Membrane inlet mass spectrometric analysis of N-isotope labelling for aquatic denitrification studies

Abstract: Techniques are described for measuring the isotope distribution in dissolved nitrate and N₂ using membrane inlet mass spectrometry, which allows several gases to be measured in a water sample without the need for any separation steps. The isotope distribution in dissolved nitrate was measured using denitrifying Pseudomonas nautica to reduce the nitrate to N₂ which was then measured by mass spectrometry. Pseudomonas nautica NCIMB 1967 was easily grown in nitrate-limited continuous culture minimising intra- or extracellular nitrate or nitrite pools, and the bioassay was tolerant of a range of salinities. The precision of the bioassay when measuring samples with high ¹⁵NO₃⁻ contents (0.5 μmol) was 0.05 atom%; with 0.1 μmol ¹⁵NO₃⁻, the precision was around 0.2 atom%. Differences in labelling of N₂ in preserved samples obtained from ¹⁵NO₃⁻ incubations of water-covered sediment cores were measured on parallel samples with membrane inlet MS and GC-MS. The membrane inlet technique was accurate but the precision on ratio measurements was lower than by GC-MS.     

Time-course of inorganic nitrogen uptake and incorporation by natural populations of freshwater phytoplankton

SUMMARY. 1. Time-course measurements of NH₄⁺ and NO₃⁻uptake were made on the natural phytoplankton populations in a eutrophic lake at a time when these nutrients were at their lowest annual concentration.
2. Both NH₄⁺ and NO₃⁻ uptake was increased at least five-fold during the first 5 min of incubation following near saturating pulses of these nutrients.
3. Elevated uptake was also observed following low level (∼2μg N 1⁻¹) pulses of NH₄⁺ and NO₃⁻, but substrate depletion during the first hour of incubation may have been partially responsible for this apparent enhancement.
4. Incorporation of ^I5N into TCA-insoluble material (protein) following the saturating NH₄⁺ pulse was increased less than total cellular ¹⁵N uptake, whereas no elevation of ¹⁵N incorporation into protein was observed following a saturating NO₃⁻pulse.
5. The percentage of ^I5N incorporated into protein, with respect to total cellular uptake, was ∼32% and ∼12% for NH₄⁺ and NO₃⁻, respectively, following 5 h of incubation.     

Nitrification and denitrification by Thiosphaera pantotropha in aerobic chemostat cultures

Abstract: Thiosphaera pantotropha has been reported to denitrify aerobically and nitrify heterotrophically. However, recent evidence has indicated that these properties (particularly aerobic denitrification) have been lost. The occurrence and levels of aerobic denitrification and heterotrophic nitrification by T. pantotropha in chemostat cultures have therefore been re-evaluated. Only low nitrate reduction rates were observed: the apparent nitrogen loss was of the same order of magnitude as the combined error in the calculated nitrogen consumption. However, ¹⁵N mass spectrometry revealed low aerobic denitrification rates (about 10% of the rates originally published by this group). Heterotrophic nitrification rates were about a third of previous observations. N₂ and N₂O were both produced from NH₄⁺, NO₃⁻ and NO₂⁻. Periplasmic nitrate reductase was present in aerobically grown cells.     

Improvement of the 15N dilution method for estimation of absorption of NOx by plants supplied with 15N-labelled fertilizer

To develop further the methods for estimation of NOx absorption by plants supplied with ¹⁵N-labelled fertilizer, we proposed a new calculation method, total N fixed method (TNF), and compared with the ¹⁵N dilution method and the classical mass balance method (MB).
Hydroponically grown soybean plants were supplied with ¹⁵N-labelled nitrate and exposed to 200–250 nl l⁻¹ NO₂ for 7 d. The proportions of the N derived from NO₂ to total N in exposed plants were estimated by the three methods.
The reported rates of NO₂ absorption by several plant species, estimated by the ¹⁵N dilution method, were recalculated using the TNF method. The results of the two methods were compared and showed that: (1) The ¹⁵N dilution method overestimated the content of NO₂-N in exposed plants compared with the MB method whilst the TNF method produced estimations of NO₂-N closer to those by the MB method when the plants were supplied with 5 m M nitrate. (2) The differences in estimations between the MB method and either the ¹⁵N dilution method or the TNF method increased with decreasing supply of ¹⁵N-labelled nitrate to roots.     

