Determination of 15N abundance of amino acids in soil hydrolysates

¹⁵N abundance of amino acids in soil hydrolysates was determined by emission spectroscopic method.     

Plant nitrate use in deciduous woodland: the relationship between leaf N, N natural abundance of forbs and soil N mineralisation

Our aim was to study whether the in situ natural abundance ¹⁵N (δ¹⁵N)-values and N concentration of understory plants were correlated with the form and amount of mineral N available in the soil. Also to determine whether such differences were related to earlier demonstrations of differences in biomass increase in the same species exposed to nutrient solutions with both and or to alone. Several studies show that the δ¹⁵N of in soil solution generally is isotopically lighter than the δ¹⁵N of due to fractionation during nitrification. Hence, it is reasonable to assume that plant species benefiting from in ecosystems without significant leaching or denitrification have lower δ¹⁵N-values in their tissues than species growing equally well, or better, on We studied the δ¹⁵N of six understory species in oak woodlands in southern Sweden at 12 sites which varied fivefold in potential net N mineralisation rate The species decreased in benefit from in the following order: Geum urbanum, Aegopodium podagraria, Milium effusum, Convallaria majalis, Deschampsia flexuosa and Poa nemoralis. Four or five species demonstrated a negative correlation between and leaf δ¹⁵N and a positive correlation between and leaf N concentration. In wide contrast, only D. flexuosa, which grows on soils with little nitrification, showed a positive correlation between and the leaf N concentration and δ¹⁵N-value. Furthermore, δ¹⁵N of plants from the field and previously obtained indices of hydroponic growth on relative to were closely correlated at the species level. We conclude that δ¹⁵N may serve as a comparative index of uptake of among understory species, preferably in combination with other indices of N availability. The use of δ¹⁵N needs careful consideration of known restrictions of method, soils and plants.     

Natural 15N and 13C abundance in Andisols influenced by long-term fertilization management in Japan

Two field experiments were conducted on Andisols in Japan to evaluate the changes in the natural ¹⁵N and ¹³C abundance in the soil profile and to determine whether the values of δ¹⁵N could be used as an indicator of fertilizer sources or fertilizer fate. The 6-year experiment conducted at the National Agricultural Research Center (NARC) consisted of the following treatments: application of swine compost (COMPOST), slow-release nitrogen fertilizer (SRNF), readily available nitrogen fertilizer (RANF), and absence of fertilization (CONTROL). Experimental plots located at the Nippon Agricultural Research Institute (NARI) received cattle compost at different rates for 12 years; a forest soil at this site was sampled for comparison. Swine compost application led to a considerable change in the δ¹⁵N distribution pattern in the soil profile, with the highest δ¹⁵N values recorded in the top 20 cm layers of the COMPOST plot, decreasing in the sequence of CONTROL >- RANF > SRNF, mainly due to the relatively high δ¹⁵N value of swine compost and its subsequent decomposition. In contrast, SRNF application resulted in the lowest δ¹⁵N values in soil, indicating the presence of negligible nitrogen losses relative to input and low nitrogen cycling rates. Values of δ¹⁵N increased with compost application rates at NARI. In the leachate collected at 1-m depth, the δ¹⁵N values decreased in the sequence of COMPOST > RANF ≥ CONTROL > SRNF. The δ¹³C values in soil peaked in the 40–60 cm layers for all the fertilizers. The δ¹³C value was lowest in forest soil due to the presence of plant residues in soil organic matter. These results indicated that the δ¹⁵N values in the upper soil layers or leachate may enable to detect pollution sources of organic or inorganic nitrogen qualitatively in Andisols.     

Enrichment of natural 15N abundance in frankia-infected nodules

Abstract

The enrichment of ¹⁵N in the nodules of some N₂-fixing leguminous plants is an interesting finding (Shearer et al. 1982). The extent of ¹⁵N enrichment differed depending on the plant species (Shearer et al. 1982; Yoneyama 1987) and bacterial strains (Steele et al. 1983), and in soybeans it was apparently related to the nitrogen fixation efficiency (Shearer et al. 1984)     

Natural N abundance of plant and soil inorganic-N as evidence for over-fertilization with compost

