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Article

Carbon Isotope Measurements to Determine the Turnover of Soil Organic Matter Fractions in a Temperate Forest Soil

1
Geographical Institute, Research Centre for Astronomy and Earth Sciences, Budaörsi út 45, H-1112 Budapest, Hungary
2
Hertelendi Laboratory of Environmental Studies, Institute for Nuclear Research, Bem tér 18/c, H-4026 Debrecen, Hungary
3
Department of Meteorology, Eötvös Loránd University, Pázmány Péter sétány 1/A, H-1117 Budapest, Hungary
4
Institute of Geography and Geoinformatics, University of Miskolc, Building A4, Miskolc-Egyetemváros, H-3515 Miskolc, Hungary
5
Institute for Geological and Geochemical Research, Research Centre for Astronomy and Earth Sciences, Budaörsi út 45, H-1112 Budapest, Hungary
6
Department of Environmental and Landscape Geography, Eötvös Loránd University, Pázmány Péter sétány 1/C, H-1117 Budapest, Hungary
*
Authors to whom correspondence should be addressed.
Agronomy 2020, 10(12), 1944; https://doi.org/10.3390/agronomy10121944
Submission received: 21 November 2020 / Accepted: 8 December 2020 / Published: 10 December 2020
(This article belongs to the Special Issue Soil Healthy in Agro-ecosystems)

Abstract

:
Soil organic matter (SOM) is a combination of materials having different origin and with different stabilization and decomposition processes. To determine the different SOM pools and their turnover rates, a silt loam-textured Luvisol from West Hungary was taken from the 0–20 cm soil depth and incubated for 163 days. Maize residues were added to the soil in order to obtain natural 13C enrichment. Four different SOM fractions—particulate organic matter (POM), sand and stable aggregate (S + A), silt- plus clay-sized (s + c) and chemically resistant soil organic carbon (rSOC) fractions—were separated and analyzed using FT-IR, δ13C, and 14C measurements. The mean residence time (MRT) of the new C and the proportion of maize-derived C in the fractions were calculated. The POM fraction was found to be the most labile C pool, as shown by the easily decomposable chemical structures (e.g., aliphatic, O-alkyl, and polysaccharides), the highest proportion (11.7 ± 2.5%) of maize-derived C, and an MRT of 3.6 years. The results revealed that the most stable fraction was the rSOC fraction which had the smallest proportion of maize-derived C (0.18 ± 2.5%) and the highest MRT (250 years), while it was the only fraction with a negative value of Δ14C (−75.0 ± 2.4‰). Overall, the study confirmed the hypothesis that the SOM associated with finer-sized soil particles decomposes the least, highlighting the significance of the fractionation process for more accurate determination of the decomposition processes of SOM pools.

1. Introduction

Soil organic matter (SOM) plays an important role in the functioning of land ecosystems. It regulates many physical, chemical, and biological functions and processes in the soil. It maintains the soil structure and porosity by aggregate formation and stabilization, increasing the water and air supply and retention in soils, and thereby enhancing the biological activity in soil environments. At the same time SOM provides a habitat and nutrient source for microorganisms. It also enhances the buffer capacity of the soil over a wide range of pH values due to the wide variety of functional groups and organic compounds (such as carboxylic, hydroxy, amine, and amide) in SOM [1].
Litter and root deposition are the two natural sources of C in the soil. Once C enters into the soil, it decomposes or stabilizes in different ways at different rates. The turnover of SOM, and thus its stabilization, is determined by (i) the variable chemical structure of SOM components and (ii) the physical protection against microbial attacks provided by aggregation and/or mineral bonding (by clay minerals and pedogenic oxides) [2]. As a consequence, the various materials making up SOM have different levels of resistance to microbial degradation. The residence time (the time the C atom spends in the soil system) of root exudates is a few hours, whereas plant residues decompose within a few weeks or months, while certain organic compounds may remain untouched for even ten thousand years [3,4,5].
The physical fractionation of soils allows the separation of these chemically and physically diverse pools. Soil fractionation results in SOM pools with different size, density, molecular, and aggregate composition, thus having various physical and chemical properties to stabilize C [6]. In general, sand-associated SOM (20 µm–2 mm) represents the active pool, whereas the silt and clay-sized fractions typifies the relatively stable C domains [7,8,9], though, several studies have produced contradictory results [10,11,12]. For example, Leifeld and Fuhrer [13] found that the mean residence time (MRT) of the S + A (sand and stable aggregate) fraction was more than 100 years, whereas the MRT of the POM (particulate organic matter) fraction was 5−7 years in meadow and pasture topsoils. In contrast, Dondini et al. [14] reported a smaller proportion of fresh carbon in the POM fraction than in the S + A fraction.
A better understanding of the properties and dynamics of SOM decomposition and sequestration is critical for predicting ecosystem responses to global change. The MRTs of the SOM fractions derived from fractionation are not just important parameters of soil ecosystem functioning, but are also significant input parameters for coupled climate-carbon models (e.g., Earth System Models), with which the size of terrestrial carbon sinks can be predicted more precisely [15,16]. In addition, the appropriate SOM management is crucial for agroecosystem health due to the above mentioned positive effects of SOM (e.g., water retention, nutrient supply, and aggregation). Nevertheless, as pointed out by Lehmann and Kleber [17] the investigation of SOM turnover is more important to gain the fertility of the soils then just to increase the number of passive carbon stocks.
The aim of this study was to quantify the stability of different SOM fractions based on the turnover characteristics derived from the 13C natural abundance and 14C methods. The C/N ratio, aromaticity, δ13C value, proportion of new maize-derived C, proportion of bomb-derived 14C, and MRT of the SOM fractions were used to test the hypothesis that the clay-sized (s + c) and the chemically resistant soil organic carbon (rSOC) fractions are the most stabile fractions. In addition, FT-IR spectroscopy was applied to characterize differences in the chemical structure of the organic matter in the separated soil fractions.

