Amino sugars as specific indices for fungal and bacterial residues in soil

Amino sugars are important indices for the contribution of soil microorganisms to soil organic matter. Consequently, the past decade has seen a great increase in the number of studies measuring amino sugars. However, some uncertainties remain in the interpretation of amino sugar data. The objective of the current opinion paper is to summarize current knowledge on amino sugars in soils, to give some advice for future research objectives, and to make a plea for the correct use of information. The study gives an overview on the origin of muramic acid (MurN), glucosamine (GlcN), galactosamine (GalN), and mannosamine (ManN). Information is also provided on measuring total amino sugars in soil but also on compound-specific δ¹³C and δ¹⁵N determination. Special attention is given to the turnover of microbial cell-wall residues, to the interpretation of the GlcN/GalN ratio, and to the reasons for converting fungal GlcN and MurN to microbial residue C. There is no evidence to suggest that the turnover of fungal residues generally differs from that of bacterial residues. On average, MurN contributes 7% to total amino sugars in soil, GlcN 60%, GalN 30%, and ManN 4%. MurN is highly specific for bacteria, GlcN for fungi if corrected for the contribution of bacterial GlcN, whereas GalN and ManN are unspecific microbial markers.     

Short-term changes in amino sugar-specific δ13C values after application of C4 and C3 sucrose

Amino sugars are useful indicators for the accumulation of microbial residues. A 14-day incubation experiment with C4 and C3 sucrose additions was carried out to investigate the relationships between amino sugar-specific shifts in δ¹³C values and those of CO₂ production, microbial biomass C, K₂SO₄ extractable C and soil organic C (SOC). High performance anion exchange chromatography (HPAEC-IRMS) was able to measure amino-sugar specific δ¹³C values for muramic acid (MurN), galactosamine (GalN), and glucosamine (GlcN) in the range of natural abundance. At day 7, the initial application of C4 sucrose significantly increased the δ¹³C value of MurN by 1.0‰ in comparison with the non-amended control treatment, whereas that of GalN and GlcN remained unchanged. This significant increase had disappeared by day 14. This means that the HPAEC-IRMS method is not useful for short-term incubation experiments in the natural abundance range, as the pool size, especially of GalN and GlcN, was too large for a significant response in δ¹³C values. The δ¹³C values significantly decreased in the order MurN (−23.2‰) > GalN (−25.7‰) > GlcN (−26.5‰) in the control treatment. Similar δ¹³C values were measured in GlcN, microbial biomass C, and SOC. MurN exhibited δ¹³C values similar to the K₂SO₄ extractable fraction. These results may be caused by differences in the access of bacteria and fungi to different SOC fractions or differences in metabolic fractionation in bacteria and fungi. C3 sucrose application without further nutrient supply seven days after C4 sucrose application together with N and P led to strong mineralization of freshly formed microbial residues.     

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.     

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.     

Microbial and plant uptake of free amino sugars in grassland soils

Amino sugars represent a major constituent of microbial cell walls (e.g. chitin, peptidoglycan) and they are present in large quantities in soil organic matter (SOM). The factors regulating their turnover in soil, however, are poorly understood. Here we investigated the turnover of glucosamine (GlcN) in comparison to glucose (Glc) and N-acetylglucosamine (GlcNAc) in two agricultural grassland soils. Over the range 0–1 mM, GlcN uptake occurred via a saturable high affinity transport systems reflecting its low solution concentrations and low rates of supply. In contrast, Glc uptake was characterised by a non-saturable much lower affinity transport system. Of the GlcN-derived carbon (C) taken into the biomass, ca. 90% was used for the production of new cell biomass rather than in respiration. Whilst temperature affected the uptake (Q₁₀ = 1.95) and mineralization (Q₁₀ = 2.32) of GlcN, it did not affect its C use efficiency within the microbial community. We calculated that the average annual flux of GlcN through the soil was 0.01–0.08 g C kg⁻¹ y⁻¹ which equated to 0.1–1.6% of total heterotrophic soil respiration. Microbial use of GlcN was significantly repressed in the presence of sugars (e.g. Glc, sucrose) and N-acetylglucosamine (GlcNAc). We ascribe this to competition at the transport level and due to internal catabolic repression of metabolic pathways involving GlcN within the microbial biomass. Maize (Zea mays L.) roots showed no capacity to take up exogenously applied GlcN at low external concentrations (10 μM) whilst GlcN was rhizotoxic at higher concentrations (EC₅₀ = 49 μM). This suggests that GlcN does not represent a significant source of dissolved organic nitrogen (DON) for plants. The presence of plants did indirectly, however, suppress the use of GlcN by the rhizosphere microbial community. Our work highlights the importance of GlcN in soil C and N cycling, however, we also raise concerns over its importance relative to that of GlcNAc which our evidence suggests plays a more prominent role in soil C and N cycling.     