To test the hypothesis that N isotope composition can be used as evidence of excessive compost application, we measured variation in patterns of N concentrations and corresponding δ¹⁵N values of plants and soil after compost application. To do so, a pot experiment with Chinese cabbage (Brassica campestris L. cv. Maeryok) was conducted for 42 days. Compost was applied at rates of 0 (SC0), 500 (SC1), 1000 (SC2), and 1500 mg N kg⁻¹ soil (SC3). Plant-N uptake linearly increased with compost application (r² = 0.956, P < 0.05) with an uptake efficiency of 76 g N kg⁻¹ of compost-N at 42 days after application, while dry-mass accumulation did not show such linear increases. Net N mineralized from compost-N increased linearly (r² = 0.998, P < 0.01) with a slope of 122 g N kg⁻¹ of compost-N. Plant-δ¹⁵N increased curvilinearly with increasing compost application, but this increase was insignificant between SC2 and SC3 treatments. The δ¹⁵N of soil inorganic-N (particularly NO₃⁻-N) increased with compost application. We found that plant-δ¹⁵N reflected the N isotope signal of soil NO₃⁻-N at each measurement during plant growth, and that δ¹⁵N of inner leaves and soil NO₃⁻-N was similar when initial NO₃⁻ in the compost was abundant. Therefore, we concluded that δ¹⁵N of whole plant (more obviously in newer plant parts) and soil NO₃⁻-N could reveal whether compost application was excessive, suggesting a possible use of δ¹⁵N in plants and soil as evidence of excess compost application.     

Natural abundance δN and δC of DNA extracted from soil

We report the first simultaneous measurements of δ¹⁵N and δ¹³C of DNA extracted from surface soils. The isotopic composition of DNA differed significantly among nine different soils. The δ¹³C and δ¹⁵N of DNA was correlated with δ¹³C and δ¹⁵N of soil, respectively, suggesting that the isotopic composition of DNA is strongly influenced by the isotopic composition of soil organic matter. However, in all samples DNA was enriched in ¹³C relative to soil, indicating microorganisms fractionated C during assimilation or preferentially used ¹³C enriched substrates. Enrichment of DNA in ¹⁵N relative to soil was not consistently observed, but there were significant differences between δ¹⁵N of DNA and δ¹⁵N of soil for three different sites, suggesting microorganisms are fractionating N or preferentially using N substrates at different rates across these contrasting ecosystems. There was a strong linear correlation between δ¹⁵N of DNA and δ¹⁵N of the microbial biomass, which indicated DNA was depleted in ¹⁵N relative to the microbial biomass by approximately 3.4‰. Our results show that accurate and precise isotopic measurements of C and N in DNA extracted from the soil are feasible, and that these analyses may provide powerful tools for elucidating C and N cycling processes through soil microorganisms.     

N2O产生法测定土壤无机态氮15N丰度

用化学方法分别将土壤中微量的铵、硝酸盐和亚硝酸盐转化为N2O气体,然后用带自动预浓缩装置的同位素比值质谱仪测定N2O中的15N丰度.N2O中的15N丰度测量值完全符合铵、硝酸盐和亚硝酸盐的15N参考值.方法快速、简单和准确,不受空气氮的污染.特别是方法的检测限很低,每批次样品中只需含5～ 20μg N.它将有助于土壤氮素的矿化作用、硝化作用和反硝化作用的研究.     

Comparison of 15N natural abundance and difference methods for estimating N2 fixation of soybeans grown in a tropical soil

Abstract

A study was carried out to compare the difference or N-yield method with the ¹⁵N natural abundance method for the estimation of the fractional contribution of biological N₂ fixation in the different plant parts of nodulating and non-nodulating isolines of soybeans. The results indicated that the δ¹⁵N values of most plant parts of soybeans were significantly lower (p<0.05) in the nodulating than in the non-nodulating isoline. However, in the case of the root+nodule component, the δ¹⁵N value was higher in the nodulating than in the non-nodulating isoline possibly due to isotopic discrimination of ¹⁵N over ¹⁴N which may have occurred in the nodules. Inoculation of soybeans with the Bradyrhizobium japonicum strain CB 1809 increased significantly (p<0.05) the δ¹⁵N value of the root+nodule component implying that the effectiveness of the soybean-rhizobium symbiosis had increased by inoculation.

