50 or 100 millimolar sorbitol; and KNO 3 at root temperatures of 30, 22, or 16 C. In all experiments, the accelerated phase of NO 3− transport had previously been induced by prior exposure to NO 3− for 10 hours. The experimental system allowed direct measurements of net NO 3− uptake and translocation, and calculation of NO 3− reduction in the root. The presence of K + resulted in small increases in NO 3− uptake, but appreciably stimulated NO 3− translocation out of the root. Enhanced translocation was associated with a marked decrease in the proportion of absorbed NO 3− that was reduced in the root. When translocation was slowed by osmoticum or by low root temperatures, a greater proportion of absorbed NO 3− was reduced in the presence of K +. Results support the proposition that NO 3− reduction in the root is reciprocally related to the rate of NO 3− transport through the root symplasm. 相似文献
20.
The effect of Ca 2+ on NO 3− assimilation in young barley ( Hordeum vulgare L. var CM 72) seedlings in the presence and absence of NaCl was studied. Calcium increased the activity of the NO 3− transporter under saline conditions, but had little effect under nonsaline conditions. Calcium decreased the induction period for the NO 3− transporter under both saline and nonsaline conditions but had little effect on its apparent Km for NO 3− both in the presence and absence of NaCl. The enhancement of NO 3− transport by Ca 2+ under saline conditions was dependent on the presence of Ca 2+ in the uptake solution along with the salt, since Ca 2+ had no effect when supplied before or after salinity stress. Although Mn 2+ and Mg 2+ enhanced NO 3− uptake under saline conditions, neither was as effective as Ca 2+. In longer studies, increasing the Ca 2+ concentration in saline nutrient solutions resulted in increases in NO 3− assimilation and seedling growth. 相似文献
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