Nitrate assimilation and nitrogen circulation in Austrian pine

Nitrate uptake, reduction and translocation were examined in 5-week-old Austrian pines ( Pinus nigra Arnold var. nigricans Host.) during exposure to 5 m M NaNO₃. The rate of nitrate uptake was linear during the 7 h light period. ¹⁵N-NO₃⁻ was detected in all parts of the pine except in the needles. By the 7th hour, 43% of the absorbed nitrate had been reduced, and this increased to 64% by the 24th hour. The major part of the total reduction occurred in the roots at this growth stage. Accumulation of ¹⁵N in reduced soluble and insoluble fractions was more prevalent in roots than in shoots. In the needles, the translocated nitrogen was mainly incorporated into the insoluble fraction. It is likely that most of the nitrogen from nitrate was transported from the roots to the aerial organs as organic nitrogen; however part of the upward nitrogen flux took place as nitrate ions.
An experiment in which an exposure for 24 h to 5 m M Na¹⁵NO₃ was followed by 13 days exposure to Na¹⁴NO₃ (pulse chase experiment) revealed a half time of about 1 day for depletion of root nitrate. A large part of this depletion was due to the loss of ¹⁵N-NO₃⁻ to the nutrient solution. The remaining pool of ¹⁵N-nitrate was partitioned between a metabolically inactive and an active pool. During the chase period, the simultaneous decrease of ¹⁵N-incorporation in the soluble N fraction and increase in the insoluble N fraction in different pine parts, particularly in the needles, suggested that protein synthesis occurred mostly in young tissues of the shoot and was the major sink of the newly absorbed ¹⁵N-NO₃.     

Effect of organic loading on nitrification and denitrification in a marine sediment microcosm

Abstract The effects of organic additions on nitrification and dentrification were examined in sediment microcosms. The organic material, heat killed yeast, had a C/N ratio of 7.5 and was added to sieved, homogenized sediments. Four treatments were compared: no addition (control), 30 g dry weight (dw) m⁻² mixed throughout the 10 cm sediment column (30M), 100 g dw m⁻² mixed throughout sediments (100M), and 100 g dw m⁻² mixed into top 1 cm (100S). After the microcosms had been established for 7–11 days, depth of O₂ penetration, sediment-water fluxes and nitrification rates were measured. Nitrification rates were measured using three different techniques: N-serve and acetylene inhibition in intact cores, and nitrification potentials in slurris. Increased organic additions decreased O₂ penetration from 2.7 to 0.2 mm while increasing both O₂ consumption, from 30 to 70 mmol O₂ m⁻² d⁻¹, and NO₃⁻ flux into sediments. Nitrification rates in intact cores were similar for the two methods. Highest rates occurred in the 30M treatment, while the lowest rate was measured in the 100S treatment. Total denitrification rates (estimated from nitrification and nitrate fluxes) increased with increased organic addition, because of the high concentrations of NO₃⁻ (40 μM) in the overlaying water. The ratio of nitrification: denitrification was used as an indication of the importance of nitrification as the NO₃⁻ supply for denitrificaion. This ratio decreased from 1.55 to 0.05 iwth increase organic addition.     