Ammonium immobilisation during chloroform fumigation

The assumption in using the chloroform fumigation technique for microbial biomass determination is that microbes are killed or at least inactivated by the treatment. Problems associated with transformations of the N released on or during fumigation have so far only been associated with the fumigation-incubation method. A laboratory and a field study were carried out to investigate the possible N transformations during biomass determination by the fumigation-extraction method. Labelled NH₄NO₃ (either the NO₃⁻, NH₄⁺ or both pools were ¹⁵N enriched) was applied to the soil and biomass determinations made at intervals subsequently. The size and enrichment of the ammonium (NH₄⁺), and nitrate (NO₃⁻) pools were determined before and after chloroform fumigation. The ¹⁵N enrichment of the NH₄⁺ pool after fumigation could only be explained if immobilisation of ammonium occurred at some time during the 24 h fumigation period. The extent of this immobilisation was calculated. In addition, there was evidence that nitrification occurred during the fumigation procedure at the start of the laboratory study and throughout the field study. The laboratory and field study differed mainly in the dynamics related to NO₃⁻ uptake and release. There was evidence for uptake of NO₃⁻ by the microbial biomass with and without utilization. We conclude that the ¹⁵N enrichment in the microbial biomass cannot be accurately determined when N transformations and release of non-utilized N occurs during fumigation. The possible immobilisation of mineral N during fumigation will affect the magnitude of the factor used to convert measured microbial biomass N to actual microbial biomass N in soil.     

Elevated CO2, but not defoliation, enhances N cycling and increases short-term soil N immobilization regardless of N addition in a semiarid grassland

Elevated CO₂ and defoliation effects on nitrogen (N) cycling in rangeland soils remain poorly understood. Here we tested whether effects of elevated CO₂ (720 μl L⁻¹) and defoliation (clipping to 2.5 cm height) on N cycling depended on soil N availability (addition of 1 vs. 11 g N m⁻²) in intact mesocosms extracted from a semiarid grassland. Mesocosms were kept inside growth chambers for one growing season, and the experiment was repeated the next year. We added ¹⁵N (1 g m⁻²) to all mesocosms at the start of the growing season. We measured total N and ¹⁵N in plant, soil inorganic, microbial and soil organic pools at different times of the growing season. We combined the plant, soil inorganic, and microbial N pools into one pool (PIM-N pool) to separate biotic + inorganic from abiotic N residing in soil organic matter (SOM). With the ¹⁵N measurements we were then able to calculate transfer rates of N from the active PIM-N pool into SOM (soil N immobilization) and vice versa (soil N mobilization) throughout the growing season. We observed significant interactive effects of elevated CO₂ with N addition and defoliation with N addition on soil N mobilization and immobilization. However, no interactive effects were observed for net transfer rates. Net N transfer from the PIM-N pool into SOM increased under elevated CO₂, but was unaffected by defoliation. Elevated CO₂ and defoliation effects on the net transfer of N into SOM may not depend on soil N availability in semiarid grasslands, but may depend on the balance of root litter production affecting soil N immobilization and root exudation affecting soil N mobilization. We observed no interactive effects of elevated CO₂ with defoliation. We conclude that elevated CO₂, but not defoliation, may limit plant productivity in the long-term through increased soil N immobilization.     

