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Humic Substances: Chemistry and Multidimensional Role in Agricultural Systems and Pollution Management

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A special issue of Agronomy (ISSN 2073-4395). This special issue belongs to the section "Soil and Plant Nutrition".

Deadline for manuscript submissions: 30 June 2025 | Viewed by 15947

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Dr. Maria Roulia

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Department of Chemistry, Inorganic Chemistry Laboratory, National and Kapodistrian University of Athens, Panepistimiopolis, 157 71 Athens, Greece
Interests: humic substances; aluminosilicate materials; fertilizers; adsorption; soil quality; clays and clay minerals; waste management; perlites
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Dear Colleagues,

Humic substances (HSs), the sophisticated and enigmatic dark-colored heterogeneous organic mixtures omnipresent in soils, sediments, and natural waters, are structurally recalcitrant compounds primarily associated with agriculture.

Humic substances are universal amphiphiles and possess unique chelating properties with both organic and inorganic species; they regulate the bioavailability of a wide range of compounds, e.g., nutrients and pollutants; they also ameliorate soils and promote plant growth, displaying auxin-like activity and improving physiological and metabolic performance; they display electron-shuttling behavior and may act as environmental signals. Therefore, HSs have multiple essential functions in water quality improvement, soil chemistry, and sustainable agriculture. However, the elusiveness regarding the molecular structures and the multidisciplinary roles of HSs remains.

In the Special Issue “Humic Substances: Chemistry and Multidimensional Role in Agricultural Systems and Pollution Management” all scientific contributions (e.g., research papers, review articles, communications, short notes, and opinions) that provide innovative insights into the related topics are welcome, and the topics include the following:

The structure and physicochemical properties of HSs;
HS benefits by shifting to more sustainable agriculture;
The impact of HSs and HS-containing materials on soils, waters, plants, crop production, and living organisms (humans, birds, animals, and fishes);
Interactions between HSs and toxic compounds, both organic and inorganic, related to environmental health;
Organic waste management techniques, e.g., composting and adsorption, connected to HSs’ fate.

Dr. Maria Roulia
Guest Editor

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Keywords

humic substances
humic acid
fulvic acid
humin
soil
water quality
pollutant sequestration
organic/inorganic nutrients
plant growth
waste management

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Published Papers (11 papers)

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Research

Jump to: Review

14 pages, 1454 KiB

Open AccessArticle

Pig Slurry Fertilization Changes the Pyrolytic Signature of Humic Substances in Calcareous Soil

by Àngela D. Bosch-Serra, Diana E. Jiménez-de-Santiago, José A. González-Pérez and Gonzalo Almendros

Agronomy 2025, 15(3), 725; https://doi.org/10.3390/agronomy15030725 - 17 Mar 2025

Viewed by 550

Abstract

The aim of this study was to determine the effect of progressive pig slurry (PS) rates, applied over a 12-year period, on the molecular composition of soil organic matter in a calcareous soil. Annual organic matter rates of PS ranged from 1.0 to 4.8 Mg ha⁻¹. Humic acids (HAs) were extracted from field plots treated with PS, including a control (no PS applied). These HAs were analysed using pyrolysis–gas chromatography–mass spectrometry. The proportions of the 122 major compounds released from the soil HAs indicated that PS stimulated humification processes, with the degree of enhancement depending on the application rate. The applied PS contained a high proportion of aliphatic compounds, but only steroids and triterpenes accumulated in the HA soil fraction, and this was only observed at low PS rates. These results suggest that the application of PS leads to a dose-dependent increase in alkyl compounds, mainly alkanes and olefins. Aromatic compounds also showed a dose-dependent increase, but not in terms of the demethoxylated compounds typical of mature humic substances found in the original soil. Instead, the increase in aromatics was observed in the form of methoxyphenols, suggesting a recent incorporation of lignin derivatives from crop residues into the HA. Full article

(This article belongs to the Special Issue Humic Substances: Chemistry and Multidimensional Role in Agricultural Systems and Pollution Management)

