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.     

Identification of Metabolically Active Rhizosphere Microorganisms by Stable Isotopic Probing of PLFA in Switchgrass

Root-derived rhizodeposits of recent photosynthetic carbon (C) are the foremost source of energy for microbial growth and development in rhizosphere soil. A substantial amount of photosynthesized C by the plants is translocated to belowground and is released as root exudates that influence the structure and function of soil microbial communities with potential inference in nutrient and C cycling in the ecosystem. We applied the ¹³C pulse chase labeling technique to evaluate the incorporation of rhizodeposit-C into the phospholipid fatty acids (PLFAs) in the bulk and rhizosphere soils of switchgrass (Panicum virgatum L.). Soil samples of bulk and rhizosphere were taken at 1, 5, 10 and 20 days after labeling and analyzed for ¹³C enrichment in the microbial PLFAs. Temporal differences of ¹³C enrichment in PLFAs were more prominent than spatial differences. Among the microbial PLFA biomarkers, fungi and Gram-negative (GM-ve) bacterial PLFAs showed rapid enrichment with ¹³C compared to Gram-positive (GM+ve) and actinomycetes in rhizosphere soil. The ¹³C enrichment of actinomycetes biomarker PLFA significantly increased along with sampling time in both soils. PLFAs indicative to fungi, GM-ve and GM+ve showed a significant decrease in ¹³C enrichment over sampling time in the rhizosphere, but a decrease was also observed in GM-ve (16:1ω5c) and fungal biomarker PLFAs in the bulk soil. The relative ¹³C concentration in fungal PLFA decreased on day 10, whereas those of GM-ve increased on day 5 and GM+ve remained constant in the rhizosphere soil. However, the relative ¹³C concentrations of GM-ve and GM+ve increased on days 5 and 10, respectively, and those of fungal remain constant in the bulk soil. The present study demonstrates the usefulness of ¹³C pulse chase labeling together with PLFA analysis to evaluate the active involvement of microbial community groups for utilizing rhizodeposit-C.     

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.     

Microbial community composition and rhizodeposit-carbon assimilation in differently managed temperate grassland soils

Rhizodeposit-carbon provides a major energy source for microbial growth in the rhizosphere of grassland soils. However, little is known about the microbial communities that mediate the rhizosphere carbon dynamics, especially how their activity is influenced by changes in soil management. We combined a ¹³CO₂ pulse-labeling experiment with phospholipid fatty acid (PLFA) analysis in differently managed Belgian grasslands to identify the active rhizodeposit-C assimilating microbial communities in these grasslands and to evaluate their response to management practices. Experimental treatments consisted of three mineral N fertilization levels (0, 225 and 450 kg N ha⁻¹ y⁻¹) and two mowing frequencies (3 and 5 times y⁻¹). Phospholipid fatty acids were extracted from surface (0-5 cm) bulk (BU) and root-adhering (RA) soil samples prior to and 24 h after pulse-labeling and were analyzed by gas chromatography-combustion-isotope ratio mass spectrometry (GC-c-IRMS). Soil habitats significantly differed in microbial community structure (as revealed by multivariate analysis of mol% biomarker PLFAs) as well as in gram-positive bacterial rhizodeposit-C uptake (as revealed by greater ¹³C-PLFA enrichment following pulse-labeling in RA compared to BU soil in the 450N/5M treatment). Mowing frequency did not significantly alter the relative abundance (mol%) or activity (¹³C enrichment) of microbial communities. In the non-fertilized treatment, the greatest ¹³C enrichment was seen in all fungal biomarker PLFAs (C16:1ω5, C18:1ω9, C18:2ω6,9 and C18:3ω3,6,9), which demonstrates a prominent contribution of fungi in the processing of new photosynthate-C in non-fertilized grassland soils. In all treatments, the lowest ¹³C enrichment was found in gram-positive bacterial and actinomycetes biomarker PLFAs. Fungal biomarker PLFAs had significantly lower ¹³C enrichment in the fertilized compared to non-fertilized treatments in BU soil (C16:1ω5, C18:1ω9) as well as RA soil (all fungal biomarkers). While these observations clearly indicated a negative effect of N fertilization on fungal assimilation of plant-derived C, the effect of N fertilization on fungal abundance could only be detected for the arbuscular mycorrhizal fungal (AMF) PLFA (C16:1ω5). On the other hand, increases in the relative abundance of gram-positive bacterial PLFAs with N fertilization were found without concomitant increases in ¹³C enrichment following pulse-labeling. We conclude that in situ¹³C pulse-labeling of PLFAs is an effective tool to detect functional changes of those microbial communities that are dominantly involved in the immediate processing of new rhizosphere-C.     