In situ mineral <Superscript>15</Superscript>N dynamics and fate of added <Superscript>15</Superscript>NH<Subscript>4</Subscript><Superscript>+</Superscript> in hoop pine plantation and adjacent native forest in subtropical Australia

Background, aim, and scope  Hoop pine (Araucaria cunninghamii) is a nitrogen (N) demanding indigenous Australia softwood species with plantations in Southeast Queensland, Australia. Soil fertility has declined with increasing rotations and comparison study of N cycling between hoop pine plantations, and adjacent native forest (NF) is required to develop effective forest management for enhancing sustainable forest production and promoting environmental benefits. Field in situ mineral ¹⁵N transformations in these two forest ecosystems have not been studied. Hence, the present study was to compare the differences in soil nutrients, N transformations, ¹⁵N fluxes, and fate between the hoop pine plantation and the adjacent native forest. Materials and methods  The study sites were in Yarraman State Forest (26°52′ S, 151°51′ E), Southeastern Queensland, Australia. The in situ core incubation method was used in the field experiments. Mineral N was determined using a LACHAT Quickchem Automated Ion Analyzer. ¹⁵N were performed using an isotope ratio mass spectrometer with a Eurovector elemental analyzer. All statistical tests were carried out by the SPSS 11.0 for Windows statistical software package. Results  Soil total C and N were significantly higher in the NF than in the 53-year-old hoop pine plantation. Concentrations of NO₃ ^– were significantly higher in the NF soil than in the plantation soil. The plantation soil had significantly higher ¹⁵N and ¹³C natural abundances than the NF soil. The NF soil had significantly lower C/N ratios than the plantation soil. NO₃ ^––N was dominated in mineral N pools in both NF and plantation soils, accounting for 91.6% and 70.3% of the total mineral N pools, respectively. Rates of net nitrification and net N mineralization were, respectively, four and three times higher in the NF soil than in the plantation soil. The ¹⁵NO₃ ^––N and mineral ¹⁵N were significantly higher in the NF soil than in the plantation soil. Significant difference in ¹⁵NH₄ ⁺–N was found in the NF soil before and after the incubation. Discussion  The NF soil had significantly higher NO₃ ^––N, mineral N, total N and C but lower δ¹⁵N, δ¹³C, and C/N ratios than the plantation soil. Moreover, the rates of soil net N mineralization and nitrification were significantly higher, but ammonification rate was lower in the NF than in the plantation. The NF soil had many more dynamic N transformations than the plantation soil due to the combination of multiple species and layers and, thus, stimulation of microbial activity and alteration of C and N pool sizes in favor of the N transformations by soil microbes. The net rate of N and ¹⁵N transformation demonstrated differences in N dynamic related to the variation in tree species between the two ecosystems. Conclusions  The change of land use and trees species had significant impacts on soil nutrients and N cycling processes. The plantation had larger losses of N than the NF. The NO₃ ^––N and ¹⁵NO₃ ^––N dominated in the mineral N and ¹⁵N pools in both forest ecosystems. Recommendations and perspectives  Native forest soil had strong N dynamic compared with the plantation soil. Composition of multiple tree species with different ecological niches in the plantation could promote the soil ecosystem sustainability. The ¹⁵N isotope dilution technique in the field can be quite useful for studying in situ mineral ¹⁵N transformations and fate to further understand actual N dynamics in natural forest soils.     

Long-term impacts of manure, straw, and fertilizer on amino sugars in a silty clay loam soil under temperate conditions

There is increasing evidence that microorganisms participate in soil C sequestration and stabilization in the form of resistant microbial residues. The type of fertilizers influences microbial activity and community composition; however, little is known about its effect on the microbial residues and their relative contribution to soil C storage. The aim of this study was to investigate the long-term impact (21 years) of different fertilizer treatments (chemical fertilizer, crop straw, and organic manure) on microbial residues in a silty clay loam soil (Udolls, USDA Soil Taxonomy). Amino sugars were used to indicate the presence and origin of microbial residues. The five treatments were: CK, unfertilized control; NPK, chemical fertilizer NPK; NPKS₁, NPK plus crop straw; NPKS₂, NPK plus double amounts of straw; and NPKM, NPK plus pig manure. Long-term application of inorganic fertilizers and organic amendments increased the total amino sugar concentrations (4.4–8.4 %) as compared with the control; and this effect was more evident in the plots that continuously received pig manure (P?<?0.05). The increase in total amino sugar stock was less pronounced in the straw-treated plots than the NPKM. These results indicate that the accumulation of soil amino sugars is largely influenced by the type of organic fertilizers entering the soil. Individual amino sugar enrichment in soil organic carbon was differentially influenced by the various fertilizer treatments, with a preferential accumulation of bacterial-derived amino sugars compared with fungal-derived glucosamine in manured soil.     

