In vivo NMR spectroscopy: in situ 15N pulse labelling NMR spectroscopy with photoautotrophic microorganisms

For studying the nitrogen metabolism in plants ¹⁵N NMR spectroscopy can be used. For in vivo ¹⁵N NMR (natural abundance of ¹⁵N: 0.37%) enrichment of the sample with the isotope ¹⁵N is compulsory. The detection of time courses of ¹⁵N assimilation from cells, which are enriched in culture is restricted in scope. Here, a method, the ¹⁵N pulse labelling NMR spectroscopy, is demonstrated, which permits labelling of different nitrogen compounds in photoautotrophic microorganisms during the NMR spectroscopic measurement. Using an effective illumination system it is possible to maintain photosynthesis in plant samples of high biomass densities in the magnet necessary for ammonia assimilation. The technique thus enables to directly observe ammonia assimilation pathways by application of a ¹⁵NO₃ ^? or ¹⁵NH₄ ^? pulses.

Für das Studium des Stickstoffstoffwechsels der Pflanzen kann die ¹⁵N-NMR-Spektroskopie herangezogen werden. Hierzu ist bei der in-vivo-¹⁵N-NMR (natürliche Häufigkeit von ¹⁵N: 0.37%) eine Anreicherung der Probe mit dem Isotop ¹⁵N unerläßlich. Eine Verfolgung der ¹⁵N-Assimilationskinetik mit Zellen, die in der Kultur angereichert wurden, ist jedoch nur bedingt möglich. In dieser Arbeit wird die ¹⁵N-Pulsmarkierungs-NMR-Spektroskopie als eine Methode vorgestellt, die es erlaubt, eine Markierung von Stickstoffverbindungen in photoautotrophen einzelligen Mikroorganismen während der NMR-Messung im Magneten vorzunehmen. Es wird ein spezielles Beleuchtungssystem verwendet, das eine für die Stickstoffassimilation ausreichende Photosyntheseleistung der Zellen unter NMR-Bedingungen bei hoher Biomassedichte ermöglicht. Diese Technik erlaubt durch die Applikation eines ¹⁵NO₃ ^?-oder ¹⁵NH₄ ⁺-Pulses eine direkte Verfolgung von Ammonium-Assimilationswegen.     

The use of stable isotopes in ecosystem research. First results of a field study with 15N

Terrestrial ecosystems, e.g. forest ecosystems, are characterized by a complex and sensitive network of biotic and abiotic factors and their interactions. By using stable isotopes (e.g. labelled nitrogen compounds), very small addition rates of highly enriched compounds can be applied, which do not change or disturb the investigated system, but provide information about single processes, their interactions and especially about their dynamics.

First results of a field study in the Fichtelgebirge, Northeast-Bavaria, Germany, are presented. The distribution of labelled nitrogen (as ¹⁵N-NH₄ ⁺ and ¹⁵N[sbnd]NO₃ ^?) within a spruce ecosystem (Picea abies (L.) Karst. in competition with understory vegetation of Vaccinium myrtillus, Calluna vulgaris and Deschampsia flexuosa) showed maximum ¹⁵N concentrations in tissues of the understory vegetation. During the first six weeks after the ¹⁵N application, the nitrogen uptake of all investigated species was higher after the ¹⁵N[sbnd]NO₃ ^? treatment than after the ¹⁵N[sbnd]NH₄ ⁺ treatment.     

Effects of fish farm loading on sea grass Posidonia oceanica at Vrgada Island (Central Adriatic): a nitrogen stable isotope study

Analyses of nitrogen stable isotopes in the marine sea grass Posidonia oceanica were used to investigate the influence of fish farming on the coastal ecosystem of Vrgada Island in the Murter Sea, Central Adriatic. The results show a statistically significant 15N enrichment (up to 4.7 per thousand at p < 0.005) in P. oceanica leaf and shoot tissues from fish cage sites with respect to the unaffected offshore reference site of Lumbarda Reef Flat (Kornati Islands). Heavy nitrogen enrichment was also detected in other benthic organisms analysed during this study and is attributed to the absorption and assimilation of 15N-enriched fish farm derived nitrogen waste.     