Linking dynamics of soil microbial phospholipid fatty acids to carbon mineralization in a C natural abundance experiment: Impact of heavy metals and acid rain

A ¹³C natural abundance experiment including GC-c-IRMS analysis of phospholipid fatty acids (PLFAs) was conducted to assess the temporal dynamics of the soil microbial community and carbon incorporation during the mineralization of plant residues under the impact of heavy metals and acid rain. Maize straw was incorporated into (i) control soil, (ii) soil irrigated with acid rain, (iii) soil amended with heavy metal-polluted filter dust and (iv) soil with both, heavy metal and acid rain treatment, over a period of 74 weeks. The mineralization of maize straw carbon was significantly reduced by heavy metal impact. Reduced mineralization rate of the added carbon likely resulted from a reduction of the microbial biomass due to heavy metal stress, while the efficiency of ¹³C incorporation into microbial PLFAs was hardly affected. Since acid rain did not significantly change soil pH, little impact on soil microorganisms and mineralization rate was found. Temporal dynamics of labelling of microbial PLFAs were different between bacterial and fungal PLFA biomarkers. Utilization of maize straw by bacterial PLFAs peaked immediately after the application (2 weeks), while labelling of the fungal biomarker 18:2ω6,9 was most pronounced 5 weeks after the application. In general, ¹³C labelling of microbial PLFAs was closely linked to the amounts of maize carbon present in the soil. The distinct higher labelling of microbial PLFAs in the heavy metal-polluted soils 74 weeks after application indicated a large fraction of available maize straw carbon still present in the soil.     

Carbon flow from C-labeled straw and root residues into the phospholipid fatty acids of a soil microbial community under field conditions

To better understand how residue quality and seasonal conditions influence the flow of C from both root and straw residues into the soil microbial community, we followed the incorporation of ¹³C-labeled crimson clover (Trifolium incarnatum) and ryegrass (Lolium multiflorum) root and straw residues into the phospholipid fatty acids (PLFA) of soil microbial biomass. After residue incorporation under field conditions in late summer (September), the ¹³C content of soil PLFA was measured in September, October, and November, 2002, and April and June, 2003. Multivariate non-metric multidimensional scaling techniques showed that the distribution of ¹³C among microbial PLFA differed among the four primary treatments (ryegrass straw and roots, clover straw and roots). Regardless of treatment, some PLFA remained poorly labeled with ¹³C throughout much of the study (16:1ω5, 10Me17:0; 0-5%), whereas other PLFA consistently contained a larger percentage of residue-derived C (16:0; 18:1ω9, 18:2ω6,9; 10-25%). The distribution of residue ¹³C among individual PLFA differed from the relative contributions of individual PLFA (mol%) to total PLFA-C, suggesting that a subset of the soil biomass was primarily responsible for assimilating residue-derived C. The distribution of ¹³C among soil PLFA differed between the sampling times, indicating that residue properties and soil conditions influenced which members of the community were assimilating residue-derived C. Our findings will provide the foundation for further studies to identify the nature of the community members responsible for residue decomposition at different times of the year, and what factors account for the dynamics of the community involved.     