2. Materials and Methods

2.1. Sampling and Laboratory Measurements Before Incubation

A soil sample was taken from a Haplic Luvisol [18] with loamy soil texture (of the A horizon) (upper 0−20 cm) in the Sopron Mountains, Hungary (N 47°40′14.6″, E 16°33′53.6″) in November 2015 with a spade from a 0.5 × 0.5 m2 area. The vegetation cover at the study site is dominated by deciduous, mainly beech (Fagus sylvatica) forest. The mean annual temperature in the area is 9.2 °C and the annual precipitation is 700–750 mm.
In the laboratory, the soil sample was air-dried, homogenized, passed through a 2-mm sieve, and stored at room temperature.
The soil texture was determined with the sieve-pipette method [19] and was classified as silt loam (with 29% sand, 54% silt, and 17% clay content). The pH was 4.31 in distilled water and 3.27 in 1 M KCl solution.

2.2. Incubation Experiment

Sieved, air-dried soil samples weighing 200 g were placed in 1 L Duran® glass bottles. After preincubation at 50% field capacity at 20 °C for two weeks, the soil was amended with maize residues (δ13C = −13.33 ± 0.04‰). One gram of the air-dried, cut into pieces, and sieved (2-mm) maize residues was thoroughly mixed with 200 g preincubated soil. There were three replicates of amended soil and one subsample for control with no residue addition. The samples were kept in an incubator (KBW400 E5.1, Binder, Tuttlingen, Germany) at 20 °C for 163 days at 70% field capacity, as this moisture content allowed the handling and compaction of the soil without the collapse of small soil aggregates [20].

2.3. Soil Fractionation

After incubation, 30 g was weighted from the amended, control, and native (without incubation and maize addition) samples and fractionated following the recommendations of Zimmermann et al. [21]. Figure 1 shows the steps of the fractionation procedure applied. The 30 g sieved soil samples were added to 150 mL distilled water and dispersed in an ultrasonic shaker with 22 J mL−1 energy for 10 s. After dispersion the suspension was wet-sieved over a 63-µm sieve until the rinsing water was clear. Of the two fractions separated by wet sieving, the >63 µm portion was dried at 40 °C and separated by NaI at a density of 1.6 g cm−3. The centrifugation (at 2275 rpm for 15 min) of the suspension resulted in a light fraction represented the POM fraction (with fresh plant residues having visible plant structure), whereas the heavy fraction contained the S + A (sand and stable aggregate) fraction. The POM and S + A parts were cleaned with distilled water to remove the NaI, dried, weighed, and subjected to further analysis.
A 0.5-µm aperture glass filter was used to filter the <63 µm fraction. The fraction retained on the filter representing the s + c (silt- and clay-associated) fraction was dried at 40 °C and weighted. The filtered suspension, which represents the dissolved organic carbon (DOC), was frozen for use in other measurements, which are not included in this paper. One gram of the s + c fraction was added to 50 mL of 6% NaOCl and kept undisturbed for 18 h at 25 °C to separate the rSOC (chemically resistant soil organic carbon) fraction from the s + c fraction. The oxidation residue was centrifuged at 2275 rpm for 15 min, decanted, washed with distilled water and centrifuged. The oxidation step was repeated twice and the residue was dried to obtain the rSOC fraction. The s + c and rSOC fractions were subjected to further analysis.