Effects of nitrate on influx, efflux and translocation of potassium in young sunflower plants

Influx, efflux and translocation of K⁺(⁸⁶Rb) were studied in the roots of sunflower seedlings ( Helianthus annuus L. cv. Uniflorus) treated with 0–4.0 m M NO₃⁻ during a 9 day growth period or a 24 h pretreatment period. Roots treated with high levels of NO₃⁻ absorbed and translocated more K⁺(⁸⁶Rb) than seedlings treated with low levels of NO₃⁻. The content of K⁺ in the shoots was, however, higher in seedlings treated with low levels of NO₃⁻, indicating a low rate of retranslocation of K⁺ in those plants. K⁺(⁸⁶Rb) efflux was highest into the low-NO₃⁻ solutions. All effects on K⁺(⁸⁶Rb)-fluxes were more obvious in high-K plants than in low-K plants. The results are discussed in relation to the Dijkshoorn-Ben Zioni hypothesis for K⁺+ NO₃⁻-uptake and translocation in plants.     

A reaction diffusion model of C-N-S-O species in some arctic sediments

Abstract A reaction diffusion model was used to simulate the mineralization processes in an Arctic sediment. The simulation and the actual sediment were compared in relation to profiles of O₂, NO₃ and NH₄⁺. The site of particulate organic matter (POM) degradation was the single most important factor in fitting the simulation profiles to those of the sediment. It was deduced that most POM degradation occurred close to the sediment surface. When a reasonably good simulation had been obtained, the sensitivity of the model to changes in other parameters was investigated. Increases in POM degradation in the upper sediment resulted in increases in concentration of NH₄⁺ and NO₃⁻, but further increases in POM degradation created anoxic conditions below 3 mm, resulting in decreases in NO₃⁻ concentrations. The model was relatively intensive to changes in POM degradation in the lower sediment layers; increases led to more anoxic conditions and to less NO₃⁻. Increases in the C/N ratio of the POM in the lower sediment layers had little effect; increases in C/N in the upper layers led to a decrease in NH₄⁺ and NO₃⁻. The model was sensitive to changes in the first order rate constant for nitrification, but not for denitrification. Decreases in the K _m for O₂ of the nitrifying bacteria had no effect on the profiles.     

Anion uptake in maize roots: Interactions between chlorate and nitrate

Effects of nitrate, chloride and chlorate ions upon nitrate and chlorate uptake by roots of maize ( Zea mays L., cv. B73) seedlings were examined. Net nitrate uptake, ³⁶ClO₃⁻ influx and ³⁶Cl⁻ influx (the latter two in a background of 0.5 m M KNO₃) displayed similar pH profiles with optima at pH 5.5 and below. External, non-labeled chloride had little effect on the accumulation of ³⁶ClO₃⁻ (both in 5 h and 20 min uptake assays), while nitrate and chlorate had almost identical, marked inhibitory effects. Nitrate pretreatment caused an apparent induction of both ³⁶ClO₃⁻ and ¹⁵NO₃⁻ uptake activities. After 5 h of treatment in nitrate, the uptake activities of chloride- and chlorate-pretreated plants increased to that of nitrate-pretreated plants. During 6 h exposure to chlorate, ³⁶ClO₃⁻ uptake activity of nitrate-pretreated plants decreased to that of chlorate- and chloride-pretreated plants. The results support the existence of a shared nitrate/chlorate transport system in maize roots which is not inhibited by external chloride, and which is induced by nitrate, but not by chlorate or chloride. The suggestion is made that selection of chlorate-resistant mutants of maize can identify nitrate uptake as well as nitrate reductase mutants.     

Simultaneous Measurement of Nitrite and Nitrate Levels as Indices of Nitric Oxide Release in the Cerebellum of Conscious Rats

Abstract: We examined the modulation of nitric oxide production in vivo by measuring levels of nitrite (NO₂⁻) and nitrate (NO₃⁻) in the dialysate of the cerebellum in conscious rats, by using an in vivo brain microdialysis technique. The levels of both NO₂⁻ and NO₃⁻ were decreased by the intraperitoneal injection of N ^G-nitro- l -arginine methyl ester, an inhibitor of nitric oxide synthase, whereas N ^G-nitro- d -arginine methyl ester had no effect. l -Arginine by itself increased NO₂⁻ and NO₃⁻ levels and diminished the reduction of their levels caused by N ^G-nitro- l -arginine methyl ester. Direct infusion of l -glutamate, N -methyl- d -aspartate, or KCl into the cerebellum through a dialysis probe resulted in an increase in NO₂⁻ and/or NO₃⁻ levels. The effects of N -methyl- d -aspartate and KCl were dependent on extracellular calcium. Furthermore, the stimulatory effects of l -glutamate and N -methyl- d -aspartate were inhibited by N ^G-nitro- l -arginine methyl ester and (±)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), an N -methyl- d -aspartate receptor antagonist. These results suggest that NO₂⁻ and NO₃⁻ levels may be related to nitric oxide production in vivo.     