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20 pages, 1887 KiB

Open AccessArticle

Alkaline Extraction in Air Enhances Antioxidant and Biological Activities of Humic Acids

by Anna Zavarzina, Irina Davydova, Natalia Kulikova, Anastasiya Nikolaeva and Olga Philippova

Agronomy 2025, 15(3), 689; https://doi.org/10.3390/agronomy15030689 - 13 Mar 2025

Viewed by 428

Abstract

Humic acids (HAs) possess diverse functionalities, endowing them with multiple applications as bioactive compounds in agriculture. Alkaline extraction is key to obtaining HAs from their source material. The presence of oxygen during extraction can lead to oxidative changes in the humic structure. The extent of HA transformation depending on their origin remains poorly understood, and the effect of alkaline extraction on the HA biological activities is yet to be estimated. Here, we compare the physicochemical properties of HAs extracted from fresh organic material, compost, in air (HA-O₂) and under nitrogen (HA-N₂). We also assess the antioxidant properties of HAs-O₂ and HAs-N₂ from compost (HAC), Retisol (HAR), and Chernozem (HACh) and relate them to the HA biological activities. Changes in the HAC properties were analyzed using the following techniques: elemental composition, ultraviolet–visible and infrared spectroscopy, ¹³C nuclear magnetic resonance (¹³C-NMR), electron paramagnetic resonance (EPR), gel filtration using Sephadex G-75 gel, and potentiometric titration. The HA antioxidant properties were explored using the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) assay (antiradical activity) and phosphomolybdenum assay (total antioxidant capacity). The HA biological activity was estimated by priming radish and wheat seeds (0.5 g L⁻¹ HAs, 25 °C, 5 h for radish and 14 h for wheat), followed by germination tests. Alkaline extraction of HAC in air vs. nitrogen resulted in a 1.2-fold increase in the O/C ratio and optical density at E₄₆₅, oxidation of aliphatic fragments, a 2-fold increase in the contents of functional groups, and a 1.2-fold increase in the number of paramagnetic centers. All HA-O₂ preparations have demonstrated an enhanced antiradical activity (1.3–1.6 times) and total antioxidant capacity (1.1–1.3 times) compared to HA-N₂. The Vigor Index of seeds primed with HA-O₂ was 1.1-to-1.8-fold higher than those treated with HA-N₂, depending on the HA origin. We demonstrate that alkaline treatment in air benefits the antiradical and biological activities of HAs, making such preparations more attractive for use as natural antioxidants and priming agents. This opens up new perspectives for using O₂-modified HAs as innovative plant stimulants in agriculture. Full article

(This article belongs to the Special Issue Humic Substances: Chemistry and Multidimensional Role in Agricultural Systems and Pollution Management)

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13 pages, 1570 KiB

Open AccessArticle

Farm-Produced Plant Biostimulant: Case Study with Passion Fruit

by Luciano P. Canellas, Natália A. Canellas, Dariellys Martinez-Balmori, Rakiely M. Silva, Raul C. C. Rosa and Fabio L. Olivares

Agronomy 2025, 15(3), 681; https://doi.org/10.3390/agronomy15030681 - 12 Mar 2025

Viewed by 520

Abstract

Plant biostimulants (PBs) have been considered the new wave for ecological intensification and sustainability, but are they sustainable? They increase nutrient use efficiency and reduce the impact of abiotic stress in plants. However, commercially available PBs based on humic substances are obtained using non-renewable sources of organic matter. At the same time, the microbial inoculants include a discussion of the properties of microorganisms and formulation design, as well as standards of purity and process control. Farmers depend on biological inputs like others to generate additional income for agribusiness. We produced a composite PB using humic substances isolated from vermicompost with KOH 5% and microbial consortia of plant growth-promoting bacteria (H. seropedicae, G. diazotrophicus, Bacillus spp.) grown in a simple medium with molasses and fishmeal as sources of C and N, respectively, in a homemade reactor at 37 °C for 36 h. The on-farm PB was applied directly in a passion fruit trial, and plant health and yield were monitored. The plants treated with the PB showed decreased visual symptoms of pests and diseases concurrent to higher activities of the enzymes used to monitor the induction of the plant resistance system (1,3-β glucanase, peroxidase, and phenylalanine ammonia-lyase). Plants treated with the PB yielded more than 50% more passion fruit than the control in soil with natural low fertility, fertilized with vermicompost. It is possible to produce PBs directly on the farm, leveraging locally available resources and simple technologies to sustainably enhance plant health and productivity. Full article

