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.