Percentage of plant N derived from atmospheric N₂ fixation (%Ndfa) estimated by the ¹⁵N natural abundance method was highly correlated (r=0.762, p<0.01) with that by the difference or N-yield method and the differences between the two methods were not statistically significant. The agreement between the two methods was closer at maturity than at the early reproductive stage.

The %Ndfa obtained by the difference method ranged from 48.4 to 92.6% whereas the %Ndfa obtained by the ¹⁵N natural abundance method ranged from 43.2 to 92.4% in the different plant parts. Based on the ¹⁵N natural abundance method, approximately 15% of the N in pod, shoot, grain, and shell was derived from the soil but in the case of stover, this fraction was about 55%.     

Natural N abundance of plants and soils under different management practices in a montane grassland

The natural ¹⁵N abundance (δ¹⁵N) of different ecosystem compartments is considered to be an integrator of nitrogen (N) cycle processes. Here we investigate the extent to which patterns of δ¹⁵N in grassland plants and soils reflect the effect of different management practices on N cycling processes and N balance. Investigations were conducted in long-term experimental plots of permanent montane meadows with treatments differing in the amount and type of applied fertilizer (0-200 kg N ha⁻¹ yr⁻¹; mineral fertilizer, cattle slurry, stable manure) and/or the cutting frequency (1-6 cuts per season). The higher δ¹⁵N values of organic fertilizers compared to mineral fertilizer were reflected by higher δ¹⁵N values in soils and harvested plant material. Furthermore, δ¹⁵N of top soils and plant material increased with the amount of applied fertilizer N. N balances were calculated from N input (fertilization, atmospheric N deposition and symbiotic N₂ fixation) and N output in harvest. ‘Excess N’—the fraction of N input not harvested—was assumed to be lost to the environment or accumulated in soil. Taking fertilizer type into account, strong positive correlations between δ¹⁵N of top soils and the N input-output balance were found. In plots receiving mineral N fertilizer this indicates that soil processes which discriminate against ¹⁵N (e.g. nitrification, denitrification, ammonia volatilization) were stimulated by the increased supply of readily available N, leading to loss of the ¹⁵N depleted compounds and subsequent ¹⁵N enrichment of the soils. By contrast, in plots with organic fertilization this correlation was partly due to accumulation of ¹⁵N-enriched fertilizer N in top soils and partly due to the occurrence of significant N losses. Cutting frequency appeared to have no direct effect on δ¹⁵N patterns. This study for the first time shows that the natural abundance of ¹⁵N of agricultural systems does not only reflect the type (organic or mineral fertilizer) or amount of annual fertilizer amendment (0-200 kg ha⁻¹ yr⁻¹) but that plant and soil δ¹⁵N is better described by N input-output balances.     

Changes in natural 15N abundance in paddy soils under different,long-term soil management regimes in the Tohoku region of Japan

Abstract

Long-term temporal changes in natural ¹⁵N abundance (δ¹⁵N value) in paddy soils from long-term field experiments with livestock manure and rice straw composts, and in the composts used for the experiments, were investigated. These field experiments using livestock manure and rice straw composts had been conducted since 1973 and 1968, respectively. In both experiments, control plots to which no compost had been applied were also maintained. The δ¹⁵N values of livestock manure compost reflected the composting method. Composting period had no significant effect on the δ¹⁵N value of rice straw compost. The δ¹⁵N values increased in soils to which livestock manure compost was successively applied, and tended to decrease in soils without compost. In soils to which rice straw compost was successively applied, the δ¹⁵N values of the soils remained constant. Conversely, δ¹⁵N values in soils without rice straw compost decreased. The downward trend in δ¹⁵N values observed in soils to which compost and chemical N fertilizer were not applied could be attributed to the natural input of N, which had a lower δ¹⁵N value than the soils. Thus, the transition of the δ¹⁵N values in soils observed in long-term paddy field experiments indicated that the δ¹⁵N values of paddy soils could be affected by natural N input in addition to extraneous N that was applied in the form of chemical N fertilizers and organic materials.     