Dinitrogen Fixation of Microbe-Plant Associations as Affected by Nitrate and Ammonium Supply

Abstract

The dinitrogen fixation activity of Azospirillum sp., and Pantoea agglomerans strains was determined by ¹⁵N₂ incorporation after incubation with ¹⁵N₂ labeled air or/and by acetylene reduction. These bacterial strains were able to fix N₂ both in pure culture and in association with wheat plants in hydroponics. Nitrogenase activity of Azospirillum sp., in pure culture was more rapidly inhibited by the addition of NH₄ ⁺ than NO₃ ^?. The N₂ fixation of P. agglomerans decreased only by NH₄ ⁺ -addition, but was stimulated by NO₃ ^?. Nitrogen fixation in association with wheat plants remained unaffected by both N compounds. However, nitrogen derived from the atmosphere (N_dfa) contributed only very little to the overall nitrogen nutrition of the plants.     

Studies of the protein and the energy metabolism in man during a wintering in Antarctica

During the 29th Soviet Antarctic Expedition in Novolazarevskaya from March 1984 to March 1985, the protein and energy metabolisms were studied in six expeditioners from the German Democratic Republic. The investigations were carried out at the beginning of the expedition (May), during the polar night (July) and during the polar day (December). The effect of a special stress situation (sledge trek in April 1984) was investigated in one subject. The stable nitrogen isotope ¹⁵N was used to study the protein metabolism. The assessment of the energy metabolism was based on the oxygen consumption, which was determined by means of a spirograph. In addition, the vital capacity, the breath minute volume, the blood pressure, etc. were measured. The following results were obtained: During the polar night, the utilisation of the dietary proteins and the whole body protein synthesis calculated by means of the ¹⁵N excretion of the total nitrogen in urine were greater (73.6±0.9 % and 3.48±0.17 g protein d^?1 kg^?1, n=3) than the respective values during the polar day (69.7±1.2, p<0.05, n=3 and 3.05±0.07, p<0.05, n=3) and at the beginning of the expedition (69.6±1.4, p<0.02, n=5 and 2.81±0.09, p<0.01, n=5). The lowest values (58.0 % and 2.43 g protein d^?1 kg^?1) were obtained in the subject after the trek. The resting metabolic rate (in kJ d^?1 m^?2) was decreased during the polar night (45.6±5.0, n=4) in comparison with the polar day (61.5±11.3, n=3) and the beginning of the expedition (52.3±9.6, n=4) with p<0.01 in both cases.     

Prediction of human dietary δ15N intake from standardised food records: validity and precision of single meal and 24-h diet data

Natural stable isotope ratios (δ¹⁵N) of humans can be used for nutritional analyses and dietary reconstruction of modern and historic individuals and populations. Information about an individual’s metabolic state can be obtained by comparison of tissue and dietary δ¹⁵N. Different methods have been used to estimate dietary δ¹⁵N in the past; however, the validity of such predictions has not been compared to experimental values. For a total of 56 meals and 21 samples of 24-h diets, predicted and experimental δ¹⁵N values were compared. The δ¹⁵N values were predicted from self-recorded food intake and compared with experimental δ¹⁵N values. Predicted and experimental δ¹⁵N values were in good agreement for meals and preparations (r?=?0.89, p?r?=?0.76, p?δ¹⁵N was mainly determined by the amount of fish, whereas the contribution of meat to dietary δ¹⁵N values was less pronounced. Prediction of human dietary δ¹⁵N values using standardised food records and representative δ¹⁵N data sets yields reliable data for dietary δ¹⁵N intake. A differentiated analysis of the primary protein sources is necessary when relating the proportion of animal-derived protein in the diet by δ¹⁵N analysis.     

