Characterization of transformations of maize residues into soil organic matter

STOTEN-21473; No of Pages 12 Science of the Total Environment xxx (2016) xxx–xxx

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Characterization of transformations of maize residues into soil organic matter Guixue Song a,b,⁎, Etelvino H. Novotny c, Jing-Dong Mao d, Michael H.B. Hayes b,⁎⁎ a

Pony Testing International Group, No. 49-3 Suzhou Street, Haidian, Beijing 100080, China Department of Chemical & Environmental Sciences, University of Limerick, Limerick, Ireland Embrapa Solos, Rua Jardim Botânico, 1024, CEP 22460-000 Rio de Janeiro-RJ, Brazil d Department of Chemistry, University of Old Dominion, Norfolk, Virginia, United States b c

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Maize (C4 plant) was incubated in a long-term cultivated soil with wheat (C3 plant). • More than half of the newly transformed organic C in the humic acid fraction. • Carbohydrate and lignin-like material were identified in SOM fractions through a variety of characterization. • Multivariate analysis was used in analysis of data from SOM fractions.

a r t i c l e

i n f o

Article history: Received 2 October 2016 Received in revised form 23 November 2016 Accepted 24 November 2016 Available online xxxx Editor: Jay Gan

a b s t r a c t An awareness of the transformation of plant residues returned to cultivated soils is vital for a better understanding of carbon cycles, the maintenance of soil fertility and the practice of a sustainable agriculture. The transformation of maize (Zea mays L) straw residues into soil organic matter (SOM) in a one year incubation experiment was studied in a soil that had been under long term cultivation with wheat (Triticum aestivum L) for N30 years. A novel sequential exhaustive extraction and fractionation procedure isolated a series of fractions of SOM. The samples were characterized by elemental and δ13C analyses, by amino acids and neutral sugars analyses, by Fourier transformed infrared (FTIR) spectrometry, and by solid state 13C nuclear magnetic resonance (NMR) spectroscopy and

⁎ Corresponding author at: Pony Testing International Group, No. 49-3 Suzhou Street, Haidian, Beijing 100080, China. ⁎⁎ Corresponding author. E-mail addresses: [email protected] (G. Song), [email protected] (M.H.B. Hayes).

http://dx.doi.org/10.1016/j.scitotenv.2016.11.169 0048-9697/© 2016 Elsevier B.V. All rights reserved.

Please cite this article as: Song, G., et al., Characterization of transformations of maize residues into soil organic matter, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.11.169

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G. Song et al. / Science of the Total Environment xxx (2016) xxx–xxx

Keywords: Plant residue transformation Soil organic matter Sequential extraction Neutral sugars Amino acids Lignin residues in humic substances Humification

with chemical shift anisotropy (CSA) –filter and dipolar dephasing (DD) spectral editing NMR techniques. The δ13C data indicated that 59% and 38% of the newly transformed organic carbon was in the humic and fulvic acid fractions, respectively, and in general a greater proportion of the transformed carbon was in the fractions isolated at the higher pH values. Results for SOM fractions from the amended soil indicate dominant contributions from carbohydrate and lignin-like material, and that can be clearly identified by FTIR, CP/TOSS, and spectral editing of CSA-filter and DD. The compositions of the fractions from the amended and non-amended soils fractions can be clearly differentiated using principal component analysis (PCA) for the data collected. The sequential extraction procedure showed that the hydrophilicity of humic fractions increased as the result of the maize amendment, and the aromaticity of the fraction decreased. The data may give some indications of transformations that take place during humification processes. © 2016 Elsevier B.V. All rights reserved.

1. Introduction The soil organic matter (SOM) content is greatly influenced by soil management and by land-use changes (Milne et al., 2007; Murty et al., 2002; Zhang et al., 2013). Biological oxidation of SOM releases carbon as CO2 and if this carbon is not replaced, the loss will contribute to atmospheric CO2 in the same way as fossil carbon. It was common practice to return crop residues to soil and the management of crop residues is a useful strategy in agronomy for the maintenance of soil structure and fertility, for the sequestration of carbon in the soil, and in mitigating greenhouse gas emissions to the atmosphere (Kumar and Goh, 2000; Jones et al., 2005). Aspects of the chemical compositions and structures of components of SOM as well as soil structure will be influenced by the effects of long term cultivation (Bayer et al., 2002; Ding et al., 2002; Gregorich et al., 1996; Novotny et al., 1999; Spaccini et al., 2000). For example, Ding et al. (2002) have reported that the humic acids (HAs) isolated from soils that had been subjected to long term cultivation where conventional tillage (CT) operational procedures were used were less aliphatic and more aromatic than HAs from soils where conservational tillage (CnT) was followed, and the aliphatic C content decreased with increasing depth in the soil profile. HAs were more reactive in the top soil (0– 5 cm) under CnT than under CT. Naturally 13C-enriched plants, especially maize (Zea mays L.), a C4 plant, are often used to trace the carbon distributions in humic fractions and to investigate turnover of plant residues, humification processes, land use changes, and the origins of SOM components, etc. (Balesdent and Mariotti, 1996; Clapp et al., 1997; Lichtfouse et al., 1995a; Lichtfouse et al., 1998; Lichtfouse et al., 1995b). When a C4 plant is grown in soil whose SOM was derived from C3 plants, or vice versa, the δ13C value of the SOM is directly related to the proportions of C3and C4-derived carbons contributing to the structures. Therefore, this natural tracer of in-situ produced organic C has proven to be a useful technique to study long-term SOM dynamics. Organic free radicals concentrations (OFRC) in SOM have been used to assess the extents of humification (or the transformation of organic residues to humic substances (HSs). The OFR signals are attributed to the semiquinone radical units, possibly conjugated to aromatic rings, although contributions from methoxybenzene and nitrogen-associated radicals cannot be excluded (Senesi, 1990). Amino N is the most abundant form of organic N in SOM, and organic N in soils is bound to peptide-like structures, and to a lesser extent in amino sugars (Knicker, 2011; Knicker et al., 2000). This organic N form can persist during the transformations in soil of the organic residues from plant, animal, and microbial tissues (Hayes et al., 1999). Polysaccharides are among the predominant components in SOM and analyses of neutral sugars (NS) and the ratios of these sugars are often used as indicators for plant or microbial origins (Clapp et al., 2005; Oades, 1984). Specific solid-state 13C nuclear magnetic resonance NMR techniques have been developed to investigate carbons connected to specific functional groups and structures, chemical shift anisotropy (CSA) and