Transformation of 15N-labelled leguminous plant material in three contrasting soils

Summary Two soils from Pakistan (Hafizabad silt loam and Khurrarianwala silt loam) and one from Illinois, USA (Drummer silty clay loam) were incubated with ¹⁵N-labelled soybean tops for up to 20 weeks at 30°C. Mineralization of soybean ¹⁵N was slightly more rapid in the Pakistani soils, and after 20 weeks of incubation, 50%, 53%, and 56% of the applied ¹⁵N was accounted for as (NH₄ ⁺+NO₃ ^–)-N in Drummer, Hafizabad, and Khurrarianwala soils, respectively. Potentially mineralizable N (determined by anaerobic incubation) varied between 1.5% and 10% of the applied ¹⁵N in the three soils at different stages of incubation; somewhat higher percentages were mineralizable in the Pakistani soils than in the Drummer soil. From 3.7% to 9% of the applied ¹⁵N was accounted for in the microbial biomass. From 10% to 32% of the applied N was recovered in the humic acid and fulvic acid fractions of the organic matter by sequential extraction with Na₄P₂O₇ and NaOH; from 12% to 49% was recovered in the humin fraction. Of the three soils, Drummer soil contained more ¹⁵N as humic and fulvic acids. In all cases, the ¹⁵N was approximately equally distributed between the humic and fulvic acid fractions. A significant percentage of the humin ¹⁵N (52%–78%, equivalent to 8%–34% of the applied ¹⁵N) occurred in non-hydrolyzable (6 N HCl) forms. Of the hydrolyzable ¹⁵N, 42%–51% was accounted for as amino acid-N followed in order by NH₃ (17%–30%), hydrolyzable unknown forms (20%–22%), and amino sugars (6%–2%). The recovery of applied ¹⁵N for the different incubation stages was 87±22%. Recovery was lowest with the Khurrarianwala soil, presumably because of NH₃ volatilization losses caused by the high pH of this soil.     

Mass spectrometric characterization of amino sugar aldononitrile acetate derivatives used for isotope enrichment assessment of microbial residues

Identifying the transformation of amino sugars in soils is essential in understanding microbial contribution to soil organic matter turnover and sequestration. Using a recently developed method, combining gas chromatograph/mass spectrometry (GC/MS) with laboratory incubation of substrates containing ¹³C or ¹⁵N isotopes, we were able to trace isotopic changes in amino sugar compounds. This allows us to quantitatively evaluate C or N enrichment in amino sugars during transformation in soils using the fragment (F) abundance ratio of m/z F + n to F (n is original skeleton atom number in each fragment). However, there is still lack of detail structural and substitutional information for each ion fragment. In order to improve the interpretation and increase our ability to study amino sugar turnover, we grew labeled amino sugars in lab-cultured organisms. We spectrometrically investigated the ion structures and original skeleton C number (mass shift n) in major ion fragments based on applying multiple representative isotope labels. Our results categorically confirm that previously made assumptions were correct regarding the substitutional number “n” of the glucosamine (He et al., 2006). Our study also added valuable structural information for aldononitrile acetate derivatized glucosamine and muramic acid upon electron impact ionization in MS.     