Leaf allocation patterns and 13C and 15N natural abundances of tropical lianas (Passiflora sp.) as dependent on external climbing support

The transformation from self-supporting lianas to host-supported climbing lianas is related to re-allocation of biomass and nutrients among plant organs. Therefore, first, variations in leaf mass per area (LMA), leaf carbon and nitrogen allocation and ¹³C and ¹⁵N natural abundances were analysed among three tropical Passiflora species (P. edulis, P. ligularis, and P. tripartita) in a greenhouse study. Second, the influence of a climbing support was considered for each species and parameter. P. ligularis leaves were most enriched in ¹³C in both treatments when compared with the other two species. This enrichment was caused by a high LMA, which is related to a high internal resistance to CO₂ diffusion. For P. edulis and P. tripartita, δ¹³C was additionally increasing with nitrogen content per area. Generally, there were no differences when considering carbon and nitrogen allocation to leaves of host-supported and self-supporting lianas. The only hints towards increased investment into leaves after the transition from self-supporting to host-supported stages could be seen by a trend to increased leaf areas and masses. δ¹³C values of supported P. edulis or P. tripartita plants were significantly increasing faster than those of non-supported plants once the interactions of leaf mass or nitrogen content per area were accounted for. Hence, the offer of a climbing support had only a minor impact on δ¹³C or δ¹⁵N values in vitro, but this could be different with increasing age of lianas in vivo.     

Nitrogen stable isotopes reveal age-dependent dietary shift in the Japanese scallop Mizuhopecten yessoensis

Ontogenetic niche shifts in diet are a consequence of changes in body size or resource partitioning between age classes. To better resolve the feeding patterns of the Japanese scallop Mizuhopecten yessoensis, we examined the relative importance of age and size in the diet of this species using stable isotope ratios of carbon (δ¹³C) and nitrogen (δ¹⁵N) from 2006 to 2009. Contribution of food sources was quantified using an isotope mixing model by comparing the muscle tissue isotope ratios to those of suspended particulate organic matter (SPOM) and their zooplankton prey (e.g. micro- and meso-zooplankton). Unlike the δ¹³C values, which remained constant with age and size, muscle δ¹⁵N values were more positively correlated with age accounting for 69?% of variations than size with only 46?%. Increasing ¹⁵N values with age suggested that shifts in diet from SPOM to micro- and meso-zooplankton occurred during ontogeny in M. yessoensis. Results of the isotope mixing model indicated that SPOM contribution to scallop’s diet decreased from 68 to 8?% while those of zooplankton increased from 15 to 50?% with increasing age. This study concludes that age-related dietary shift explains the enrichment of ¹⁵N, as a result of predation on zooplankton by M. yessoensis.     

Stable carbon and nitrogen isotope discrimination and turnover in a small-bodied insectivorous lizard

Laboratory experiments are useful for estimating the carbon and nitrogen isotope discrimination factors and turnover rates that are critical for drawing field-based inferences on consumer diets using stable isotopes. Although the utility of these discrimination factors is widely recognized, work in terrestrial systems has largely been limited to studies involving mammals and birds. In contrast, scant attention has been paid to the application of isotopic techniques to reptiles, despite their broad diversity in terms of numbers of species as well as their trophic roles. Here we estimate carbon and nitrogen isotope discrimination factors and turnover for the tree lizard (Urosaurus ornatus) using a diet-switch experiment. Lizards were collected from a C₄-dominated grassland and then switched to C₃-based diet (crickets) in the laboratory. We estimated discrimination by lizard claw tissue as Δ¹³C?=?1.2?±?0.1?‰ for carbon and Δ¹⁵N?=?0.7?±?0.1?‰ (mean?±?1 SE) for nitrogen, with 95?% turnover occurring after ～15.5 days. These estimates should be appropriate for use in trophic studies of U. ornatus, and possibly other related small-bodied insectivorous lizards.     

Soil δ15N patterns in old-growth forests of southern Chile as integrator for N-cycling

Old-growth forests of southern Chile represent an important reserve of temperate (rain) forests in the world. Wetter and colder forest ecosystems appear to be more efficient in conserving and recycling N such that mostly non-plant available N species are lost, which could be indicated by more depleted δ¹⁵N values of the soil and plants. Hydrological N loss from the old-growth forests in southern Chile occurs mainly via dissolved organic nitrogen and not via dissolved inorganic N. Forest disturbances (e.g. fire, clear-cutting or enhanced N deposition) cause (abrupt) changes in ecosystem N-cycling processes. In this study, we hypothesized that δ¹⁵N signatures of soil profiles under old-growth forests could be used as an integrator for ecosystem N-cycling, and changes of these δ¹⁵N profiles could be valuable to assess ecosystem resilience towards disturbances. Six old-growth forests were selected in the phytogeographical region of the Valdivian rain forest in southern Chile. One of the sites has been partly burned in February 2002. First, we observed that ecosystems with higher mean annual precipitation and lower mean annual temperature were relatively more depleted in ¹⁵N. Secondly, we found that a forest fire caused a 100-fold increase of the nitrate export and induced an enrichment of the soil δ¹⁵N signal in the upper 20 cm.     

