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Keywords:

  • amino sugars;
  • biogeochemistry;
  • compound-specific stable isotope analysis;
  • nitrogen deposition;
  • soil density fractionation;
  • soil organic matter

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgments
  7. Author contribution
  8. References

Atmospheric nitrogen (N) deposition has frequently been observed to increase soil carbon (C) storage in forests, but the underlying mechanisms still remain unclear. Changes in microbial community composition and substrate use are hypothesized to be one of the key mechanisms affected by N inputs. Here, we investigated the effects of N deposition on amino sugars, which are used as biomarkers for fungal- and bacterial-derived microbial residues in soil. We made use of a 4-year combined CO2 enrichment and N deposition experiment in model forest ecosystems, providing a distinct 13C signal for ‘new’ and ‘old’ C in soil organic matter and microbial residues measured in density and particle-size fractions of soils. Our hypothesis was that N deposition decreases the amount of fungal residues in soils, with the new microbial residues being more strongly affected than old residues. The soil fractionation showed that organic matter and microbial residues are mainly stabilized by association with soil minerals in the heavy and fine fractions. Moreover, the bacterial residues are relatively enriched at mineral surfaces compared to fungal residues. The 13C tracing indicated a greater formation of fungal residues compared to bacterial residues after 4 years of experiment. In contradiction to our hypotheses, N deposition significantly increased the amount of new fungal residues in bulk soil and decreased the decomposition of old microbial residues associated with soil minerals. The preservation of old microbial residues could be due to decreased N limitation of microorganisms and therefore a reduced dependence on organic N sources. This mechanism might be especially important in fine heavy fractions with low C/N ratios, where microbial residues are effectively protected from decomposition by association with soil minerals.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgments
  7. Author contribution
  8. References

Atmospheric nitrogen (N) deposition increased three- to fivefold within the last century due to human activities (Denman et al., 2007). The main sources of reactive N in the atmosphere are combustion of fossil fuels and fertilizer application including animal manure (Davidson, 2009). Atmospheric N is mainly deposited to terrestrial ecosystems by rainfall in readily bioavailable forms, which has a fertilizing effect. Ecosystems that typically do not receive additional amounts of N and especially forests in industrialized regions are affected by increased N deposition in several ways. N deposition stimulates photosynthesis and enhances the allocation shift to more aboveground vs. belowground plant growth, thereby reducing organic matter inputs into the rhizosphere (Janssens et al., 2010). Although N additions have frequently been found to suppress heterotrophic respiration, the response of soil microbial communities is not yet well understood (Fog, 1988; Janssens et al., 2010). Changes in microbial community composition following increased N deposition were often observed and ultimately affect key processes of the soil carbon (C) cycle which have ecosystem-level implications (Zak et al., 2011). It is hypothesized that N deposition causes a shift from fungal- to bacterial-dominated microbial communities (Strickland & Rousk, 2010). Possible reasons are inhibition of fungal enzymes by N deposition (Fog, 1988), and outcompeting of less efficient fungi that require less N by highly efficient, but N limited, bacteria (Ågren et al., 2001). This might be due to stoichiometric differences between fungi and bacteria because fungi have higher C/N ratios than bacteria and therefore are expected to have lower N demands. Consequently, a shift toward bacterial biomass is expected when N limitation is reduced due to atmospheric N deposition while C sources remain equally accessible (Strickland & Rousk, 2010).

Amino sugars are components of microbial cell walls, which are stabilized in soil after cell death and are therefore used as biomarkers to assess fungal and bacterial residues in soil (Parsons, 1981; Amelung, 2001; Joergensen & Wichern, 2008). Amino sugars were used as a proxy for both, living and dead microbial biomass in soil. However, the living microbial biomass contributes only a small amount to the total amino sugars found in soil (Guggenberger et al., 1999). Therefore, amino sugars not only reflect the microbial community at the time point of sampling, but can also be used to monitor medium-to-long-term changes in microbial community (Glaser et al., 2004). Although more than 26 amino sugars have been identified, only three amino sugars are found in considerable amounts in soil: glucosamine, galactosamine, and muramic acid. Glucosamine mainly originates from chitin of fungal cell walls, which is a polymer of N-acetyl-glucosamine units. However, it is also present in bacterial cell walls as part of peptidoglycan, where N-acetyl-glucosamine is alternately linked with N-acetyl-muramic acid. Muramic acid is exclusively found in bacterial peptidoglycan, making it a highly specific biomarker for bacteria. Higher contents of peptidoglycan were found in gram-positive compared to gram-negative bacteria (Guggenberger et al., 1999; Amelung, 2001). Although galactosamine was found in fungi and bacteria (Engelking et al., 2007) and its origin is less clearly defined, it was often used as an indicator for bacterial tissue. The ratios of glucosamine to galactosamine and of glucosamine to muramic acid are frequently used indicators for the relative contribution of fungi vs. bacteria to microbial residues in soil (Amelung, 2001). Although many studies used the composition of amino sugars as a proxy for microbial residues in soil, little is known about the formation and stabilization of amino sugars in soil (Joergensen & Wichern, 2008). Recently developed compound-specific stable isotope analysis of individual amino sugars offers the possibility to elucidate these processes (Bodé et al., 2009).

Stabilization of organic matter in soil is often defined as protection of organic matter against decomposition and is considered mainly driven by three mechanisms: (i) selective preservation of organic matter, (ii) spatial inaccessibility of organic matter, and (iii) association of organic matter with soil minerals (Sollins et al., 1996; Six et al., 2002; Von Lützow et al., 2006). Selective preservation of organic matter is based on the concept that certain ‘recalcitrant’ organic compounds persist in soil due to their molecular properties (Kleber, 2010). Plant-derived lignin was thought to be ‘recalcitrant’ and therefore more effectively stabilized in soil than ‘labile’ compounds like microbially derived sugars (Baldock & Skjemstad, 2000). Although this was often observed in short-term litter decomposition experiments, there is growing evidence that the long-term stabilization of soil organic matter is not governed by the selective preservation of ‘recalcitrant’ organic matter (Von Lützow et al., 2006; Marschner et al., 2008; Schmidt et al., 2011; Dungait et al., 2012). Spatial inaccessibility of organic matter from decomposition is attributed to occlusion of organic matter within soil aggregates. Aggregate occluded organic matter is protected from decomposition by reduced access of microbes and reduced diffusion of their enzymes into soil aggregates. Also, the diffusion of oxygen into aggregates is reduced and further limits aerobic decomposition (Von Lützow et al., 2006). Association of organic matter with soil minerals is thought to stabilize organic matter more effectively than aggregation. Stabilization of organic matter is accomplished by interactions of organic matter with clay mineral surfaces and complexation with ions of calcium, iron, and aluminum (Von Lützow et al., 2006; Kögel-Knabner et al., 2008).