A novel GC/MS technique to assess N and C incorporation into soil amino sugars

Amino sugars have been used as biomarker to indicate microorganism contribution to soil organic matter turnover and sequestration. However, there is no direct gas chromatograph mass spectrometry (GC/MS) approach to assess microbial synthesis of amino sugars in soil. We developed a novel method which combines laboratory incubation of substrate containing ¹⁵N or ¹³C and a GC/MS technique to trace ¹⁵N or ¹³C isotope changes in three amino sugars, glucosamine, galactosamine, and muramic acid. Sample preparation followed the procedure of Zhang and Amelung (1996) [Zhang, X., Amelung, W., 1996. Gas chromatographic determination of muramic acid, glucosamine, galactosamine, and mannosamine in soils. Soil Biology and Biochemistry 28, 1201-1206.]. The GC/MS determination was conducted using a full scan mode with both electronic ionization (EI) and chemical ionization (CI) sources. The CI source was suitable for all of the three amino sugars, while the EI source was not applicable to muramic acid due to its low sensitivity in the determination as well as low concentration of muramic acid in soil. The enrichment of ¹⁵N or ¹³C in amino sugars during incubation was estimated by calculating the atom percentage excess (APE). ¹⁵N incorporation was evaluated according to fragment (F) abundance ratio of mass F+1 to F, whilst ¹³C incorporation was estimated according to the ratio of mass F+n to F (n is skeleton carbon number in the fragment). This novel method was assessed by using two soil samples (a Kandiudult and a Udoll) incubated with either ¹⁵N-amonium or U-¹³C-glucose. The results indicate that the GC/MS determination is reproducible, thus this technique is useful in detecting the microbial synthesis of amino sugars in soil, and especially it should be possible when looking at the position or how much labeled carbon and nitrogen atoms have been incorporated.     

Depth dynamics of soil N contents and natural abundances of 15N after 43 years of long-term fertilization and liming in sub-tropical Alfisol

The aim of this study was to understand impacts of long-term (43 years) fertilization on soil aggregation, N accumulation rates and δ¹⁵N in surface and deep layers in an Alfisol. Soil samples from seven treatments were analysed for N stocks, aggregate-associated N in 0–30 cm and the changes in δ¹⁵N in 0–90 cm depths. The treatments were: unfertilized control (control); recommended N dose (N); recommended N and phosphorus doses (NP); recommended N, P and potassium doses (NPK); 150% of recommended N, P and K doses (150% NPK); NPK + 10 Mg FYM ha^?1 (NPK + FYM) and NPK + 0.4 Mg lime ha^?1 (NPK + L). Results revealed that plots under NPK + FYM had ~39% higher total N concentrations than NPK + L in 0–30 cm soil layers. In NPK + L, macro-aggregates had 35 and 11% and microaggregates had 20 and 9% lower δ¹⁵N values than NPK + FYM in 0–15 and 15–30 cm soil layers, respectively. However, plots receiving NPK + FYM had ~39% greater deep soil (30–90 cm) N accumulation than NPK + L. These results would help understanding N supplying capacity by long-term fertilization and assist devising N management strategies in sub-tropical acidic Alfisols.     

Fate of Fertilizer-15N to a Maize Crop Grown in Northern Zambia

Abstract

A field study with maize (Zea mays L.) was conducted in the 1988/89 cropping season to investigate the fate of ¹⁵NO₃-N-labelled NH₄ ¹⁵NO₃ applied at 40, 80 and 120 kg N ha^?1 (unlabelled N applied at 0, 80, 160 and 240 N ha^?1) with and without lime. The investigations were conducted in northern Zambia at Misamfu Regional Research Centre, Kasama on a Misamfu red sandy loam soil. The experimental design was a split plot arrangement with four replications with main plots receiving 0 and 2 Mg ha^?1 dolomitic limestone, while subplots received fertilizer N at various rates. Significant (p < 0.001) grain and DM yield responses to applied N up to 160 kg ha^?1 were observed. At higher rates little or no crop responses were observed and fertilizer use efficiency declined. Partitioning of amounts of total N and ¹⁵N in plants was in the order of seed = tassel > leaf> cob = earleaf> stem. Fertilizer N rates showed a highly significant (p < 0.001) effect on plant uptake of labelled N. Lime and its interaction with N rates had no effect on all measured parameters. Leaching of NO₃-N fertilizer to lower soil depths was in proportion to the rate of N applied, with highly significant (p < 0.001) differences among soil depths. Although higher concentrations of fertilizer-¹⁵N were recovered in the 0–20 cm depth the recovered portion at lower soil depths was still significant. Total recovery of labelled N by plant and by soil after crop harvest averaged 75, 55 and 54% of originally applied fertilizer-¹⁵N at 40, 80 and 120 kg N ha^?1, respectively. Corresponding unaccounted for ¹⁵N was 25, 45 and 46%. The most probable loss mechanism could have been by leaching to depths greater than 60 cm, gaseous losses to the atmosphere and root assimilation.     