Conference Reports

Abstract

One purpose of new land use concepts for degraded fens (organic soils with high N content) is the reduction of the mineralization process due to very high groundwater levels. However, knowledge of nitrogen mineralization process (net and gross) in degraded fen soils affected by reflooding is very small. Therefore, the objectives of our study were (a) to evaluate the suitability of ¹⁵N pool dilution method for measurements of gross mineralization rates in degraded fen soils and (b) to investigate how the reflooding of a degraded fen affects the net and gross nitrogen mineralization in a short-term incubation experiment. The usability of the ¹⁵N pool dilution method was diminished by the low recovery of the applied ¹⁵NH₄ ^? at time zero. The recovery of the added ¹⁵NH₄ ^? in the extractable soil NH₄ ^? pool was only 13.5% for the drained soil and 59.6% for the reflooded soil. However, the gross mineralization rates were similar for both soils and exceeded always the net rates substantially. The cumulative net mineralization rate was higher for the reflooded soil (1.58 μg N?cm^?3?d^?1) than for the drained soil (-0.67 μg N?cm^?3?d^?1). Differences between the two soils were also found in the nitrification intensity and the loss of ¹⁵N. This was probably one reason for the higher net mineralization rate in the reflooded soil.     

Ion-implanted planar resistors

Abstract

Boron-ions have been implanted into Silicon to form p-type planar resistors. The resistors have Boron-diffused contacts and the energetic ions were implanted through an approximately 0.2 micron thick thermally grown SiO₂-film, on top of which a pattern was etched in an evaporated aluminum layer to define the areas to be implanted. The ion-doses were in the range 10¹² cm^?2 to 10¹⁵ cm^?2 with ion-energies 120 keV and 200 keV. All implantations were performed at room temperature and the annealing took place for 15 min in a nitrogen ambient in the temperature range 300–950 °C. Among the results of the investigations are the obtainable range of sheet resistivities, the large-area-homogeneity and the temperature coefficient of resistance (TCR) as a function of the above mentioned parameters.     

Feeding level and diet quality influence trophic shift of c and n isotopes in nile tilapia (oreochromis niloticus (L.))

Many scientists use naturally occurring stable isotopes to reconstruct the diets of animals. However, isotopic ratios may be affected not only by the composition of the diet but also by the amount of food consumed. Thus, an experiment using tilapia (Oreochromis niloticus) was carried out to test the influence of feeding level on δ ¹³C and δ ¹⁵N of fish given a semi-synthetic wheat gluten/wheat starch based diet. In addition, the effect of diet quality was tested by comparing tilapia given this feed with tilapia fed a fish meal/wheat meal based diet. Forty-four tilapia were reared individually. After a prefeeding phase, fish were randomly assigned to five groups, four on the semi-synthetic diet at different feeding levels and one group on the fish meal/wheat meal based diet fed at the equivalent of the highest level of the semi-synthetic diet. The experiment lasted eight weeks. Proximate composition, gross energy content and δ ¹³C and δ ¹⁵N values were determined in feed and fish, for δ ¹³C separately in the lipids and the lipid-free matter. δ ¹³C in the lipids and the lipid-free matter and δ ¹⁵N of tilapia fed the semi-synthetic diet decreased significantly with increasing feeding rate. The absolute values of the trophic shift in fish fed the semi-synthetic wheat based diet were significantly higher than in fish fed the fish meal/wheat meal based diet. The different δ ¹³C and δ ¹⁵N values in tilapia fed the same diet at different feeding levels and the influence of feed quality on the trophic shift add to the uncertainty involved in the use of stable isotopes in ecological research.     

