Spectroscopic characterization of humic and fulvic acids in soil aggregates, Brazil.

Agricultural management methods affect organic compounds in the soil. The aim of this study was to characterize humic and fulvic acids using infrared spectroscopy and nuclear magnetic resonance (NMR) in aggregates collected from areas under different crop and soil management systems. Soil samples were collected at depths of 0.00-0.10 m in a trial set up in 1989 with four management systems (no-till, no-till with chiseling every three years, disk harrowing and heavy disking) under two production systems (succession and rotation). In the laboratory, the soil was sieved and separated into two fractions: particle size of 2 mm or more, and less than 2 mm. From each of the samples an aliquot was extracted and the humic and fulvic acids purified for FTIR, UV-VIS and NMR-1H analysis. The results obtained showed that the structural characterization of the humic and fulvic acid fractions were linked to organic matter left on the soil surface, especially where the agricultural system involved species diversity. Aggregates ≥2 mm produce higher NMR, UV-VIS and FTIR spectral magnitudes in aromatic and aliphatic structures than those <2 mm. Aggregates collected from soil under no-till retained their aromatic and aliphatic chemical structures, resulting in higher spectral amplitude.

Introduction

pan class="Chemical">Pn>roblems caused by agriculturpan class="Chemical">al managemepan class="Chemical">nt can lead to exaggerated responses, which can cause soil loss and degradation, nutrient leaching and loss of organic matter, which result productivity decline (Bispo et al., 2017).

Soil organic matter (SOM) is one of the agepan class="Chemical">ntn>s responsible for stabilizing soil aggregates, supn>plying nutrients and maintaining soil microbiological balance (Huang et pan class="Chemical">al., 2010). SOM dynamics are driven by adding organic compounds, which are transformed as time progresses by a combination of chemical, physical and biological factors (Ver et al., 1999; Gude et al., 2012). These transformations occur as the organic matter decomposes, changing the size of the molecules, chemical composition, structure and functional groups, as well as the polyelectrolytic characteristics of the added material (Kononova, 1966; Stevenson, 1994; Loss et al., 2016).

In chemicpan class="Chemical">aln> terms, organic matter consists of three fractions of pan class="Chemical">humic substances (HS): pan class="Chemical">humin, fulvic acids and humic acids (Santos and Camargo, 1999). Each of these substances has a specific function in the soil: for example, fulvic and humic acids play a role in transporting cations between exchange organic-mineral complexes and also affect cation-exchange capacity (CEC) of organic origin (Senesi et al., 1994; Janos, 2003; Benites et al., 2010).

The pan class="Chemical">humic substancesn> evepan class="Chemical">ntually form between 80 and 90% of all SOM. They consist of heterogeneous molecular compounds containing different functional groups (Chen et al., 2002). They are dark in color, with no defined property, hydrophilic and stable, and their constituents can vary as follows: 40–60% C; 30–50% O; 0–4% N; 2–4% H; 0–2% S (Moraes et al., 2011).

pan class="Chemical">Humic substancesn> ipan class="Chemical">nteract with ions to form complexes of differepan class="Chemical">nt stabilities and structural characteristics (Clemente et al., 2013). All these characteristics can indicate the soil management stage in agricultural areas. Some authors have used humic substances to indirectly evaluate soil management quality based on the degree of SOM humification (Canellas et al., 2004, 2007; Rosa et al., 2005).

SOM resulting from organic residues boosts pan class="Chemical">carbonn> copan class="Chemical">ntepan class="Chemical">nt, depending on the existing structures, generally with carboxyl and aromatic bonds, and there is a drop in carbon associated with polysaccharides (Zech et al., 1997; Canellas et al., 2007). Thus, humic fractions tend to increase particle cohesion and aggregate stability, and are associated with the distribution of aggregate particle sizes and conservation of carbon in the soil (Huang et al., 2010; Loss et al., 2016). Examining the spectral characteristics of separate fractions of organic matter can provide important information on how to improve soil quality (Oades et al., 1987; Rigobello et al., 2017).

The use of agriculturpan class="Chemical">aln> managemepan class="Chemical">nt with species diversity significapan class="Chemical">ntly increases the organic matter content, thus acting effectively on the relationship between soil aggregates. As consequence, it changes the soil chemical structure, and humic and fulvic acids increase as part of soil quality (Sarkhot et al., 2007).