Fate of C- and N-labelled rhizodeposition of Lolium perenne as function of the distance to the root surface

A greenhouse rhizobox experiment was carried out to quantify the incorporation of ¹³C- and ¹⁵N-labelled rhizodeposits into different soil pools, especially into the rhizosphere microbial biomass, with increasing distances to the root surface of Lolium perenne. Five layers were analysed over 0-4.2 mm distance to an artificial root surface. C and N derived from rhizodeposition were 4.2% of total C and 2.8% of total N in soil at 0-1.0 mm distance and decreased rapidly with increasing distance. Microbial biomass C and N increased significantly towards the roots. At 0-1.0 mm distance microbial biomass C and N accounted for 66% and 29% of C and N derived from rhizodeposition, respectively. These percentages declined with increasing distance to the roots, but were still traceable up to 4.2 mm distance. Only small amounts of root released C and N were found in the 0.05 M K₂SO₄-extractable fraction. Extractable C and N derived from rhizodeposition varied around means of 4% of total C and N derived from rhizodeposition and increased only marginally with increasing distance to the roots. C derived from rhizodeposition in the non-extractable soil organic matter increased from 65 to 89% of total C derived from rhizodeposition at 0-3.4 mm distance. Conversely, microbial biomass C derived from rhizodeposition decreased from 33 to 4%. N derived from rhizodeposition in the non-extractable soil organic matter increased from 61 to 79% of total N derived from rhizodeposition at 0-2.6 mm distance, followed by a decline to roughly 55% in the two outer layers. Microbial biomass N decreased from 37 to 16% at 0-2.6 mm distance, followed by an increase to roughly 41% in the two outer layers. The C/N ratio of total C and N derived from rhizodeposition as well as that of extractable C and N derived from rhizodeposition increased with increasing distance to the roots to values above 30. In contrast, the C/N ratio of incorporated rhizodeposition C and N into the microbial biomass decreased to values less than 5 at 2.6-4.2 mm distance. The data indicate differential microbial response to C and N derived from rhizodeposition at a high spatial resolution from the root surface. The turnover of C and N derived from rhizodeposition in the rhizosphere as a function of the distance to the root surface is discussed.     

Isotope 15N enrichment of soil-ammonium N as a reference to estimate N2 fixation by stem and root-inoculated S. rostrata

Summary A pot experiment in the greenhouse was conducted to compare the contribution of N derived from the atmosphere or from biological N₂ fixation by Sesbania rostrata inoculated with Azorhizobium caulinodans, applied either to roots or to roots and stems (single or multiple stem inoculation). Two subsequent crops were grown for 50 days under flooded conditions. N derived from air was estimated by ¹⁵N dilution using ¹⁵N enrichment of soil NH _inf4 ^sup+ -N and of Echinochloa crusgalli as the non-N₂-fixing reference datum and compared with estimates obtained by the N-difference method. The first crop was grown to stabilize the ¹⁵N into the soil organic N fraction. The ¹⁵N enrichment of soil NH _inf4 ^sup+ -N in the second crop declined slowly. The extractability ratio (¹⁵N enrichment of extractable soil N to ¹⁵N enrichment of total soil N) decreased from 4.8 to 4.1 50 days after planting. The enrichment of soil NH _inf4 ^sup+ -N was comparable to that of E. crus-galli, resulting in similar estimates of N derived from air when either soil NH _inf4 ^sup+ -N or enrichment of E. crus-galli was used as a non-fixing reference. The N-difference method did not always provide reliable estimates of N derived from air; percentages ranged from 75 to more than 80 by 50 days after planting in both crops and did not differ among treatments. The study demonstrates the potential of using ¹⁵N enrichment of soil NH _inf4 ^sup+ -N as a non-N₂-fixing reference for reliable BNF estimates of crops in lowland puddled soil.     

Natural N abundances in a tallgrass prairie ecosystem exposed to 8-y of elevated atmospheric CO2

