Soil Humic Substances Research Articles

Soil–root cross‐talking: The role of humic substances

AbstractThe biological activity of humic substances (HS) has been elucidated in the last 40 years. Growth enhancement from HS has been demonstrated in several plants in the laboratory and the field. Morphogenesis effects have also been investigated and include induction of lateral root formation and root hair initiation in intact plants and stimulation of root and shoot development in treated cell calluses. HS enhance nutrient use efficiency, aiding assimilation of both macro and microelements and promoting plant growth by the induction of carbon, nitrogen, and secondary metabolism. The review aims are to: (1) shed light on the mechanism by which plants “talk” with soil through humic substances, (2) elucidate the plant responses to the stimulatory effects of HS, the regulatory circuits that allow plants to cope with humus, and the feedback between plant community structures, and (3) show (in light of recent debate about the alkaline extraction of soil humic substances) the plant capability to acquire biologically active substances from soil. It will be shown that plants modify soils, creating and maintaining favorable habitats for growth and survival. Therefore, organic substances exuded by roots are not a wasteful loss of carbon and energy. They represent an evolved strategy by which plants “talk” to the soil. The mobilization of bioactive organic/humic substances from bulk soil or bulk humus is critical to plant and soil health.

Comparison of bacterial growth in response to photodegraded terrestrial chromophoric dissolved organic matter in two lakes.

Terrestrial chromophoric dissolved organic matter (CDOM) could subsidize lake food webs. Trophic state and altitude have a pronounced influence on the CDOM concentration and composition of a lake. The impact of future changes in solar radiation on high-altitude lakes is particularly alarming because these aquatic ecosystems experience the most pronounced radiation variation worldwide. Photodegradation experiments were conducted on terrestrial CDOM samples from oligotrophic alpine Lake Tiancai and low-altitude eutrophic Lake Xiaohu to investigate the response of bacterial growth to photodegraded CDOM. During the photo-irradiation process, the fluorescent CDOM intensity evidently decreased in an inflowing stream of Lake Tiancai, with the predominance of humic-like fluorescence. By contrast, minimal changes were observed in the riverine CDOM of Lake Xiaohu, with the predominance of protein-like fluorescence. The kinetic constants of photodegradation indicated that the degradation rate of terrestrial (soil) humic acid in Lake Tiancai was significantly higher than that in Lake Xiaohu (p<0.001). Soil humic and fulvic acids irradiated in the simulated experiment were applied to incubated bacteria. The specific growth rate of bacteria incubated with soil humic substances was significantly higher in Lake Tiancai than in Lake Xiaohu (p<0.05). Furthermore, the utilizing rate of dissolved oxygen (DO) confirmed that the DO consumption by bacteria incubated with terrestrial CDOM in Lake Tiancai was significantly greater than that in Lake Xiaohu (p<0.05). In summary, the exposure of terrestrial CDOM to light significantly enhances its availability to heterotrophic bacteria in Lake Tiancai, an oligotrophic alpine lake, which is of importance in understanding bacterial growth in response to photodegraded terrestrial CDOM for different types of lakes.

Soil humic acid and aggregation as restoration indicators of a seasonally flooded riparian forest under buffer zone system

A degraded riparian forest that has lost vegetation and has suffered periodic flooding was rehabilitated using native species under buffer zone system which was stratified according to the distance from the river (Zones I, II, III). To restore the soil stabilisation and water flow we compared the soil aggregation, porosity and soil humic substances of the experimental site (ES) with those of a disturbed site (DS) and a preserved site (PS). Six years post-transplantation, soil aggregation and micro-porosity were improved in the ES relative to the DS, but only micro-porosity achieved a pattern similar to that of the PS in Zone III. Differences in soil aggregation between ES and PS were correlated with the negative effect promoted by a high aliphatic-humic acid composition in Zone I and by an undifferentiated distribution of fulvic acid among sites which were attributed to the flooding effect. Similarities among ES and PS were related to aromatic:vinylhumic acid ratio, allowing for the clustering of samples from Zones II and III of both the PS and the ES, via a principal component analysis. Samples from Zones I of ES and PS were separated driven by aliphatic and fulvic acid contributions, while all of the DS samples formed an isolated group under the influence of aliphatic species and macro-porosity. Based on these attributes, the ES is evolving towards the PS, and the rehabilitation process has attained an intermediate phase of restoration. The zones system adopted for riparian forest restoration allowed the use of soil humic substances, aggregates and micro-porosity as indicators of restoration.