dipolar dephasing (DD) experiments provide excellent examples of such specific tools. Multivariate analyses, such as principal component analysis (PCA), have been applied to infrared spectra, EPR, NMR spectra of HAs, toxic elements and the physical properties of soils, etc., in order to reduce the parameters or variables and to show the signal correlations (Aguiar et al., 2013; Borůvka et al., 2005; Guo et al., 2013; Levi and Rasmussen, 2014; Novotny et al., 2008). The first few principal components (PCs), the transformed new set of variables, retain most of the variation present in all of the original variables. Lignin is considered to have a high resistance to degradation, but there is some evidence to suggest that, in less than one year, it is largely decomposed as CO2, or transformed into other non-lignin products (Rasse et al., 2006). However, we do not know how components of plant residues transform to SOM and we lack information about the extents of the transformation. In order to better understand the nature of the OM transformations, we incubated maize (Zea mays L) straw residues (stover) for 12 months in a soil that had been under long term cultivation to wheat for N30 years. A series of humic fractions were isolated using a sequential and exhaustive extraction procedure (Song et al., 2008, 2011), the SOM fractions were characterized by means of elemental and δ13C analyses, electron paramagnetic resonance (EPR), amino acids (AA) and NS analyses, Fourier transformed infrared spectrometry (FTIR), and solid state 13C NMR spectrometry. The data are compared with those for similar isolates from the control soil (unamended soil). 2. Material and methods 2.1. Soil and experimental design Air-dried maize from the Teagasc Research Farm at Oak Park (52o51.364′, N, 6o54, 727′W′), Co. Carlow, Ireland, was chopped, ground to b4 mm, thoroughly blended and mixed (~7.9% dry weight in the total soil mixture) with 32.5 kg of the Oak Park cultivated soil. The soil is a gravely brown earth of coarse sandy loam with clay, silt, and sand contents of about 11%, 22% and 67%, respectively, and with an organic carbon content of 56 g·kg− 1 (Ryan and Fanning, 1999). NPK fertilizer (400 g 10-10-20) was mixed with the organic amended soil mixture, transferred to a plastic barrel (100 cm diameter, 150 cm depth) and incubated in the laboratory at ambient temperatures (which fluctuated between 15 and 25 °C during the course of the incubation). The soil was mixed and watered regularly during the course of the incubation, and distilled water was added at intervals to keep the moisture at about 20%. 2.2. Extractions of humic substances A soil sample was taken after incubation for 12 months. The soil was air-dried and sieved (b 2 mm), and about 1 kg soil was adjusted to pH 1.5 using 1 M HCl in a soil/solution ratio of 1:3. The suspension was shaken for 1 h, then allowed to settle for 12 h, and the supernatant

Please cite this article as: Song, G., et al., Characterization of transformations of maize residues into soil organic matter, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.11.169

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was collected. The procedure was repeated, and the residue was washed with distilled water until chloride free. The supernatant is part of the fulvic acids (FAs) fraction, it was saved and mixed with the FA isolated at pH 7. The soil residue was brought to pH 7 using 0.1 M NaOH, and the soil/ solution ratio was 1:5, and exhaustive extraction was carried out as described by Hayes and Graham (2000) and by Song et al. (2011). Similar exhaustive extractions of the soil were continued at pH 10.6, at pH 12.6 (0.1 M NaOH), and with 0.1 M NaOH + 6 M urea. Extractions in alkaline conditions were carried out in an atmosphere of N2 gas. 2.3. Fractionation of extracts After passage through 0.2 μm cellulose acetate membrane, the SOM extracts isolated at pH 7, 10.6, and 12.6 were acidified with 6 M HCl to pH 1– 1.5, then allowed to stand for 12– 16 h to separate the precipitate from the supernatant. The precipitated HAs were transferred to a visking dialysis tubing (100 kDa), dialyzed against distilled water, then freeze-dried. The supernatant was pumped through XAD-8 and XAD-4 resin column in tandem following the procedure described by (Hayes et al., 2008), and fulvic FAs (from the eluate from XAD-8) and XAD-4 acids (from the XAD-4 eluate) were obtained. The pH value of the base/urea extracts was lowered to 1 to 1.5 using 6 M HCl to separate the precipitate from the supernatant. The precipitated humic acid-like material (HALM) was re-dissolved in 0.1 M NaOH, and diluted to a weak colour. The pH was carefully adjusted to 2.5 (because a significant precipitate can occur at pH ~2.0, but no distinct precipitation took place at pH 2.5 or higher). The solution was pumped on to an XAD-8 resin column, the column was washed with distilled water to remove urea, and the column was then back eluted in 0.1 M NaOH. The eluate was H+-exchanged by passing through an IR-120 column, and freeze-dried to give the HALM fraction. The supernatant (Fulvic acids-like material, FALM) was recovered by using the XAD-8 resin technique. The XAD-4 acids were not in sufficient amounts to warrant recovery. Humic samples and XAD-4 acids isolated at different pH values and from the base/urea solvent, are named as HAs or FAs + pH values, e.g., HAs pH 7, HAs pH 10.6, FAs pH 12.6, XAD-4 acids pH 12.6, XAD-4 acids base/urea). 2.4. Elemental and δ13C analyses Carbon and nitrogen contents, and the δ13C values were measured on an elemental analyzer (Carlo Erba, model NA 1500, Milan, Italy) with a stable isotope ratio mass spectrometer (Fisons, Optima model) continuous flow system. Results of the isotope analyses are expressed in terms of δ values (‰):
δ C ¼ Rsample =Rstandard −1  1000 13

ð1Þ

where R = ratio of 13C/12C. The δ13C values were calculated relative to the Pee Dee Beleminite (PDB) as an original standard. A HA standard from the International Humic Substances Society (IHSS) served as working standard for accuracy. Solid samples were weighted about 1– 2 mg (accurate to 2 μg). The approximation equation (Balesdent and Mariotti, 1996) X ¼ ðδ–δ3 Þ  100=ðδ4 –δ3 Þ

ð2Þ

was used, where: X is the percent of the contribution from the maize; δ is the δ13C value of the sample; δ3 is the δ13C value of the corresponding sample or fraction from the C3 reference soil; δ4 is the δ13C value of the C4 plant residue. A determination was made of the ratio of maize derived C in the humic fractions. The average δ13C of maize −12‰ was used; δ3 is usually measured in the reference plot kept bare of any vegetation, and

3

does not vary throughout the experiment. The average δ13C values of HAs −28.39‰ and of FAs −27.91‰, were used for the δ3 of the control soil.

2.5. EPR spectroscopy The EPR measurements were carried out following the method in described by Novotny and Martin-Neto (2002). The relative concentration of organic free radicals (spin concentration) was obtained using a ruby crystal (as a secondary standard) calibrated with strong pitch reference of known free radical content, according to Singer's method (Martin-Neto et al., 1994; Rosa et al., 2005; Singer, 1959). The HSs samples were dried in desiccators over silica gel and the results obtained were corrected for the sample's carbon content [spins (g C)−1].

2.6. Neutral sugars (NS) analyses NS analyses were carried out based on that described in literature (Hayes et al., 2008; Oades, 1984) on the SOM isolates from the maize amended and the non-amended (control) soils. Seven neutral sugars were analyzed: glucose (Glu), galactose (Gal), mannose (Man), rhamnose (Rha), fucose (Fuc), xylose (Xyl) and arabinose (Ara).

