A new 15N tracer method to determine N turnover and denitrification of Pseudomonas stutzeri

Although denitrification is one of the key processes of ecosystem N turnover, the understanding of the regulation of the denitrification pathway is still limited due to the lack of feasible methods for the quantification of N₂ formation. Based on the previously developed isotope pairing method, we present a new in vitro ¹⁵N tracer method for the quantification of N₂ released from denitrification by bacterial cultures. The application of the new method was enabled by replacing the background air in the sample flasks with a gas mixture of He and O₂ with an approximately 50-fold reduced N₂ background (1.7% v/v), allowing for a direct and sensitive quantification of N₂ formation with isotope-ratio mass spectrometry after ¹⁵N-labelling on the one hand, but leaving the method relatively insensitive to intrusion of ambient N₂ on the other hand. The method was tested on bacterial cultures of Pseudomonas stutzeri grown at different oxygen levels. Additionally, NO and N₂O formation were determined with a chemoluminescence analyser and a gas chromatograph, respectively. Following labelling with ¹⁵N-ammonium and ¹⁵N-nitrate, it could be shown that P. stutzeri used ammonium preferably for biomass build-up, and nitrate preferably as electron acceptor. Between 84–107% of the total available N could be recovered. Due to the high sensitivity of the new method only low levels of ¹⁵N tracer were necessary, minimising substrate-induced effects and making this method also an appropriate tool for the use on soil cores. By that it offers a new method for studying denitrification in terrestrial ecosystems.     

Diet–consumer nitrogen isotope fractionation for prolonged fasting arthropods

Nitrogen acquisition for cellular metabolism during diapause is a primary concern for herbivorous arthropods. Analyses of naturally occurring stable isotopes of nitrogen help elucidate the mechanism. Relevant articles have cited (58 times up to mid-June 2011) anomalously elevated δ¹⁵N (per mil deviation of ¹⁵N/¹⁴N, relative to atmospheric nitrogen=0 ‰) values (diet–consumer nitrogen isotope fractionation; up to 12 ‰) for a prolonged fasting raspberry beetle (Byturus tomentosus Degeer (Coleoptera: Byturidae)), which feeds on red raspberries (Rubus idaeus: δ¹⁵N=～+2 ‰). Biologists have hypothesised that extensive recycling of amino acid nitrogen is responsible for the prolonged fasting. Since this hypothesis was proposed in 1995, scientists have integrated biochemical and molecular knowledge to support the mechanism of prolonged diapausing of animals. To test the validity of the recycling hypothesis, we analysed tissue nitrogen isotope ratios for four Japanese arthropods: the shield bug Parastrachia japonensis Scott (Hemiptera: Cydnidae), the burrower bug Canthophorus niveimarginatus Scott (Hemiptera: Cydnidae), leaf beetle Gastrophysa atrocyanea Motschulsky (Coleoptera: Chrysomelidae) and the Japanese oak silkworm Antheraea yamamai (Lepidoptera: Saturniidae), all of which fast for more than 6 months as part of their life-history strategy. Resulting diet–consumer nitrogen isotope discrimination during fasting ranged from 0 to 7‰, as in many commonly known terrestrial arthropods. We conclude that prolonged fasting of arthropods does not always result in anomalous diet–consumer nitrogen isotope fractionation, since the recycling process is closed or nearly closed with respect to nitrogen isotopes.     

Source attribution of agriculture-related deposition by using total nitrogen and δ15N in epiphytic lichen tissue,bark and deposition water samples in Germany

Compared with physico-chemical deposition measurement methods, lichens are able to identify the long-term overall effects of high N pollution concentrations in the air. In addition, the natural abundances of the stable isotope of N, ¹⁵N, are being widely used in research on N cycling in ecosystems. They can also be used as instruments for source attribution. In this study, epiphytic lichens were tested to determine whether their respective N content and δ¹⁵N ratios can be used to estimate N deposition rates and to locate various sources of N compounds. Epiphytic lichen and bark samples were collected from around various deposition measurement field stations at different sites in the western part of Germany. The N content of epiphytic lichens reflects the species-specific, agriculture-related circumstances of N deposition at various sites in Germany. At the same time, δ¹⁵N signatures of the different investigated epiphytic lichen species and bark samples are highly depleted in ¹⁵N under high ammonium deposition. The different surface types of lichens and barks exhibit different concentrations of N and δ¹⁵N ratios, despite being exposed to similar N deposition rates. The verification of highly negative δ¹⁵N ratios at sites with local and regional emitters shows that source attribution is possible by comparing different δ¹⁵N signatures in areas with a wide range of different N deposition types and the corresponding differences in δ¹⁵N among various source N pools. Especially nitrophytic lichens can support the on-site instrumentation measuring N deposition by qualification and quantification.     

Spatial variations in larch needle and soil δ15N at a forest–grassland boundary in northern Mongolia

The spatial patterns of plant and soil δ¹⁵N and associated processes in the N cycle were investigated at a forest–grassland boundary in northern Mongolia. Needles of Larix sibirica Ledeb. and soils collected from two study areas were analysed to calculate the differences in δ¹⁵N between needle and soil (Δδ¹⁵N). Δδ¹⁵N showed a clear variation, ranging from ?8?‰ in the forest to ?2?‰ in the grassland boundary, and corresponded to the accumulation of organic layer. In the forest, the separation of available N produced in the soil with ¹⁵N-depleted N uptake by larch and ¹⁵N-enriched N immobilization by microorganisms was proposed to cause large Δδ¹⁵N, whereas in the grassland boundary, small Δδ¹⁵N was explained by the transport of the most available N into larch. The divergence of available N between larch and microorganisms in the soil, and the accumulation of diverged N in the organic layer control the variation in Δδ¹⁵N.     