Soil density fractionation is frequently used to study the stabilization mechanisms of organic matter by separating qualitatively distinct organic matter fractions that should correspond to different stabilization mechanisms (Christensen, 1992, 2001; Von Lützow et al., 2007). Light density fractions mainly consist of recent, only partially decomposed organic matter that is either physically unprotected or occluded with soil aggregates, whereas heavy density fractions consist of more decomposed organic matter that is associated with soil minerals (Golchin et al., 1994; Crow et al., 2007). Therefore, C concentrations, C/N ratios, and mean residence times of organic C usually decrease from light to heavy density fractions (John et al., 2005; Dorodnikov et al., 2011). The application of promising new analytical techniques like compound-specific stable isotope analysis of biomarkers in soil density fractions will provide new insights into stabilization processes of organic matter in soil (Glaser, 2005; Amelung et al., 2008; Simpson & Simpson, 2012; Gleixner, 2013).

This study made use of model forest ecosystems that had been isotopically labeled with 13C-depleted CO2 and treated with two levels of N deposition. We investigated the composition and dynamics of total organic C and microbial residues in bulk soil and density fractions using compound-specific stable isotope analysis of individual amino sugars. By linking state-of-the-art biomarker analysis with soil density fractionation, we wanted to gain insight into the formation and stabilization of soil organic matter in general and of microbial residues in particular. More specifically, we tested the hypothesis that microbial residues are mainly stabilized in fine mineral soil fractions. Furthermore, we tested the hypotheses that increased N deposition decreases the contribution of fungal-derived residues in soil and that mainly ‘new’, experiment-derived, microbial residues are affected in contrast to ‘old’, preexperimental microbial residues.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgments
  7. Author contribution
  8. References

Experimental setup

We analyzed archived soil samples from a combined N deposition and elevated CO2 experiment that was conducted over four growing seasons between 1994 and 1998. Model forest ecosystems were established in 12 open-top chambers (Egli et al., 1998). An acidic soil with sandy loamy texture (Haplic Alisol) was transferred from a natural beech–spruce forest site into gravitational lysimeters. Soils were planted with beech (Fagus sylvatica) and spruce (Picea abies) trees as well as five typical understory species (Carex sylvatica, Geum urbanum, Ranunculus ficaria, Viola sylvatica, and Hedera helix). Beech trees were 2–3 years old and spruce trees were 4 years old at planting. Ecosystems were treated with ambient CO2 (370 μmol mol−1; δ13C = −8.3‰) and elevated CO2 (570 μmol mol−1; δ13C = −29.8‰) in combination with two levels of N fertilizer (low N: 7 kg NH4NO3-N ha−1 yr−1; high N: 70 kg NH4NO3-N ha−1 yr−1). The following four treatments were applied: ambient CO2 + low N; elevated CO2 + low N; ambient CO2 + high N; elevated CO2 + high N. The treatments were arranged in a Latin square design with three replicates for each CO2 × N treatment. After 4 years of treatment, soils were sampled from 0 to 10 cm depth prior to tree harvest. Soil samples were dried at 60 °C for 48 h immediately after sampling. Visible dead and living plant residues were removed from the soils by hand and soils were sieved through a 2 mm sieve. Samples were stored in a fully climatized archive (17 °C) with low air humidity (<40%).

Soil fractionation

Density fractionation was carried out as described by Golchin et al. (1994). Bulk soil was separated into free light fraction (fLF), occluded light fraction (oLF), and heavy fraction (HF) by density fractionation and ultrasonic dispersion. HF material was further separated at 20 μm by particle-size fractionation (Fig. 1). The selection of density and dispersion energy should be based on preceding experiments that show which parameters produce the maximum C content of light fractions (Cerli et al., 2012; Griepentrog & Schmidt, 2013). We chose 1.6 g cm−3 as a density cutoff because it was the most suitable density to separate maximum C content in light fractions (Cerli et al., 2012). Dispersion energies should be determined for every soil because they vary with aggregate stability. Therefore, we sequentially sonicated a soil sample to determine the dispersion energy which produces maximum C content of the occluded light fraction (Cerli et al., 2012). Results showed that 250 J ml−1 was the most suitable dispersion energy for our soil.

image

Figure 1. Soil fractionation scheme. Bulk soil was separated into free light fraction (fLF), occluded light fraction (oLF), and heavy fraction (HF) by density fractionation and ultrasonic dispersion. Heavy fraction was further particle size fractionated at 20 μm into a coarse (HF > 20 μm) and a fine-textured heavy fraction (HF < 20 μm).

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Sodium polytungstate (SPT 0, TC Tungsten Compounds, Grub am Forst, Germany) solution was adjusted to a density of 1.6 g cm−3 by weighing out defined volumes of the solution. During the whole procedure, SPT solutions were collected and recycled with the method described by Six et al. (1999). Dried and sieved (<2 mm) bulk soil samples were suspended in SPT solution (soil : solution ratio 1 : 5) and subsequently centrifuged at 4500 g for 10 min. fLF material was collected by careful removal of the floating material and subsequent filtration using glass fiber filters (GF 6, Whatman, Dassel, Germany). fLF material was rinsed with deionized water until the electrical conductivity was <50 μS cm−1, to remove residual SPT. The remaining soil material was resuspended in SPT solution (density 1.6 g cm−3, soil : solution ratio 1 : 5) and ultrasonically dispersed with 250 J ml−1. The ultrasonic equipment (UW 3400, Bandelin, Berlin, Germany) was calorimetrically calibrated according to Schmidt et al. (1999). oLF material was collected after centrifugation by careful removal of the floating material and filtration using glass fiber filters. oLF material was rinsed with deionized water until the electrical conductivity was <50 μS cm−1, to remove residual SPT. The remaining soil material with a density >1.6 g cm−3 (HF) was washed three times with deionized water following centrifugation each time. HF material was further separated at 20 μm by wet sieving. The coarse heavy fraction larger 20 μm (HF > 20 μm) was collected from the sieve, whereas the fine heavy fraction smaller 20 μm (HF < 20 μm) was collected after sedimentation. All fractions were freeze dried and milled prior to analysis. All soil samples were fractionated in triplicate and fractions were pooled for further analysis. On average, we recovered >99% of the initial sample masses.