2.7. Amino acids analyses Amino acids were determined using RP-HPLC, based on procedures in the literature (Hayes et al., 2008; Turnell and Cooper, 1982). Each sample was derivatized with o-phthalaldehyde (OPA) and analyzed by a TSP HPLC system (Thermo Separation Product, USA) with pre-column derivation. A standard containing known concentrations of individual amino acids (ca. 1000 nmol for each AA standard) and the internal standard was injected before and after every fifth sample in the series.

2.8. Infrared spectrometry Infrared spectra of freeze-dried samples in KBr pellets were obtained using a Bomen FTIR (model Amwen/32, Canada) spectrometer, scanning from 4000 to 400 cm−1, averaging 20 scans at 1.0 cm−1 interval, and with a resolution of 4.0 cm−1. For comparative purposes, all spectra after acquisition were normalized to a maximum absorbance of 1.0.

2.9. Solid-state NMR spectroscopy All the experiments used a Bruker DSX400 spectrometer at a 13C frequency of 100 MHz. HAs and FAs were packed in 7 mm sample rotors. The 13C CP/TOSS experiments were run at a spinning speed of 5 kHz, a CP time of 1 ms, and a 1H 90° pulse length of 4 μs to obtain qualitative information with good sensitivity (Novotny et al., 1999). A 13C CSA filter was inserted into the 13C CP/TOSS in order to select signals of sp3 hybridized carbons. In particular, this process can separate signals of anomeric carbons from those of aromatic carbons (Mao and SchmidtRohr, 2004). The CSA was also combined with short CP to obtain selective spectra of protonated anomerics (Mao et al., 2011a). In order to detect non-protonated anomeric carbons, this filter was combined with a dipolar dephasing time of 40 μs. The CSA was also combined with short CP to obtain selective spectra of protonated anomerics. A total of 4096 scans were averaged for spectra of CSA filter and CSA filter coupled with short CP, with a recycle delay of 0.5 s, while 8192 scans were averaged for the spectrum of CSA filter coupled with dipolar dephasing, with a recycle delay of 0.5 s (Mao et al., 2011b). In all NMR experiments, during detection, two-pulse phase modulated (TPPM) decoupling was applied.

Please cite this article as: Song, G., et al., Characterization of transformations of maize residues into soil organic matter, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.11.169

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2.10. Principal component analysis Principal component analysis (PCA) was applied to NS content, AA content, C content, N content, δ13C values, OFRC, and relative intensity integrations of NMR spectra for those samples isolated from the maize amended and control soils. The OFRC of HAs pH 7 (maize amended soil) is used to evaluate the value of HAs pH 10.6 due to lack of the value for this fraction. The data set consisted of a matrix of fourteen variables, with each row representing each humic sample. A software package of SPSS Statistics (version 22.0, IBM, USA) was used, and the factor analysis in the SPSS was used for PCA calculation. The SPSS will normalize the raw data before the factor analysis. The primary component loading was calculated from the component loading in the component matrix by dividing by the square root of corresponding component's eigenvalue. For the NMR data, the scores of the first three PCs accounted for 40.1%, 30.8% and 9.8%, respectively, for percentages of the total variances. The scores of primary components were calculated from factor scores by multiplying the square root of the corresponding variance. 3. Results and discussion 3.1. Yields of SOM fractions The resin fractionation procedure allowed HAs, FAs, and XAD-4 acids to be isolated from the SOM. The total mass yields isolated accounted for about 1.44% of the total soil mass. Based on the conventional definition for HSs (Clapp et al., 2005), the HALM fraction would be in the humin category. However, once isolated from the soil it has the solubility criteria for HSs, and we tentatively describe this fraction as humic acid-like (HALM) or fulvic acid-like (FALM) materials. Because the HALM and FALM have the solubility characteristics of conventional HAs and FAs, these are often discussed in the context of the conventionally defined samples in this paper. The HAs isolated at pH 12.6 accounted for 25.4% of the total fractions isolated, and a further 23.7% (w/w) of the isolates arose from the base/ urea solvent (sum of FALM, HALM and XAD-4 acids). The HAs plus HALM, were the major contributors to the whole of the SOM extracted. The sum of the XAD-4 acids, which are composed mainly sugar- and amino acid-containing substances, and other polar materials, accounted for about 10.4% of the total mass yield. Although the HAs isolated at pH 12.6 was the major fraction (25.4% of the total fractions isolated), the fractions isolated by the base/urea solvent (sum of FALM, HALM and XAD-4 acids) accounted for 23.7% (w/w), and that was 1.4% more than was extracted from a Mollisol using the same procedure (Song et al., 2008). The HAs plus HALM, were the major contributors to the whole of the SOM extracted. The sum of the XAD-4 acids, which are composed mainly sugar- and amino acid-containing substances, and other polar materials, accounted for about 10.4% of the total mass yield. 3.2. Elemental analyses and δ13C values The elemental analyses, C/N ratios, δ13C values, and the maize contribution to the humic samples isolated from the amended and control (unamended) soils are shown in Table 1. The C and N contents are calculated on a dry, ash-free basis. The C contents of the fractions from the amended soil varied from 51% to 63%, and the N contents ranged from 3.2% to 5.8% and these values were invariably greater than those of the control. The average C contents of HAs and FAs samples (including HALM and FALM) at about 60% and 57%, respectively, are significantly greater than those from the control. The N content in HALM was the greatest, which might partially result from the interactions with the urea in the solvent. However, the N content of the FALM was lower than that of the FAs isolated at pH 12.6. Nevertheless, based on the average results for C/N ratios, the

Table 1 Elemental contents of C and N, C/N ratios, δ13C values and maize contribution to the humic fractions isolated from the maize amended and control soils. Samples

C (%)

N (%)

C/N ratio

δ13C ‰

Maize contrib. (%)a

HAs pH 7 HAs pH 10.6 HAs pH 12.6 HALM FAs pH 7 FAs pH 10.6 FAs pH 12.6 FALM HAs pH 7 control HAs pH 12.6 control FAs pH 7 control FAs pH 10.6 control

57.5 59.3 63.5 59.8 56.1 51.0 61.5 59.2 50.0 55.3 49.8 52.2

4.46 4.05 5.33 5.85 3.60 4.18 3.51 3.22 2.58 5.81 3.89 4.49

15.0 17.1 13.9 11.9 18.2 14.2 20.4 21.4 22.6 11.1 14.9 13.6

−20.19 −19.24 −17.97 −18.04 −22.40 −23.46 −20.58 −20.63 −28.42 −28.36 −27.85 −27.97

51.6 57.2 64.7 64.3 34.6 28.0 46.1 45.8

C and N contents were calculated on a dry, ash-free basis. Control = the unamended soil. a Maize contri = the contribution from maize organic carbons for each fraction was calculated from the Eq. (2).