Regulation of K+ and NO3− fluxes in roots of sunflower (Helianthus annuus) after changes in light intensity

Shoot activity has been reported to affect rates of ion uptake by plant roots in other ways than merely through supply of assimilates. To elucidate the mechanisms by which a signal from the upper part of the plant controls the rate of K⁺ and NO₃⁻ uptake by roots, both uptake of K⁺ and NO₃⁻ and secretion into the xylem of young sunflower plants ( Helianthus annuus L.) were measured after changes in light intensity.
No close correlation was observed between the uptake of NO₃⁻ and that of K⁺; an increase in light intensity produced a much greater stimulation of NO₃⁻ uptake than of K⁺ uptake. On the other hand, secretion of NO₃⁻ into the xylem was tightly coupled to that of K⁺, and this coupling was strongly disturbed by excision of the root. The results suggest the involvement of the K₂-malate shuttle on the regulation by the shoot of K⁺ and NO₃⁻ secretion in the xylem, which is linked to NO₃⁻ uptake, while K⁺ uptake is independent of this regulation mechanism.     

Induction of nitrate reductase in needles of Scots pine seedlings by NOX and NO3−

The possibility to induce nitrate reductase (NR; EC 1.6.6.2) in needles of Scots pine ( Pinus sylvestris L.) seedlings was studied. The NR activity was measured by an in vivo assay. Although increased NR activities were found in the roots after application of NO₃⁻, no such increase could be detected in the needles. Detached seedlings placed in NO₃⁻ solution showed increasing NR activities with increasing NO₃⁻ concentrations. Exposure of seedlings to NO_x (70–80 ppb NO₂ and 8–12ppb NO) resulted in an increase of the NR activity from 10–20 nmol NO₂⁻ (g fresh weight)⁻¹ h⁻¹ to about 400 nmol NO₂⁻ (g fresh weight)⁻¹ h⁻¹. This level was reached after 2–4 days of exposure, thereafter the NR activity decreased to about 200 nmol NO₂⁻ (g fresh weight)⁻¹ h⁻¹. Analyses of free amino acids showed low concentrations of arginine and glutamine in NO_x-fumigated seedlings compared to corresponding controls.     

Distribution of nitrate reductase activity in Vicia faba: Effect of nitrate and plant genotype

The short term effect of NO₃⁻ (12 mM) on nitrate reductase (NR. EC 1.6.6.1) activity has been studied in the roots, nodules and leaves of different genotypes of Vicia faba L. at the end of vegetative growth. Root and leaf NR activity responded positively to NO₃⁻ while nodule activity, where detected, proved to he strongly inhibited. The withdraw of this NO₃⁻ from the solution consistently reduced activity in the roots and leaves but surprising, promoted a significant increase in nodule activity, which matched or surpassed that of control plants On the other hand, nodules developed in the presence of 8 mM NO₃⁻ expressed an on average 141% higher level of NR activity than did controls. This effect was observed even in nodules with negligible control activity. In any case, a naturally occurring mutant (VF17) lacking root and nodule NR activity is described. The results indicate that in V. faba. the effects of NO₃⁻ and plant genotype on NR activity depended on plant organ and time of NO₃⁻ application, hut the distribution of NO₃⁻ reduction through the plain was mainly dependent on plant genotype, and to a lesser extent on NO: supply and plant age.     