Use of C abundance to study short-term pig slurry decomposition in the field

Applying pig slurry (PS) on agricultural soils is a common practice. However, its impact on soil organic C dynamics is not clear. This experiment investigated the use of natural ¹³C abundance to study the short-term C mineralization of anaerobically stored PS under field conditions. Measurements of δ¹³C-CO₂ were made on soil air samples obtained from a bare sandy loam during 22 d following incorporation of either PS alone, PS+barley straw, or barley straw alone; an unamended treatment was used as a control. Slurry C was enriched in ¹³C (−20.0‰) because of the high corn (Zea mays L.) content of the animal diet. This value contrasted with δ¹³C of −28.4‰ for the soil organic matter and of −29.0‰ for the barley straw. A peak of high δ¹³CO₂ values (average of −9.2‰) was observed on the day of PS application and was attributed to the dissociation of PS carbonates when mixed with the relatively acidic soil. After this initial burst, 36% of the evolved CO₂ originated from the decomposing PS. After 22 d of incubation, approx. 20% of the PS-C had been lost as CO₂. This short-term field study did not show any priming effect of PS on the mineralization of straw or native soil C. Due to its heterogeneity, the use of the isotopic composition of the evolved CO₂ for estimating PS decomposition requires precaution either through the use of a specific experimental design involving comparable C3 and C4 treatments, or calculations to account for the presence of ¹³C-enriched inorganic C in the PS.     

Microbial community response to nitrogen deposition in northern forest ecosystems

The productivity of temperate forests is often limited by soil N availability, suggesting that elevated atmospheric N deposition could increase ecosystem C storage. However, the magnitude of this increase is dependent on rates of soil organic matter formation as well as rates of plant production. Nonetheless, we have a limited understanding of the potential for atmospheric N deposition to alter microbial activity in soil, and hence rates of soil organic matter formation. Because high levels of inorganic N suppress lignin oxidation by white rot basidiomycetes and generally enhance cellulose hydrolysis, we hypothesized that atmospheric N deposition would alter microbial decomposition in a manner that was consistent with changes in enzyme activity and shift decomposition from fungi to less efficient bacteria. To test our idea, we experimentally manipulated atmospheric N deposition (0, 30 and 80 kg NO₃⁻-N) in three northern temperate forests (black oak/white oak (BOWO), sugar maple/red oak (SMRO), and sugar maple/basswood (SMBW)). After one year, we measured the activity of ligninolytic and cellulolytic soil enzymes, and traced the fate of lignin and cellulose breakdown products (¹³C-vanillin, catechol and cellobiose).In the BOWO ecosystem, the highest level of N deposition tended to reduce phenol oxidase activity (131±13 versus 104±5 μmol h⁻¹ g⁻¹) and peroxidase activity (210±26 versus 190±21 μmol h⁻¹ g⁻¹) and it reduced ¹³C-vanillin and ¹³C-catechol degradation and the incorporation of ¹³C into fungal phospholipids (p<0.05). Conversely, in the SMRO and SMBW ecosystems, N deposition tended to increase phenol oxidase and peroxidase activities and increased vanillin and catechol degradation and the incorporation of isotope into fungal phospholipids (p<0.05). We observed no effect of experimental N deposition on the degradation of ¹³C-cellulose, although cellulase activity showed a small and marginally significant increase (p<0.10). The ecosystem-specific response of microbial activity and soil C cycling to experimental N addition indicates that accurate prediction of soil C storage requires a better understanding of the physiological response of microbial communities to atmospheric N deposition.     

The effect of diet on isotopic turnover in Collembola examined using the stable carbon isotopic compositions of lipids

Combined compound-specific stable carbon isotopic methods and fatty acid abundance determinations have been used to examine feeding preferences and C allocation in organisms where direct observation of feeding is difficult. In order to examine the effect of differing diets on the δ¹³C values of fatty acids and sterols of Collembola, the diets of two collembolan species, Folsomia candida and Proisotoma minuta, were switched from a yeast diet to one of four isotopically distinct diets, and the δ¹³C values of the lipids monitored over the next 39 d. Cholesterol remained the only sterol detected in both collembolan species, despite the diets containing widely differing sterol compositions. The δ¹³C values of collembolan lipids recorded after long term feeding were often different to those of the same components in the diet, indicating that fractionation or partitioning occurs during digestion, assimilation and biosynthesis within the Collembola, thereby shifting consumer lipid δ¹³C values away from those of the corresponding dietary components. The rates of change of δ¹³C values differed among compounds, with half-lives ranging between 29 min and 14 d. Some of these differences appear to be related to the abundance of dietary components, such that fatty acids present in high abundance in the diet (e.g. 18:2(n−6)) were rapidly assimilated in high proportions into collembolan lipids, leading to a rapid change in δ¹³C values. Similarly, isotopic turnover in the 16:1(n−7) fatty acid, present in the newly presented diets in only low abundances, was significantly correlated to the rate of removal of this component from the consumer fatty acid pool. The rates of change of δ¹³C values in P. minuta lipids did not vary significantly with diet, whilst the rates of change of δ¹³C values of lipids in F. candida were affected by the diets the Collembola consumed. Results of an experiment providing F. candida and P. minuta with two diets of different quality demonstrated that F. candida responded to the high quality diet with increased growth and fecundity, whilst P. minuta responded with increased fecundity only. Thus, the abilities of the two species to respond to diets of varying quality, amongst other factors, is concluded to lead to differences in the rates of change of δ¹³C values reflecting differences in lipid turnover.     