ratio for the humic samples was lower than that for the control, whereas the C/N ratio of the fulvic samples was greater than that for the FAs in the control samples, (14.5 vs 16.8, 18.6 vs 14.2, respectively). In the case of the maize amended soil, the δ13C values of fractions were enriched in 13C, as expected (with the values ranging from − 18.04‰ to −23.45‰, and the average values for the HA and FA fractions were −18.86‰ and −21.77‰, respectively). In general, the 13C enrichment in the humic fractions increased as the pH of the extractant increased. That reflects the relative amounts of plant inputs in the less oxidized fractions isolated at the higher pH values. The δ13C of the HAs were more enriched in 13C than the corresponding FAs, and again that reflects the relative extents of biological transformations of the plant source materials. The δ13C values of HAs and FAs of the control soil were close, in the range of − 27.85‰–28.42‰ (Table 1), though the δ13C values of FAs were slightly more enriched in 13C than the HAs, (as was observed also for similar samples from other soils (Hayes et al., 2008). That might be attributable to selective microbial utilization of plant residues, or to a different bioavailability of the fractions. Fungal biomass is 13C enriched relative to plant biomass (δ13C values of Mycorrhizal fungi −26.5‰ to −24.0‰, Saprotrophic fungi −24.5‰ to −21.7‰). However, soil respiration (roots and microbial) is also 13C enriched; that implies the selective loss of the 13C of the SOM and the depletion of the 13 C of the SOM (Gleixner et al., 2005). FAs are considered to have significant inputs from microbial sources, and materials of plant origins to have greater inputs to HAs. According to the δ13C results, the general increase of the maize contribution to the extracts was as follows: (fractions isolated at) pH 7 b pH 10.6 b pH 12.6 ≈ base/urea. The materials isolated at the lower pH values reflect the most highly (negatively) charged and transformed components of SOM, and reflect the losses of biologically oxidizable organic carbon during the humification process. In other words, oxidation leads to depletion in 13C; therefore, greater transformations of the hydrophilic fraction results in lesser 13C enrichment. Our NS data (Table 3) strengthen that hypothesis. The hydrophilic fractions (XAD-4 acids) can be expected to be largely of microbial origins, as evidenced by the δ13C values of that fraction isolated from a grassland soil (Hayes et al., 2008). 3.3. EPR results The organic free radicals concentrations (OFRC) from EPR are given in Table 2. It is clear that the free radical contents of the FAs were inevitably less than those for the HAs. This is in line with the lesser aromaticity (as shown by NMR) and the phenolic or O-aryl functionalities of the FAs, as would be expected for the more highly oxidized cultivated soil. Because phenols oxidize to quinones via a free radical mechanism,

Please cite this article as: Song, G., et al., Characterization of transformations of maize residues into soil organic matter, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.11.169

5 a XAD-4 acids base/urea isolated from 0.1 M NaOH + 6 M urea; Neutral sugars: glucose (Glu), galactose (Gal), mannose (Man), rhamnose (Rha), fucose (Fuc), xylose (Xyl) and arabinose (Ara); Neutral sugars contents (as relative molar percentages of the total sugars).

7.8 7.5 8.2 7.3 4.3 14.4 12.6 12.6 2.8 5.4 5.4 2.8 6.3 2.6 3.3 1.7 1.7 1.7 1.8 1.4 1.6 1.3 1.3 1.3 0.9 0.9 2.4 1.7 3.0 2.5 0.2 0.2 0.2 0.2 0.4 0.3 0.2 0.2 0.7 0.6 0.6 0.4 0.4 0.4 0.5 0.5 0.5 0.5 0.5 1.1 1.0 0.5 0.5 1.8 0.9 0.9 1.1 1.3 1.8 1.6 42.3 69.8 44.5 45.3 51.1 72.0 57.5 67.6 135.6 202.3 223.1 40.5 78.7 53.9 51.3 25.8 42.6 27.1 27.6 30.3 42.7 34.9 41.0 79.6 120.8 133.3 24.0 47.8 32.9 31.2 18.1 17.6 17.2 16.9 19.2 24.7 21.9 21.5 18.6 19.3 18.9 14.7 15.9 16.8 16.7 12.9 12.9 12.9 12.9 18.3 16.4 13.2 13.1 22.0 15.3 15.2 19.5 25.4 21.6 23.9 11.0 11.2 11.3 11.5 15.4 15.2 9.4 9.5 17.1 14.6 14.8 17.3 15.4 21.6 17.8 32.8 32.4 32.2 31.6 18.9 19.3 27.5 27.1 11.7 17.9 17.6 21.3 18.5 13.6 14.0 17.0 17.7 18.4 19.1 13.2 13.6 19.6 20.5 9.9 14.4 15.0 13.2 13.0 10.3 12.4 1.7 1.7 1.6 1.8 4.2 1.1 1.0 1.0 8.0 2.8 2.8 6.9 4.0 8.4 7.2

Gal/Fuc Man/Rha (Fuc + Rha)/(Ara + Xyl)

6.6 6.6 6.5 6.4 10.8 9.7 7.3 7.3 12.7 15.6 15.6 7.1 8.8 7.1 7.2

The NS contents of samples isolated from the maize amended soil are presented in Table 3. In general, the contribution of total NS to the compositions of the fractions ranged from 42–72 μg mg−1. The amounts of total NS in the humic samples and HALM were less than those of fulvic samples and FALM, averaging 50– 62 μg mg− 1, respectively. These total NS contents for the HSs from the maize amended soils were similar to those for the control soil, and are significantly less than those of the HSs isolated from IHSS standard soils. The distribution of NS in the HAs isolated at pH 12.6 and in the HALM, in the FAs isolated at pH 12.6 and in the FALM are very similar. In the case of the maize-amended HSs samples, Xyl, Ara, Glu and Gal were most abundant, and in the order: Xyl N Ara N Glu N Gal. That reflects the sugar abundances in the maize amendment, in which hemicellulose is a major component of the stalks, and with Xyl composing at least half of the NS (Chen et al., 2008; Xiao et al., 2001). The abundance of Xyl is less evident for the compositions of the FAs from the amended soil, though it clearly dominated the NS compositions in the cases of the FAs pH 12.6 and the FALM, or the less transformed HS materials. Xyl was the major NS of the FA pH 7 extract from the control soil, whereas Gal and Man were the major sugar components of the FA pH 12.6 fraction. The XAD-4 acids fraction (resolved using the XAD-4 resin) was, as expected, the most abundantly enriched in sugars (Table 3) because it is the most hydrophilic of the components in SOM. The NS contents in these samples ranged from 135–223 μg mg−1. The ratio values further emphasize the prevalence of microbial synthesis for these fractions. Significantly higher ratios (Man + Gal)/(Ara + Xyl) ≤ 0.9–1.8 and (Fuc +

HAs pH 7 HAs pH 10.6 HAs pH 12.6 HALM FAs pH 7 FAs pH 10.6 FAs pH 12.6 FALM XAD-4 pH 7 XAD-4 pH 12.6 XAD-4 base/ureaa FAs pH 7 control FAs pH 12.6 control HAs pH 7.0 control HAs pH 12.6 control