Commercial Humates in Agriculture: Real Substance or Smoke and Mirrors?

Soil humic substances (HS) are known to be beneficial for soils and plants, and most published studies of HS and humates, usually conducted under controlled conditions, show benefits. However, the value of commercial humate application in the field is less certain. This review attempts to answer the question: How effective are commercial humates in the field? Commercial humates, especially K humate, are used widely in agriculture today as “soil conditioners”. A wide range of benefits is claimed, including growth of beneficial soil microbes; deactivation of toxic metals; improvements in soil structure including water retention capacity, enhanced nutrient and micronutrient uptake and photosynthesis; resistance to abiotic stress, including salinity; and increased growth, yield and product quality. Despite this, there is a surprising lack of solid evidence for their on-farm effectiveness and findings are often inconsistent. The industry relies largely on anecdotal case studies to promote humates, which are often applied at unrealistically low levels. It is recommended that products should be well characterised, physically and chemically, and that careful field studies be conducted on foliar humate application and pelletised humates at realistic rates, targeted to the seedling rhizosphere, for a variety of crops in a range of soils, including low C sandy and saline soils.

Fate and fouling characteristics of fluorescent dissolved organic matter in ultrafiltration of terrestrial humic substances

Ultrafiltration (UF) membrane fouling caused by terrestrial input of dissolved organic matter (DOM), especially during high flood periods, is poorly understood. In this study, we examined the fouling characteristics of three different terrestrial humic substances (HS) on regenerated cellulose (RC) UF membranes with the pore sizes of 30 k–3 kDa via conventional bulk HS measurements as well as an advanced fluorescence spectroscopy. The fluorescence excitation-emission matrix coupled with parallel factor analysis (EEM-PARAFAC) identified one protein-like (C1) and three humic-like fluorescent components (C2–C4) from soil and leaf-derived HS. The fate of the different fluorescent components was individually tracked for the UF processes. The higher removal rates were found generally on the order of high molecular weight (HMW) C1 to smaller sized humic-like components (C4 > C3 > C2) regardless of the HS sources, implying the importance of HS molecular sizes on the UF operation. Among the humic-like components, C2 was the most associated with irreversible fouling, while other two humic-like components contributed more to reversible fouling. For soil-derived HS, C4 can be suggested as a good surrogate for membrane fouling, as evidenced by the highest correlation between the removal rates and the total fouling indices among the tested HS variables including conventional bulk parameters. Our study demonstrated a promising application of EEM-PARAFAC for probing membrane fouling of terrestrial DOM, which provided additional insight into the fate of different fluorescent components on the UF processes.

In memoriam Prof. F.J. Stevenson and the Question of humic substances in soil

In memoriam Prof. F.J. Stevenson and the Question of humic substances in soil

A humic substance analogue AQDS stimulates Geobacter sp. abundance and enhances pentachlorophenol transformation in a paddy soil

Soil humic substances can be used as redox mediators in accelerating the biotransformation of organic pollutants, and humus-respiring bacteria are widely distributed in soils. However, the impact of humic substances on the soil microbial community during the biotransformation of organic pollutants is expected to be crucial while remains to be unclear. In this study, the biostimulation of indigenous microbial communities and the consequent effects on anaerobic transformation of pentachlorophenol (PCP) by a model humic substance, anthraquinone-2,6-disulfonate (AQDS), were systematically investigated in a paddy soil. The addition of AQDS was observed to increase the production of HCl-extractable Fe(II) and enhance the PCP transformation rates consequently. The pseudo-first-order rate constants of the PCP transformation showed a positive exponential relationship with the AQDS dosage. The terminal restriction fragment length polymorphism (T-RFLP) results indicated the substantial effect of added AQDS on soil microbial community. The enhanced abundance of Geobacter sp. was disclosed to be most critical for accelerated PCP transformation when with AQDS, in which Geobacter sp. functioned for promoting the generation of active Fe(II) and consequently enhancing the PCP transformation rates. The transformation rates of PCP were exponentially correlated with the abundance of Geobacter sp. positively. The findings are expected to improve the understanding of diversity and ubiquity of microorganisms in humic substances-rich soils for accelerating the transformations of soil chlorinated pollutants.

Stimulation of Tetrabromobisphenol A Binding to Soil Humic Substances by Birnessite and the Chemical Structure of the Bound Residues.

