3. Results and discussion

3.1. Elemental composition

The soils analyzed showed varied HM contents ranging from 37.09 to 65.05% of TOC (Table 2), which confirms the great diversity of the soils analyzed. These data are in good agreement with results obtained by other authors (Grasset and Ambles, 1998; Zhang et al., 2019), highlighting the large share of the HM in SOM. The elemental composition of the HM is presented in Table 3. The ash content in the HM ranged from 22.89 to 54.50%, which is within the typical range for mineral soils (Stevenson, 1994; Tan, 2014) and was probably due to the different strengths of organo-mineral bonds, nevertheless confirming a strong association of the HM with mineral colloids probably related to the processes of their stabilization in the soil (Hayes et al., 2017; Pham et al., 2021; Simpson and Johnson, 2006; Tan, 2014; Zhang et al., 2015). The ash content was not correlated with clay fraction (< 0.002 mm) nor with TOC, indicating that its share depends mainly on the strength of organo-mineral bonds, protecting the mineral fractions from destruction during the treatment of the sample with HF/HCl (Weber et al., 2022).
The content of elements varied in the range of 42.02–58.86% for C, 3.88–4.77% for H, and 2.52–3.68% for N, and this affected the atomic ratios given in Table 4. It is generally assumed that the increase in humification is associated with a decrease in C/N, H/C, and O/C ratios in response to a relative enrichment with total N relative to organic C, as well as a residual enrichment of more recalcitrant aromatic and aliphatic compounds (Zaccone et al., 2018). The low H/C ratio indicated the dominance of aromatic structures (Rice and MacCarthy, 1988; Zhang et al., 2015) in the investigated HM samples.
The degree of internal oxidation (ω) is another parameter that clearly indicates the quantitative predominance of hydrocarbon structures over functional groups containing oxygen and nitrogen (Table 4). This parameter is considered significant for the analysis of diagnostic changes in HS that are related to degradation and oxidation reactions (Tan, 2014). The HM of the analyzed soils showed different values of this parameter, ranging from 0.020 to 0.672. These differences may indicate diversity in aerobic conditions related to soil pedogenesis. The HM with the lowest degree of oxidation was isolated from the Gleyic Phaeozem (8C) and developed under weak drainage conditions. Haplic Chernozem (7T) is a well-drained soil with much better aerobic conditions, which favors the oxidation of humic substances (Cieslewicz et al., 2008; Tan, 2014), resulting in the highest (0.672) ω value. However, not all Gleyic Phaeozems (6M, 10PY, and 11K) exhibited a comparatively low degree of internal oxidation.