New method to estimate root biomass in soil through root-derived carbon

Quantification of root biomass through the conventional root excavation and washing method is inefficient. A pot experiment was conducted to estimate root-derived carbon (C) in soil. Spring wheat (Triticum aestivum L. cv. ‘Quantum’) was grown in plastic containers (6 L) filled with sterilized sandy soil in a greenhouse. Plants were enriched with ¹³CO₂ in a glass chamber twice at growth stages GS-37 and GS-59 for 70 min at each time. In one treatment, roots were separated from soil at crop maturity, washed and dried for the determination of biomass. Isotope ratios were then separately analyzed for roots and soil. In a second treatment, roots were thoroughly mixed with the whole soil and representative samples were analyzed for ¹³C abundance at crop maturity. Control plants were untreated with ¹³C, in which roots were separated from soil. The root biomass was calculated based on the root-derived C, which was measured through ¹³C abundance in the soil and root mixed samples. A substantial amount of root-derived C (24%) was unaccounted while separating the roots from soil. Similarly, about 36% of the root biomass was underestimated if conventional root excavation and washing method is used. It has been shown that root biomass can be estimated more accurately from the root-derived C using ¹³C tracer method than the estimates made by the conventional excavation and washing method. We propose this as an alternative method for the estimation of root-derived C in soil, based on which root biomass can be estimated.     

Arbuscular mycorrhizal fungi contribute to C and N enrichment of soil organic matter in forest soils

Increasing evidence suggests that accretion of microbial turnover products is an important driver for isotopic carbon (C) and nitrogen (N) enrichment of soil organic matter (SOM). However, the exact contribution of arbuscular mycorrhizal fungi (AMF) to soil isotopic patterns remains unknown. In this study, we compared ¹³C and ¹⁵N patterns of glomalin-related soil protein (GRSP), which includes a main fraction derived from AMF, litter, and bulk soil in four temperate rainforests. GRSP was an abundant C and N pool in these forest soils, showing significant ¹³C and ¹⁵N enrichment relative to litter and bulk soil. Hence, cumulative accumulation of recalcitrant AMF turnover products in the soil profile likely contributes to ¹³C and ¹⁵N enrichment in forest soils. Further research on the relationship between GRSP and AMF should clarify the exact extent of this process.     

Health of remnant woodlands in fragments under distinct grazing regimes

Fragmented remnant woodlands in agricultural landscapes are of high conservation value world-wide. Many eucalypts in agricultural landscapes of Australia are in decline. We aimed to investigate nutrient enrichment as a process that may contribute to eucalypt decline. We studied remnant woodlands that had been exposed to distinct recent and current livestock grazing treatments: Currently Intense Grazed; Recently Intense Grazed (until 3 years ago); Recent Intermediate Grazed; and Recent Lightly Grazed by livestock. We assessed soil nutrient status and penetrability, eucalypt foliar nutrition and stable isotope ratios for N and C, attributes of understorey vegetation, and tree health. Soils of the Currently Intense Grazed treatment had high levels of ammonium and Colwell-P. Total N, P, C:N ratio and soil penetrability were generally high in Currently Intense Grazed and Recently Intense Grazed treatments relative to Recent Intermediate Grazed and Recent Lightly Grazed treatments. Foliar N, N stable isotope ratios, P and carbon stable isotope ratios (δ¹³C) were generally higher (less negative δ¹³C) in trees on Currently Intense Grazed and Recently Intense Grazed treatments than in trees on Recent Intermediate Grazed and Recent Lightly Grazed treatments. Soil surface litter, tall and low shrubs and rock were positively correlated with tree health. Grasses and eucalypt foliar N, P and δ¹³C were negatively correlated with tree health. Soil nutrient enrichment increased with increasing grazing intensity and was associated with increased weed invasion and with poor tree health that was in turn correlated to increased foliar N and P and less negative δ¹³C in woodland trees in this study. We argue that minimising soil nutrient enrichment of fragmented remnant woodlands is important, given the association of elevated soil nutrition with poor tree health, to ensure the persistence of eucalypts in agricultural landscapes.     