A GC/MS method for the assessment of N and C incorporation into soil amino acid enantiomers

Identifying the transformation process of amino acid enantiomers was essential to probe into the fate, turnover and aging of soil nitrogen due to their important roles in the biogeochemical cycling. If this can be achieved by differentiating between the newly biosynthesized and the inherent compounds in soil, then the isotope tracer method can be considered most valid. We thereby developed a gas chromatography/mass spectrometry (GC/MS) method to trace the ¹⁵N or ¹³C isotope incorporation into soil amino acid enantiomers after being incubated with ¹⁵NH₄⁺ or U-¹³C-glucose substrates. The most significant fragments (F) as well as the related minor ions were monitored by the full scan mode and the isotope enrichment in amino acids was estimated by calculating the atom percentage excess (APE). ¹⁵NH₄⁺ incorporation was evaluated according to the relative abundance increase of m/z F+1 to F for neutral and acidic amino acids and F+2 to F (mass 439) for lysine. The assessment of ¹³C enrichment in soil amino acids was more complicated than that of ¹⁵N due to multi-carbon atoms in amino acid molecules. The abundance ratio increment of m/z F+n to F (n is the original skeleton carbon number in each fragment) indicated the direct conversion from the added glucose to amino acids, but the total isotope incorporation from the added ¹³C can only be calculated according to all target isotope fragments, i.e. the abundance ratio increment summation from m/z (Fa+1) through m/z (Fa+T) represented the total incorporation of the added ¹³C (Fa is the fragment containing all original skeleton carbons and T is the carbon number in the amino acid molecule). This method has a great advantage especially for the evaluation of high-abundance isotope enrichment in organic compounds compared with GC/C/IRMS. And in principle, this technique is also valid for amino acids besides enantiomers if stereoisomers are not concerned. Our assessment approach could shine a light on investigating the biochemical mechanism of microbial transformation of N and C in soils of terrestrial ecosystem.     

C and N natural abundance of the soil microbial biomass

Stable isotope analysis is a powerful tool in the study of soil organic matter formation. It is often observed that more decomposed soil organic matter is ¹³C, and especially ¹⁵N-enriched relative to fresh litter and recent organic matter. We investigated whether this shift in isotope composition relates to the isotope composition of the microbial biomass, an important source for soil organic matter. We developed a new approach to determine the natural abundance C and N isotope composition of the microbial biomass across a broad range of soil types, vegetation, and climates. We found consistently that the soil microbial biomass was ¹⁵N-enriched relative to the total (3.2 ‰) and extractable N pools (3.7 ‰), and ¹³C-enriched relative to the extractable C pool (2.5 ‰). The microbial biomass was also ¹³C-enriched relative to total C for soils that exhibited a C3-plant signature (1.6 ‰), but ¹³C-depleted for soils with a C4 signature (−1.1 ‰). The latter was probably associated with an increase of annual C3 forbs in C4 grasslands after an extreme drought. These findings are in agreement with the proposed contribution of microbial products to the stabilized soil organic matter and may help explain the shift in isotope composition during soil organic matter formation.     

Seasonal Changes of Shoot Nitrogen Concentrations and 15N/14N Ratios in Common Reed in a Constructed Wetland