C/N (isotope) analysis

The C and N contents and the δ13C values of bulk soil and soil density fractions were determined with an automated element analyzer – continuous flow isotope ratio mass spectrometer (EA-1110, Carlo Erba, Fisons, Italy, interfaced with a ConFlo II to a Delta-S, Thermo Finnigan MAT, Bremen, Germany). Results of the C isotope analysis are expressed in δ units (‰):

  • display math(1)

where R = 13C/12C for both, sample and standard. The Vienna Pee Dee Belemnite (VPDB) standard was used as reference. Fraction of new soil C derived from plant input during the experimental period was calculated from δ13C values using a simple mixing model equation (Balesdent et al., 1988):

  • display math(2)

where δsoil,depleted and δsoil,ambient are the δ13C values of bulk soil or soil fractions for treatments with 13C-depleted CO2 and ambient CO2, respectively. δplant,depleted and δplant,ambient are the δ13C values of plant litter input for treatments with 13C-depleted CO2 and ambient CO2, respectively. Concentrations of total organic C in the control treatments are within the range of that from before the experiment (Hagedorn et al., 2001) and therefore the assumptions of Balesdent et al. (1988) should be valid for our calculation.

As the input of aboveground vs. belowground plant litter into soil was not known, we took the mean of the δ13C values of leaves, needles, and fine roots for δplant. This assumption is reasonable, as the differences between δ13C values of leaves, needles, and fine roots were negligible compared to the large shift in δ13C values due to 4 years of treatment with 13C-depleted CO2. Plant biomass grown under 13C-depleted CO2 and ambient CO2 differed on average by 10.5‰ in their δ13C values (Hagedorn et al., 2001).

Treatments with elevated CO2 did not significantly affect total organic C concentrations in bulk soil and soil fractions compared to treatments with ambient CO2 (data not shown).

Amino sugar (isotope) analysis

Extraction of amino sugars from bulk soil and soil density fractions was adapted from the method described by Zhang & Amelung (1996). Therefore, amounts of sample material containing 0.3 mg of N were hydrolyzed by adding 6 m HCl (20 ml g−1 of sample material) and heating at 105 °C for 8 h. Samples were filtered over glass fiber filters (GF/C, Whatman, Dassel, Germany) and the filtrate was evaporated to dryness at 40–45 °C under reduced pressure to remove HCl. Dried filtrate was redissolved in Milli-Q water (Direct-Q 3 System, Millipore, Billerica, MA, USA), transferred in a 2 ml tube (Eppendorf, Hamburg, Germany) and centrifuged. The supernatant was added onto a cation exchange resin (AG 50W-X8, Bio-Rad Laboratories, Hercules, CA, USA). After rinsing the resin with Milli-Q water to remove neutral and negatively charged compounds, the fraction containing amino sugars was eluted with 0.5 m HCl and again evaporated to dryness to remove HCl. Dried amino sugars were redissolved in Milli-Q water and transferred in a 2 ml tube. After desiccation using a centrifugal vacuum concentrator (SpeedVac, Thermo Scientific, Langenselbold, Germany), samples were stored at −18 °C until analysis.

Compound-specific stable isotope analysis of amino sugar extracts was performed according to the method described by Bodé et al. (2009). Therefore, we used a high pressure liquid chromatography (HPLC) system existing of an autosampler (Surveyor Autosampler Plus, Thermo Electron, Bremen, Germany) and a HPLC pump (Surveyor MS-Pump Plus, Thermo Electron) with an analytical anion-exchange column (PA20 CarboPac, 3 × 150 mm, 6.5 μm) that was coupled through a wet oxidation interface (LC Isolink, Thermo Electron) to an isotope ratio mass spectrometer (IRMS; DELTAPLUS XP, Thermo Electron).

Bacterial- and fungal-derived amino sugar carbon (AS-C) was calculated according to Van Groenigen et al. (2007). Based on the assumption that all galactosamine and muramic acid are produced by bacteria and that glucosamine and muramic acid occur in equal amounts in bacteria

  • display math(3)

The remainder of the amino sugar C pool is derived from fungi and therefore

  • display math(4)

Treatments with elevated CO2 did not significantly affect amino sugar concentrations in bulk soil and soil fractions compared to treatments with ambient CO2 (data not shown).

Statistical analysis

For replicate measurements, the mean of three field replicates is given along with the standard error (Webster, 2001). Effects of CO2 treatment, N deposition, and their interactions were tested for significant differences between and within fractions and bulk soil by analysis of variance (Webster, 2007).

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgments
  7. Author contribution
  8. References

Concentrations of organic carbon and nitrogen

Highest C concentrations were found in both light fractions, while occluded light fractions showed slightly higher C concentrations than free light fractions (Table 1). Both light fractions together stored 29% of the soil organic C, whereof around three quarters were stored in free light fractions and one quarter in occluded light fractions (Fig. 2a). Heavy fractions had substantially lower C concentrations than light fractions. Here, fine heavy fractions showed higher C concentrations than coarse heavy fractions (Table 1). Most of the soil organic C was recovered in fine heavy fractions (62%), whereas coarse heavy fractions contributed only 9% to soil organic C (Fig. 2a). C/N ratios were highest in both light fractions, decreased in coarse heavy fraction and were lowest in the fine heavy fraction (Table 1).

Table 1. Mass, organic carbon (C), total nitrogen (N), and C/N ratios in bulk soil and soil fractions (free light fraction, fLF; occluded light fraction, oLF; total heavy fraction, HF; coarse heavy fraction, HF >20 μm; fine heavy fraction, HF <20 μm) for treatments with low and high N deposition. Total C was separated into new (experiment-derived) C and old (preexperiment) C using isotope mixing model
 Mass (gfr kgsoil−1)Organic carbon (gCfr kgfr−1)Nitrogen (gNfr kgfr−1)C/N (-)
Total CNew COld C
  1. Asterisks denote significant N treatment effects: n.s. = not significant (P > 0.05), *P < 0.05, **P < 0.01, ***P < 0.001.

