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.