Studies have shown the main fate of the flame retardant tetrabromobisphenol A (TBBPA) in soils is the formation of bound residues, and mechanisms on it are less-understood. This study investigated the effect of birnessite (δ-MnO2), a naturally occurring oxidant in soils, on the formation of bound residues. (14)C-labeled TBBPA was used to investigate the pH dependency of TBBPA bound-residue formation to two soil humic acids (HAs), Elliott soil HA and Steinkreuz soil HA, in the presence of δ-MnO2. The binding of TBBPA and its transformation products to both HAs was markedly increased (3- to 17-fold) at all pH values in the presence of δ-MnO2. More bound residues were formed with the more aromatic Elliott soil HA than with Steinkreuz soil HA. Gel-permeation chromatography revealed a uniform distribution of the bound residues within Steinkreuz soil HA and a nonuniform distribution within Elliott soil HA. (13)C NMR spectroscopy of (13)C-TBBPA residues bound to (13)C-depleted HA suggested that in the presence of δ-MnO2, binding occurred via ester and ether and other types of covalent bonds besides HA sequestration. The insights gained in this study contribute to an understanding of the formation of TBBPA bound residues facilitated by δ-MnO2.

Copper binding to soil fulvic and humic acids: NICA-Donnan modeling and conditional affinity spectra

Binding of Cu(II) to soil fulvic acid (JGFA), soil humic acids (JGHA, JLHA), and lignite-based humic acid (PAHA) was investigated through NICA-Donnan modeling and conditional affinity spectrum (CAS). It is to extend the knowledge of copper binding by soil humic substances (HS) both in respect of enlarging the database of metal ion binding to HS and obtaining a good insight into Cu binding to the functional groups of FA and HA by using the NICA-Donnan model to unravel the intrinsic and conditional affinity spectra. Results showed that Cu binding to HS increased with increasing pH and decreasing ionic strength. The amount of Cu bound to the HAs was larger than the amount bound to JGFA. Milne’s generic parameters did not provide satisfactory predictions for the present soil HS samples, while material-specific NICA-Donnan model parameters described and predicted Cu binding to the HS well. Both the ‘low’ and ‘high’ concentration fitting procedures indicated a substantial bidentate structure of the Cu complexes with HS. By means of CAS underlying NICA isotherm, which was scarcely used, the nature of the binding at different solution conditions for a given sample and the differences in binding mode were illustrated. It was indicated that carboxylic group played an indispensable role in Cu binding to HS in that the carboxylic CAS had stronger conditional affinity than the phenolic distribution due to its large degree of proton dissociation. The fact was especially true for JGFA and JLHA which contain much larger amount of carboxylic groups, and the occupation of phenolic sites by Cu was negligible. Comparable amounts of carboxylic and phenolic groups on PAHA and JGHA, increased the occupation of phenolic type sites by Cu. The binding strength of PAHA-Cu and JGHA-Cu was stronger than that of JGFA-Cu and JLHA-Cu. The presence of phenolic groups increased the chance of forming more stable complexes, such as the salicylate-Cu or catechol-Cu type structures.

Fluorescence Analysis of Cu(II), Pb(II) and Hg(II) Ion Binding to Humic and Fulvic Acids

Abstract. The aim of this work was to study molecular and quantitative aspects of metal ion binding to humic substances (HS). The object of our study was characterization of two standards of humic substances (Elliott Soil standard HA 1S102H and Elliott Soil standard FA 2S102F). All samples of IHSS standards HS were characterized by elemental analysis (EA), ultraviolet-visible spectroscopy (UV/Vis), Fourier transform infrared spectroscopy (FTIR) and steady-state fluorescence spectroscopy. Chemical parameters on the complexation of Cu (II), Pb(II) and Hg(II), including the conditional stability constants and the percentage of fluorophores participating in the complexation, were estimated by the modified Stern–Volmer equation. The stability constants (log Ka) of Me(II)–ESHS complexes range from 3.70 to 5.15 in the order: Hg–ESHA&gt;Cu–ESHA&gt;Pb–ESHA&gt;Cu–ESFA&gt;Pb–ESFA. With respect to the ESHA, ESFA, which showed the smallest contents of O-containing functional groups (e.g. hydroxyl, carbonyl, ester, especially carboxyl groups on the aromatic ring) and the lowest humification degree, the ESFA was characterized by much smaller stability constants. Our findings suggest that soil HS belongs to class of important organic ligands for complexation with heavy metal ions and may significantly affect the chemical forms, mobility, bioavailability and ecotoxicity of heavy metals in the soil environment.