Modification of the original chloroform fumigation extraction technique to allow measurement of δC of soil microbial biomass carbon

In studies of the soil microbial biomass C by the chloroform fumigation extraction (CFE) technique, biomass C is routinely extracted using 0.5 M K₂SO₄ solution. The excessive amounts of salts contained in the extracting solution pause a significant challenge in using ¹³C isotope techniques to study the nature of C in the soil microbial biomass. This is because the salts can affect the oxidation process and therefore hamper accurate mass spectromic analysis of dried extracts. In spite of this, no standard protocol exists for preparing the K₂SO₄ extracts for ¹³C isotope analysis. We have modified the original CFE method to allow measurement of the δ¹³C of soil microbial biomass C by using 2 M KCl instead of the usual 0.5 M K₂SO₄ solution to extract biomass C. Excess salts were removed by dialysis in 100 molecular weight cut off membranes, after which the extracts were freeze-dried and their δ¹³C measured using a mass spectrometer. The soil microbial biomass C and δ¹³C of 2 M KCl extracts were compared with those of 0.5 M K₂SO₄ extracts. There was excellent agreement between organic C and δ¹³C estimates for dialyzed 2 M KCl and 0.5 M K₂SO₄ extracts, but the speed of dialysis for the latter was very slow, making use of the former more rapid. These results suggest that in procedures where oxidation with potassium dichromate is not critical to analysis of soluble C, 2 M KCl may be used in place of 0.5 M K₂SO₄ to extract soil microbial biomass C for δ¹³C measurements. The new procedure is relatively easy and rapid for obtaining indices for both pool sizes and turnover rates of soil microbial biomass C and provides a promising approach to study soil organic C.     

Use of 13C and 15N natural abundance techniques to characterize carbon and nitrogen dynamics in composting and in compost-amended soils

Isotope fractionation during composting may produce organic materials with a more homogenous δ¹³C and δ¹⁵N signature allowing study of their fate in soil. To verify this, C, N, δ¹³C and δ¹⁵N content were monitored during nine months covered (thermophilic; >40 °C) composting of corn silage (CSC). The C concentration reduced from 10.34 to 1.73 g C (g ash)⁻¹, or 83.3%, during composting. Nitrogen losses comprised 28.4% of initial N content. Compost δ¹³C values became slightly depleted and increasingly uniform (from −12.8±0.6‰ to −14.1±0.0‰) with composting. Compost δ¹⁵N values (0.3±1.3 to 8.2±0.4‰) increased with a similar reduced isotope variability.The fate of C and N of diverse composts in soil was subsequently examined. C, N, δ¹³C, δ¹⁵N content of whole soil (0-5 cm), light (<1.7 g cm⁻³) and heavy (>1.7 g cm⁻³) fraction, and (250-2000 μm; 53-250 μm and <53 μm) size separates, were characterized. Measurements took place one and two years following surface application of CSC, dairy manure compost (DMC), sewage sludge compost (SSLC), and liquid dairy manure (DM) to a temperate (C₃) grassland soil. The δ¹³C values and total C applied (Mg C ha⁻¹) were DM (−27.3‰; 2.9); DMC (−26.6‰; 10.0); SSLC (−25.9‰; 10.9) and CSC (−14.0‰; 4.6 and 9.2). The δ¹³C of un-amended soil exhibited low spatial (−28.0‰±0.2; n=96) and temporal (±0.1‰) variability. All C₄ (CSC) and C₃ (DMC; SSLC) composts, except C₃ manure (DM), significantly modified bulk soil δ¹³C and δ¹⁵N. Estimates of retention of compost C in soil by carbon balance were less sensitive than those calculated by C isotope techniques. One and two years after application, 95 and 89% (CSC), 75 and 63% (SSLC) and 88 and 42% (DMC) of applied compost C remained in the soil, with the majority (80-90%) found in particulate (>53 μm) and light fractions. However, C₄ compost (CSC) was readily detectable (12% of compost C remaining) in mineral (<53 μm) fractions. The δ¹⁵N-enriched N of compost supported interpretation of δ¹³C data. We can conclude that composts are highly recalcitrant with prolonged C storage in non-mineral soil fractions. The sensitivity of the natural abundance tracer technique to characterize their fate in soil improves during composting, as a more homogeneous C isotope signature develops, in addition to the relatively large amounts of stable C applied in composts.     