3.4. Neutral sugars (NS) analyses

(Man + Gal)/(Ara + Xyl)

it can be expected that the greater the extent of humification the lesser will be the free radicals content. That reasoning holds for the FAs, but not for the HAs from the maize amended soil. The OFRC of humic and fulvic samples isolated from the maize amended soil ranged from 7.43 × 1016 to 2.62 × 1017 spin (g C)−1 and 1.80 × 1016 to 4.29 × 1016 spin (g C)−1, respectively, whereas the OFRC values of the HA and FA samples from the control soil ranged from 1.21 × 1017 to 2.08 × 1017 spin (g C)− 1 and 6.01 × 1016 to 7.97 × 1016 spin (g C)−1, respectively. The OFRC average values for the maize amended fulvic samples (including FALM) were lower than those from the control soils (3.31 × 1016 spin (g C)−1 vs 6.91 × 1016 spin (g C)−1, respectively), whereas the average of the humic samples (including HALM) were similar (1.60 × 1017 spin (g C)−1 vs 1.61 × 1017 spin (g C)−1, respectively). The greater abundance of free radicals in the humic samples isolated at pH 12.6 from this soil would support the view that the humification process is still active compared to the HAs isolated from the control at pH 12.6.

Total sugar (μg mg−1)

“−”: The samples were not tested.

Total sugar (nmol mg−1)

1016 1016 1017 1017 1017 1016 1016 1016

Glu

× × × × × × × ×

Gal

1016− 1017 1017 1016

Man

× × × ×

Xyl

− 7.43 2.62 1.44 4.29 – 3.83 1.80 1.21 1.54 2.08 6.01 7.97 6.74

Ara

HAs pH 7 HAs pH 10.6 HAs pH 12.6 HALM FAs pH 7.0 FAs pH 10.6 FAs pH 12.6 FALM HAs pH 7.0 control HAs pH 10.6 control HAs pH 12.6 control FAs pH 7.0 control FAs pH 10.6 control FAs pH 12.6 control

Fuc

Organic free radicals (spin·g−1)

Rha

Fraction

Sample

Table 2 Spin concentration of organic free radicals for fractions from the maize amended and the control soils.

Table 3 Neutral sugars contents (as relative molar percentages of the total sugars) and (Man + Gal)/(Ara + Xyl), (Fuc + Rha)/(Ara + Xyl), Man/Rha and Gal/Fuc ratios of the humic acids (HAs), fulvic acids (FAs) and XAD-4 acids and humic-like (HALM) and fulvic-like (FALM) materials from the maize amended and the control soils.

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Rha)/(Ara + Xyl) ≥ 0.6) for the XAD-4 acids isolated at neutral pH value reflect the importance of microbial synthesis. Microbial contributions to this fraction are greater for the XAD-4 acids isolated at pH 12.6 and for the base/urea solvent isolates. These trends are also revealed by the ratios for Man/Rha and Gal/Fuc (Table 3). 3.5. Amino acids (AA) analyses A total of 14 amino acids (AA) were quantitatively determined for the samples isolated (Table 4). Some S-containing AA (e.g. L-methionine) were not determined due to their very low concentrations in soil HS samples (Hayes et al., 2008). The amino acids can be divided into four major groups: total acidic (TA) (Asp, Glu); total basic (TB) (Arg, His, and Lys); total neutral hydrophilic (TNHi) (Thr, Ser, Gly, and Ala); and total neutral hydrophobic (TNHo) (Val, Ileu, Leu, Tyr, Phe). The data in Table 4 show that the AA contents of humic fractions ranged from 5.6%–6.0%, whereas those of fulvic fractions from 1.9%–2.4%; the AA contents of the HAs were normally 2.5– 2.9 times greater than those of the FAs. The AA contents in XAD-4 acids were 3.0%–3.9%, less than those for the HAs but more than those for the FAs. It is of interest to note that the AA contents of the HALM and of the FALM are much greater than those of the HAs or of the FAs (for example, the HALM content was 62% higher than that of HAs isolated at pH 12.6, and that for the FALM was 100% higher than that for FAs pH 12.6 isolate. That resulted might not be expected but it is likely that it reflects the extents to which the AA components were held in the soil by secondary forces. Generally, the contents of TA, TB, THo and THi AAs in the different humic fractions and in the HALM followed the order: TNHi ≫ TNHo N ≈TA N TB, while the order followed for the fulvic fractions and for the FALM was: TNHi N TA N TNHo N TB. In the case of HA or FA samples from the control soil, the trend followed the same order for FAs or FALM isolated from the maize amended soil. 3.6. FTIR spectra The FTIR spectra of HSs fractions from the incubated are shown in Fig. 1. Though the FTIR spectra of the HSs samples are normally very broad, they can provide some useful compositional information. The

spectra for the HAs and HALM are qualitatively similar, but some differences in detail are evident for the FAs and FALM samples. Based on assignments in the literature (Faix et al., 1989, Telysheva et al., 2007), the peaks at 1510 and 1463 cm− 1, and at 1270 and 1220 cm−1 indicate the presence of syringyl units and that evidence is also supported by the strong band at 1330 cm− 1 (C − O vibration syringyl units). An aromatic skeletal vibration (1509 cm−1), methoxyl groups (1465 cm− 1), C-N in amides, aromatic skeletal vibrations, or methoxyl C\\O stretch (1426 cm−1) are also observed. These three peaks were generally present in humic and fulvic fractions. The lignincarbohydrate complexes or lignocellulosic structures in HSs may be ascribed to C\\O stretch bands from ether or phenols at 1228 cm−1, C\\O stretching of carbohydrate at 1036 cm−1, as supported by the data for sugars in Table 3 and by the NMR spectra (discussed below). With regard to the HALM, the FTIR spectrum of the HAs isolated at pH 12.6 and that of the HALM are similar (Fig. 1). These show distinct bands at 1652, 1503, 1452, 1416, 1220, and 1036 cm−1. The higher intensities at 1510 cm−1 and at 1268 cm−1 may indicate predominant guaiacyl units in the samples. However, the spectra were not well resolved at 1240 cm−1, and that may be attributable to overlaps of signals from polysaccharides and lignin (from C\\O stretch and C\\H bends vibrations). The FTIR spectra of FAs isolated at pH 12.6 and the FALM are different. The bands at 1715 cm− 1 and at 1168–1030 cm− 1 for the FALM suggested that the absorption could be due to carbonyl functionality in undissociated COOH and COOR groups. There were distinct peaks in the ‘finger print region’ indicating strong contributions from lignin structures that originated from transformed maize residues: an aromatic skeletal vibration (1509 cm−1); C\\H deformation, partly in methoxyl groups (1465 cm−1); C\\N in amides, aromatic skeletal vibrations, or methoxyl C\\O stretch (1426 cm−1). These three peaks were generally present in humic and fulvic fractions. The ester carbonyl peak at 1714 cm−1 (shoulder), together with the shoulder at 1166 cm− 1 and the band at 835 cm− 1 may suggest the presence of etherified p-coumaric units in the lignin core. The lignin-carbohydrate complexes or lignocellulosic structures in HS may be ascribed to: the C\\O stretch bands from ether or phenols (1228 cm− 1), C\\H in-plane deformation from syringyl units (1120 cm− 1), and aromatic C\\H in-plane deformation or C\\O stretching of carbohydrate (1036 cm−1).