2.4. Total C, N, and δ13C Measurements

The SOM fractions were analyzed with a Flash 2000 Elemental Analyzer (Thermo Scientific, Rhodano, Italy) using an internal TCD detector to determine the elemental percentages of C and N. The SOM fractions were also analyzed with the Flash 2000 Organic Elemental Analyzer (Thermo Scientific, Rhodano, Italy) coupled to a continuous flow isotope ratio mass spectrometer (Delta V Advantage, Thermo Finnigan, Bremen, Germany) to measure the stable carbon isotopes. The stable carbon isotope data are presented using the δ13C notation [22], where differences in the 13C/12C isotope ratio are expressed as per mil (‰) relative to the Vienna-PDB international standard. The IAEA-CH-7 (δ13C = −32.51‰) and IAEA-CH-6 (δ13C = −10.45‰) reference standards [23] were used for calibration. Samples were measured in three replicates.
The proportion of new added carbon (maize residues) to the total C content was calculated using δ13C values in a mixing model for the separated SOM fractions as follows [24]:
F = (δAB − δA)/δB − δA),
where F is the proportion of C derived from maize residues, δA and δAB is the δ13C value of the control and maize-amended soils, respectively, and δB is the δ13C value of maize residues.
The mean residence time (MRT) of C in the separated fractions was calculated from F as follows [25]:
MRT = −t/ln(1 − F),
where t is the incubation time.

2.5. 14C Measurements

14C analysis was carried out on the native samples (bulk soil and SOM fractions) before incubation. Since the pH of the bulk soil was 4.31 (distilled water) and 3.27 (KCl solution), no other carbonate removal was applied, but the pH of the samples was adjusted to 3 with 4% HCl prior to analysis. A two-step combustion method was applied for the bulk soil sample: first, combustion at low temperature (at 400 °C for 40 min, “L” fraction), followed by high temperature combustion on the same sample (at 800 °C for 20 min, “H” fraction) in the presence of high-purity oxygen gas in a quartz tube [26,27]. Because of the low organic content of the sample (2.97%), 1–2 g of sample had to be combusted in the on-line combustion system to provide at least 0.2 mg C/sample, allowing the production of graphite targets and normal accelerator mass spectrometry (AMS) analysis. The resulting CO2 gas was then collected and purified separately to form “L“ and “H” fractions using an on-line combustion system line and then converted to graphite using the sealed tube Zn-graphitization method [28]. IAEA–C9 (fossil wood) standards were treated and measured parallel to the sample to check the quality of sample preparation.
The separated SOM fractions were dried at 50 °C. Then, CO2 was generated from the samples by combustion at 550 °C for at least 12 h in the presence of MnO2 [29]. A specially designed vacuum line was used for the extraction and purification of the resulting CO2 gas. The pure CO2 gas was sealed by flame in glass tubes (with Zn and TiH catalysts and iron powder) and graphitized [28]. Finally, the graphitized samples were pressed into aluminum targets and subjected to EnvironMicadas AMS analysis to measure the carbon isotopic composition of the samples [30]. The “Bats” data reduction software was used [31] for data evaluation, and percent Modern Carbon (pMC) units were used in the calculations [32]. The radiocarbon data were expressed in Δ14C units (‰) according to Stuiver and Polach [33]:
Δ14C = (ASN/AABS − 1) × 1000,
where ASN is the activity of 14C in the sample, normalized for δ13C isotopic fractionation and decay-corrected between 1950 and the time of analysis (2018) and AABS is the absolute international standard activity of oxalic acid.

2.6. FT-IR Measurements

All the fractionated samples were powdered and dried at 40 °C directly before FT-IR measurement to remove the absorbed water which can significantly affect the vibration of OH groups and H2O molecules [34]. The FT-IR measurements were carried out on a Bruker Vertex 70 spectrometer with an RT-DLaTGS detector. For each sample a spectral range of 4000–400 cm−1, a resolution of 4 cm−1, 16 scans, and three replicates were recorded. The spectra were corrected (atmospheric water and CO2 correction), normalized, subjected to 17-point smoothing, and baseline corrected.
For the detection of relative changes in the spectra and spectral comparison, an aromaticity index (IAR) and relative absorbances (rA) were calculated. The aromaticity index derived from Inbar et al. [35] was calculated by dividing the intensity of absorption at 1640 cm−1 (aromatic C = C) by the intensity of absorption at 2920 cm−1 (aliphatic C–H) as follows:
IAR = A1640 cm−1/A2920 cm−1.
Relative absorbances [36,37,38] were calculated by dividing the peak height of each of the peak of the six peaks (2920, 2850, 1730, 1640, 1515, or 1420 cm−1) by the sum of the absorbances of all six peaks and multiplying it by 100. For example:
rA1420 = (A1420/∑A2920–1420 cm−1) × 100.