Denitrification and nitrate-reducing bacterial populations in abandoned and reclaimed minesoils

Abstract Microbial populations, nitrogen mineralization potentials, and denitrification enzyme activities were examined in two abandoned carbolithic minesoils. Numbers and activities of bacteria and fungi were lower in nonamended than in lime and/or fly ash amended sites. Rates of aerobic NO₃⁻ production (3 to 38 μg-N kg⁻¹ h⁻¹) and anaerobic NO₃⁻ reduction to N₂O (5 to 68 μg-N kg⁻¹ h⁻¹) were measured. Organisms capable of N₂O production under anaerobic soil conditions were present in low numbers, and their activity was restricted in part by low soil pH. Nondenitrifying nitrate-reducing bacteria were more diverse and in greater numbers than respiratory denitrifiers and may have been responsible for N₂O production in assays measuring denitrification enzyme activity.     

Isolation, characterization, and nitrification potential of a methylotroph and two heterotrophic bacteria from a consortium showing methane-dependent nitrification

Abstract A methanotrophic nitrifying consortium was previously obtained from a humisol which showed CH₄-dependent nitrification. Although the methanotroph could not be obtained in pure culture, three other members of the consortium have been isolated: An obligately methylotrophic Methylobacillus (Is-1) which grows only on CH₃OH and does not nitrify; a Pseudomonas (Is-2) which grows on Is-1 culture filtrate and produces NO₂⁻, NO₃⁻ and N₂O from NH₂OH, and NO₃⁻ from NO₂⁻; and a second Pseudomonas (Is-3) which produces NO₃⁻ from NH₄⁺ or NO₂⁻, and N₂O from NH₂OH. A model is proposed for the trophic relations and nitrogen transformations in the consortium which may apply to some natural systems.     

Net uptake and partitioning of nitrogen and potassium in cultivars of barley during ageing

Experiments were carried out with barley cultivars ( Hordeum vulgare L.) grown in both pot- and water-culture. Net uptake of NO3₋ and K⁺ in the roots was followed in two barley cultivars grown on water-culture for 85 days. After an initial period of low net uptake of both ions, uptake increased until a maximum was reached after 30 to 45 days. Thereafter, net uptake of NO₃⁻ and K⁺ steadily decreased. In the pot experiments, effects of different mineral supply on day 4 to 18 upon the development of five barley cultivars of various earliness were investigated. The effect of earliness on fresh weight production was largest when mineral supply on day 4 to 18 was limited. The influence of limited mineral supply on day 4 to 18 on K-economy was independent of earliness of the cultivars. The maximal N-content was reached at the same time as maximal fresh and dry weight in fairly late cultivars; in early cultivars maximum N-level was reached later than maximum fresh and dry weight. Overall, maximal N-content was higher in the fairly late cultivars than in the early cultivars. The highest rate of ¹⁵N-transport was attained later in two of three fairly late cultivars than in early cultivars. Partitioning of dry weight, N and K in the shoots changed during ageing, ears being an important sink. Varietal differences in partitioning depended on the earliness of the cultivars. The largest fraction of recently supplied ¹⁵N, supplied as nitrate, and K⁺ (⁸⁶Rb) were found in the stems. In the oldest plants of the early cultivars the transport to the ears of these isotopes was gradually impaired, reflecting the decreasing function of the long distance transport system.     

Methane-dependent nitrate production by a microbial consortium enriched from a cultivated humisol

Abstract A consortium was enriched from a humisol incubated with 3.6 kPa CH₄ and NH₄⁺. This consortium oxidized NH₄⁺ to NO₂⁻ and NO₃⁻ (NO₃⁻/NO₂⁻ ratio about 20) with smaller amounts of N₂O. This oxidation stopped in the stationary phase after depletion of CH₄. CH₃OH or CO₂ did not support oxidation. Growth and resting cell experiments suggested that nitrification was associated with methanotrophic activity and that chemoautotrophic nitrifiers were absent.     