Table 4 Amino acids contents of humic acids (HAs), fulvic acids (FAs), XAD-4 acids and humic-like (HALM) and fulvic-like (FALM) materials from the maize amended and the control soils. Sample

HAs pH 7.0 HAs pH 10.6 HAs pH 12.6 HALM FAs pH 7 FAs pH 10.6 FALM XAD-4 pH 7 XAD-4 pH 12.6 XAD-4 base/urea HAs pH 7.0 Control HAs pH 12.6 Control FAs pH 7.0 Control FAs pH 12.6 Control

Acidic (TA)

Basic (TB)

Neutral hydrophobic (TNHo)

Neutral hydrophilic (TNHi)

Total AA Total (nmol mg−1) AA (%)

N (%)

Asp

Glu

Total acidic

Arg

His

Lys

Total basic

Val

Ile

Leu

Tyr

Phe

Total NHo

Thr

Ser

Gly

Ala

Total NHi

9.2 53.3 28.6 103.1 38.6 24.3 49.2 37.2 20.8

49.1 35.7 33.1 89.8 24.1 16.2 38.5 18.7 13.9

58.3 89.0 61.6 192.9 62.7 40.5 87.7 55.8 34.7

25.2 21.1 23.3 30.6 4.8 4.8 2.7 5.0 14.6

9.6 6.5 8.7 8.7 0 0.8 0 0 4.6

0 20.7 29.0 7.4 8.8 6.2 20.3 2.1 36.0

34.8 48.3 61.0 46.6 13.6 11.8 23.0 7.1 55.1

32 25.4 31.5 53.8 10.0 9.3 18.4 5.6 9.3

15.2 13.1 18.9 31.5 3.3 3.8 10.1 1.0 1.4

20.3 20.6 34.8 60.5 4.8 6.7 18.4 4.4 7.5

7.5 7.1 10.5 18.3 1.4 1.5 5.4 0 0.6

14.6 11.6 17.6 29.4 2.7 3.2 7.7 0.5 1.4

89.7 77.7 113.3 193.5 22.2 24.5 60.2 11.5 20.1

46.4 41.1 36.2 57.4 14.3 11.5 19.4 21.8 23.7

40.5 28.8 31.7 53.3 12.4 10.2 18.7 15.7 18.7

188.2 147.0 143.0 162.3 57.6 47.9 76.1 126.5 148.7

38.3 32.2 43.4 60.3 13.3 12.2 22.6 15.5 23.5

313.3 249.0 254.4 333.4 97.6 81.8 136.8 179.5 214.6

496.1 464.1 490.3 766.5 196.1 158.6 307.7 254.0 324.5

6.0 5.6 5.9 9.2 2.4 1.9 3.7 3.0 3.9

21.5 20.6 16.8 23.3 9.5 6.6 16.3 9.0 13.3

53.9

33.8

87.7

11.6 1.9

3.1

0.9

2.7

22.3

27.3 18.8 118.9 23.0

187.9

324.0

3.9

12.7

240.6

53.3 22.2 22.0 97.5

30.5 19.7 25.5 7.5

14.6 97.8

87.4 77.2 155.6 66.9

387.1

823.0

9.6

32.9

241.6 124.4 366.0

83.0 24.1 54.9 162.0

63.9 35.6 48.6 19.3 52.3 219.7

71.9 92.7 146.2 112.5 423.3

1171

14.6

39.7

192.9 141.4 334.3

31.3 7.2

33.5 72.0

30.5 17.3 21.9 5.5

10.4 85.6

75.4 78.7 99.8

828.9

10.1

38.9

234.3 153.9 388.2

60.1 9.4

39.8 109.3

61.7 31.9 39.0 8.1

22.2 162.9

116

1173.2

14.2

45.9

147.6 93.0

12.5 26.0

13.2 2.4

103

83.1

337.0

160.8 133.1 512.8

Amino acids contents expressed as relative molar percentages of the total amino acids contents; AA% refers to the mass percentage of the humic sample present as amino acids. Values for total AA% are evaluated on a dry and ash-free basis. Amino acids: Threonine (Thr), Serine (Ser), Glycine (Gly), Alanine (Ala), Aspartic (Asp) Glutamic (Glu), Valine (Val), Isoleucine (Ile), Leucine (Leu), Tyrosine (Tyr), and Phenylalanine (Phe), Arginine (Arg), Histidine (His), and Lysine (Lys). FAs pH 12.6 was not analyzed due to not enough amount. N% refers to the percentage of N in the sample which can be accounted for as amino acid nitrogen; XAD-4 = XAD-4 acids.

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Fig. 1. Infrared spectra of humic acids (HAs), fulvic acid (FAs), the HALM and FALM from the maize-amended soil and the HAs and FAs isolated from the control soil. HALM = Humic acidlike material; FALM = Fulvic acid-like material.

It is clear that the FTIR spectra of the humic samples from the maizeamended soil have signals that can be traced to the amending maize, when compared with spectra for the FAs and HAs isolated from the control soil. The signals for the FAs also display some contributions from lignin and polysaccharides, and although the lignin signals are distinct in FAs isolated at pH 12.6, these are not well resolved in the other fulvic samples. The C\\O stretches from polysaccharides (at 1222 and 1075 cm−1) are predominant for the FA pH 7 and FA pH 10.6 samples. These data that indicate enrichments from the amending maize are consistent with the δ13C values for the SOM fractions (Table 1), and indicate that significant amounts of the organic C of the maize residues had been transformed into HSs, and more than half of HAs, and about one third of FAs could have originated from the maize transformations. 3.7. Solid-state NMR data The 13C NMR spectra of the humic and fulvic samples include CP/ TOSS (Fig. 2A) and the corresponding editing spectra, CSA filter (Fig. 2B), dipolar dephasing (Fig. 2C) and the CSA filtered spectra after

dipolar dephasing experiments (Fig. 2D). The CP/TOSS 13C NMR spectra of FAs and HAs from the control soil and the corresponding editing spectra (CAS-filter and dipolar dephasing) are presented in Fig. 3. The data for the integration areas of the CP/TOSS 13C NMR spectra are given in Table 5. The FTIR spectra have provided evidences for lignin-derived materials in the humic and fulvic fractions isolated from the maize amended soil. That evidence is emphasized in the NMR spectra by: (1) The sharp peak at 55 ppm which can be ascribed to the methoxyl C of lignin, and this is confirmed by its presence after dipolar dephasing (Fig. 2C, i to l), and by the CSA filtered spectrum after DD (Fig. 2D, m to p): (2) The peak near 130 ppm, ascribed to aromatic C, could also have contributions attributable to the syringyl unit; (1) A forked peak (148 and 153 ppm), distinct in a and c of Fig. 2A, can be indicative of lignin-derived residues with the peak (or shoulder in the cases of b and d) at 148 ppm, possibly