After 8-y of elevated CO₂, we previously detected greater amounts of total soil nitrogen, suggesting that rates of ecosystem N flux into or out of tallgrass prairie had been altered. Denitrification and associative N fixation rates are the two primary biological processes that are known to control N loss and accumulation in tallgrass prairie soil. Therefore, our objective was to assess the natural abundance of plant and soil ¹⁵N isotopes as a cumulative index of potential change in efflux or influx of N into and out of the tallgrass prairie after 8-y of exposure to elevated CO₂. Aboveground plant delta ¹⁵N values of Andropogon gerardii were close to zero and more positive as a result of elevated CO₂, but whole-soil values at the 5-30 cm depth were significantly reduced (6.8 vs 7.3; P<0.05) under elevated CO₂-chamber (EC) relative to ambient CO₂- chamber (AC). Total, aboveground plant biomass, root-in-growth, extractable N, microbial biomass N, and soil pools collectively exhibited a range of delta ¹⁵N values from −2.8 to 7.3. Measurements of surface soil ¹⁵N indicate that a change in N inputs and outputs has occurred as a result of elevated atmospheric CO₂. In addition to possible changes in denitrification and N₂ fixation, other sources of N such as the re-translocation of N to the surface from deeper soil layers are needed to explain how soil N accrues in surface soils as a consequence of elevated CO₂. Our results support the notion that C accrual may promote N accrual, possibly driven by high plant and microbial N demand amplified by soil N limitation.     

Short-term competition between crop plants and soil microbes for inorganic N fertilizer

Agricultural systems that receive high amounts of inorganic nitrogen (N) fertilizer in the form of either ammonium (NH₄⁺), nitrate (NO₃⁻) or a combination thereof are expected to differ in soil N transformation rates and fates of NH₄⁺ and NO₃⁻. Using ¹⁵N tracer techniques this study examines how crop plants and soil microbes vary in their ability to take up and compete for fertilizer N on a short time scale (hours to days). Single plants of barley (Hordeum vulgare L. cv. Morex) were grown on two agricultural soils in microcosms which received either NH₄⁺, NO₃⁻ or NH₄NO₃. Within each fertilizer treatment traces of ¹⁵NH₄⁺ and ¹⁵NO₃⁻ were added separately. During 8 days of fertilization the fate of fertilizer ¹⁵N into plants, microbial biomass and inorganic soil N pools as well as changes in gross N transformation rates were investigated. One week after fertilization 45-80% of initially applied ¹⁵N was recovered in crop plants compared to only 1-10% in soil microbes, proving that plants were the strongest competitors for fertilizer N. In terms of N uptake soil microbes out-competed plants only during the first 4 h of N application independent of soil and fertilizer N form. Within one day microbial N uptake declined substantially, probably due to carbon limitation. In both soils, plants and soil microbes took up more NO₃⁻ than NH₄⁺ independent of initially applied N form. Surprisingly, no inhibitory effect of NH₄⁺ on the uptake and assimilation of nitrate in both, plants and microbes, was observed, probably because fast nitrification rates led to a swift depletion of the ammonium pool. Compared to plant and microbial NH₄⁺ uptake rates, gross nitrification rates were 3-75-fold higher, indicating that nitrifiers were the strongest competitors for NH₄⁺ in both soils. The rapid conversion of NH₄⁺ to NO₃⁻ and preferential use of NO₃⁻ by soil microbes suggest that in agricultural systems with high inorganic N fertilizer inputs the soil microbial community could adapt to high concentrations of NO₃⁻ and shift towards enhanced reliance on NO₃⁻ for their N supply.     

Plant-N incorporation into microbial amino sugars as affected by inorganic N addition: A microcosm study of N-labeled maize residue decomposition

Carbon (C) and/or nitrogen (N) in plant residues can be assimilated into microbial biomass during the plant residue decomposition before incorporation into SOM in the form of microbial residues. Yet, microbial transformation of plant residue-N into microbial residues and the effects of inorganic N inputs on this process have not been well documented. Here, we undertook a 38-week incubation with a silt loam soil amended with a ¹⁵N-labeled maize (Zea mays L.) residue to determine how the transformation of maize residue-N into soil amino sugars was affected by rates of inorganic N addition. The newly metabolized amino sugars derived from maize residue-N were differentiated and quantified by using an isotope-based gas chromatography-mass spectrometry technique. We found that greater amounts of maize residue-N were transformed into amino sugars with lower inorganic N addition at the early stages of the plant residue degradation. However, the trend was reversed during later stages of decay as greater percentage of maize residue-N (8.6-9.4%) were enriched in amino sugars in the N_med and N_high soils, as compared with N₀ and N_low (7.5-8.2%). This indicated that higher availability of inorganic N could delay the transformation process of plant-N into microbial residues during the mineralization of plant residues. The dynamic transformations of the plant residue-N into individual amino sugars were compound-specific, with very fast incorporation into bacterial MurA_M-new found during the initial weeks, while the dynamics of maize residue-derived GluN exhibited a delayed response to assimilate plant-N into fungal products. The findings indicated differential contributions of maize residue decomposing microorganisms over time. Moreover, we found no preferential utilization of inorganic N over plant residue-N into amino sugars during the incubation course, but inorganic N inputs altered the rate of plant-N accumulation in microbial-derived organic matters. Our results indicated that higher N availability had a positive impact on the accumulation or stabilization of newly-produced microbial residues in the long term.     