Bulk soil n.s.n.s.n.s.n.s.n.s.*
Low nitrogen1000.0 ± 0.014.9 ± 0.53.2 ± 0.411.6 ± 0.41.0 ± 0.015.6 ± 0.3
High nitrogen1000.0 ± 0.014.7 ± 0.43.1 ± 0.211.6 ± 0.21.0 ± 0.014.7 ± 0.2
Density fractionation
fLFn.s.n.s.n.s.n.s.******
Low nitrogen8.7 ± 0.4323.6 ± 4.7192.0 ± 19.4131.6 ± 19.49.8 ± 0.133.2 ± 0.8
High nitrogen9.0 ± 0.4326.6 ± 6.9195.2 ± 14.0131.4 ± 14.011.6 ± 0.228.2 ± 0.6
oLFn.s.*n.s.n.s.n.s.***
Low nitrogen2.3 ± 0.2393.1 ± 10.383.4 ± 13.6309.7 ± 13.612.1 ± 0.232.4 ± 0.7
High nitrogen2.6 ± 0.1353.1 ± 8.373.4 ± 10.7279.8 ± 10.712.4 ± 0.228.4 ± 0.4
Total HF*n.s.n.s.n.s.***
Low nitrogen979.2 ± 0.89.5 ± 0.11.0 ± 0.18.5 ± 0.10.7 ± 0.013.0 ± 0.1
High nitrogen982.8 ± 0.99.7 ± 0.21.3 ± 0.28.4 ± 0.20.8 ± 0.012.6 ± 0.1
Particle-size fractionation
Coarse HF (>20 μm)n.s.n.s.*n.s.n.s.n.s.
Low nitrogen669.8 ± 2.91.4 ± 0.00.2 ± 0.01.2 ± 0.00.1 ± 0.014.0 ± 0.4
High nitrogen661.7 ± 2.61.6 ± 0.10.4 ± 0.01.1 ± 0.00.1 ± 0.015.5 ± 0.7
Fine HF (<20 μm)n.s.n.s.n.s.n.s.n.s.n.s.
Low nitrogen301.8 ± 3.322.9 ± 0.51.9 ± 0.220.9 ± 0.22.0 ± 0.111.2 ± 0.2
High nitrogen308.2 ± 3.023.3 ± 0.32.5 ± 0.220.8 ± 0.22.1 ± 0.011.3 ± 0.1
image

Figure 2. Mass distribution of recovered (a) soil mass, total organic carbon, total nitrogen, and (b) amino sugars (glucosamine, GlcN; galactosamine, GalN; muramic acid, MurA) between soil fractions (free light fraction, fLF; occluded light fraction, oLF; total heavy fraction, HFtotal; coarse heavy fraction, HF > 20 μm; fine heavy fraction, HF < 20 μm). Recoveries after fractionation procedure were (a) 99.2 ± 0.1% of mass, 89.3 ± 1.8% of carbon, 87.9 ± 1.6% of nitrogen and (b) 94.5 ± 2.8% of GlcN, 90.4 ± 4.6% of GalN, 67.9 ± 8.9% of MurA. For amino sugars (b), coarse heavy fraction was not analyzed and therefore only total heavy fraction is shown. Average data for the four experimental treatments are shown.

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High C concentrations and C/N ratios in light density fractions are commonly observed, showing that they consist of fresh organic matter, which is in agreement with the concept of the density fractionation approach (Cerli et al., 2012). Organic matter in free light fractions mainly originates from plant residues, whereas organic matter in occluded light fractions consists of more fragmented plant residues and materials with less recognizable structure, indicating that organic matter in occluded light fractions is more degraded than in free light fractions (Golchin et al., 1994; Crow et al., 2007; Wagai et al., 2009; Dorodnikov et al., 2011). Higher C concentrations in occluded compared to free light fractions can be attributed to increased protection of organic matter from degradation due to aggregation and were also observed by other studies (Golchin et al., 1994; John et al., 2005; Dorodnikov et al., 2011).

Lower C concentrations and C/N ratios in heavy fractions compared to light fractions are attributed to an increasing degree of organic matter degradation and incorporation of organic matter into microbes (John et al., 2005). Higher C concentrations in fine heavy fractions compared to coarse heavy fractions are attributed to enhanced protection of organic matter by mineral surfaces in the fine heavy fractions and interactions with clay minerals and Fe and Al oxides are thought to be most relevant for organic matter stabilization (Kögel-Knabner et al., 2008). Several studies found that a major part of soil organic C was associated with heavy fractions (John et al., 2005; Dorodnikov et al., 2011).

Increased N deposition decreased the C/N ratios of both light fractions (Table 1). In free light fractions the decrease in C/N was caused by a significant increase in N concentrations (Table 1), very likely reflecting higher N concentrations and decreased C/N ratios after N additions in foliage (Hagedorn et al., 2000) and fine roots (Hagedorn et al., 2001) of this experiment. In contrast to free light fractions, the decrease in C/N ratio in occluded light fractions was the consequence of a significant decrease in C concentrations (Table 1). However, also more material was recovered and thus the total C pool of occluded light fractions did not change for both N treatments.

Isotopic composition of total organic carbon

Density fractionation separated individual soil fractions with significantly different C isotopic composition. Under both, ambient and 13C-depleted CO2 treatments, 13C abundance increased in the order: free light fraction < occluded light fraction < coarse heavy fraction < fine heavy fraction (Fig. 3), as observed in other studies (John et al., 2005; Dorodnikov et al., 2011). Organic C in bulk soil is generally enriched in 13C compared to that in light fractions, indicating degradation of plant residues (Ehleringer et al., 2000). Enrichment of organic matter in 13C is generally attributed to isotope fractionation during microbial degradation or preferential substrate utilization by microorganisms (Blagodatskaya et al., 2011), but also to gradual shifts in the relative contribution of microbial vs. plant components (Ehleringer et al., 2000). Using 13C CP/MAS NMR, Golchin et al. (1994) could also show that occluded light fractions and heavy fractions contained organic matter that was further degraded than in free light fractions.

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Figure 3. Carbon isotope ratios (δ13C) of total organic carbon in bulk soil and soil fractions (free light fraction, fLF; occluded light fraction, oLF; coarse heavy fraction, HF > 20 μm; fine heavy fraction, HF < 20 μm) for treatments with ambient and 13C-depleted CO2 as well as the fraction of new carbon synthesized during 4 years of experiment. Average data for the two nitrogen deposition treatments are shown.