Differepan class="Chemical">ntn> managemepan class="Chemical">nt systems may affect the structurpan class="Chemical">al formation of humic and fulvic acids. This fact is the basis for the hypothesis that either the type of soil management and the aggregate particle size affect may the quality and spectra of humic substances. Thus, the aim of this study was to characterize humic and fulvic acids by means of infrared spectroscopy and nuclear magnetic resonance in aggregates collected from soils under different management and cropping systems.

Material and methods

Experimental area

Samples were collected in February, 2018, at location 23°11′ S, 51°11′ W (Brazil) in a dystroferric Red Latosol (Oxisol) area. The area was split up into fully randomized expn>erimen>an class="Chemical">ntal units of 225 m2 (7.5 m × 30 m), with four replicates per treatment. The experiment was divided in four agricultural management types (no-till; no-till chiseled every three years; disk harrowing; heavy disking) and two production systems (rotation and succession crops).

The following soil managemepan class="Chemical">ntn> systems were studied: No-Till (pan class="Chemical">NT), in which seeds are sown in the waste of the previous crop by simply opening a narrow furrow; No-till, chiseled (NTC) with winter chiseling every 3 years using a chiseler with five tines spaced at 0.35 m, working to an average depth of 0.25 m; Disk Harrowing (DH), in which the soil is prepared every year before summer cropping to an average depth of 0.20 m, followed by leveling harrowing; Heavy Disking (HD) in which the soil is plowed to an average depth of 0.15 m followed by light harrowing prior to summer planting. Under the DH and HD systems, this procedure is also implemented prior to winter cropping. All soil management systems were assessed with crop rotation and soybean/wheat succession. Under NTC, the most recent chiseling operation had been carried out three years prior to the evaluation.

At the beginning of the tripan class="Chemical">aln>, plots were sown with wheat and winter cover (white lupin or black oats) in April. Summer crops (pan class="Species">soybean and maize) were sown in November.

Soil collection and characterization

Soil samples were collected at den class="Chemical">pths of 0.0–0.10 m, then air dried and sieved through a 2 mm mesh ready for chemical ann>an class="Chemical">alysis and clay content evaluation, following the methods described in Pavan et al. (1992) and Claessen (1997) (Table 1). The following parameters were determined: potential acidity (H + Al) by SMP; Ca; Mg and Al extracted by 1N KCl and titrated with EDTA (Ethylenediamine tetraacetic acid) and NaOH; P and K by Melhich-1 extraction and spectrophotometry at 630 nm (P) and flame photometry (K); and carbon by oxidation with Cr2O72- and titration with FeSO4. Clay content was determined in 20 g of fine oven dried soil using the pipette method with slow stirring (180 rpm) for 16 h and 1N NaOH as a chemical dispersant. After drying, the material was weighed and the following formula used: Clay (%) = ((Weight or mass of clay x 500) – 2).

Table 1

Chemical properties and clay-content evaluated after 26 years of trials.

Depth	Mgmt.	Clay	C	H + Al	pH	Ca	Al	Mg	P	K
0.00–0.10 m	HD	786	12.0	4.87	4.76	4.15	0.08	1.85	22.8	0.65
	DH	780	11.7	5.46	4.64	3.92	0.07	1.49	17.3	0.56
	NTC	742	12.1	5.58	4.73	4.11	0.10	1.85	37.9	0.74
	NT	710	16.6	4.22	5.26	5.36	0.01	2.56	38.5	0.94

Depth in m. pH determined by CaCl2; H + Al determined by SMP; Ca, Mg and Al extracted by KCl; P and K extracted by Mehlich-1; C extracted by K2Cr2O2; and clay determined by the slow stirring method. H + Al; Ca; Mg; Al; K in Cmolc dm−3; P in mg dm−3; C in g kg−1; Clay in g kg−1. Mgmt.: Manegement; HD: heavy disking; DH: disk harrowing; NTC: no-till chiseled; NT: no-till.

Chemicpan class="Chemical">al propn>erties and clay-con>an class="Chemical">ntent evaluated after 26 years of tripan class="Chemical">als.