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Fig. 2. The 13C NMR spectra of fractions of the maize amended soil: (A) 13C CP/TOSS NMR; (B) CSA filter; (C) dipolar dephasing; and (D) CSA after dipolar dephasing.

attributable to the C-3 and C-4 of guaicyl units, and with the 153 ppm resonance assignable to the O-aryl C (C-3 and C-5) in syringyl units (Balesdent et al., 1987; Hatcher, 1987).

mobile sp3 carbons of humic fractions can be obtained. This is important for discriminating between ketose and aldose sugars. 3.8. Aromaticity and the hydrophobicity (HB)/hydrophilicity (HI) ratios

3.7.1. CSA filtered spectra and DD spectra The distinct abundances of carbohydrate are evident in the spectra of all samples, as shown by the resonance at 72 ppm and that of the anomeric C at 103 ppm. The anomeric C is highlighted, especially in the CSA filtered spectra, e.g. and h in Fig. 2B. In the cases the CP/TOSS spectra for a, c, and d (Fig. 2A), the ratios of the areas of the O-alkyl C to those of the anomeric C resonances were, 2.4, 1.9, and 2.1, respectively, whereas these values were 6.1, 7.8, and 5.1, respectively in the cases of the data for the CSA filtered samples (e, g, and h, respectively, Fig. 2B). These values are close to the ratios for hexose (5:1). The CSA-filter technique is often used to separate the non-protonated aromatic from the sp3-hydridized O-C-O carbon (unprotonated ketal, or protonated acetal, anomeric C in sugar rings), and to overcome the overlapping between aryl C and alkyl C resonances around 90– 120 ppm in CPMAS spectra (Mao and Schmidt-Rohr, 2004; Xu et al., 2017). The size of the 13C CSA, which reflects carbon bonding symmetry, is different for sp2 hybridized aromatic and sp3-hybridized alkyl C. In using this technique, the aromatic C is dephased totally because of the decay under the influence of the recoupled CSA, whereas the O-C-O and other alkyl signals are retained. The CSA based dephasing is especially useful for distinguishing the anomeric C observed at ca. 103 ppm as shown in Fig. 2B (e–h). By means of a combination of the CSA filter and the DD technique sub-spectra of non-protonated and of

The hydrophobicity index (HB/HI) and aromaticity were determined to show the trend of changes of functional groups in the maize amended and the control soils. The sum of integrated areas of polar carbons [(44– 110 + 160–220)] ppm resonances (Table 5) represent the hydrophilicity (HI) parameter of the HSs, and hydrophobicity (HB) is related to the sum of areas of apolar carbons [(0–44 + 110–160)] ppm resonances (Piccolo, 2002; Simpson et al., 2002). The integrated area percentages of aromatic carbons (110–142) + (142–160) are considered to represent the aromaticity. The ratio values may be considered as a general indication of the potential reactivities of the HSs. For HAs and HALM, the HB/HI ratios were gradually decreased from 0.99 to 0.87; and the aromaticity (%) decreased from 30.1% to 27.1% (Table 5). Compared with the corresponding values for the control, The HB/HI values of the HSs from the maize-amended soil are generally greater. That indicates that the hydrophilicity of HSs increased following maize amendment and the aromaticity is decreased. The hydrophobicity and aromaticity followed the order: FAs b HAs; HAs base/ urea b HAs pH 12.6 b HAs pH 10.6 b HAs pH 7. The HB/HI index suggests that the HSs extracted from the maizeamended soil have greater hydrophilicity, with the FAs, as expected, having greater hydrophilicity than the HAs. The increased hydrophilicity of the HALM may be attributable mainly to the increase of methoxyl and of O/N-alkyl carbons. The contribution arises from the less humified maize residues extracted in the alkaline medium with 6 M urea. It is

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Fig. 3. The 13C NMR spectra of fractions of the control soil, (A) CP/TOSS 13C NMR, (B) CSA filter; (C) dipolar dephasing.

Table 5 Distribution (%) of the relative signal intensities of 13C CP/TOSS NMR spectra of fractions from the control and maize amended soils. Fraction (ppm) Alkyl-C (0–44)

Methoxyl, N-alkyl (44–62)

O/N-alkyl (62–90)

di-O-alkyl (90–110)

Aryl-C (110–142)

O/N-aryl (142–160)

Carboxyl, amide, ester (160–185)

Carbonyl (185–220)

HB/HIa Arom. (%)b

FAs pH 7 HAs pH 7 HAs pH 12.6 HALM FAs pH 7 control FAs pH 12.6 control HAs pH 7 control HAs pH 12.6 control)

18.3 15.2 17.0 15.5 17.8

16.4 16.6 19.6 20.4 6.3

19.6 16.3 16.5 18.1 17.5

8.0 8.9 8.5 8.5 6.9

17.1 18.6 18.3 18.2 17.9

7.8 11.5 8.9 8.9 8.8

10.5 11.0 9.4 9.2 21.4

2.3 1.8 1.9 1.2 3.5

0.89 0.99 0.93 0.87 0.80

24.9 30.1 27.2 27.1 26.7

16.6

9.1

29.4

7.9

11.5

5.1

17.0

3.3

0.50

16.6

16.7

6.3

12.3

5.4

22.0

9.6

20.1

3.5

1.02

31.5

24.7

8.8

16.6

5.7

18.5

7.3

16.0

2.4

1.02

25.8

HALM = Humic acid-like material. a HB/HI (hydrophobicity index): [(0–44 + 110–160)]/[(44–110 + 160–220)]. b Aromaticity (%) = (110–142) + (142–160).

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expected that the base/urea medium released additional hydrophilic humic components (of lower HB/HI value) entrapped in the “humin” matrix and recovered as HALM. 3.9. Principal component analysis PCA scores and loadings of NMR integration of major functional groups are presented in Figs. 4 and 5. Scores of PC1, PC2 and PC3 were obtained from total NS, total AA, carbon and nitrogen contents, δ13C values, OFRC, and relative intensity integrations of NMR spectra from the maize amended and control soils. The values were normalized. The scores clearly differentiate the humic samples from that of the control according to maize amendment. The greater scores were generally associated with the relative higher pH extraction. The scores clearly differentiate the humic samples isolated from the maize amended soil from those from the control soil. The higher scores were generally associated with the extracts from the greater pH extractants. The HSs from the maize-amended soil are clearly grouped whereas those from the control are relatively scattered. In Fig. 5, the PC1 (40.8%) is characterized mainly by loadings of carboxyl C, O/N-alkyl C, anomeric C (di-O-Alkyl), δ13C value, AA, NS, and negative loadings of methoxyl, aryl C, alkyl C, and C content. In another words, positive loading for carboxyl (including amide and esters), carbohydrate, negative loading for lignin-like substance and aliphatic carbon. The PC2 (34.8%) showed mainly a high positive loading for anomeric C in carbohydrate, and O/N-aryl in lignin-like substance, organic free radicals, as well as C and N contents, negative for carbonyl and aromatic C. The PC1 alone discriminated the control from the amended soil samples, whereas the PC2 discriminated the humic samples, probably those from the amended soil. It could be argued that the amended samples had more lignin and unaltered cellulose (based on anomeric C) (Mao et al., 2011b) and less cellulose oxidized to glucuronic acid functionalities (O-alkyl and carboxyl). As the loadings of the PC2 are positive for carbohydrates (O-alkyl and di-O-alkyl, and negative for aryl C in lignin-like substance, whereas the loading of O/N aryl (an indicator of presence of lignin-like structure) is positive. At the same time, the PC2 showed a high negative loading for alkyl groups, possibly mainly from amorphous polymethylene signals at around 30 ppm and methyl carbons at around 20 ppm as indicated by CSA filtered and the CSA filtered after dipolar dephasing NMR spectra (Fig. 2B and D).