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Four years of treatment with 13C-depleted CO2 significantly decreased δ13C values compared to treatment with ambient CO2 for bulk soil (−2.3 ± 0.1‰) and individual density fractions (Fig. 3). The difference in δ13C values between treatments with ambient and 13C-depleted CO2 is a measure for the incorporation of isotopic label during the experimental period and hence for the fraction of ‘new’ plant-derived C. As free light fractions mainly consist of recent plant residues, they had the highest percentage of newly formed organic matter (60%). Occluded light fractions contained considerably less organic matter from the experimental period (21%), showing that the transfer of organic matter from free light to occluded light fractions and the replacement of the organic matter pool in occluded light fractions are rather slow. This might be due to the stabilization of organic matter by aggregation in occluded light fractions (Sollins et al., 1996; Six et al., 2002). The percentage of new C was not significantly different between occluded light and coarse heavy fractions. This could be due to a rather slow replacement of the organic matter pool in coarse heavy fractions or a fast transfer of organic matter from occluded light to coarse heavy fractions. Due to continuous renewal of soil aggregates (Six et al., 2000) a close interaction between occluded materials and coarse mineral fraction can be assumed and therefore an equal stabilization of organic matter in both fractions. Fine heavy fractions showed the lowest percentage of new organic matter (10%). Here, the slow replacement of the organic matter pool is attributed to the large C pool in this fraction (Fig. 2a) and also to the strong interaction between organic matter and soil minerals, which was shown to be a major control of organic matter stabilization in soils (Kögel-Knabner et al., 2008).

Increased N deposition significantly increased the amount of new, experiment-derived C in coarse heavy fractions (Table 1). However, due to the small amount of C stored in coarse heavy fractions (Fig. 2a), this increase might not be relevant for total soil C sequestration. The result is consistent with a study from the same experiment that also found significantly greater amounts of new C in coarse sand fractions after particle-size fractionation (Hagedorn et al., 2003). Nevertheless, increased N deposition did not affect the amounts of old vs. new C in the other soil fractions significantly (Table 1). This shows that increased N deposition did not substantially alter how new and old C was distributed between the different soil fractions in our study. In contrast to our study, Hagedorn et al. (2003) found after particle-size fractionation that increased N deposition reduced the amount of old C in silt and clay fractions that was mineralized during the experimental period. In line, Neff et al. (2002) also observed that N additions significantly increased C in mineral-associated soil fractions. Mechanisms that could explain the retarded decomposition of old C under increased N deposition are changes in the microbial decomposer community (Janssens et al., 2010) or a preferential substrate use of decomposer organisms (Liljeroth et al., 1990).

Concentrations of individual amino sugars

Total amino sugar C contributed 1.4% to total soil organic C (Fig. 4a). In bulk soil and density fractions, the concentrations of individual amino sugars decreased in the order: glucosamine > galactosamine > muramic acid (Fig. 4a). In bulk soil, glucosamine, galactosamine, and muramic acid contributed 63%, 26%, and 11% to the total amino sugar C, respectively. Similar amounts of total amino sugars and a comparable distribution of individual amino sugars are typically observed (Van Groenigen et al., 2007, 2010; Bai et al., 2013). Individual amino sugar concentrations differed significantly among density fractions and increased in the order: free light fraction < occluded light fraction < coarse heavy fraction < fine heavy fraction (Fig. 4a). Also on a mass balance, most amino sugars were recovered in heavy fractions. 84%, 92%, and 97% of glucosamine, galactosamine, and muramic acid were found in heavy fractions, respectively (Fig. 2b). Our results are in agreement with other studies showing that amino sugar concentrations increased from coarse to fine fractions and that most of the total amino sugars are found in silt- and clay-sized fractions (Zhang et al., 1999; Solomon et al., 2001; Turrión et al., 2002). Also analysis of noncellulosic neutral sugars indicated that microbially derived sugars are accumulating in heavy density fractions (Rumpel et al., 2010) and fine particle-size fractions (Kiem & Kögel-Knabner, 2003; Jolivet et al., 2006; Spielvogel et al., 2008). The high amino sugar concentrations found in the heavy fractions further corroborate that microbial residues are typically associated with fine-textured soil minerals. Therefore, association with soil minerals seems to be the most significant stabilization mechanism for microbial residues in soil (Guggenberger et al., 1999).

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Figure 4. Amino sugar carbon normalized to total organic carbon (a: glucosamine, GlcN; galactosamine, GalN; muramic acid, MurA) and fungal-to-bacterial ratios (b: GlcN/GalN and GlcN/MurA) in bulk soil and soil fractions (free light fraction, fLF; occluded light fraction, oLF; total heavy fraction, HF; fine heavy fraction, HF < 20 μm). Average data for the four experimental treatments are shown.

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Both amino sugar ratios, glucosamine to galactosamine and glucosamine to muramic acid, decreased in the order: free light fraction > occluded light fraction > total heavy fraction > fine heavy fraction (Fig. 4b). Changes in amino sugars ratios of glucosamine to galactosamine and of glucosamine to muramic acid are used to qualitatively determine relative increases or decreases of fungal vs. bacterial residues. However, due to the unspecific origin of glucosamine, changes in both ratios should be consistent (Amelung, 2001). In our study, both fungi-to-bacteria ratios consistently decreased by a factor of three from free light to fine heavy fractions, indicating a relative shift from fungal to bacterial residues in this direction. Another way to express the contribution of fungal- vs. bacterial-derived residues is based on the stoichiometric differences between fungal-derived chitin and bacterial-derived peptidoglycan. The calculation is based on the assumptions that galactosamine is solely originating from bacteria, whereas bacterial glucosamine is solely originating from peptidoglycan and that glucosamine and muramic acid occur in a constant ratio in peptidoglycan (Van Groenigen et al., 2007). Here, fungal- and bacterial-derived amino sugar C both increase from free light to fine heavy fractions, whereas the fungal-to-bacterial ratio decreases in the same direction (Fig. 5), which is consistent with the observation from glucosamine-to-galactosamine and glucosamine-to-muramic acid ratios (Fig. 4b) and further corroborates the relative enrichment of bacterial residues in fine heavy fractions. In line, other studies on amino sugars observed that fungal-to-bacterial ratios decreased from coarse sand to clay fractions (Zhang et al., 1999; Solomon et al., 2001; Turrión et al., 2002). Potential reasons for preferential association of bacterial residues with soil minerals could be that in contrast to bacteria, most fungi are obligatory aerobes and are restricted to air-filled spaces in soil. Fungal hyphae were found to dominate in the outer regions of macroaggregates, whereas bacteria dominate in the center of microaggregates (Chenu & Stotzky, 2002).

image

Figure 5. Fungal- and bacterial-derived amino sugar carbon (AS-C) as well as their ratio calculated after Van Groenigen et al. (2007) and normalized to total organic carbon in bulk soil and soil fractions (free light fraction, fLF; occluded light fraction, oLF; total heavy fraction, HF; fine heavy fraction, HF < 20 μm). Average data for the four experimental treatments are shown.