Abstract

Nitrogen (N) concentrations and stable N isotope abundances (δ¹⁵N) of common reed (Phragmites australis) planted in a constructed wetland were measured periodically between July 2001 and May 2002 to examine their seasonal variations in relation to N uptake and N translocation within common reed. Nitrogen concentrations in P. australis shoots were higher in the growing stage (7.5 to 24.8 g N kg^?1) than in the senescence stage (4.2 to 6.8 g N kg^?1), indicating N translocation from shoots to rhizomes. Meanwhile, the corresponding δ¹⁵N values were higher in the senescence stage (+12.2 to +22.4‰) than in the growing stage (+5.1 to +11.3‰). Coupled with the negative correlation (R²=0.24, P<0.05, n=18) between N concentrations and δ¹⁵N values of shoots in the senescence stage, our results suggested that shoot N became enriched in ¹⁵N due to N isotopic fractionation (with an isotopic fractionation factor, α_s/p, of 1.012) during N translocation to rhizomes. However, the positive correlation between N concentrations and δ¹⁵N values in the growing stage (R²=0.19, P<0.001, n=54) suggested that P. australis relies on N re‐translocated from rhizome in the early growing stage and on mineral N in the sediment during the active growing stage. Therefore, seasonal δ¹⁵N variations provide N‐isotopic evidence of N translocation within and N uptake from external N sources by common reed.     

The use of 15N natural abundance variation to examine plant and soil organic fractions in pasture under different management practises

 Pasture systems lack the complexity of natural grasslands and have undisturbed soil profiles relative to arable monocultures. With controlled nitrogen (N) fertilizer inputs and measurable outputs (e.g. grazing and leaching), they can be used to investigate ¹⁵N natural abundance variation as a tool for the study of soil-N processes. In the present study, four pastures of different sward composition and N inputs were examined. Plant shoots and a range of soil fractions, categorized by size, were sampled in May prior to any major N additions, and again in July after initial N inputs had of been made. Samples were analyzed for ¹⁵N natural abundance (δ¹⁵N) and total N (εN). In the May sample plant and soil fractions varied in both ¹⁵N and εN between treatments. The 0.5 mm and 0.2 mm soil factions were comparable within treatments, as were the silt and clay fractions. Between May and July changes were apparent in the δ¹⁵N and εN of shoots and some soil fractions within each plot these corresponded to N inputs or sward type. Changes in silt-N especially, were similar to those occurring in the shoots. No comparable changes were seen in the larger fractions. Not all measured variation was explicable in this study. The inadequacies of the approach are highlighted and suggested improvements discussed. Received: 9 February 1998     

Fate of nitrogen derived from 15N-labeled plant residues and composts in rice-planted paddy soil

Pot experiments that lasted for 3 y were conducted to investigate the dynamics of nitrogen derived from plant residues (rice root, hull, straw, corn root, and rapeseed pod-wall), and composts (rice straw compost, cattle manure compost, and cattle manure sawdust compost), which were labeled with ¹⁵N. The rates of nitrogen uptake by rice (=N efficiency), denitrification, and immobilization derived from the organic materials incorporated before the first year of cultivation were investigated throughout 3 y of cultivation. At the end of the first year of cultivation, relatively high rates of N efficiency were obtained for rapeseed pod-wall (24.6%), rice straw (19.1%), and rice hull (18.6%), while corn root and cattle manure sawdust compost displayed a noticeably high denitrification rate. Corn root, cattle manure sawdust compost, rice hull, and rapeseed pod-wall exhibited remarkably high N mineralization rates ranging from 60 to 75% of the organic materials N applied. Cumulative rates of N efficiencies from the organic materials applied before the first year of cultivation fitted well to a first-order kinetic model and their asymptotes were compared among the organic materials. The asymptotic rates of N efficiency tended to depend on the rates at the end of the first year of cultivation.     

Interference by amino acids during the determination of N ammonium in soil

In the study of terrestrial N cycling, NH₄⁺ concentration and ¹⁵N enrichment are routinely determined by colorimetric continuous flow analysis and microdiffusion methods. Amino acids can interfere in these determinations; consequently the aim of the present study was to evaluate the significance of the interference. Glycine and glutamine are key amino acids in soil and were therefore used as ‘models’. Both glycine and glutamine interfered during continuous flow analysis, whereas interference during microdiffusion was of little importance. The effects of interference can be significant, e.g. estimates of gross mineralisation rate were reduced up to 33%, where we allowed for amino acid interference during determination of NH₄⁺ concentration. The potential influence of amino acid interference emphasises that development of continuous flow analysis to increase NH₄⁺ specificity is needed.     

Incorporation of 14CO2 and 15NH4 into amino acids of the two subunits of fraction 1 protein in spinach leaves

In the investigation of nitrogen metabolism in plants, it is important to deal with proteins, which are the end-products of nitrogen metabolism.