Changes in Structure and Content Humic Substances in Soil During the Laboratory Simulated Fires

Abstract The paper deals with the process of thermal degradation of humic substances in soil samples exposed to increased temperature. To determine the basic properties of humic substances, humic and fulvic acids are used conventional fractionation chemical laboratory methods. To determine changes in the chemical structure, the method of use of FT-IR ATR spectroscopy technique.

Organic plum cultivation in the Mediterranean region: The medium-term effect of five different organic soil management practices on crop production and microbiological soil quality

Organic farming practices recommended by European regulations offer an alternative to conventional soil management, but these practices should be analyzed in greater depth to assess their effectiveness on different plants and in different local conditions. Accordingly, this study evaluates the efficacy of five different organic management strategies on an organic plum farm in Southeastern Spain. This study was conducted for a period of six years and the five different organic treatments were applied in triplicate in 256m2 plots randomly distributed in three blocks (15 plots in total); each plot had 16 plum trees. Every year crushed pruning wastes were incorporated into the soil in all the experimental plots. One triplicated plot only received this aforementioned treatment (crop biomass treatment, CB). The rest of the plots also received one of the following treatments: (i) the addition of a commercial product for organic agriculture based on bacteria (biofertilizer), composed of Azospirillum brasilense (bacteria fixing N) and Pantoea dispersa (bacteria with a capacity for solubilising phosphates and stimulating plant growth) (biofertilizer treatment, B); (ii) the annual addition of 20t/ha of certified commercial organic compost (annual compost treatment, AC); (iii) the biennial addition of 20t/ha of certified commercial organic compost (biennial compost treatment, BC); and (iv) the sowing and subsequent incorporation of a mixture of 60% Avena sativa and 40% Vicia sativa (green manure treatment, GM). Each year the following indicators of ecosystem sustainability related to soil microbiological characteristics and carbon fixation were measured after harvest, in addition to plum yield: organic carbon (Corg), water soluble C, humic substances and humic acid C, microbial biomass C, respiration, ATP, dehydrogenase activity and hydrolase enzymes (alkaline phosphatase, urease and beta-glucosidase) activities. The two way ANOVA of the results indicated that all the above parameters were significantly affected (p≤0.001) by both, treatment and time. The interaction of treatment and time also significantly affected all of these parameters, except for yield and dehydrogenase activity. Composts not only produced better results than green manure cover crops and biofertilizer treatments in terms of plum yields, but they also brought about higher increases in the soil C pool and greater improvements in soil microbiological characteristics. In the six years of the study, compost-treated soils showed higher average plum yields and higher organic carbon, humic substance and humic acid contents than the rest of treatments. The size and activity of microbial population was also greater in the compost-treated soils, reflected in the higher values found in these soils for microbial biomass C, soil respiration, ATP (adenosine-tri-phosphate) content and dehydrogenase and hydrolases activity. Nevertheless, yields when compost was added biennially (BC) tended to be higher than in the annual compost treatment (AC) in most years as well as on average, although the differences were usually not significant. Yields in the sixth crop showed a high positive correlation with soil humic substances and humic C content (p≤0.01), as well as with Corg, microbial biomass C and phosphatase and urease activity (p≤0.05).

土・水環境に遍在するフミン物質の構造化学的特徴とその多様性

土・水環境に遍在するフミン物質の構造化学的特徴とその多様性

Spectroscopic Investigations of the Oxidative Polymerization of Hydroquinone in the Presence of Hexavalent Chromium

Polyphenol polymerization receives much scientific attentions because it is an important reaction of humification processes. Through an oxidative polymerization reaction (OPR), hydroquinone (H2Q), a naturally occurring compound, has been found to be a vital precursor of humic substances in soils. Chromate (Cr(VI)), a strong oxidant, is widely distributed in the environment due to the inappropriate disposal of Cr-containing wastes. The OPR of H2Q in the presence of Cr(VI) may occur naturally. In the study, it was found that 400 mg L−1H2Q could be polymerized at pH 3.0 with 105 mg L−1Cr(VI). The results of FT-IR revealed the presence of both hydroquinone and benzoquinone nuclei in the polymer. The spectra of X-ray absorption near edge structure (XANES) demonstrated a decrease in Cr(VI) content during the OPR of H2Q. Based on the results of linear combination fitting (LCF) for the spectra of XANES, the proportions of Cr species in the precipitates mainly consisted of Cr(OH)3, and Cr-carboxylate contents increased gradually from 15.3 to 25.5% over the reaction time. After acidizing the supernatants at pH 1.0 for 1 d, Cr(III)-HA dominated Cr(III) species. This suggested that humic-like substances were formed during OPR of H2Q, possibly through the formation of Cr(III)-organic complex.