Depth in m. pH determined by pan class="Chemical">CaCl2; H + n>an class="Chemical">Al determined by SMP; Ca, Mg and Al extracted by KCl; P and K extracted by Mehlich-1; C extracted by K2Cr2O2; and clay determined by the slow stirring method. H + Al; Ca; Mg; Al; K in Cmolc dm−3; P in mg dm−3; C in g kg−1; Clay in g kg−1. Mgmt.: Manegement; HD: heavy disking; DH: disk harrowing; NTC: no-till chiseled; NT: no-till.

Aggregate particle size

The aggregates were sieved through a 2 mm mesh to separate particles ≥2 and <2 mm.

Humic substance extraction and purification

The method used to extract the pan class="Chemical">humic substancesn> was as described in Giovanela et pan class="Chemical">al. (2004) and Lamar et pan class="Chemical">al. (2014), with some adaptations. Five grams of sampled material were washed in 0.145 mol l−1 HCl for an hour with mechanical stirring in order to eliminate any minerals present. The suspension was then left for 24 h at 4 °C. The supernatant was separated by centrifuging at 5000 rpm for 20 min. The precipitate was washed in 0.113 mol l−1 NaOH for 4 h under an inert atmosphere of N2 and stirred constantly. The samples were then left for 24 h at 4 °C. The supernatant was separated from the precipitate by centrifuging. The alkaline extract was acidified by adding 6.034 mol l−1 HCl to obtain a pH of approximately 1.203, and the humic (HA) and fulvic (FA) acids separated. The acids extracted were left for 24 h at 4 °C and then centrifuged.

The HA precipitate was washed in 0.141 mol l−1 pan class="Chemical">HCl/0.324 mol l−1 HF (n>an class="Chemical">hydrofluoric acid) for 12 h with stirring. The suspension was washed in deionized water until the quantity of Cl− dropped, as verified by 0.142 mol l−1 AgNO3. The humic acids were dissolved in 0.113 mol l−1 NaOH and eluted using a column packed with Amberlite IRA 200 resin, and the extract frozen and then lyophilized.

The supernatan class="Chemical">pan class="Chemical">nt copn>an class="Chemical">ntaining fulvic acids was eluted a number of times using Supelite XAD-8 resin, previously purified and acidified with 0.145 mol l−1 HCl. The fulvic acids were adsorbed and the column washed with deionized water to eliminate any salts present and eluted in 0.113 mol l−1 NaOH. The elutant was run several times through a column of Amberlite IRA 200. The concentrate was frozen and then lyophilized.

Characterization of samples the humic and fulvic acids

Fourier transform infrared Spectroscopy

The purified, lyophilized samples were prepared in tablets and compressed with KBr for anpan class="Chemical">alysis. The FTIR spn>ectra at 400 to 4000 cm−1 were obtained using a Bruker Vertex 70 FTIR spn>ectropn>hotometer with n>an class="Chemical">Platinum ATR (attenuated total reflection) module. A resolution of 4 cm−1 and 10 scans were used to obtain the 10 spectra, which were analyzed using Origin software (5.0, 2007).

1H nuclear magnetic resonance

Five hundred microliters of pan class="Chemical">deuterium oxiden> (pan class="Chemical">D2O-99,9%, Sigma Aldrich®) were added to 100 mg of the purified, lyophilized sample. The spectra were obtained at the spectroscopy laboratory (SPEC) of the State University of Londrina using a Bruker Avance III spectrometer set to 400 MHz for 1H, with 5 mm multinuclear probes. The zg30 program was used for 1H, solvent suppression at 4.7 ppm with zgpr. Number of scans 64000 for 1H.

UV/VIS Spectrophotometry

In the AJX-1600 spectrophotometer, two milligrams of purified sample were added to 10 ml 0.05 mol pan class="Chemical">NaHCO3 and the E4/E6 ratio determined by reading 465 and 665 nm absorbance in order to determine the degree of aromaticity.