Relationships between C and N availability, substrate age, and natural abundance C and N signatures of soil microbial biomass in a semiarid climate

Soil microbial organisms are central to carbon (C) and nitrogen (N) transformations in soils, yet not much is known about the stable isotope composition of these essential regulators of element cycles. We investigated the relationship between C and N availability and stable C and N isotope composition of soil microbial biomass across a three million year old semiarid substrate age gradient in northern Arizona. The δ¹⁵N of soil microbial biomass was on average 7.2‰ higher than that of soil total N for all substrate ages and 1.6‰ higher than that of extractable N, but not significantly different for the youngest and oldest sites. Microbial ¹⁵N enrichment relative to soil extractable and total N was low at the youngest site, increased to a maximum after 55,000 years, and then decreased slightly with age. The degree of ¹⁵N enrichment of microbial biomass correlated negatively with the C:N mass ratio of the soil extractable pool. The δ¹³C signature of soil microbial biomass was 1.4‰ and 4.6‰ enriched relative to that of soil total and extractable pools respectively and showed significant differences between sites. However, microbial ¹³C enrichment was unrelated to measures of C and N availability. Our results confirm that ¹⁵N, but not ¹³C enrichment of soil microbial biomass reflects changes in C and N availability and N processing during long-term ecosystem development.     

应用15N稀释法测定土壤氮素初级转化速率的一些关键技术

本文综合评述了应用~(15)N库稀释法测定土壤氮素初级转化速率的一些关键技术,即~(15)N标记土壤氮库的方法、~(15)N的加入量、丰度和标记物种类的选择,以及初始取样时间的确定。只有合理地运用这些关键技术,才能更准确地测定土壤氮素初级转化速率,进而更真实地表征土壤氮素的实际周转状况。     

Decomposition of N-labelled maize leaves in soil affected by endogeic geophagous Aporrectodea caliginosa

A microcosm experiment was carried out for 56 days at 12 °C to evaluate the feeding effects of the endogeic geophagous earthworm species Aporrectodea caliginosa on the microbial use of ¹⁵N-labelled maize leaves (Zea mays) added as 5 mm particles equivalent to 1 mg C and 57 μg N g⁻¹ soil. The dry weight of A. caliginosa biomass decreased in the no-maize treatment by 10% during the incubation and increased in the maize leaf treatments by 18%. Roughly 5% and 10% of the added maize leaf-C and leaf-N, respectively, were incorporated into the biomass of A. caliginosa. About 29% and 33% of the added maize leaf-C were mineralised to CO₂ in the no-earthworm and earthworm treatments, respectively. The presence of A. caliginosa significantly increased soil-derived CO₂ production by 90 μg g⁻¹ soil in the no-maize and maize leaf treatments, but increased the maize-derived CO₂ production only by 40 μg g⁻¹ soil. About 10.5% of maize leaf-C and leaf-N was incorporated into the soil microbial biomass in the absence of earthworms, but only 6% of the maize leaf-C and 3% of the maize leaf-N in the presence of earthworms. A. caliginosa preferentially fed on N rich, maize leaf-colonizing microorganisms to meet its N demand. This led to a significantly increased C/N ratio of the unconsumed microbial biomass in soil. The ergosterol-to-microbial biomass C ratio was not significantly decreased by the presence of earthworms. A. caliginosa did not directly contribute to comminution of plant residues, as indicated by the absence of any effects on the contents of the different particulate organic matter fractions, but mainly to grazing of residue-colonizing microorganisms, increasing their turnover considerably.     

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.