Investigation of the effect of pH, ionic strength, foreign ions, temperature, soil humic substances on the sorption of 152+154Eu(III) onto NKF-6 zeolite

The sorption of 152+154Eu(III) on NKF-6 zeolite is investigated by batch technique under ambient conditions. The results indicate that the sorption of Eu(III) is affected by ionic strength at pH 8.0. The sorption is dominated by ion exchange or outer-sphere surface complexation at low pH and by inner-sphere surface complexation at high pH. The thermodynamic parameters suggest that the sorption is an endothermic and spontaneous process. The results show that the NKF-6 zeolite is a suitable adsorbent for the preconcentration of Eu(III) from large volumes of nature aqueous solutions.

Land use change affecting soil humic substances in three semi-arid agro-ecosystems in South Africa

Productivity of cropland soils, especially in semi-arid to arid zones, is declining due to increasing losses of stable organic matter. Therefore, it was found important to evaluate alternative management systems that can control, and if possible combat, these escalating losses of recalcitrant fractions of soil organic matter (SOM). The aim of this study was to quantify the influence of land use changes on humic substances of the semi-arid Plinthustalfs. Topsoil samples from distinctive agro-ecosystems at Harrismith, Tweespruit and Kroonstad were collected in the virgin, cultivated and restored Plinthustalfs and analysed for C and N in crude humic substances (Cs and Ns), extractable humic substances (Ce and Ne), humic acids (Ch and Nh) and fulvic acids (Cf and Nf). The results indicated that cultivation depleted the former virgin soils of crude humic substances, extractable humic substances, humic acids and fulvic acids across the three agro-ecosystems, with the exception of fulvic acids, which were higher in the cultivated soils than in the virgin soils of Kroonstad. Restored soils had substantial amounts of these humic fractions, except fulvic acids that behaved differently in the Kroonstad agro-ecosystem. Some correlations were observed between climate variables and humic fractions. In general, mean annual rainfall (MAR) and aridity index (AI) contributed to the loss of crude humic substances, extractable humic substances, humic acids and fulvic acids, while mean annual temperature (MAT) did not. These three climate variables did not influence the buildup of humic fractions. No trends were observed regarding the C/N ratios of the examined humic fractions; however, most were found to be closer to a range of 10–12, suggesting that SOM was approaching equilibrium state. Although it is known that humic substances are recalcitrant in nature, our results revealed that management practices that prohibit soil disturbance are needed to complement the refractory nature of humic substances against biological oxidation, and hence reduce C and N losses into the atmosphere.

Effects of heating on composition, degree of darkness, and stacking nanostructure of soil humic acids

Wildfires and prescribed burning can affect both the quality and the quantity of organic matter in soils. In this study, we investigated qualitative and quantitative changes of soil humic substances in two different soils (an Entisol from a paddy field and an Inceptisol from a cedar forest) under several controlled heating conditions. Soil samples were heated in a muffle furnace at 200, 250, or 300°C for 1, 3, 5, or 12h. The humic acid and fulvic acid contents of the soil samples prior to and after heating were determined. The degree of darkness, elemental composition, carbon and nitrogen stable isotope ratios, 13C nuclear magnetic resonance spectra, and X-ray diffraction patterns of humic acids extracted from the soils before and after heating were measured. The proportion of humic acids in total carbon decreased with increasing heating time at high temperature (300°C), but increased with increasing heating time at ≤250°C. The degree of darkness of the humic acids increased with increasing heating time and temperature. During darkening, the H/C atomic ratios, the proportion of aromatic C, and the carbon and nitrogen stable isotope ratios increased, whereas the proportions of alkyl C and O-alkyl C decreased. X-ray diffraction analysis verified that a stacking nanostructure developed by heating. Changes in the chemical structure of the humic acids from the heated soils depended on the type of soil. The major structural components of the humic acids from the heated Entisol were aromatic C and carboxylic C, whereas aliphatic C, aromatic C, and carboxylic C structural components were found in the humic acids from the heated Inceptisol. These results suggest that the heat-induced changes in the chemical structure of the humic acids depended on the source plant.