3.2. 13C CP MAS NMR studies

The NMR spectra obtained (Figure 1) demonstrated the classical pattern observed for the HS (Hayes et al., 2017; Swift, 1996). All recorded spectra were very similar to each other and indicated several functional groups typical for organic substances. The alkyl region (18–57 ppm) includes peaks assigned to saturated hydrocarbons, from primary to quaternary, including alkanes and most of the alkyl groups. In the case of the HS, these peaks can be attributed to methylene C in organic acids (Almendros et al., 2018). In this region, a sharp peak typical for the methyl –CH3groups was detected in all observed spectra. The region between 57 and 73 ppm is assigned to methoxyl carbon, but also to N-substituted alkyl C, such as the Cα of amino acids (Almendros et al., 2016). In the presented HM in this ppm range, one weaker but broad peak at 65 ppm was detected from alkylamine C and O-alkyl C derived primarily from lignin (Swift, 1996). A more intense peak at 82 ppm was typical for carbohydrates (Preston, 1996; Preston et al., 1994; Schnitzer et al., 2006). Peaks in the region between 102 and 116 ppm can be assigned to anomeric C, sp3 carbon bonded to two O atoms (Swift, 1996). In all HM studied, the peak at about 104 ppm was relatively weak (carbohydrate shift) and exists more as a hump on the much more intense peak of aromatic C (116–151 ppm). At the highest chemical shift (~185 ppm) in all the studied samples spectra, the C peak from the carboxylic group was found.
The NMR spectra of the HM samples were integrated with the areas characteristic of particular functional groups. This indicates the predominance of aromatic structures over other organic components (Table 5), according to the results of the elemental composition, in which the H/C ratio confirms the high aromaticity of the investigated fraction.
The discussed spectra differ from those obtained by Song et al. (2011), who used an alkaline solvent enriched with urea (0.1 M NaOH + 6 M urea) and acidified DMSO solvent (DMSO: H2SO4; 94:6 v/v). These HM samples were devoid of organo-mineral bounds and showed a predominance of aliphatic structures with only a negligible presence of aromatic compounds. However, as studied by these authors, the fraction of the HM insoluble in DMSO obtained from Terra Preta soil exhibited higher amounts of aromatic structures and was more similar to the HM described in this work. The findings of Song et al. (2011) suggest that the HM obtained from various soils may differ in properties, and this confirms the need to analyze this fraction obtained from many soils developed under different environmental conditions. On the other hand, the dominance of aromatic structures in the HM studied indicates the presence of bound-HA strongly associated with the mineral fraction that protected them during NaOH extraction (Weber et al., 2022). This suggests the mutual protective effect of organic and mineral colloids (clay minerals, metal oxides, and hydroxides) present in organo-mineral bounds. Moreover, these observations confirm the processes of organic matter transformation and the interpenetration of the HA to the stable HM as a result of complexation processes (Yang et al., 2017). These components could be removed by alternating treatments with NaOH and HF/HCl to give a pure organic HM completely free of alkali-soluble components. Nevertheless, the HM samples obtained in this way would not have represented the HM naturally occurring in the soil environment, where they are strongly bound to the mineral fraction. These strong organo-mineral bonds of the HM represent the most resistant fraction of the SOM, which plays a key role in carbon sequestration in the soil environment.

3.3. FTIR spectroscopy

The FTIR spectra of the investigated HM (Figure 2) represented a similar pattern, typical for SOM (Helal et al., 2011; Mastrolonardo et al., 2013; Stevenson, 1994; Swift 1996; Tatzber et al., 2007; Ukalska-Jaruga et al., 2023). They are good indicators of qualitative characteristics, with typical bands distinguishing the humic materials. They consisted of the same peaks, with some differences observed in the region below the wavenumber of 700 cm-1. This can be attributed to various compositions of inorganic constituents of the studied HS (Chen et al., 2015).
Although the organic parts of the spectra followed a similar pattern, it was possible to find differences in their intensities. Based on the transmittance, five peaks were chosen for quantitative analysis of their absorbance spectra integrated areas: 1190–1300 cm-1 (–C, –O, and –OH deformations of COOH), 1570–1677 cm-1 (–C=C– stretching of aromatics), 1677–1800 cm-1 (–C=O stretching of COOH), 2796–2850 cm-1(–CH2– symmetric stretching), 2877–2985 cm-1 (–CH2– asymmetric stretching) (Machado et al., 2020; Swift, 1996; Tatzber et al., 2007). Among all the HM examined, the contents of aromatic structures dominated over aliphatic ones, ranging from 7.05 to 10.32% (Table 6). The 9H sample was characterized by the largest area under the spectral curve associated with aromatic structures. The investigated HM exhibited different shares of particular areas, but the differences were too scarce to show their dependence on the properties of soil material and/or soil genesis.
Studies of 13C-labeled HA (Spaccini et al., 2002) showed that, during incubation, the methyl group was oxidized to a carboxyl group, indicating that despite its hydrophilicity, the resulting carboxylic carbon was sequestered in the hydrophobic domain in soil. These results suggest that microbial mineralization of unstable organic compounds can be effectively reduced in soil by humified SOM, which in turn can increase the biological stability of SOM soil, and thus contribute to a significant reduction of CO2emissions from agricultural soils.