Results and discussion

FTIR spectroscopy

The FTIR spectra for pan class="Chemical">humic and pan class="Chemical">fulvic acids extracted from aggregates of ≥2 mm or <2 mm are shown in Figures 1 and 2, and Table 2 gives absorption bands for each component according to Stevenson (1994); González Péres et al. (2004) and Moraes et al. (2011). The spectra of the main chemical structures (OH stretch; C=O; C=C aromatic; C-H and polysaccharides) are similar (Figures 1 and 2). This is due to the complex composition of humic and fulvic acids and the diversity of kinds of organic matter from which they are formed (Moraes et al., 2011).

Figure 1

Wave numbers in the middle infrared with Fourier Transforms (FTIR) of fractions of humic acids for the following treatments: NT- No-till; NTC - No-till, chiseled; DH - Disk harrowing; HD - Heavy disking; R - crop rotation; S - crop succession. (A) humic acid fractions (HAF) extracted from aggregates <2 mm under crop rotation; (B) HAF extracted from aggregates <2 mm under crop succession; (C) HAF extracted from aggregates ≥2 mm under crop rotation; (D) HAF extracted from aggregates ≥2 mm under crop succession.

Figure 2

Wave numbers in the middle infrared with Fourier Transforms (FTIR) of fractions of fulvic acids for the following treatments: NT- No-till; NTC - No-till, chiseled; DH - Disk harrowing; HD - Heavy disking; R - crop rotation; S - crop succession. (A) Fulvic acid fractions (FAF) extracted from aggregates < 2mm under crop rotation; (B) FAF extracted from aggregates < 2mm under crop succession; (C) FAF extracted from aggregates ≥2 mm under crop rotation; (D) FAF extracted from aggregates ≥2 mm under crop succession.

Table 2

Proposed attributions for wavelengths (cm−1) of FTIR spectra.

Wavelengthcm−1	Proposed attribution
3700–3620	Free OH stretch
3400–3200	OH stretch, inter- and intra-molecular N-H, amine NH and phenol OH
1660–1630	Amide C=O stretch; quinone C=O and/or C=O bonded to H in paired ketones; C=C aromatic
1600–1580	C = C aromatic group stretching
~1430	Stretching of methyl and methylene C-H bonds
1400–1380	Aliphatic C-H stretch
<1000	C-O stretches; aromatic C-H corresponding to polysaccharides and carbohydrates

Stevenson (1994); González Péres et al. (2004) and Moraes et al. (2011).

Wave numbers in the middle infrared with Fourier Transforms (FTIR) of fractions of pan class="Chemical">humic acidsn> for the following treatmepan class="Chemical">nts: pan class="Chemical">NT- No-till; NTC - No-till, chiseled; DH - Disk harrowing; HD - Heavy disking; R - crop rotation; S - crop succession. (A) humic acid fractions (HAF) extracted from aggregates <2 mm under crop rotation; (B) HAF extracted from aggregates <2 mm under crop succession; (C) HAF extracted from aggregates ≥2 mm under crop rotation; (D) HAF extracted from aggregates ≥2 mm under crop succession.

Wave numbers in the middle infrared with Fourier Transforms (FTIR) of fractions of pan class="Chemical">fulvic acidsn> for the following treatmepan class="Chemical">nts: pan class="Chemical">NT- No-till; NTC - No-till, chiseled; DH - Disk harrowing; HD - Heavy disking; R - crop rotation; S - crop succession. (A) Fulvic acid fractions (FAF) extracted from aggregates < 2mm under crop rotation; (B) FAF extracted from aggregates < 2mm under crop succession; (C) FAF extracted from aggregates ≥2 mm under crop rotation; (D) FAF extracted from aggregates ≥2 mm under crop succession.

pan class="Chemical">Proposed attributions for wavelengths (cm−1) of FTIR spn>ectra.

Stevenson (1994); González pan class="Chemical">Péres et pan class="Chemical">al. (2004) and Moraes et pan class="Chemical">al. (2011).

Wavelengths in the 3700–3620 cm−1 band denote the appearance of OH, alcohols and free n>an class="Chemical">phenols, which can be seen for all treatments in humic acids (Figure 1), but in lower incidence in fulvic acids (Figure 2). These bands can denote impurities associated with minerals or water, or show the complexity of the chains in humic acids (Stevenson, 1994) resulting from the kind of management system applied, and humic acids are more recalcitrant than fulvic acids.