2.7. Statistical Analysis

Statistical analysis was conducted using IBM SPSS Statistics 22.0 (Armonk, NY, USA). One-way analysis of variance (ANOVA) with post hoc Tukey’s HSD test was used to evaluate differences among the treatment categories—(i) native soil without incubation and maize addition; (ii) maize-amended soil with incubation; and (iii) control soil with incubation but without maize residue addition—within the SOM fractions (POM, S + A, s + c, and rSOC).

3. Results and Discussion

3.1. Total C and N Concentration in the Soil Fractions

The total C and N concentrations in the different fractions are presented in Table 1, which shows that the POM fraction had the highest and the rSOC fraction the smallest C and N concentrations in the native, amended, and control samples. The S + A and s + c fractions had similar C and N values in all the samples with concentrations between those of the POM and rSOC fractions.
Incubation and maize addition had the least influence on the total C and N concentrations of the POM fraction, whereas the other SOM fractions had significant total C and N concentration differences (p < 0.05) between the native and the incubated samples (Table 1). The decrease in the C and N concentrations of POM in the amended sample compared to the control sample (Table 1, p < 0.05) can be attributed to the increased mineralization of C caused by the maize residues [39,40,41].
The fact that C/N ratios decreased from the POM to the s + c fraction (Table 1) is consistent with the literature [42,43,44] showing that the C/N ratio decreased as the density of the fractions increased. The C/N ratio was found to be the widest in the rSOC fraction in the present study. Leifeld and Fuhrer [13] also recorded the widest C/N ratios (17.5–22.2) for the rSOC fractions at almost all soil depths of two soils, which they explained by the presence of black carbon in the samples. Similar to black carbon, the rSOC fraction consists high carbon content organic compounds most resistant to oxidation, whereas N-containing carbohydrates and amino acids decompose most rapidly during NaOCl oxidation [45], resulting in a C enrichment and a N depletion of this fraction.

3.2. Quality Assessment of SOM Fractions Using FT-IR Spectroscopy

The FT-IR absorbance of the soil fractions (Figures S1–S3) was evaluated by establishing the ratios between the main absorbance peaks of the spectra and calculating relative absorbance values (Table 2). The bands at 2920 and 2850 cm−1, associated with aliphatic C−H stretching [36,46], had the highest relative absorbance in the POM fraction and the lowest in the s + c and rSOC fractions in all the soils.
The POM fraction also had by far the highest relative absorbance for the carboxylic substituents of aromatic esters in the band at 1730 cm−1 [46] in all the soil samples regardless of the treatment (Table 2), indicating a high proportion of aromatic compounds. The relative absorbance of the band at 1640 cm−1, characteristic of aromatic C and carboxylic acid [47], was also high in the POM fraction. The relative absorbance of the band at around 1515 cm−1, which is assigned to aromatic C = C vibrations [46], was found to be the highest in the POM fraction and the lowest in the rSOC fraction in the native soil spectrum (Table 2). The high proportion of aromatic compounds can be attributed to the fact that the POM sample is composed of relatively undecomposed or partly degraded plant residues that contain both aliphatic and aromatic (lignin-derived) structures.
The levels of carboxyl and aromatic carbon was found to be low in the NaOCl-resistant rSOC fraction at 1660–1620 cm−1 [45], in agreement with our results (Table 2). As shown in Table 2, there were significant differences (p < 0.05) between the incubated samples and the native sample in all SOM fractions (except for the rSOC fraction) in the relative absorbance at 1420 cm−1, which can be attributed to the aromatic ring stretching of COO [35]. The rA1420 of the rSOC fraction showed high values and no difference (p < 0.05) between the treatments, which can be attributed to the fact that NaOCl has high pH (~10), which may cause the deprotonation of most of the carboxylic groups in organic matter in every treatment.
The aromaticity index of the soil fractions differed among the fractions (Table 2). The POM fractions had the lowest aromaticities, while the s + c and rSOC fractions had high values. Baldock et al. [48] also found that the content of aromatic carbon was highest in the intermediate-sized fractions (2–53 µm). Incubation had a great effect on the aromaticity of the soil fractions: the aromaticity of rSOC increased (p < 0.05) during incubation in the amended soil, probably due to the enhanced biological activity, which may have caused a decrease in aliphatic compounds.