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Increased N deposition did significantly increase the total content of glucosamine in fine heavy fractions (Table 2) and consequently the total fungal-derived amino sugars in fine heavy fractions (Table 3). These results are consistent with a previous study from the same experiment, showing that N deposition significantly increased fungal biomass (determined by ergosterol content) associated with fine roots and in root-free soil (Wiemken et al., 2001a), while it did neither affected total microbial biomass (determined by fumigation extraction) nor phospholipid fatty acids (Wiemken et al., 2001b). However, ergosterol and phospholipid fatty acids rapidly decompose after cell death and mainly represent the living microbial biomass at the time of sampling. In contrast, amino sugars are substantially stabilized in soil after death of microbial biomass (Glaser et al., 2004) and therefore their abundance might be a more useful indicator for long-term microbial responses (Van Groenigen et al., 2007). Our results in a forest soil contrast with the study by Van Groenigen et al. (2007) in a grassland ecosystem, where N addition had significant effects on amino sugars with high rates of N fertilizer decreasing fungal residues. However, their study lasted 10 years and used a 10 times higher N input added in single high doses as compared to our study applying lower amounts of N (70 kg N ha−1 yr−1) continuously. Nevertheless, a study in a forest ecosystem that used lower amounts of N (15 kg N ha−1 yr−1) over a longer period (14 years) also showed that N additions resulted in a significant reduction in fungal biomass (Frey et al., 2004). In contrast, Dörr et al. (2010) studied the impact of reduced N deposition in a N-saturated forest ecosystem and did not find changes in amino sugar composition after 14.5 years. Initial effects could be shown at the plant level, but Dörr et al. (2010) argued that the experimental period was too short to detect effects on soil organic matter, which generally has higher mean turnover times (Amelung et al., 2008).

Table 2. Amino sugar carbon (C) in bulk soil and soil fractions (free light fraction, fLF; occluded light fraction, oLF; total heavy fraction, HF; fine heavy fraction, HF <20 μm) normalized to total organic C content, for treatments with low and high nitrogen (N) deposition. Total C was separated into new (experiment-derived) C and old (preexperiment) C using isotope mixing model
 Glucosamine (gC kgCfr−1)Galactosamine (gC kgCfr−1)Muramic acid (gC kgCfr−1)
Total CNew COld CTotal CNew COld CTotal CNew COld C
  1. Asterisks denote significant N treatment effects: n.s. = not significant (P > 0.05), *P < 0.05, **P < 0.01.

Bulk soil n.s.*n.s.n.s.n.s.n.s.n.s.n.s.*
Low nitrogen8.4 ± 0.31.5 ± 0.36.9 ± 0.33.5 ± 0.30.2 ± 0.23.4 ± 0.21.5 ± 0.20.2 ± 0.01.3 ± 0.0
High nitrogen9.2 ± 0.62.8 ± 0.36.4 ± 0.33.7 ± 0.30.4 ± 0.13.3 ± 0.11.6 ± 0.20.1 ± 0.01.5 ± 0.0
Density fractionation
fLFn.s.n.s.n.s.n.s.n.s.**n.s.n.s.n.s.
Low nitrogen3.7 ± 0.42.2 ± 0.31.5 ± 0.30.6 ± 0.10.2 ± 0.00.4 ± 0.00.2 ± 0.10.1 ± 0.10.1 ± 0.1
High nitrogen4.1 ± 0.32.3 ± 0.21.8 ± 0.20.7 ± 0.10.2 ± 0.00.5 ± 0.00.2 ± 0.00.1 ± 0.00.1 ± 0.0
oLFn.s.n.s.n.s.n.s.n.s.n.s.n.s.n.s.n.s.
Low nitrogen5.9 ± 0.41.0 ± 0.34.9 ± 0.31.2 ± 0.10.2 ± 0.11.0 ± 0.10.3 ± 0.00.0 ± 0.10.2 ± 0.1
High nitrogen5.7 ± 0.61.0 ± 0.64.8 ± 0.61.2 ± 0.10.3 ± 0.20.9 ± 0.20.3 ± 0.1−0.1 ± 0.00.4 ± 0.0
Total HFn.s.n.s.n.s.n.s.n.s.n.s.n.s.n.s.n.s.
Low nitrogen9.6 ± 0.51.3 ± 0.08.3 ± 0.04.1 ± 0.20.2 ± 0.23.9 ± 0.21.1 ± 0.20.0 ± 0.11.1 ± 0.1
High nitrogen10.0 ± 0.51.4 ± 0.68.5 ± 0.63.9 ± 0.20.1 ± 0.13.8 ± 0.11.6 ± 0.30.4 ± 0.11.2 ± 0.1
Particle-size fractionation
Fine HF (<20 μm)**n.s.*n.s.n.s.*n.s.n.s.n.s.
Low nitrogen11.2 ± 0.51.8 ± 0.39.4 ± 0.35.5 ± 0.30.4 ± 0.25.1 ± 0.21.5 ± 0.20.1 ± 0.21.3 ± 0.2
High nitrogen13.1 ± 0.41.7 ± 0.511.4 ± 0.56.0 ± 0.20.1 ± 0.15.8 ± 0.11.4 ± 0.20.1 ± 0.11.4 ± 0.1
Table 3. Fungal- and bacterial-derived amino sugar (AS) carbon (C) in bulk soil and soil fractions (free light fraction, fLF; occluded light fraction, oLF; total heavy fraction, HF; fine heavy fraction, HF <20 μm) normalized to total organic C content, for treatments with low and high nitrogen (N) deposition. Fungal and bacterial AS-C was calculated after Van Groenigen et al. (2007). Total C was separated into new (experiment-derived) C and old (preexperiment) C using isotope mixing model
 Fungal AS-C (gC kgCfr−1)Bacterial AS-C (gC kgCfr−1)
Total CNew COld CTotal CNew COld C
  1. Asterisks denote significant N treatment effects: n.s. = not significant (P > 0.05), *P < 0.05, **P < 0.01.