The relative isotopic abundance (δ13C, δ15N) during composting of agricultural wastes in relation to compost quality and feedstock

Variations in the relative isotopic abundance of C and N (δ¹³C and δ¹⁵N) were measured during the composting of different agricultural wastes using bench-scale bioreactors. Different mixtures of agricultural wastes (horse bedding manure?+?legume residues; dairy manure?+?jatropha mill cake; dairy manure?+?sugarcane residues; dairy manure alone) were used for aerobic–thermophilic composting. No significant differences were found between the δ¹³C values of the feedstock and the final compost, except for dairy manure?+?sugarcane residues (from initial ratio of ?13.6?±?0.2?‰ to final ratio of ?14.4?±?0.2?‰). δ¹⁵N values increased significantly in composts of horse bedding manure?+?legumes residues (from initial ratio of +5.9?±?0.1?‰ to final ratio of +8.2?±?0.5?‰) and dairy manure?+?jatropha mill cake (from initial ratio of +9.5?±?0.2?‰ to final ratio of +12.8?±?0.7?‰) and was related to the total N loss (mass balance). δ¹³C can be used to differentiate composts from different feedstock (e.g. C₃ or C₄ sources). The quantitative relationship between N loss and δ¹⁵N variation should be determined.     

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.     

Tracing changes in the diet and habitat use of black-tailed godwits in Western France,using a stable isotope approach

Western France is at the crossroads of the migratory routes of two subspecies of black-tailed godwit, Limosa limosa. After leaving Iceland, the godwit L.l. islandica Icelandic black-tailed godwit (IBTG) winters on the coast of western Europe, while the continental black-tailed godwit (CBTG) L.l. limosa can stop in France when migrating between Iberia or Africa and their main breeding grounds in the Netherlands. In this study, we analysed δ¹⁵N and δ¹³C from flight feathers and whole blood throughout the non-breeding period to trace variations in habitat use for both subspecies on the western French coast. Adults and juveniles of IBTG adopt the same feeding habitats as soon as they arrive in the study area, progressively losing the Icelandic freshwater habitat signal, and becoming clearly restricted to marine habitats in winter. Some individuals begin to move locally to freshwater habitats, joining CBTG in a stopover at the end of the wintering period in preparation for northward migration.     

Nitrogen isotope fractionation factors (α) measured and estimated from the volatilisation of ammonia from water at pH 9.2 and pH 8.5

ABSTRACT

This paper examines the nitrogen isotope fractionation factors (α) associated with the volatilisation of ammonia from water under controlled conditions at two pH values (8.5 and 9.2). This experiment assumed the continuous removal of ammonia at a single purge rate of 10?mL air min^?1. The fractionation resulting from the removal of total ammonia from the water into an acid trap was named the observed isotope fractionation factor (α_obs), and it was measured as 1.019 (±0.0025) at pH 8.5 and 1.030 (±0.0025) at pH 9.2. The observed isotope fractionation factor includes the equilibrium isotope fractionation factor (α_eq) and the kinetic isotope fractionation factor (α_kin), each one mathematically derived from the experimental data. The equilibrium and kinetic isotope fractionation factors were estimated as α_eq?=?1.036 (±0.0014) and α_kin?=?1.050 (±0.003), respectively. Our results are compared to other previously measured and estimated fractionation factors.     

Stable isotopes of H,C and N in mice bone collagen as a reflection of isotopically controlled food and water intake*

²H/¹H ratios in animal biomass reflect isotopic input from food and water. A 10-week controlled laboratory study raised 48 mice divided in two generations (8 mothers Mus musculus and their offspring). The mice were divided into four groups based on the combination of ²H, ¹³C, ¹⁵N-enriched and non-enriched food and water. Glycine, the most common amino acid in bone collagen, carried the ²H, ¹³C, ¹⁵N-isotopic spike in food. ANOVA data analysis indicated that hydrogen in food accounted for ～81?% of the hydrogen isotope inventory in collagen whereas drinking water hydrogen contributed ～17?%. Air humidity contributed an unspecified amount. Additionally, we monitored ¹³C and ¹⁵N-enrichment in bone collagen and found strong linear correlations with the ²H-enrichment. The experiments with food and water indicate two biosynthetic pathways, namely (i) de novo creation of non-essential amino acids using hydrogen from water, and (ii) the integration of essential and non-essential amino acids from food. The lower rate of isotope uptake in mothers’ collagen relative to their offspring indicates incomplete bone collagen turnover after ten weeks. The variance of hydrogen stable isotope ratios within the same cohort may limit its usefulness as a single sample proxy for archaeological or palaeoenvironmental research.     

The effect of different fish feed compositions on δ13C and δ15N signatures of sea bass and its potential value for tracking mariculture-derived nutrients

Increasing demand for fish and seafood calls for an expansion of aquaculture production. At the same time, the status of the marine environment must not be jeopardised. Stable isotopes are potential markers for tracking feed-based nutrient flows from aquaculture into marine biota. Here, we demonstrate how four experimental diets (main protein components: fishmeal, soya protein concentrate, wheat gluten, and Jatropha kernel meal) and a commercial diet induce characteristic δ¹³C and δ¹⁵N signals in sea bass (Dicentrarchus labrax) during nine weeks of laboratory feeding under replicate conditions. The plant-protein-based diets containing wheat gluten and soya, and the commercial feed consistently induced the largest isotopic differentiation of the fish, both from the feed source and from the pre-experimental condition. The large difference of the fish on plant-protein-based diets compared to the range of natural isotopic variation in the macrozoobenthos of the North Sea lends support to the idea that plant-based feeds are suitable for tracing mariculture-derived organic matter under practical conditions. The commercial feed had a similar effect as the experimental feeds and would be a cost-effective option for an offshore aquaculture experiment.