In pan class="Chemical">all pan class="Chemical">humic and pan class="Chemical">fulvic acid spectra, there are wide bands between 3400 and 3200 cm−1, indicating COOH stretch on H bridges. The strength of intra-molecular bonds is related to stretching and widening: the greater the width the higher the number of bridges and the more acid the material (Oliveira Dias et al., 2009).

In the pan class="Chemical">humic acid fractions (pan class="Disease">HAF) of aggregates ≥2 mm (Figure 1) under no-till with pan class="Disease">crop rotation and fulvic acid fractions (FAF) in the no-till chiseled there was a greater intensity, and under disk harrowing with succession a lower intensity of peaks in FAF, which could be correlated with the management systems and cropping in the area. Dobbss et al. (2009) observed intense bands in HA than in FA, corroborating the spectra found in this study. These authors also state that narrow bands denote a low degree of humic substance association.

For pan class="Chemical">aln>l treatmepan class="Chemical">nts, aromatic characters were observed (mainly in pan class="Chemical">humic acids) in the 1580 cm−1 region. This aromatic region indicates less labile structures in humic acids, in contrast to fulvic acids extracted from aggregates ≥2 mm, where these bands were not found, showing that these compounds were not predominant. In fulvic acids extracted from aggregates <2 mm, and humic acids extracted from aggregates < and ≥2 mm, the carbon is retained in the soil, even under the effects of waste decomposition in more intense management systems and the presence of biological activity. Working on isolated soil humic acids, alginites, lignites, organic compounds and commercial chemicals, Pospíšilová et al. (2015) found similar values and stated that the characteristics of humic acids are linked to soil type and origin from management, as well as the kind of organic matter input and this organic matter is degraded. Exposure to enzymatic attack increases humification, therefore more molecular structures can be observed in humic acids (Figures 1 and 2). Biological activity is essential for chemically degrading organic matter (Silva et al., 2014). Thus, chemical forms are more marked in the aromatic region.

Low-ipan class="Chemical">ntn>ensity bands were observed at 1645 cm−1, corresponding to C=O stretches in n>an class="Chemical">carboxyl groups. This wavelength was found under all treatmepan class="Chemical">nts. However, comparing bands at shorter wavelengths, it can be seen that, irrespective of the treatment, humic acids contain more C=O, -COO-, ketone and polysaccharide group molecules, due to the amount of peaks present.

In the pan class="Chemical">fulvic acidn> fraction extracted from aggregates <2 mm, there were neither N-H stretch bands nor C=N stretch at 1430 cm−1. However, these bands were observed in aggregates ≥2 mm, and could indicate the presence of methyl and methylene groupn>s. The solubility of n>an class="Chemical">fulvic acids could have affected absorption in these bands (fulvic acids are less soluble and easier to remove), indicating the presence of lignin at this amplitude, therefore eliminating the amplitude of 1430 cm−1. With aggregates breakdown and soil drying, exposing organic compounds, decomposition increases in the aerobic environmepan class="Chemical">nt and the humification rate, causing bands of lower peaks for some treatments (DiDonato et al., 2016).

Bands related to pan class="Chemical">aln>iphatic C-H stretching were observed in the 1380 cm−1 band, with high intensity of fulvic acids extracted from aggregates < and ≥2 mm for all the treatments evaluated, except DH-S for aggregates ≥2 mm, in which there was a low incidence. This low incidence could be linked to the breakup of the aggregates and exposure of organic matter to the environment, preventing reads in the spectrum. Working with humic substances in soil under degraded pasture, Santana et al. (2011) observed the same wavelength, but at lower intensity, showing that degradation influences wavelength. The use no-till and rotation crops have positive effect in C-H stretching in fulvic and humic acids fractions.

For pan class="Chemical">humic acidsn> extracted from aggregates ≥2 mm under crop succession and extracted from aggregates <2 mm under cropn> succession and rotation, there were spn>ecific peaks at wavelengths below 1000 cm−1, correspn>onding to CO stretching in n>an class="Chemical">polysaccharides, and aromatic or out-of-plane C-H. However, for fulvic acids there were few of these peaks under both treatments (Figure 2). Since fulvic acid fractions are less stable, they show peaks at low incidence in the region below 1000 cm−1 in aggregates both larger and smaller than 2 mm, obtained under the crop rotation and succession conditions evpan class="Chemical">aluated.