3.3. Evaluation of C Isotope Changes in SOM Fractions and Turnover Time of the SOM Fractions

Based on the results of stable C isotope measurements, it can be seen that the δ13C values of the S + A fraction (−26.32 ± 0.04‰ for the native soil, −26.48 ± 0.03‰ for the control soil, and −25.98 ± 0.09‰ for the amended soil) were closest to that of the unfractionated bulk soil (−26.28 ± 0.06‰ for the native soil, −26.50 ± 0.09‰ for the control soil, and −25.94 ± 0.08‰ for the amended soil) in all the soils (Figure 2). The s + c fraction was slightly enriched in 13C compared to the bulk soil in all the soils. In contrast, 13C was depleted in the POM and rSOC fractions compared to the bulk soil, regardless of incubation or maize addition. The negative δ13C values of the POM fractions in the native (−27.47 ± 0.01‰) and control (−27.76 ± 0.04‰) soils could be attributed to the fact that the organic matter of fresh plant residues entering the soil under forest vegetation is depleted in 13C [49]. Since Fagus sylvatica is the main vegetation cover in the study site, the C isotopic signature of this tree species may be reflected in the POM fraction. Keitel et al. [50] reported that the δ13C values of the total leaf organic matter of Fagus sylvatica varied from −26.5 to −32‰.
In general, δ13C values increase with an increasing degree of mineral–organic association in soil fractions [43,51,52,53]. This trend could be detected for the POM, S + A, and s + c fractions of native, control, and amended soils in the present study. The most negative δ13C value recorded for the rSOC fraction in all the soils (Figure 2) could be the consequence of chemical oxidation. Zimmermann et al. [45] also revealed that NaOCl-resistant fractions were depleted in δ13C, which they explained by the fact that oxidation with NaOCl dissolves 13C-enriched compounds (cellulose, hemi-cellulose, carboxyl, and amino acids) and leaves the rSOC fraction depleted in 13C. After incubation, the fractions of the control samples had more negative δ13C values than the native samples, except for the s + c fraction, where the values were similar. This can be explained by the preferential use of 13C-enriched substrates (including sugars, starch, cellulose, etc.) by microorganisms during decomposition, resulting in the depletion of 13C in the remaining SOM [54]. Microbial metabolism, taking place during the incubation, results 13C-depleted CO2 and 13C-enriched SOM [55], which would cause more positive δ13C value of the SOM fractions. Thus the fractionation effect of the preferential use of 13C-enriched substrates and the microbial metabolism are contradictory, but according to Šantrůčková et al. [56] the effect of preferential substrate utilization is stronger than that of the microbial metabolism, which causes the more negative δ13C value of the SOM fractions of the control sample compared to the SOM fractions of the native sample.
The Δ14C values of the fractions in the studied soil (Table 3) showed the highest values for the bulk soil (78.2 ± 1.7‰) and the POM fraction (75.2 ± 1.9‰), similar values for the S + A (51.0 ± 2.1‰) and s + c (49.7 ± 1.9‰) fractions, and the lowest value for the rSOC fraction (−75.0 ± 2.4‰), thus showing the highest proportion of bomb-derived 14C in the bulk soil and the POM fraction, as positive values of Δ14C indicate the presence of bomb-derived 14C [43,57]. The >100 pMC values of the bulk soil and the POM, S + A, and s + c fractions (Table 3) also suggested a significant amount of bomb-derived 14C [58]. The lowest and only negative Δ14C value measured in the rSOC fraction indicated that the 14C in the sample was not in contact with the atmosphere, so significant radioactive decay could have occurred [57]. This is in accordance with the findings of Kögel-Knabner et al. [59] who also reported the smallest 14C activities (99.3 and 90.7 pMC) for the heavy (>1.6 g cm−3), NaOCl-resistant fractions in the “A” horizons of a Cambisol and a Podzol, respectively.
The fraction of C derived from the maize residues was the highest (11.7 ± 2.5%) for the POM fraction, followed by 3.8 ± 2.8 and 2.7 ± 3.0% for the S + A and s + c fractions, respectively, and 0.18 ± 2.5% for the rSOC fraction (Table 3). The fact that the POM fraction had the highest proportion of maize-derived C and the highest 14C activity/Δ14C value with the fastest turnover (Table 3) confirmed that this fraction was the most labile C pool in the Luvisol investigated in the present study. This is consistent with the statement that this fraction is predominantly plant-derived and sensitive to soil disturbances such as land use and management [21]. Further evidences are the following: (1) the aliphatic and aromatic (lignin-derived) structures of the POM fractions, determined by FT-IR spectroscopy, showed that these fractions were composed of partly degraded plant residues; (2) the δ13C value of POM was similar to that of plant litter; and (3) the MRT determined for POM in the present study was (3.6 years) in the MRT range (2–5 years) reported by Gaudinski et al. [60] for recognizable leaf litter.
The MRTs of the S + A (11.5 years) and s + c (16.1 years) fractions were similar, as were the proportions of maize-derived C and the 14C values (Table 3). The fast turnover of the S + A fraction can be explained by the fact that this fraction also contains POM particles attached to sand grains [61]. In addition, fresh C imbedded in aggregates represents an active pool with short turnover, while the size of the aggregates plays a significant role in C stabilization. For example, Yamashita et al. [62] reported that occluded POM with a density of 1.6–2.0 g cm−3 within the aggregates had considerably shorter MRTs (20–34 years) for the mega- and large macroaggregates than for the microaggregates (51 years) or <53 µm aggregates (166 years).
The oxidation using NaOCl (resulted in the rSOC fraction) changed the C isotope values of the s + c fraction in the present study. Although Poeplau and Don [61] found that the rSOC fraction was almost as sensitive to land use changes as the total SOC, the highest MRT of 249.7 years was found for the rSOC fraction in the present study. It could be explained by the fact that this fraction was free of plant residues, POM, and aggregates [21]. Many studies [11,63] demonstrated that NaOCl oxidation can be used to obtain a stable C pool with high MRTs and radiocarbon ages.