Fig. 5. PCA loading of PC1 and PC2 for neutral sugars content (NS), amino acids content (AA), C%, N%, δ13C values, OFRC, and relative intensity integrations of NMR spectra for isolates from the maize-amended soil and the control soil.

The loadings of PC1 and PC2 indicated the important compositional influences of carbohydrate components (altered and unaltered cellulose) and of lignin-derived materials. Such could be typical of newly formed maize-derived HSs, and might be associated with the degree of humification; the greater the scores the greater the humification (Fig. 4).

4. Conclusions

Fig. 4. PCA scores (PC1, PC2, PC3) for fractions isolated from the maize amended and the control soils. Where, the maize amended, 1 = FAs pH 7, 2 = HAs pH 7, 3 = HAs pH 12.6, 4, HALM; and the control, 5 = FAs pH 7, 6 = FAs pH 12.6, 7 = HA pH 7, 8 = HAs pH 12.6.

The objective of this study was to observe the contributions to soil organic matter (SOM) of additions of maize (Zea mays) stover 12 months after incubation in a coarse sandy loam soil that had been under long term cultivation to wheat (Triticum aestivum) for N30 years. Humic substances (HSs) components were isolated on the basis of charge density differences at pH 7, pH 10.6, and pH 12.6 (0.1 M NaOH) and in 0.1 M NaOH + 6 M urea to isolate components held by secondary forces in the organic matter – soil mineral matrix. The base-urea system isolated an additional 24% of soluble material that would be regarded as humin in the classical definitions, but these extracts satisfy the classification requirements of humic (HAs) and fulvic (FAs) acids and are referred to as humic acid (HALM) and fulvic

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acid (FALM)-like materials. The non-amended soil was extracted and fractionated in the same way. Fractions were subjected to elemental, δ13C, neutral sugar (NS) and amino acids (AA) analyses, to Electron Paramagnetic Resonance (EPR), to CPMAS 13C NMR and NMR spectral editing techniques, and FTIR spectroscopies, and Principal Component Analysis (PCA) was applied to the data. The results have shown that: 1. Based on the abundance of natural 13C, the maize was transformed preferentially into the humic acids (HAs) rather than into fulvic acids (FAs) fractions. On an average, about 59% of the turnover C was contained in the HAs and 38% in the FAs. 2. The results of for neutral sugars (NS) and amino acids (AA) analyses discriminate the organic fractions from maize-amended soil significantly from those of the control soil. The xylose contents in NS analysis constitute about 19% to 33% of total monosaccharides of the fractions; this may suggest that components from hemicellulose material in maize residues transformed to newly formed humic substances. 3. FTIR, 13C NMR and NMR spectral editing techniques, such as CSA filter and dipolar dephasing experiments, indicate that the contributions of lignin material to the fractions increased with the steps of sequential extraction (isolates at pH 7 b pH 10.6 b pH 12.6 ≈ base/ urea). 4. The fractions isolated in the base/urea solvent system display characteristics of conventional humic acids and fulvic acids, which are released and recovered from entrapment in the humin matrix. According to the conventional definition, the HALM and FALM fractions would be components of humin. 5. The combination of results and their multivariate analyses have shown clearly that the contributions of lignin and of carbohydrate play key roles in the maize transformation process. The PCA analysis also indicates the contributions to the transformation products of carbohydrate and lignin-like materials. 6. The HSs extracted from the maize-amended soil have greater hydrophilicity compared with the control. A plot of PCA data emphasize the differences between the fractions from the two soils. Acknowledgments We thank Prof Schmidt-Rohr K and Dr Julieta Ferreira for their kind helps and contribution. We acknowledge the support from Science Foundation Ireland (SFI) (WRMDS1-SI2008 and GEOF833), the Environmental Protection Agency (EPA) Ireland (G2001S/CD-(3/3)), and the Irish Research Council for Science, Engineering and Technology (IRCSET). References Aguiar, N.O., Novotny, E.H., Oliveira, A.L., Rumjanek, V.M., Olivares, F.L., Canellas, L.P., 2013. Prediction of humic acids bioactivity using spectroscopy and multivariate analysis. J. Geochem. Explor. 129, 95–102. Balesdent, J., Mariotti, A., 1996. Measurement of soil organic matter turn over using 13-C natural abundance. In: Boutton, T.W., Yamasaki, S.I. (Eds.), Mass Spectrometry of Soils. Marcel Dekker, New York, pp. 83–111. Balesdent, J., Mariotti, A., Guillet, B., 1987. Natural 13C abundance as a tracer for studies of soil organic matter dynamics. Soil Biol. Biochem. 19, 25–30. Bayer, C., Martin-Neto, L., Mielniczuk, J., Saab, S.C., Milori, D.M.P., Bagnato, V.S., 2002. Tillage and cropping system effects on soil humic acid characteristics as determined by electron spin resonance and fluorescence spectroscopies. Geoderma 105, 81–92. Borůvka, L., Vacek, O., Jehlička, J., 2005. Principal component analysis as a tool to indicate the origin of potentially toxic elements in soils. Geoderma 128, 289–300. Chen, M., Zhao, J., Xia, L., 2008. Enzymatic hydrolysis of maize straw polysaccharides for the production of reducing sugars. Carbohydr. Polym. 71, 411–415. Clapp, C.E., Layese, M.F., Hayes, M.H.B., Huggins, D.R., Alimaras, R.R., Wilson, W.S., 1997. Natural abundances of 13C in soils and waters. In: Hayes, M.H.B. (Ed.), Humic Substances, Peats and Sludges. Health and Environmental Aspects. The Royal Society of Chemistry, Cambridge, pp. 158–175. Clapp, C.E., Hayes, M.H.B., Simpson, A.J., Kingery, W.L., 2005. Chemistry of soil organic matter. In: Tabatabai, M.A., Slarks, D.L. (Eds.), Chemical Processes in Soils. Soil Science Society of America, Madison, WI, pp. 1–150.

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Please cite this article as: Song, G., et al., Characterization of transformations of maize residues into soil organic matter, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.11.169