3.4. EPR measurement

The 13C NMR and FTIR analyses showed that the highest content of aromatic groups was in the 9H sample. The same sample was also characterized by the highest concentration of radicals (Table 7) measured by EPR spectroscopy, which confirms that radicals in the HM are known to have an aromatic structure (semiquinones, structures with an aromatic ring substituted with O).
The similar and low values of the g -parameter ranged from 2.0029 to 2.0031, pointing out the very similar structure of the radicals in all the investigated HM and their predominantly aromatic structure. The values of theg -parameter of the investigated HM were smaller than those usually observed for the HA and FA (Jerzykiewicz et al., 1999; Jezierski et al., 2000), which is evidence of an unpaired electron situated on the more aromatic moieties (Gerson and Huber, 2003). Small differences in the structures (reflected by small g-parameter change) depend on the stability of the aromatic core of the semiquinone matrix (Jerzykiewicz et al., 1999; Jezierski et al., 2000; Gerson and Huber, 2003). It can therefore be assumed that the reactivity of the HM will be similar and dependent on the functional groups located at the aromatic rings. This indicates that the differences in the HM structures occur mainly in the forms of the aromatic core stabilizing the structure of this fraction, which is only to a small extent capable of chemical interactions.

3.5. HPLC analyses

The HPLC analysis of the HM resulted in its separation into hydrophilic and hydrophobic fractions (Table 8). The amounts of hydrophilic fractions in all investigated HM were low; thus, they indicated a significant share of hydrophobic fractions, ranging from 77.41 to 80.83%. The high share of hydrophobic fractions resulted in low values of the HI/HB parameter at the level of 0.237–0.292. Since the HM has not been a frequent subject of HPLC studies on the hydrophobicity index, it is difficult to compare our results with those known in the literature. This revealed that the HI/HB ratios for HM are lower than those known for the HA (Dębska et al., 2010), indicating a higher content of hydrophobic components in the HM than in other HS (Rusanov and Anilova, 2009). On the other hand, the observed hydrophobicity coefficients are consistent with the data obtained from 13C NMR. Nevertheless, the aromatic compounds probably derived from the bound-HA fraction are in the form of HA occluded in HM structures – following the theory of interfacial SOM phases (Schauman 2006a, 2006b; Pignatello 2012).
Hydrophobicity can also be calculated from NMR spectra (Xu et al., 2017) or FTIR spectra (Matejkova and Simon, 2012). The observed hydrophobicity coefficients obtained from chromatography are consistent with the data obtained from 13C NMR. However, calculations of hydrophobicity based on the FTIR spectra do not appear to be correct (data not shown). In this method, only two intensities (at approximately 1600 and 3000 cm-1) are considered. In the obtained spectra, bounds at 1600 cm-1 (considered hydrophilic) are always more intense than those at 3000 cm-1, suggesting the dominance of hydrophilic groups over hydrophobic ones. This contradicts our other results and general knowledge about HM properties. The problem is most likely related to the limited number of regions chosen to determine the hydrophobicity index. This approach assumes that only aliphatic groups (3000 cm-1) are included in calculations, whilst aromatic ones are ignored, which especially in the case of HM may result in a great falsification of the results. In addition, aromatic group bands may occur in several places and could overlap slightly with the band around 1622 cm-1 (Machado et al., 2020; Swift, 1996; Tatzber et al., 2007), which is considered exclusively hydrophilic in this method (carboxylic group vibrations). Hydrophobicity calculated from13C CP MAS NMR spectra for different carbons gives results much closer to those obtained for HPLC, although it is difficult to correlate them.
The lowest HI/HB ratio for the 9H sample supports the results discussed above, distinguishing this particular HM sample as having the most aromatic and condensed structure (Tables 5 and 6). In general, mutual proportions of the hydrophilic and hydrophobic fractions in the HM indicate the direction of SOM transformations, which may affect the balance of organic carbon in the environment (de Aguiar et al., 2022) and contribute to its sequestration (Song et al., 2018). Spaccini et al. (2002) indicated that the higher the hydrophobicity of the humic material, the larger the sequestration of organic carbon in the soil, thus contributing to a significant reduction in CO2emissions. Furthermore, a large share of hydrophobic fraction in the investigated compounds indicated a sufficient content of substances to form stable soil aggregates, which was noted by Capriel et al. (1990), who obtained a positive correlation between the hydrophobic components and the stability of the aggregates.