4. Conclusions

The results from the use of combined physical and chemical fractionation, in combination with FT-IR data and stable and radioactive isotope measurements, agree with the consensus about SOM fractions representing different stabilities. The results revealed that the POM fraction in this Luvisol was the most labile C pool, with easily degradable chemical structures (e.g., aliphatic, O-alkyl, and polysaccharides), depleted 13C values, the highest proportion of new C, and the fastest turnover rate, confirming that this fraction contained non-stabilized organic matter which was easily available for decomposition. The analysis of the POM, S + A, and s + c fractions showed that, in general, with decreasing grain size distribution: (1) the C/N ratio decreases; (2) the aromaticity increases; (3) the δ13C value increases; (4) the proportion of new maize-derived C decreases; (5) the proportion of bomb-derived 14C decreases; and (6) the MRT of C increases, suggesting an increasing degree of mineral–organic association, leading to decreased degradability and greater resistance to decomposition. Therefore, the hypothesis that the smallest fraction is the most stabile was confirmed. In addition, the fact that the rSOC fraction represents a relatively inert C pool was also demonstrated by the much smaller proportion of maize-derived C and bomb-derived 14C and by far the longest MRT, confirming the ability of NaOCl oxidation to separate a C fraction which is less transformed by microorganisms in the native forest soil investigated. Nevertheless, the fact that the rSOC fractions unexpectedly had the widest C/N ratio, a decrease in relative aromaticity and the most negative δ13C value, regardless of incubation or maize addition, highlighted the effect of chemical oxidation by NaOCl.

Supplementary Materials

The following are available online at https://www.mdpi.com/2073-4395/10/12/1944/s1, Figure S1: The FT-IR spectra of the native samples, Figure S2: The FT-IR spectra of the control samples, Figure S3: The FT-IR spectra of the amended samples.

Author Contributions

Conceptualization, D.Z., T.F., G.J., and Z.S.; data curation, D.Z and T.F.; methodology, M.M., T.K., C.K., I.H., and L.G.; resources, G.J. and M.M.; supervision, T.F., G.J., and Z.S.; visualization, D.Z.; writing—original draft, D.Z.; writing—review & editing, T.F., G.J., and Z.S. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the European Union and the State of Hungary, co-financed by the European Regional Development Fund in the project of GINOP-2.3.2-15-2016-00009 ‘ICER’ and by the ÚNKP-17-3 New National Excellence Program of the Ministry of Human Capacities, Hungary, and by the Development and Innovation Fund of Hungary [No. NKFIH 123953].