Synergistic and antagonistic interactions among organic amendments of contrasted stability, nutrient availability and soil organic matter in the regulation of C mineralisation

Organic amendments to agricultural soils may increase soil organic matter reserves. However, manure amendment may stimulate soil microbial activity and soil organic matter decomposition. The stability of manure and soil organic matter and the availability of nutrients are crucial for the regulation of soil organic matter decomposition. Therefore, we aimed to study the effects of adding stabilised or unstabilised manures combined with mineral nutrients (N–NO3, P–PO4, K and S–SO4) on carbon mineralisation in two agricultural (sandy loam) soils with contrasted organic matter stocks and N availability. These two soils were incubated alone and after adding unstabilised farmyard manure (FYM) or stabilised manure (compost), combined with the addition of mineral nutrients. Mineralisation of C (CO2 evolution) increased after FYM addition while it decreased after compost addition as compared to the control soils. When mineral nutrients were added to soils amended with FYM, C mineralisation synergistically increased only in soils with low nitrogen availability. In these soils, the addition of nutrients may promote the mobilization of soil C, while in soils with high N availability the synergistic mobilization of C on adding FYM may be reduced. In contrast, the depressed C mineralisation after the addition of compost was little affected by nutrient availability. We suggest that recalcitrant compounds in compost may be stabilised in soil particles and humic substances with little effects of nutrient availability. In contrast, the incorporation of FYM was mainly driven by microbial transformations and affected by nutrient availability. Finally, we conclude that the application of stabilised organic matter may be a good practice for the built up of humus in a wide range of arable lands with particular significance for land reclamation in organic matter depleted soils.

From humic substances to soil organic matter–microbial contributions. In honour of Konrad Haider and James P. Martin for their outstanding research contribution to soil science

Many decades of research have shown that humic compounds as part of the soil organic matter (SOM) are essential for the stability and ecosystem services of soils. James P. Martin and Konrad Haider based on several pioneers in humus research improved the basis for the current knowledge of key processes in the soil environment, in particular structure and formation of humus triggered mainly by soil fungi. Other major contributions are briefly described but not in the focus of this article, such as their innovative tracer experiments with isotope-labelled xenobiotic chemicals and natural litter to study their fate. Both scientists inspired generations of younger researchers to advance their approaches and laid the cornerstone of the current understanding of soil organic matter formation. This article values the key experiments of Martin and Haider in this field and the related follow-up research finally resulting in the current view on formation mechanisms of SOM and non-extractable residues (NER) of xenobiotics. The improved understanding of these processes considering necromass with tissue residues of plants, microbes and animals challenges the traditional views of humic matter as macromolecular organic matrix, which according to the research of the last years represents only a variable part of the total organic matter besides associates of low molecular weight molecules. We discuss views on soil organic matter and humic substances that are nowadays considered not to differ in molecular diversity. We will start by demonstrating the understanding of humus characteristics and humus formation three decades ago closely related to the findings of Martin and Haider. Methodological approaches to characterize relevant structural and mechanistic pictures of SOM such as the priming effect, clay mineral catalysed reactions and the various mechanisms by which natural and xenobiotic chemicals are protected in soil are briefly illustrated by examples. Fungal activities in producing secondary metabolites like polyketides and their probably minor contributions to SOM formation are presented. Open research questions stimulated by these two soil scientists are sketched which are nowadays possible to address by new sophisticated high-resolution techniques.

Short-column anion-exchange chromatography for soil and peat humic substances profiling by step-wise gradient of high pH aqueous sodium ethylenediaminetetraacetate

Novel anion-exchange liquid chromatographic method with step gradient of aqueous EDTA4− based mobile phase elution has been developed to profile available Slovak soil humic substances and alkaline extracts of various soils. The method utilize short glass column (30mm×3mm) filled in with hydrolytically stable particles (60μm diameter) Separon HEMA-BIO 1000 having (diethylamino)ethyl functional groups. Step gradient was programmed by mixing mobile phase composed of aqueous solution of sodium EDTA (pH 12.0; 5mmolL−1) and mobile phase constituted of aqueous solution of sodium EDTA (pH 12.0, 500mmolL−1). The FLD of HSs was set to excitation wavelength 480nm and emission wavelength 530nm (λem). Separation mechanism was studied by use of selected aromatic acids related to humic acids with the aid of UV spectrophotometric detection at 280nm. The proposed method benefits from high ionic strength (I=5molL−1) of the end mobile phase buffer and provides high recovery of humic acids (98%). Accurate and reproducible profiling of studied humic substances, alkaline extracts of various types of soils enables straightforward characterization and differentiation of HSs in arable and forest soils. Selected model aromatic acids were used for separation mechanism elucidation.