2. Materials and methods
2.1. Soil location and characteristics
Soil samples were collected from the upper 30 cm of eight mollic
horizons of Chernozems and Phaeozems developed from different parent
rocks. They were used as arable soils with different plants grown in
various regions of Poland (Table 1). The HM samples were labelled 11K,
10PY, 9H, 8C, 7T, 6M, 3Z, and 1PS, which refer to the sampling location
by the number and first letter(s) of the names of the nearest towns or
villages.
Investigated soils were developed under diverse agroecological
conditions and exhibited different basic properties (Table 2). They
indicated different pH (5.64 - 7.71), TOC contents (13.3 – 41.7 g
kg−1), and CEC values (21.6 – 53.2 cmol(+)
kg-1) as well as textures (from sandy loam to clay),
thus representing various soils cultivated in temperate climatic
conditions.
2.2. Isolation of humin
The detailed isolation procedure of the HM was described in our previous
work (Weber et al., 2022). Briefly, the applied method was based on the
classic separation of the HS by eliminating the alkali-soluble SOM
fractions (HA and FA) from the soil matrix by exhaustive 0.1 M NaOH
extraction. Next, the remaining mineral fraction was digested with a
10% HF-HCL mixture. The collected HM was freeze-dried and then
subjected to spectral studies to assess its molecular characteristics.
All analyses were performed on solid HM samples (excluding HPLC
analyses).
2.3. Elemental composition
The elemental composition of the HM was analyzed with the CHNS Vario EL
Cube analyzer (Elementar; Langenselbold, Germany) in three repetitions
to obtain the highest precision of results. The contents of C, H, N, and
S were measured as percentages of the ash-free mass, while the oxygen
concentration was calculated from the difference. The atomic ratios H/C,
O/C, O/H, and N/C were calculated to show the relationship of the
elements in the composition of the HM. Furthermore, the degree of
internal oxidation (ω) was determined from the atomic percentage
contents (Tan, 2014), according to the formula: ω = [(2O + 3N) –
H]/C.
2.4. 13C CP MAS NMR
The 13C CP MAS NMR spectra were acquired with a Bruker
Avance III spectrometer (Bruker Inc., Germany) at 300 MHz, equipped with
a 4-mm MAS probe and operating in a 13C resonance
sequence at 75.48 MHz. The HM samples were placed in a rotor (sample
holder) of zirconium dioxide (ZrO2) with Kel-F caps,
with a rotation frequency of 10 kHz. The spectra were obtained by
collecting 4994 data points from the same number of scans at an
acquisition time of 50 ms and with a 4 s recycle delay. Spectral
collection and elaboration were performed using Bruker Topspin 3.6
software. The areas of characteristic functional groups were calculated
by integration of appropriate parts of the spectra using Bruker Topspin
4.1.1. software. Hydrophobicity (HB) was calculated as follows:
HB = [(0–57 ppm) + (116–151 ppm)] / [(57–116 ppm) + (151–220
ppm)] (Xu et al., 2017).
2.5. FTIR
The FTIR spectra were measured using a Bruker Vertex 70 FTIR
spectrometer with KBr pellets (approximately 1 mg of sample in 400 mg of
KBr). Collected spectra were presented as transmittance. For the chosen
peaks: –C, –O, and –OH deformations of COOH (1190–1300
cm-1), aromatic –C=C– (1570–1677
cm-1), –C=O of COOH (1677–1800
cm-1), –CH2– symmetric stretch
(2796–2850 cm-1), and –CH2–
asymmetric stretch (2877–2985 cm-1) integrations were
performed (Machado et al., 2020; Swift, 1996; Tatzber et al., 2007). The
integral areas were calculated from absorbance spectra using OriginPro
2016 and OriginPro 9.5 software.
2.6. EPR
The X-band EPR spectra were obtained with a Bruker Elexsys E500
spectrometer at room temperature with the use of the double rectangular
cavity resonator devoted to quantitative measurements. More details
could be found in the work of Pospíšilová et al. (2020). The HA standard
of Pahokee peat (1S103H) and the Leonardite standard HA (1S104H)
distributed by the International Humic Substances Society, in addition
to the Bruker alanine pill, were used as quantitative standards. To
quantitatively analyze the spectra of the HM radicals, double
integration of the signals was performed using WinEPR by Bruker.
2.7. HPLC
The hydrophilic-hydrophobic properties of the HM were determined using a
Perkin-Elmer HPLC Series 200 liquid chromatograph equipped with a
fluorescence detector. Chromatographic separation was carried out on an
analytical column X-Terra C18 with a particle size of 5 μm and
dimensions of 250 × 4.6 mm ID. The HM samples (5 mg) were dissolved in 2
cm3 of DMSO–H2SO4mixture (94:6 v/v) and filtered through a 0.45-μm PVDF syringe filter.
For the chromatographic separation, an eluent of acetonitrile-water and
a gradient elution program were used. The injection was 10 μL, and the
detection was at excitation/emission wavelength (λex/λem) 270/330 nm.
Based on the determined areas under the peaks, the share of hydrophilic
(HI) and hydrophobic fractions (HB) as well as the HI/HB parameter were
calculated.
2.8. SEM EDS
Scanning electron microscopy (SEM) analysis of the HM ash was performed
with a Hitachi S-3400N SEM, equipped with a tungsten cathode attached
with energy-dispersive X-ray spectroscopy. Measurements were made at a
pressure of 30 Pa and an accelerating voltage of 30.0 kV. The images
were captured using backscattered electrons to better visualize the
material contrast. The EDS analyses were performed with a Noran System7
analyzer with a Thermo Scientific Ultra Dry detector, with a resolution
of 129 eV.
2.9. Statistical analysis
OriginPro 2016 and OriginPro 9.5 software were used for the elemental
analysis and the concentration of EPR radicals. The software package
Statistica (Dell Statistica, version 13.1) was used for the principal
component analysis (PCA) and cluster analysis. These analyses were used
to investigate the relationships among the determined humin molecular
characteristics. In order to identify the main correlation structure of
the measured variables, their inter-relationships and hidden structures
were evaluated with PCA, whereas cluster analysis was performed to
identify dependent groups.