The objective of this paper was to investigate the molecular characterization of soil organic matter fractions (humic substances (HS): fulvic acids-FAs, humic acids-HAs, and humins-HNs), which are the most reactive soil components. A wide spectrum of spectroscopic (UV–VIS and VIS–nearIR), as well as electrochemical (zeta potential, particle size diameter, and polydispersity index), methods were applied to find the relevant differences in the behavior, formation, composition, and sorption properties of HS fractions derived from various soils. Soil material (n = 30) used for the study were sampled from the surface layer (0–30 cm) of agricultural soils. FAs and HAs were isolated by sequential extraction in alkaline and acidic solutions, according to the International Humic Substances Society method, while HNs was determined in the soil residue (after FAs and HAs extraction) by mineral fraction digestion using a 0.1M HCL/0.3M HF mixture and DMSO. Our study showed that significant differences in the molecular structures of FAs, Has, and HNs occurred. Optical analysis confirmed the lower molecular weight of FAs with high amount of lignin-like compounds and the higher weighted aliphatic–aromatic structure of HAs. The HNs were characterized by a very pronounced and strong condensed structure associated with the highest molecular weight. HAs and HNs molecules exhibited an abundance of acidic, phenolic, and amine functional groups at the aromatic ring and aliphatic chains, while FAs mainly showed the presence of methyl, methylene, ethenyl, and carboxyl reactive groups. HS was characterized by high polydispersity related with their structure. FAs were characterized by ellipsoidal shape as being associated to the long aliphatic chains, while HAs and HNs revealed a smaller particle diameter and a more spherical shape caused by the higher intermolecular forcing between the particles. The observed trends directly indicate that individual HS fractions differ in behavior, formation, composition, and sorption properties, which reflects their binding potential to other molecules depending on soil properties resulting from their type. The determined properties of individual HS fractions are presented as averaged characteristics over the examined soils with different physico-chemical properties.

Background: Humic substances (HS) are compounds with a complicated structure, present in the humus soil layer, water, lake sediments, peat, brown coal and shales. Due to their similar physicochemical properties to DNA, they may have an adverse effect on the subsequent use of the isolated material. The main aim of this research was to examine the effect of HS on DNA isolation depending on the soil type and land use, taking into account the spectroscopic full characteristics of HS fractions. Methods: The research was conducted on eight types of soil sample. Soils represented the most important Soil Reference Groups for temperate climates: Fluvisols, Regosols, Cambisols, Arenosols, Histosols and Luvisols. Soil samples were also collected from areas diversified in terms of use: arable land, grassland and forest. The extraction of HS fractions was performed using the procedure recommended by the International HS Society. The fractional composition of HS was characterized by UV–Vis and fluorescence methods. Soil DNA is extracted by direct cell lysis in the using a CTAB-based method with a commonly-used commercial soil DNA isolation kit. The basis for assessing the quantity and quality of extracted DNA was the Polymerase chain reaction (PCR) reaction since the analysis of soil DNA often relies on the use of PCR to study soil microorganisms. Results: Based on the results, it can be concluded that in the presence of a high concentration of HS, the isolated DNA was low quality and the additional purification procedure was necessary. Despite the differentiation of the internal structure of HS fractions, the decisive factor in the efficiency of DNA isolation from soil samples was the total carbon content in HS. Reduced DNA yields can significantly constrain PCR detection limits to levels inadequate for metagenomic analysis, especially from humus-rich soils.