Vibrations between 1090-995 cm−1 could pan class="Chemical">aln>so be due to pan class="Chemical">aluminosilicate impurities (Si-O stretch) (Madejová and Komadel, 2001) that, during the extraction process, were not completely removed from the pan class="Chemical">humic acids (Pernet-Coudrier et al., 2011). In this case, ascribing these bands to aluminosilicate impurities is based on several factors, including the additional presence of bands at 890 cm−1 (AlAlOH stretch) and 530 cm−1 (Al-O-Si stretch) (Rodríguez et al., 2016).

1H nuclear magnetic resonance

Differences in the spectrn class="Chemical">pan class="Chemical">al ipan class="Chemical">ntensities were observed in line with the diversity and activity of the soil management systems (Figures 3 and 4). Peaks were interpreted based on Rumjanek (2005), Khan et al. (2006) and Rodríguez et al. (2016). The region close to 5 ppm, corresponding to D2O. Although there are similarities in the chemical structures shown by the spectra, peak magnitudes do vary.

Figure 3

¹H NMR spectra for humic acid fractions extracted from aggregates ≥2 mm under different management systems. NT: no-till; NTC: no-till, chiseled; DH: disk harrowing; HD: heavy disking; R: rotation; S: succession.

Figure 4

¹H NMR spectra for humic acid fractions extracted from aggregates <2 mm under different management systems. NT: no-till; NTC: no-till, chiseled; DH: disk harrowing; HD: heavy disking; R: rotation; S: succession.

¹H NMR sn class="Chemical">pectra for humic acid fractions extracted from aggregates ≥2 mm under differen>an class="Chemical">nt management systems. NT: no-till; NTC: no-till, chiseled; DH: disk harrowing; HD: heavy disking; R: rotation; S: succession.

¹H NMR sn class="Chemical">pectra for humic acid fractions extracted from aggregates <2 mm under differen>an class="Chemical">nt management systems. NT: no-till; NTC: no-till, chiseled; DH: disk harrowing; HD: heavy disking; R: rotation; S: succession.

Figure 3 shows more pan class="Chemical">aln>iphatic structure than aromatic in n>an class="Chemical">humic acids fractions, with peak distribution between 0 and 4 ppm. Methyl (CH3-R) and methylene (R-CH2-R) were observed for all treatmepan class="Chemical">nts, but with greater intensity under NT and NTC with crop rotation and succession (Figure 3). Clement et al. (2013) point out that these structures are typical of molecules which have high and low molecular weight and form part of the polymer lipids, cutin and suberin.

The same structure (CH3-R) was found in the pan class="Disease">HAFn> extracted from aggregates <2 mm, but with lower ipan class="Chemical">ntensity under pan class="Chemical">NTC-R, NTC-S, DH-R and HD-R. The soil under NT, however, showed the highest intensities for aliphatic chains (0–4 ppm) (Figure 4). This result indicates that the diversity of species grown on the land boosts the presence of polysaccharides from the cell walls, formed by chains of polysaccharides, proteins and phenolic compounds, which can be very resistant to decomposition. The fraction of humic acids extracted from aggregates ≥2 mm, resulted in higher intensities of aliphatic and aromatic chains. This aggregate particle size could help conserve intra and inter-aggregate organic material, preventing its rapid decomposition. It could, therefore, be indicative of structural changes in systems in which the aggregates are broken down during soil preparation and in which agricultural systems like rotation are used, and the quality of the material depends on recalcitrant organic matter. Khan et al. (2006) found similarity between 1H spectra for all areas incubated with Oryza sativa, Quercus dendata and Cryptomeria japônica.