Development of non-lethal sampling of carbon and nitrogen stable isotope ratios in salmonids: effects of lipid and inorganic components of fins

The preferred tissue for analyses of fish stable isotope ratios for most researchers is muscle, the sampling of which typically requires the specimen to be sacrificed. The use of non-destructive methods in fish isotopic research has been increasing recently, but as yet is not a standard procedure. Previous studies have reported varying levels of success regarding the utility of non-lethally obtained stable isotope materials, e.g. fins, but none have accounted for the potential compounding effects of inorganic components of fin rays or lipids. Comparisons of carbon (δ¹³C) and nitrogen (δ¹⁵N) stable isotope ratios of muscle with adipose and caudal fin of two salmonids, Atlantic salmon (Salmo salar L.) and brown trout (Salmo trutta L.), revealed that caudal fin can be used as a non-destructive surrogate for muscle in stable isotope analysis, but that adipose fin, where available, is a better proxy. The use of a published model to inexpensively counteract the confounding effect of lipids, which are depleted in ¹³C, greatly improved the relationship between fish muscle and fins. However, efforts to account for the inorganic components of fin rays were counterproductive and required twice the biomass of fins clipped from each fish. As this experiment was conducted on wild fish, controlled laboratory studies are required to confirm these field observations.     

The Fate of [15N]Ammonium and [15N]Nitrate in the Soil of a 140-Year-Old Spruce Stand (Picea Abies) in the Fichtelgebirge (NE-Bavaria)

Abstract

A ¹⁵N tracer-experiment was carried out in a 140-year-old spruce stand (Picea abies (L.) Karst.) in the Fichtelgebirge (NE-Bavaria, Germany). Highly enriched (98 at%) [¹⁵N]ammonium and [¹⁵N]nitrate were applied as tracers by simulation of a deposition of 41.3 mol N ha^?1 with 11 water m^?2. To examine seasonal variations of uptake by spruce and understorey vegetation, different plots were labelled in spring, summer and autumn 1994.

One aim of the present study was to perfect a method of preparation of soil extracts for isotope ratio mass spectrometry (IRMS) measurements. Ammonium and nitrate from soil extracts were prepared for IRMS measurements by steam distillation and subsequent freeze drying. Additionally, tracer distribution and transformations in the soil nitrogen pools were examined. Ammonium, nitrate and total nitrogen were examined in the organic layer and the upper 10 cm of the mineral soil during 3 months after the first tracer application in spring 1994.

In July 1994, three months after tracer application, 40% of the [¹⁵N]ammonium label and 29% of the [¹⁵N]nitrate label, respectively, were recovered in the total N pool of the investigated soil horizons. In the organic layer the L/Of horizon retained most of the recovered tracers. Nitrification, immobilisation and mineralisation occurred even under the conditions of high soil acidity at the study site.     

Aufnahme von 15NO2 und Einbau des 15NO2-Stickstoffs in verschiedene Stickstofffraktionen bei Sonnenblumen (Uptake of 15NO2 and Metabolic Transfer of the 15NO2 Nitrogen to Various Nitrogen Fractions of Sunflowers)

Abstract

Sunflowers were exposed to ¹⁵NO₂ at a range between 4,9 and 42,0 ppb. The ¹⁵NO₂ uptake was quantified and the fate of the ¹⁵N in different parts and pools of nitrogen was investigated. The very high δ¹⁵N-values of the free amino acid pool can't give an answer to the question whether the NO₂-nitrogen is incorporated by a different pathway, compared to the nitrogen which derives from root uptake. Or does a compartmentation of the nitrate pool in the plant cell cause a dilution of the ¹⁵N enrichment in the mineral nitrogen pool during sample preparation?     

Optical properties and radiation effects in layered crystals of (CnH2n+nNH3)2MCl4: n = 1, 2, 3; M = Cd,Cu, Mn

Abstract

A new absorption band has been found at 5.10 eV in (C _n H_{2n + 1}NH₃)₂CdCl₄: n = 1, 2, 3 in addition to the absorption bands of CdCl₂ whose electronic structure resembles the former crystals. The energy of the additional peak shifts with temperature by as much as 0.38 eV from 5.10eV at room temperature (RT) to 5.48 eV at liquid nitrogen temperature. This large peak shift is attributed to a structural phase transition between these two temperatures. A new type of electron center has been found in these crystals (M = Cd, Mn; n = 1, 2, 3) irradiated with X-rays at 15 K in addition to the Cl₂ ^?. This shows optical absorption bands (IR bands) in the infrared region of 10 ～ 20 kcm ^?1. The IR bands are assigned to an electron center where an electron is trapped at an ammonium site in the neighborhood of a Cl^? vacancy.     