Transfer of nitrogen from a tropical legume tree to an associated fodder grass via root exudation and common mycelial networks

Symbiotic dinitrogen fixation by legume trees represents a substantial N input in agroforestry systems, which may benefit the associated crops. Applying ¹⁵N labelling, we studied N transfer via common mycelial networks (CMN) and root exudation from the legume tree Gliricidia sepium to the associated fodder grass Dichantium aristatum . The plants were grown in greenhouse in shared pots in full interaction (treatment FI) or with their root systems separated with a fine mesh that allowed N transfer via CMN only (treatment MY). Tree root exudation was measured separately with hydroponics. Nitrogen transfer estimates were based on the isotopic signature of N ( δ ¹⁵N) transferred from the donor. We obtained a range for estimates by calculating transfer with δ ¹⁵N of tree roots and exudates. Nitrogen transfer was 3.7–14.0 and 0.7–2.5% of grass total N in treatments FI and MY, respectively. Root δ ¹⁵N gave the lower and exudate δ ¹⁵N the higher estimates. Transfer in FI probably occurred mainly via root exudation. Transfer in MY correlated negatively with grass root N concentration, implying that it was driven by source-sink relationships between the plants. The range of transfer estimates, depending on source δ ¹⁵N applied, indicates the need of understanding the transfer mechanisms as a basis for reliable estimates.     

Oxygen control prevents denitrifiers and barley plant roots from directly competing for nitrate

Abstract Two denitrifying bacteria ( Pseudomonas chlororaphis and P. aureofaciens ) and a plant (barley, Hordeum vulgare ) were used to study the effect of O₂ concentration on denitrification and NO₃⁻ uptake by roots under well-defined aeration conditions. Bacterial cells in the early stationary phase were kept in a chemostat vessel with vigorous stirring and thus a uniform O₂ concentration in the solution. Both Pseudomonads lacked N₂O reductase and so total denitrification could be directly measured as N₂O production.
Denitrification decreased to 6–13% of the anaerobic rate at 0.01% O₂ saturation (0.14 μM O₂) and was totally inhibited at 0.04% O₂ saturation (0.56 μM O₂). In this well-mixed system denitrification was 10-times more oxygen sensitive than stated in earlier reports. Uptake of nitrate by plants was measured in the same system under light. The NO₃⁻ uptake rate decreased gradually from a maximum in 21% O₂-saturated medium (air saturated) to zero at 1.6% O₂ saturation (22.4 μM O₂). Owing to the very different non-overlapping oxygen requirements of the two processes, direct competition for nitrate between plant roots and denitrifying bacteria cannot occur.     

Metabolism of nitric oxide and nitrous oxide during nitrification and denitrification in soil at different incubation conditions

Abstract NO production and consumption rates as well as N₂O accumulation rates were measured in a loamy cambisol which was incubated under different conditions (i.e. soil moisture content, addition of nitrogen fertilizer and/or glucose, aerobic or anaerobic gas phase). Inhibition of nitrification with acetylene allowed us to distinguish between nitrification and denitrification as sources of NO and N₂O. Under aerobic conditions untreated soil showed very low release of NO and N₂O but high consumption of NO. Fertilization with NH₄⁺ or urea stimulated both NO and N₂O production by nitrification. Addition of glucose at high soil moisture contents led to increased N₂ and N₂O production by denitrification, but not to increased NO production rates. Anaerobic conditions, however, stimulated both NO and N₂O production by denitrification. The production of NO and N₂O was further stimulated at low moisture contents and after addition of glucose or NO₃⁻. Anaerobic consumption of NO by denitrification followed Michaelis-Menten kinetics and was stimulated by addition of glucose and NO₃⁻. Aerobic consumption of NO followed first-order kinetics up to mixing ratios of at least 14 ppmv NO, was inhibited by autoclaving but not by acetylene, and decreased with increasing soil moisture content. The high NO-consumption activity and the effects of soil moisture on the apparent rates of anaerobic and aerobic production and consumption of NO suggest that diffusional constraints have an important influence on the release of NO, and may be a reason for the different behaviour of NO release vs N₂O release.