In the pan class="Chemical">humic acidn> fraction (pan class="Disease">HAF) from aggregates <2 mm, the spectra obtained were similar for all treatments (Figure 4). NT-R, NT-S and of the spectra for DH-S and HD-S showed similar magnitudes; the same was true of NTC-R; NTC-S; DH-R and HD-R. Working with organic soils, Enev et al. (2014) observed the presence of aliphatic and aromatic structure in soils with organic compounds. This corroborates the values in Table 3, especially for NT in which the crop waste is left on the soil surface. Tadini et al. (2015) report that the cell walls contain cutin and suberin, resistant aliphatic biopolymers that are derived from lignin, cellulose and hemicellulose, and affect the 1H NMR spectra, mainly in no-till.

Table 3

Ratio of E465 to E665, (E4/E6) obtained by UV/VIS spectrophotometry for different management systems in humic and fulvic acid fractions extracted from aggregates < and ≥2 mm.

E4/E6 ratio
	HAF		FAF
System	≥2 mm	<2 mm	≥2 mm	<2 mm
NT-R	7.0	15.3	9.9	6.8
NT-S	4.3	15.0	11.2	6.0
NTC-R	8.5	42.0	7.7	7.1
NTC-S	4.6	29.0	9.2	15.3
DH-R	11.4	19.0	5.3	9.5
DH-S	8.7	13.3	6.3	14.6
HD-R	18.0	17.3	14.3	5.6
HD-S	11.7	11.6	14.0	5.9

NT: no-till; NTC: no-till, chiseled; DH: disk harrowing; HD: heavy disking; R: rotation; S: succession.

Ratio of E465 to E665, (E4/E6) obtained by UV/VIS spectrophotometry for differepan class="Chemical">nt managemen>an class="Chemical">nt systems in humic and fulvic acid fractions extracted from aggregates < and ≥2 mm.

n class="Chemical">n class="Chemical">NT: no-till; pn>an class="Chemical">NTC: no-till, chiseled; DH: disk harrowing; HD: heavy disking; R: rotation; S: succession.

Table 3 shows the E4/E6 ratio for absorbance between 465 and 665 nm that defines the degree of aromaticity in pan class="Chemical">humic substancesn>. According to Rumjanek (2005), the higher the E4/E6 ratio, the higher the degree of aromaticity. The vpan class="Chemical">alues in Table 4 corroborate with the spectrometry results. The degree of aromaticity is dependepan class="Chemical">nt on the humification of humic substances and represented by a low E4/E6 ratio, showing that little humificated residue is present (Saab and Martin-Neto, 2007).

Rigobello et pan class="Chemical">aln>. (2017) reported that the E4/E6 ratio is influenced by molecule size, environmen>an class="Chemical">ntal pH, levels of pan class="Chemical">oxygen, carbon and carboxyl groups, and the age of the humic material. This affects the formation of humic substances, which, according to Stevenson (1994), occurs through three mechanisms: a) microbiological activity releases nitrogenated compounds that react with lignins; b) oxidation of polyphenolic compounds to form quinones that are condensates in reactions with aminated compounds, and c) reduction of sugars that react with aminated compounds and change into macropolymers of dark in color and with high molecular mass. Microorganisms play a fundamental role in forming the structures of HAF and FAF, releasing compounds and enzymes, in addition to degrading lignin, which binds the chains (Silva et al., 2014).

The differepan class="Chemical">ntn> E4/E6 ratios and spectra observed according to aggregate particle size could be related to the quan>an class="Chemical">ntity of organic matter incorporated into the soil, such as lignins, pan class="Chemical">carbohydrates, lipids, polysaccharides, etc. These compounds are degraded and form complexes such as lignin-carbohydrate that is amphiphilic and has a tendency to form aggregates, especially mycelial aggregates (Canellas et al., 2007).

Another poin class="Chemical">pan class="Chemical">nt to be considered is that the E4/E6 ratio is related to aggregate aromatic condensation and the copan class="Chemical">ntent of phenol and benzene-carboxyl groups (Rubenacker et al., 2012). A low E4/E6 ratio suggests that humic substance particles are large with high molecular masses and degrees of humification (Traversa et al., 2014) but it depends on the type of organic matter to be inserted into the agricultural system. The degree of humification could have affected the fulvic acid fraction (FAF), whereas the humic acid fraction contains highly humificated organic compounds that are resistant to degradation, and therefore tend to persist for longer. Thus, management systems like NT directly influence the various structural compounds. The length of the trial and the diversity of the species grown could be another factor that influenced the structures, with lower impact on humic acids and higher on fulvic acids.

pan class="Chemical">Fulvic acid fractionsn> were similar (Figures 5 and 6). Under pan class="Chemical">NTC-R, pan class="Chemical">DH-S and HD-S, they were chemically more aromatic, corroborating the values in Table 3, with peaks between 7 and 9 ppm. E4/E6 ratios and magnitudes are related to the formation process and the structural chain, since fulvic acids are more soluble and tend to be rapidly degraded.