不同施肥处理对光合碳在花生-土壤系统中分配的影响

通过室内花生盆栽,设置NPK(常规氮磷钾施肥)、NPKS(常规氮磷钾加玉米秸秆)、NPKA(常规氮磷钾加腐殖酸)和CK(不施肥对照)4个不同的施肥处理,采用3次~(13)CO2脉冲标记的方法对不同施肥处理下光合碳在花生-土壤系统中的分配进行定量研究。结果表明:不同施肥处理对标记期内花生总生物量影响不显著,但是NPKA处理显著提升了花生根系生物量,较CK、NPK和NPKS分别高22.04%、19.47%和53.38%。NPKS处理地上部~(13)C丰度最高,但土壤中~(13)C丰度最低,NPKA处理土壤中~(13)C丰度最高。各处理地上部的~(13)C含量无显著差异,NPKA处理根系的~(13)C含量显著高于NPK且土壤~(13)C含量显著高于其他处理。NPKA处理地上部的~(13)C分配比例最低而土壤中分配比例最高,根系~(13)C分配比例与其他处理无显著差异,根系与土壤~(13)C分配比例之和显著高于其他处理。本研究表明腐殖酸能显著促进花生光合碳向地下部的转运。     

Stable isotopes revisited: Their use and limits for oribatid mite trophic ecology

In this review we summarize our knowledge of using stable isotopes (¹⁵N/¹⁴N, ¹³C/¹²C) to better understand the trophic ecology of oribatid mites. Our aims are (a) to recapitulate the history of stable isotope research in soil animals with a focus on oribatid mites, (b) to present new stable isotope data for oribatid mites and overview the current state of knowledge of oribatid mite trophic niche differentiation, (c) to compile problems and limitations of stable isotope based analyses of trophic relationships and (d) to suggest future challenges, questions and problems that may be solved using stable isotope analyses and other novel techniques for improving our understanding on the trophic ecology of soil invertebrates. We conclude that (1) in addition to ¹⁵N/¹⁴N ratios, ¹³C/¹²C ratios contribute to our understanding of the trophic ecology of oribatid mites, allowing, e.g. separation of lichen- and moss-feeding species, (2) there likely are many lichen but few moss feeding oribatid mite species, (3) oribatid mite species that are endophagous as juveniles are separated by their stable isotope signatures from all other oribatid mite species, (4) fungivorous oribatid mite species cannot be separated further, e.g. the fungal taxa they feed on cannot be delineated. A particular problem in using stable isotope data is the difficulty in determining signatures for basal food resources, since decomposing material, fungi and lichens comprise various components differing in stable isotope signatures; ¹³C/¹²C ratios and potentially other isotopes may help in identifying the role of these resources for decomposer animal nutrition.     

A liquid chromatographic/mass spectrometric method to evaluate <Superscript>13</Superscript>C and <Superscript>15</Superscript>N incorporation into soil amino acids

Purpose  

Amino acids are highly associated with biogeochemical cycling and represent an important potential source and sink of carbon (C) and nitrogen (N) in terrestrial ecosystems. Tracing the isotope dynamics of amino acids can improve the understanding of the origin and transformation of amino acids in soil matrix at process-levels; hence, the liquid chromatographic/mass spectrometric (LC/MS) method to evaluate ¹³C or ¹⁵N enrichment in amino acids is necessary to be established.     