Acknowledgments

The authors are grateful for the help of Dóra Kesjár in the FT-IR measurements.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Schematic diagram of the fractionation procedure applied after Zimmermann et al. [21]. POM = particulate organic matter; S + A = sand and stable aggregate fraction; DOC = dissolved organic carbon; s + c = silt- plus clay-sized fraction; rSOC = chemically resistant SOC fraction.
Figure 1. Schematic diagram of the fractionation procedure applied after Zimmermann et al. [21]. POM = particulate organic matter; S + A = sand and stable aggregate fraction; DOC = dissolved organic carbon; s + c = silt- plus clay-sized fraction; rSOC = chemically resistant SOC fraction.
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Figure 2. δ13C values of the bulk forest soil and of the soil fractions (POM = particulate organic matter; S + A = sand and stable aggregate fraction; s +c = silt plus clay-sized fraction; rSOC = chemically resistant SOC fraction) separated from the native soil before incubation and from the control and maize-amended samples after the 163-day incubation. Line bars indicate standard error. Different letters indicate significant differences (ANOVA, p < 0.05) among the treatment categories within an SOM fraction.
Figure 2. δ13C values of the bulk forest soil and of the soil fractions (POM = particulate organic matter; S + A = sand and stable aggregate fraction; s +c = silt plus clay-sized fraction; rSOC = chemically resistant SOC fraction) separated from the native soil before incubation and from the control and maize-amended samples after the 163-day incubation. Line bars indicate standard error. Different letters indicate significant differences (ANOVA, p < 0.05) among the treatment categories within an SOM fraction.
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Table 1. Total C and N content and C/N ratio of the different soil fractions in the Luvisol studied.
Table 1. Total C and N content and C/N ratio of the different soil fractions in the Luvisol studied.
Fraction 1Treatment 2TOC
(g kg−1)
TN
(g kg−1)
C/N Ratio
POMNative245 ± 25.4 3 a 49.84 ± 1.28 ab25.2 ± 1.31 a
Amended229 ± 16.4 a8.02 ± 0.552 a28.5 ± 0.43 b
Control341 ± 3.39 b11.9 ± 0.099 b28.7 ± 0.21 b
S + ANative21.6 ± 0.13 b1.53 ± 0.009 b14.1 ± 0.03 a
Amended17.5 ± 0.36 a1.12 ± 0.023 a15.7 ± 0.05 c
Control17.6 ± 0.11 a1.15 ± 0.006 a15.3 ± 0.11 b
s + cNative23.0 ± 0.18 c1.69 ± 0.002 c13.6 ± 0.10 a
Amended19.4 ± 0.16 a1.44 ± 0.011 a13.5 ± 0.05 a
Control20.3 ± 0.03 b1.49 ± 0.008 b13.7 ± 0.06 a
rSOCNative7.72 ± 0.07 c0.21 ± 0.001 b36.5 ± 0.49 b
Amended5.05 ± 0.17 a0.17 ± 0.004 a30.2 ± 0.65 a
Control6.56 ± 0.09 b0.20 ± 0.006 b33.2 ± 1.43 a
Bulk recovery (mass %)Native80.5 ± 0.39 a113.6 ± 0.33 b
Amended134.3 ± 8.14 b101.6 ± 3.28 a
Control93.9 ± 0.20 a98.0 ± 0.31 a
1 POM = particulate organic matter fraction; S + A = sand plus stable aggregate fraction, s + c = silt plus clay fraction; rSOC = chemically resistant soil organic carbon fraction. 2 Native = native soil without incubation and maize addition; amended = maize-amended soil with incubation; control = control soil with incubation but without maize residue addition. 3 Mean ± standard error. 4 Means with different letters indicate significant differences (ANOVA, p < 0.05) among the treatment categories within an SOM fraction.
Table 2. Relative absorbance as a % of the sum of all selected peak heights, and aromaticity index (IAR) derived from the FT-IR spectra of the soil fractions in the Luvisol studied.
Table 2. Relative absorbance as a % of the sum of all selected peak heights, and aromaticity index (IAR) derived from the FT-IR spectra of the soil fractions in the Luvisol studied.