Figure 5

¹H NMR spectra for fulvic acid fractions extracted from aggregates ≥2 mm under different management systems.

Figure 6

¹H NMR spectra for fulvic acid fractions extracted from aggregates <2 mm under different management systems.

¹H NMR spectra for pan class="Chemical">fulvic acid fractions extracted from aggregates ≥2 mm under differen>an class="Chemical">nt management systems.

¹H NMR spectra for pan class="Chemical">fulvic acid fractions extracted from aggregates <2 mm under differen>an class="Chemical">nt management systems.

The pan class="Chemical">1Hn> NMR spectra for n>an class="Chemical">fulvic acids extracted from aggregates <2 mm were very similar under NT-R and S; pan class="Chemical">NTC-R and S; DH-R and HD-R. The influence of various rotated crops and their decomposition is clearly visible, since the magnitudes of spectra are between 7 and 9 ppm, showing the aromatic nature of the structures. These spectral magnitudes correlate with the values in Table 3.

In the light of the silviculture work carried out by Moraes et pan class="Chemical">aln>. (2011), a number of hypotheses can be considered regarding the formation of aliphatic and aromatic structures. Under no-tillage systems, the organic matter cycle tends to be constant throughout a given period, favoring the formation of labile C chains in the pan class="Chemical">fulvic and humic acid fractions. Therefore, the aromatic nature of the fulvic acids could be related to exposure of the material to microbial attack due to the disruptions of the soil, boosting oxidation of the organic matter and forming more labile organic structures.

According to Gonzáles-pan class="Chemical">Pn>éres et pan class="Chemical">al. (2008), some chemicpan class="Chemical">al structures, such as aromaticity, could be related to the fresh organic matter or the slow process of decomposition on superficial horizons. However, for aliphatic structures, increments of lignin directly influence these structures.

Ikeya et pan class="Chemical">al. (2015) reported that n>an class="Chemical">humic substances, especially the pan class="Chemical">humic acid fraction, are a mixture of many molecules that can vary according to the origin, age and degree of humification. It is difficult to establish a structural parameter for humic substances in view of environmental influences.

Conclusion

No-till soil managemen class="Chemical">pan class="Chemical">nt systems pn>roduced pan class="Chemical">aliphatic chemicpan class="Chemical">al structure and the no-till method maintained the stability of the chemical structures in the soil and increased the magnitude and amplitude of the 1H NMR and FTIR spectra.

Aggregates ≥2 mm conserved more organic matter, therefore copan class="Chemical">ntn>ain a greater diversity of chemicpan class="Chemical">al compounds.

Both pan class="Chemical">humicn> fractions presented aliphatic and aromatic character but with greater represepan class="Chemical">ntativeness in humic acids and aggregates ≥2 mm. The diversity of the crop species grown in the trial area influenced the structure of HAF and FAF, especially in the presence of open-chain structures, helping to increase the magnitude of spectral peaks.

Declarations

Author contribution statement

W. Machado: Conceived and designed the experimen class="Chemical">pan class="Chemical">nts; pan class="Chemical">Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.

J. C. Franchini: Conceived and designed the experimen class="Chemical">pan class="Chemical">nts; pan class="Chemical">Performed the experiments; Contributed reagents, materials, analysis tools or data.

M. Guimarães: Conceived and designed the experimen class="Chemical">pan class="Chemical">nts; Anpn>an class="Chemical">alyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.

J. Tavares Filho: Conceived and designed the experimen class="Chemical">pan class="Chemical">nts; pan class="Chemical">Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Funding statement

This work was supported by .

Competing interest statement

The authors declare no conflict of ipan class="Chemical">nterest.

Additional information

No additionpan class="Chemical">al information is available for this paper.