Differential acquisition of amino acid and peptide enantiomers within the soil microbial community and its implications for carbon and nitrogen cycling in soil

l-isomeric amino acids and oligopeptides are thought to represent a key nitrogen (N) source for plants and soil microorganisms, bypassing the need to take up inorganic N, whilst self-cycling of d-enantiomers within peptidoglycan-containing bacteria may provide a further short circuit within the N cycle. Here we use stable isotope profiling (SIP) to identify the fate of organic N within soil microbial communities. We followed the incorporation of ¹³C-labelled d- or l-labelled amino acids/peptides into phospholipid fatty acids (PLFAs). l-alanine and its peptides were taken up more rapidly than d-enantiomers by Gram-positive bacteria with ¹³C incorporation being predominantly into anteiso- and iso-fatty acids typically associated with Gram-positive bacteria. d-enantiomer uptake was found not to differ significantly between the microbial groups, providing little support for the view that soil bacteria may self-cycle d-forms of amino acids and peptides. There was no consistent association between peptide chain length and incorporation. The concentrations of l- and d-isomeric amino acids in soil solution were 866 nM and 72 nM, respectively. We conclude that Gram-positive bacteria appear to be the primary competitors for l-enantiomeric forms of amino acids and their peptides, but that both d- and l-enantiomers are available N and C sources for bacteria and fungi.     

Effect of continuous compost application on humus composition and nitrogen fertility of soils in a field subjected to double cropping

We investigated the effect of continuous compost application on humus composition and N fertility of soils in a field subjected to double cropping (paddy rice and barley) for 25 years. Soil samples were collected from three different plots: (a) No-NF, fertilizer containing P and K but no N; (b) F, fertilizer containing N, P, and K; and (c) F+C, fertilizer plus compost. The amounts of total humus, extracted humus, and humic and fulvic acids increased in the order No-NF<F≪F+C. The amounts of humic and fulvic acids were 2.7 and 1.7 times larger in the F+C plot than in the F plot, respectively. The degree of humification of the humic acids decreased in the order No-NF<F<F+C. The absorption curves and ¹³C-NMR spectra (TOSS method) of the humic acids indicated the presence of lignin-like structure, and its degree was the strongest in the F+C plot. The ¹³C-NMR spectra showed distinct differences in the distribution of carbon species between humic and fulvic acids. In humic acids, the content of aromatic-C, ranging from 37 to 44%, was the highest among carbon species. In fulvic acids, the content of O-alkyl-C, ranging from 45 to 51%, was the highest. The amounts of phosphate buffer-extractable N (PEON) and total N (TN) increased in the order No-NF<F<F+C. The amounts of PEON and TN were 1.2 and 1.7 times larger in the F+C plot than in the F plot, respectively. Present and previous findings indicated that continuous compost application could improve various properties of soils in a field subjected to long-term double cropping.     

Long-term nitrogen additions increased surface soil carbon concentration in a forest plantation despite elevated decomposition

Forests cover one-third of the Earth’s land surface and account for 30-40% of soil carbon (C). Despite numerous studies, questions still remain about the factors controlling forest soil C turnover. Present understanding of global C cycle is limited by considerable uncertainty over the potential response of soil C dynamics to rapid nitrogen (N) enrichment of ecosystems, mainly from fuel combustion and fertilizer application. Here, we present a 15-year-long field study and show an average increase of 14.6% in soil C concentration in the 0-5 cm mineral soil layer in N fertilized (defined as N+ hereafter) sub-plots of a second-rotation Pinus radiata plantation in New Zealand compared to control sub-plots. The results of ¹⁴C and lignin analyses of soil C indicate that N additions significantly accelerate decomposition of labile and recalcitrant soil C. Using an annual-time step model, we estimated the soil C turnover time. In the N+ sub-plots, soil C in the light (a density < 1.70 g cm⁻³) and heavy fractions had the mean residence times of 23 and 67 yr, respectively, which are lower than those in the control sub-plots (36 and 133 yr in the light and heavy fractions, respectively). The commonly used lignin oxidation indices (vanillic acid to vanillin and syringic acid to syringaldehyde ratios) were significantly greater in the N+ sub-plots than in the control sub-plots, suggesting increased lignin decomposition due to fertilization. The estimation of C inputs to forest floor and δ¹³C analysis of soil C fractions indicate that the observed buildup of surface soil C concentrations in the N+ sub-plots can be attributed to increased inputs of C mass from forest debris. We conclude that long-term N additions in productive forests may increase C storage in both living tree biomass and soils despite elevated decomposition of soil organic matter.