Soil organic matter (SOM) and its heterogeneous nature constitutes the main factor determining the fate and transformation of organic chemicals (OCs). Thus, the aim of thus research was to analyze the influence of the molecular chemodiversity of a stable SOM (S-SOM) on the sorption potential of different groups of OCs (organochloride pesticides—OCPs, and non-chlorinated pesticides—NCPs, polycyclic aromatic hydrocarbons—PAHs). The research was conducted as a batch experiment. For this purpose, a S-SOM was separated from six soils (TOC = 15.0–58.7 gkg⁻¹; TN = 1.4–6.6 gkg⁻¹, pH in KCl = 6.4–7.4 and WRB taxonomy: fluvisols, luviosols, leptosols) by alkaline urea and dimethylsulphoxide with sulfuric acid. Isolated S-SOM fraction was evaluated by UV–VIS, FT-IR and EEM spectroscopy to describe molecular diversity, which allowed the assessment of its potential sorption properties regarding OCs. In order to directly evaluate the sorption affinity of individual OCs to S-SOM, the mixture of the 3 deuterated contaminants: chrysene (PAHs), 4,4′DDT (OCPs) atrazine (NCPs) were applied. The sorption experiment was carried out according to the 106 OECD Guidelines. The OCs concentration was analyzed by gas chromatography triple mass spectrometry (GC-MS/MS). OCs were characterized by different sorption rates to S-SOM fractions according to the overall trend: atrazine (87.5–99.9%) > 4,4′DDT (64–81.6%) > chrysene (35.2–79.8%). Moreover, atrazine exhibited the highest saturation dynamic with fast bounding time amounting to 6 h of contact with S-SOM. Proportionally, the chrysene showed the slowest binding time achieving an average of 55% sorption after 78 h. Therefore, S-SOM isolated from different soils demonstrated varying binding capacity to OCs (CoV = 21%, 27% and 33% for atrazine, DDT and chrysene, respectively). Results indicate that each sample contains S-SOM with different degrees of transformation and sorption properties that affect the OCs availability in soil. Spectroscopic analyses have shown that the main component of S-SOM are biopolymers at various stages of transformation that contain numerous aromatic–aliphatic groups with mostly hydrophilic substituents.

Humic substances, including humin fraction, play a key role in the fate of organic and inorganic xenobiotics contaminating the environment. Humin is an important fraction of humic substances, which has been the least studied to date. This is due to the difficulties connected with its isolation that pose a number of methodological problems. Methods of humin fraction isolation can be divided into following main groups: (1) digestion of mineral soil components with HF/HCl followed by alkali extraction of HA and FA; (2) alkali extraction of HA and FA followed by extraction of humin by different organic solvents; and (3) alkali extraction of HA and FA followed by HF/HCl digestion of mineral soil components. Nevertheless, each of these methods has different limitations. We described in detail a useful procedure of humin isolation, in which this fraction was not extracted, but isolated from the soil by removing its soluble organic and mineral components. A modified method of HA and FA extraction with 0.1 M NaOH, according to the International Humic Substances Society, was used in the first step. Then, the mineral components in the residue were digested with the 10% HF/HCl. Unlike the procedures oriented to increase the concentration of organic matter, samples were treated several times with the HF/HCl mixture until the mineral fraction was almost completely digested. The main assumption of the method modification was to obtain the highest yield with the lowest possible ash content, but without affecting humin chemical structure. The results showed that the proposed procedure is characterized by a high efficiency and recovery and, therefore, it can be used to isolate high amounts of humin from soil.

Humic substances (HS) are the most important natural biostimulant of plants. However, the relationship between their structure and biological activity in plants is still not well recognized. The objective of this paper was to assess the influence of molecular fractions of humic acids (HA) (HA < 30 kDa and HA > 30 kDa) on reducing negative effects of drought stress in soybean (Glycine max (L.) Merr.) seedlings of Progres and Nawiko cultivars. Drought stress was induced in laboratory conditions by the addition of polyethylene glycol 6000 (PEG 6000) to make a water potential of −0.5 MPa. HA were extracted according to the International Humic Substances Society procedure, and then were separated into two molecular fractions by membrane filtration. The following physiological and biometric parameters were determined: chlorophyll content, photosynthesis activity, electrical conductivity, fresh and dry mass of overground and roots, and plant length. The enzyme activity and ion contents were also measured. Differences in response to drought stress with the addition of HA < 30 kDa and HA > 30 kDa or not to the Hoagland’s solution were observed among studied cultivars. Drought stress caused a decrease in the most physiological parameters and increase in peroxidase activity in the case of both studied cultivars. However, the results of biometric measurements showed that the Progres cultivar appears to have better tolerance to drought stress. The significant influence of water deficit on most macroelement content in dry matter leaves of both studied cultivars was not observed, while its effect on microelement uptake by soybean plants was concluded. In the case of the Progres cultivar, the results showed a significant decrease in microelement content in the dry matter of leaves, whereas in the leaves of Nawiko cultivar there was a significant increase. The influence of HA > 30 kDa and HA < 30 KDa fractions on physiological features of both studied cultivars was varied. HA > 30 kDa fraction better up-regulated the antioxidant defense system. Unfortunately, no effect of either HA fraction on the macro- and micronutrients uptake system of both studied cultivars was observed.