Bulk soil n.s.*n.s.n.s.n.s.n.s.
Low nitrogen7.0 ± 0.41.3 ± 0.35.6 ± 0.36.5 ± 0.40.5 ± 0.16.0 ± 0.1
High nitrogen7.6 ± 0.62.7 ± 0.35.0 ± 0.36.9 ± 0.40.6 ± 0.26.3 ± 0.2
Density fractionation
fLFn.s.n.s.n.s.n.s.n.s.n.s.
Low nitrogen3.5 ± 0.32.1 ± 0.21.4 ± 0.21.1 ± 0.10.4 ± 0.10.7 ± 0.1
High nitrogen3.9 ± 0.12.2 ± 0.21.6 ± 0.21.1 ± 0.00.3 ± 0.10.8 ± 0.1
oLFn.s.n.s.n.s.n.s.n.s.n.s.
Low nitrogen5.6 ± 0.30.9 ± 0.34.7 ± 0.31.7 ± 0.10.3 ± 0.21.4 ± 0.2
High nitrogen5.4 ± 0.51.0 ± 0.64.4 ± 0.61.8 ± 0.20.2 ± 0.31.7 ± 0.3
Total HFn.s.n.s.n.s.n.s.n.s.n.s.
Low nitrogen8.5 ± 0.61.2 ± 0.17.3 ± 0.16.3 ± 0.40.3 ± 0.26.0 ± 0.2
High nitrogen8.4 ± 0.71.0 ± 0.87.3 ± 0.87.1 ± 0.30.9 ± 0.26.2 ± 0.2
Particle-size fractionation
Fine HF (<20 μm)**n.s.*n.s.n.s.n.s.
Low nitrogen9.8 ± 0.31.7 ± 0.48.1 ± 0.48.4 ± 0.20.7 ± 0.57.6 ± 0.5
High nitrogen11.7 ± 0.51.6 ± 0.510.1 ± 0.58.8 ± 0.30.2 ± 0.18.6 ± 0.1

Our results could have been biased by drying the soils at 60 °C and storing them for 15 years before analysis. To our knowledge, there had not been a thorough assessment of long-term soil storage on amino sugars. However, the study of Zelles et al. (1991) showed that storing soils for 1, 2, and 20 months at different temperatures (+21, +4, −18, and −140 °C) did not have significant effects on amino sugar compositions (see statistical evaluation by Amelung, 2001). We also do not expect that the relatively high drying temperature of 60 °C affected our results, as amino sugar polymers are relatively stable (Amelung, 2001) and the differences among soil fractions were large and consistent with other studies conducted with fresh soil samples (e.g., Guggenberger et al., 1999). However, the drying step could potentially have a beneficial effect with respect to sample storage, as it will degenerate most proteins and be fatal for large parts of the living soil microorganisms. Moreover, soil samples used in our study have been stored in a fully climatized archive at air humidity below 40%, which further prevents microbial activity. Finally, our measurements of C contents (Table 1) and δ13C values (Fig. 3) are well consistent with that of Hagedorn et al. (2004) who measured the same samples about 10 years ago, which indicates that sample storage had no major effects on organic matter composition in our soil samples.

Carbon isotopic composition of individual amino sugars

In bulk soil and density fractions, amino sugars were enriched in 13C compared to total organic C at natural abundance. Enrichment of 13C increased in the order: total organic C < glucosamine < galactosamine < muramic acid (Fig. 6a and b). Differences between δ13C values of total organic C and microbial residues result from isotopic fractionation during the biochemical synthesis of individual compounds (e.g., amino sugars) and from different δ13C values of the substrates used for biosynthesis (Hobbie et al., 1999). Gleixner et al. (1993) found that fungi were 4‰ enriched in 13C compared to their substrates in wood. Chitin in their cell walls was enriched by 2‰ relative to wood cellulose. They further argued that the hexose units of chitin (amino sugars) must be even more enriched in 13C because the polymer has substitution by acetyl groups, which should be 13C-depleted (Melzer & Schmidt, 1987). These findings are consistent with our observation that microbial residues are generally enriched in 13C compared to total organic C for both, ambient and 13C-depleted CO2 treatments. In contrast to our results and the results presented above, Glaser et al. (2006) determined 13C isotopic signatures of individual amino sugars in a grassland ecosystem and found that galactosamine was depleted in 13C compared to total organic C. However, the applicability of their method (GC-IRMS) to determine δ13C values of amino sugars in soil at natural abundances was recently questioned because of high analytical errors during derivatization (Decock et al., 2009).

image

Figure 6. Carbon isotope ratios (δ13C) of total organic carbon (TOC) and individual amino sugars (glucosamine, GlcN; galactosamine, GalN; muramic acid, MurA) in bulk soil and soil fractions (free light fraction, fLF; occluded light fraction, oLF; total heavy fraction, HF; fine heavy fraction, HF < 20 μm) for treatments with (a) ambient CO2 and (b) 13C-depleted CO2 after 4 years of experiment. Average data for the two nitrogen deposition treatments are shown.

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Among soil fractions, C isotope ratios of individual amino sugars showed the same pattern as total organic C. Amino sugars in heavy fractions were enriched in 13C compared to amino sugars in light fractions (Fig. 6a and b). 13C enrichment of amino sugars in heavy fractions is possibly attributed to isotope fractionation during decomposition of amino sugars, a mechanism which is frequently discussed for organic matter in general (Galimov, 2006). This would suggest that amino sugars in heavy fractions are decomposed to a higher proportion than in light fractions. Another possibility for the 13C enrichment of amino sugars in heavy fractions is that the C source for amino sugar formation is taken from the 13C-enriched C already present in the fraction.