3.6. SEM-EDS study

To characterize the mineral fraction bound to the HM more precisely, ash samples were analyzed with SEM-EDS. Analyses revealed that the ash consisted mainly of Ti (10–25 weight%), Al (4–22 weight%), Mg (3–14 weight%), and Zr (0.05–14 weight%), depending on the sample area studied (Figure 3). This indicates the dominance of oxide forms with a small share of silicates in the mineral part of the investigated HM.
To obtain more information on the mineralogical composition of the inorganic components of the HM, isolated fraction was analyzed by X-ray diffraction. The results of the XRD analysis (data intended for a separate publication, not presented in the work) confirmed that the mineralogical composition of the HM organo-mineral complex consisted mainly of anatase (TiO2), rutile (TiO2) and a smaller amount of sillimanite (Al2SiO5). These minerals are known to be stable, the most resistant to weathering processes.

3.7. Mutual relations between determined molecular characteristics of HM

Due to the numerous parameters obtained during HM analyses, cluster analysis and principal component analysis were performed to assess their mutual relationships (Figure 4). The determined HM properties were concentrated in two groups of interrelated factors: group 1 covered qualitative factors and was related to the molecular properties of the HM; group 2 covered quantitative factors, which are dependent on the fractional composition and shares of individual fractions in the total content of organic carbon. According to the statistical data (Table 9), the parameters of one group are not dependent on the parameters of the second group, i.e. the SOM fractional composition does not affect the molecular properties of the HM. Therefore, it can be assumed that the molecular properties of the HM depend on the factors affecting the quality of soil organic matter, like humin precursors and/or the soil’s biological activity. These are individual features of each microspace where humification processes take place.
In the PCA analysis, the first three factors were significant and dominated the overall assessment of the mutual influence of the investigated HM properties, which was assessed on the base of the scree curve. Generally, the first three principal components accounted for 84% of the total variance of the results, while up to 66% of the variance was connected with the first two factors (Figure 5 and Table 9). The first PCA component (PCA 1) that explained 37% of the variance, was significantly correlated with elemental composition and their proportions in the HM structures (r = 0.79, r = 0.68, and r = 0.74 for H/C, O/C, and N/C, respectively), aromaticity, and related functional groups. such as COOH, O–C–O, and CH–OH (r = -0.80, r = 0.73, r = 0.88, and r = 0.61, respectively) as well as the internal oxidation parameter (r = 0.59) and the g parameter (r = -0.82). The second PCA component (PCA 2), representing merely 29% of data variance, indicated significant correlation with radical concentration (r = 0.73), as well as parameters of hydrophobicity and hydrophilicity (r = -0.75, r = 0.75, and r = 0.74, for HI, HB, and HI/HB proportion, respectively). Moreover, this component was also related to hydrophobic functional groups and ω. The third component (PCA 3) accounted for 18% of the variation and was related to reactive carbon functional groups such as CH–OH (r = 0.68), OCH3 (0.69), CH3 (r = 0.82), and COOH (r = 0.68). The observed correlations indicate that the individual HM properties are interdependent and determine the total chemodiversity of this fraction.
Moreover, the obtained results allowed to identify homogeneous groups (Figure 6) with similar properties, i.e., 8C, 3Z, and 1PS (first group), 10PY and 11K (second group), 6M and 7T (third group), and 9H (fourth group). These identified HM groups with similar molecular properties were isolated from soils with different characteristics, but no correlation was found between the individual specific properties of these soils. This confirms that the individual properties of HM may depend more on humification processes than on soil characteristics. Nevertheless, the number of soils to be analyzed should be extended to reliably assess the distinctive features of the HM.