Fraction 1TreatmentrA2920rA2850rA1730rA1640rA1515rA1420IAR
POMNative12.7 ± 0.89 2 a 38.7 ± 0.62 a24.7 ± 0.87 ab23.1 ± 0.33 b11.2 ± 0.93 b10.9 ± 1.6 b1.9 ± 0.15 b
Amended13.6 ± 0.15 a8.8 ± 0.07 a24.1 ± 0.54 a23.5 ± 0.22 b7.0 ± 0.30 a6.8 ± 0.41 a1.7 ± 0.02 b
Control17.8 ± 0.18 b12.6 ± 0.21 b26.4 ± 0.33 b20.7 ± 0.36 a8.5 ± 0.18 a5.4 ± 0.51 a1.2 ± 0.03 a
S + ANative10.8 ± 0.11 b6.7 ± 0.09 b7.1 ± 0.31 a21.6 ± 0.22 a5.7 ± 0.30 a12.7 ± 0.57 a2.0 ± 0.01 a
Amended8.9 ± 0.37 a5.5 ± 0.26 a6.5 ± 0.31 a22.2 ± 0.22 ab8.5 ± 0.70 ab19.8 ± 1.6 b2.5 ± 0.10 b
Control8.1 ± 0.40 a4.9 ± 0.24 a6.2 ± 0.32 a23.5 ± 0.72 b9.0 ± 1.2 b20.7 ± 1.7 b2.9 ± 0.14 c
s + cNative7.9 ± 0.19 ab4.5 ± 0.13 ab7.0 ± 0.18 a24.7 ± 0.29 a7.5 ± 0.74 b20.7 ± 1.3 b3.1 ± 0.08 a
Amended8.4 ± 0.23 b5.0 ± 0.16 b6.4 ± 0.19 a24.4 ± 0.48 a5.1 ± 0.29 a13.0 ± 0.78 a2.9 ± 0.12 a
Control7.4 ± 0.29 a4.4 ± 0.18 a6.9 ± 0.26 a24.5 ± 0.67 a5.4 ± 0.43 a15.5 ± 1.5 a3.3 ± 0.16 a
rSOCNative8.3 ± 0.21 b5.3 ± 0.13 b4.0 ± 0.40 c18.0 ± 0.28 a11.3 ± 0.37 a21.9 ± 1.0 a2.2 ± 0.03 a
Amended3.5 ± 0.14 a2.5 ± 0.09 a2.1 ± 0.13 a20.0 ± 0.74 a15.3 ± 0.37 b18.7 ± 2.1 a5.8 ± 0.30 b
Control7.4 ± 0.52 b4.9 ± 0.36 b3.2 ± 0.16 b20.2 ± 0.75 a11.1 ± 0.49 a18.2 ± 1.9 a2.8 ± 0.24 a
1 POM = particulate organic matter fraction; S + A = sand plus stable aggregate fraction, s + c = silt plus clay fraction; rSOC = chemically resistant soil organic carbon fraction. 2 Mean ± standard error. 3 Means with different letters indicate significant differences (ANOVA, p < 0.05) among the treatment categories within an SOM fraction.
Table 3. Proportion and mean residence time of C derived from the C4−C (maize residues) input, 14C activity and Δ14C for the soil fractions separated from the Luvisol studied.
Table 3. Proportion and mean residence time of C derived from the C4−C (maize residues) input, 14C activity and Δ14C for the soil fractions separated from the Luvisol studied.
Fraction 1Fmaize 2(%)MRT 3
(Years)
14C activity
(pMC)
Δ14C
(‰)
POM11.7 ± 2.5 43.6108.4 ± 0.1975.2 ± 1.9
S + A3.8 ± 2.811.5106.0 ± 0.2151.0 ± 2.1
s + c2.7 ± 3.016.1105.8 ± 0.1949.7 ± 1.9
rSOC0.18 ± 2.5249.793.3 ± 0.24−75.0 ± 2.4
Bulk soil4.3 ± 2.910.2108.7 ± 0.1778.2 ± 1.7
1 POM = particulate organic matter fraction; S + A = sand plus stable aggregate fraction; s + c = silt plus clay fraction; rSOC = chemically resistant soil organic carbon fraction. 2 Fmaize = proportion of C derived from the C4−C (maize residues) input. 3 MRT = mean residence time of C derived from the C4−C (maize residues) input. 4 Means with ± standard error.
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Zacháry, D.; Filep, T.; Jakab, G.; Molnár, M.; Kertész, T.; Király, C.; Hegyi, I.; Gáspár, L.; Szalai, Z. Carbon Isotope Measurements to Determine the Turnover of Soil Organic Matter Fractions in a Temperate Forest Soil. Agronomy 2020, 10, 1944. https://doi.org/10.3390/agronomy10121944

AMA Style

Zacháry D, Filep T, Jakab G, Molnár M, Kertész T, Király C, Hegyi I, Gáspár L, Szalai Z. Carbon Isotope Measurements to Determine the Turnover of Soil Organic Matter Fractions in a Temperate Forest Soil. Agronomy. 2020; 10(12):1944. https://doi.org/10.3390/agronomy10121944

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Zacháry, Dóra, Tibor Filep, Gergely Jakab, Mihály Molnár, Titanilla Kertész, Csilla Király, István Hegyi, Lilla Gáspár, and Zoltán Szalai. 2020. "Carbon Isotope Measurements to Determine the Turnover of Soil Organic Matter Fractions in a Temperate Forest Soil" Agronomy 10, no. 12: 1944. https://doi.org/10.3390/agronomy10121944

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