Adsorption and isotopic exchange of nitrogen on polycrystalline iron films between 273 and 600 K

The adsorption of nitrogen on evaporated iron films under uhv conditions has been studied between 273 and 600 K by means of direct volumetric and pressure measurements. The dissociative adsorption occurs with sticking probabilities in the range of 10^?8 to 10^?7 and apparent activation energies between ?1 and 5 kJ mol^?1 depending on the structure of the film. A mechanism for the dissociative adsorption of nitrogen is proposed taking into account molecularly adsorbed nitrogen as a precursor. After the adsorption of nitrogen enriched in ¹⁵N₂ the establishing of the iso topic equilibrium was observed using isothermal desorption.     

Seasonal variation in natural abundance of δ13C and 15N in Salicornia brachiata Roxb. populations from a coastal area of India

High and fluctuating salinity is characteristic for coastal salt marshes, which strongly affect the physiology of halophytes consequently resulting in changes in stable isotope distribution. The natural abundance of stable isotopes (δ¹³C and δ¹⁵N) of the halophyte plant Salicornia brachiata and physico-chemical characteristics of soils were analysed in order to investigate the relationship of stable isotope distribution in different populations in a growing period in the coastal area of Gujarat, India. Aboveground and belowground biomass of S. brachiata was collected from six different populations at five times (September 2014, November 2014, January 2015, March 2015 and May 2015). The δ¹³C values in aboveground (?30.8 to ?23.6?‰, average: ?26.6?±?0.4?‰) and belowground biomass (?30.0 to ?23.1?‰, average: ?26.3?±?0.4?‰) were similar. The δ¹³C values were positively correlated with soil salinity and Na concentration, and negatively correlated with soil mineral nitrogen. The δ¹⁵N values of aboveground (6.7–16.1?‰, average: 9.6?±?0.4?‰) were comparatively higher than belowground biomass (5.4–13.2?‰, average: 7.8?±?0.3?‰). The δ¹⁵N values were negatively correlated with soil available P. We conclude that the variation in δ¹³C values of S. brachiata was possibly caused by soil salinity (associated Na content) and N limitation which demonstrates the potential of δ¹³C as an indicator of stress in plants.     

Estimation of Urea Production Rate With [15N2]Urea and [13C]Urea to Measure Catabolic Rates in Diabetes Mellitus

Abstract

For verifying catabolic states in insulin-dependent patients and dogs the method estimating urea production rates with ¹³C and with doubly ¹⁵N labeled urea, respectively, has been established. For a fast steady state of urea tracer dilution, a prime of 600 times the continuous infusion rate had to be injected. Urea was isolated from plasma samples by protein precipitation and cation exchange chromatography with a consecutive derivatization of the dried urea fraction (trimethylsilyl derivatives). The masses of the fragment ions m/z 189 (¹⁴N¹⁴N), 190 (¹⁴N¹⁵N) and 191 (¹⁵N¹⁵N) urea are monitored to estimate the [¹⁵N₂]urea frequency in the overall body urea pool in mol percent excess (MPE). 1 to 15 ng of derivatized urea were measured efficiently. An excellent correlation between expected standard and measured MPE (r = 0.9977) was achieved from solutions containing 1 to 7% [¹⁵N₂]urea. The interassay coefficient of variation amounted to < 10% for a [¹⁵N₂]urea portion of ≥ 3%.

Normoglycemic diabetic patients who were treated with insulin overnight showed significantly higher urea production compared to healthy controls (9.22 ± 2.07 vs. 5.4 ± 0.32 μmol·kg^?1 · min^?1; p < 0.05). Measurements in chronic diabetic dogs proved an increased rate of amino acid catabolism (+ 20% urea production) in systemic versus portal application of insulin in paired studies. This increased nitrogen load in diabetics may accelerate progression of diabetic nephropathy. - Thus, the established stable isotope technique may serve as a sensitive and useful indicator of amino acid catabolism in clinical and experimental research.