Adding 13C-depleted CO2 for 4 years significantly decreased δ13C values of amino sugars compared to treatments with ambient CO2 (Figs. 6a and b). The fraction of newly formed amino sugars followed the pattern of total organic C, with the highest fraction of new C in free light fractions and considerably lower fractions of new C in occluded light and heavy fractions (Fig. 7). This suggests that the replacement of amino sugar pools in these fractions is slower than in free light fractions, which might be due to stabilization of amino sugars by aggregation in occluded light fractions and association with soil minerals in fine heavy fractions. However, also the total pool size of amino sugars is larger in heavy than in light fractions and hence it takes much longer until this pool is replaced. Nevertheless, we do not know, where new amino sugars are actually formed, as they could be formed in situ within soil fractions but also transferred between fractions. We also do not know on which timescales the in situ formation of amino sugars in soil fractions and the transfer of amino sugars between soil fractions occur.

image

Figure 7. Fraction of new fungal- and bacterial-derived amino sugar carbon (AS-C) as well as total organic carbon (TOC) in bulk soil and soil fractions (free light fraction, fLF; occluded light fraction, oLF; total heavy fraction, HF; fine heavy fraction, HF < 20 μm) after 4 years of experiment. Average data for the two nitrogen deposition treatments are shown.

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The fraction of newly formed fungal amino sugars was in the range of that of total organic C for both bulk soil and soil fractions (Fig. 7). Generally, the percentage of newly formed fungal amino sugars was higher than that of newly formed bacterial amino sugars in bulk soil and soil fractions (Fig. 7), which indicates that more fungal amino sugars were formed during the experimental period than bacterial amino sugars (Table 2). This is furthermore supported by the soil incubation experiment with isotopically labeled crop residues by Bai et al. (2013) showing that glucosamine had shorter mean production times (2.1–5.0 days) than galactosamine (2.5–9.3 days).

Increased N deposition affected the distribution of both new (experiment-derived) and old (preexperimental) amino sugar C in bulk soil and density fractions in several ways. High N treatment significantly increased new, experiment-derived, glucosamine in bulk soil (Table 2) and consequently, also the amount of new fungal-derived C (Fig. 8). Hence, fungal biomass increased after N additions, which is also supported by increased ergosterol concentrations under high N in our experiment (Wiemken et al., 2001a). This contradicts the hypothesis that increased N deposition leads to a decrease in fungal biomass, due to outcompeting of less efficient fungi that require little N, by high efficient bacteria that are N limited (Strickland & Rousk, 2010). According to the hypothesis, a shift toward bacteria is expected, when N is not limited, but access to C remains equivalent. The likely reason for the larger amount of new fungal-derived C at high N inputs is the N-induced growth stimulation in our experiment, significantly increasing the fine root biomass and associated ectomycorrhizal fungi (Wiemken et al., 2001a). However, longer term studies often show that fungal biomass decreases after N additions (Treseder, 2008) and our study may represent an early response to increased N deposition.

image

Figure 8. New and old fungal-derived amino sugar carbon (AS-C) in bulk soil and fine heavy fractions (HF < 20 μm) under low and high nitrogen (N) treatments. Fungal-derived amino sugar carbon was calculated after Van Groenigen et al. (2007) and normalized to total organic carbon content in bulk soil and fine heavy fractions, respectively. Asterisks denote significant N treatment effects (*P < 0.05).

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Our results also showed that under increased N deposition, old, preexperimental glucosamine, and galactosamine in fine heavy fractions were significantly higher than in the control (Table 2). Hence, the decomposition of old amino sugar C was reduced under high N additions (Fig. 8). A possible explanation could be that microorganisms are relying less on decomposition of amino sugars, which are rich in N but difficult to decompose, if they receive additional inorganic N. Under high N conditions, microorganisms that need inorganic N to decompose substrates outcompete microorganisms that mine soil organic matter for N (Fontaine et al., 2011). Therefore, mining of soil organic matter increases, when N availability is low. In contrast, mining of soil organic matter is low when N availability is high, which leads to sequestration of C (Fontaine et al., 2011). This mechanism might be especially important in fine heavy fractions because here organic matter is additionally protected from decomposition by association with soil minerals, which furthermore impedes mining of soil organic matter.

In summary, microbial residues were mainly found in fine heavy fractions, which contained the least amount of new microbial residues. Thus, association with soil minerals seems to be the key process for the stabilization of microbial residues in soil. Furthermore, a consistent decrease in fungal-to-bacterial ratios between free light and fine heavy fractions suggests that bacterial residues are relatively enriched at mineral surfaces compared to fungal residues. In soil fractions and bulk soil, the percentage of new glucosamine was in the range of that of new organic C and more glucosamine was formed during the experimental period compared to galactosamine and muramic acid. Thus, more fungal-derived residues were formed compared to bacterial-derived residues. High N deposition significantly increased new glucosamine in bulk soil and therefore also the amount of new fungal-derived C, which contradicts the hypothesis that increased N deposition decreases the contribution of fungal-derived residues in soil. Furthermore, old glucosamine and old galactosamine in fine heavy fractions were significantly higher under increased N deposition compared with control. Thus, the decomposition of old microbial residues was reduced under high N deposition, which contradicts the hypothesis that mainly new, experiment-derived, microbial residues are affected as compared to old, preexperiment, microbial residues. The retarded decomposition of old microbial residues could be due to declined N limitation of microorganisms and therefore reduced dependence on organic N sources. This might be especially important in fine heavy fractions because of the high interaction and stabilization of microbial residues with soil minerals.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgments
  7. Author contribution
  8. References

E. Gillis (Ghent University) gave laboratory assistance during amino sugar extraction and U. Graf (WSL) did bulk elemental (isotope) analyses. M.P.W. Schneider, D.B. Wiedemeier and G.L.B. Wiesenberg (all University of Zurich) gave constructive comments on outline and figures of the manuscript. The study was funded by the Swiss National Science Foundation (SNF) and Marco Griepentrog received a travel grant from the European Science Foundation (ESF-MOLTER) to visit the ISOFYS laboratory at Ghent University.

Author contribution

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgments
  7. Author contribution
  8. References

A.H., F.H. and M.W.I.S. initially proposed the study. P.B. and S.B. made the compound-specific stable isotope analysis of amino sugars possible and gave conceptual and technical support. M.G. conducted the laboratory work, analyzed the data, and wrote the manuscript. All authors contributed with constructive comments to the final version of the manuscript.

References

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  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgments
  7. Author contribution
  8. References
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