(This article belongs to the Special Issue Humic Substances: Chemistry and Multidimensional Role in Agricultural Systems and Pollution Management)

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18 pages, 11061 KiB

Open AccessArticle

Humic Acid Enhances Antioxidant and Glyoxalase Systems to Combat Copper Toxicity in Citrus

by Wei-Tao Huang, Xu-Feng Chen, Wei-Lin Huang, Qian Shen, Fei Lu, Ning-Wei Lai, Jiuxin Guo, Lin-Tong Yang, Xin Ye and Li-Song Chen

Agronomy 2025, 15(1), 99; https://doi.org/10.3390/agronomy15010099 - 1 Jan 2025

Viewed by 755

Abstract

Most commercial citrus fruits are grown in acidic soils with high copper (Cu) and low organic matter levels in China. Sweet orange (Citrus sinensis (L.) Osbeck cv. Xuegan) seedlings were treated with 0 (HA0), 0.1 (HA0.1), or 0.5 (HA0.5) mM humic acid (HA) and 0.5 (Cu0.5) or 400 (Cu400 or Cu excess) μM CuCl₂ for 24 weeks. The purpose was to validate the hypothesis that HA reduces the oxidative injury caused by Cu400 in roots and leaves via the coordination of strengthened antioxidant defense and glyoxalase systems. Copper excess increased the superoxide anion production rate by 27.0% and 14.2% in leaves and by 47.9% and 33.9% in roots, the malonaldehyde concentration by 199.6% and 27.8% in leaves and by 369.4% and 77.4% in roots, and the methylglyoxal concentration by 18.2% and 6.6% in leaves and by 381.8% and 153.3% in roots, as well as the H₂O₂ production rate (HPR) by 70.5% and 16.5% in roots, respectively, at HA0 and HA0.5. Also, Cu400 increased the leaf HPR at HA0, but not at HA0.5. The addition of HA reduced the Cu400-induced production and accumulation of reactive oxygen species and methylglyoxal and alleviated the impairment of Cu400 to the antioxidant defense system (ascorbate-glutathione cycle, antioxidant enzymes, sulfur-containing compounds, and sulfur-metabolizing enzymes) and glyoxalase system in roots and leaves. The HA-mediated amelioration of Cu toxicity involved reduced oxidative injury due to the coordination of strengthened antioxidant defense and glyoxalase systems. These findings highlight the promise of HA for sustainable citrus cultivation in heavy metal (Cu)-polluted soils. Full article

(This article belongs to the Special Issue Humic Substances: Chemistry and Multidimensional Role in Agricultural Systems and Pollution Management)

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20 pages, 2640 KiB

Open AccessEditor’s ChoiceArticle

Humic Substances from Waste-Based Fertilizers for Improved Soil Fertility

by Angela Maffia, Federica Marra, Francesco Canino, Santo Battaglia, Carmelo Mallamaci, Mariateresa Oliva and Adele Muscolo

Agronomy 2024, 14(11), 2657; https://doi.org/10.3390/agronomy14112657 - 11 Nov 2024

Cited by 5 | Viewed by 1569

Abstract

This research explores how different organic waste transformation methods influence the production of humic substances (HSs) and their impact on soil quality. Using olive and orange wastes as substrates, the study compares vermicomposting, composting, and anaerobic digestion processes to determine which method produces the most humic-substance-rich products. The characterization of HSs in each product included analyses of total organic carbon (TOC), humic and fulvic acid content, humification rate, humification degree, and E4/E6 ratio, with HSs extracted using potassium hydroxide (KOH) and analyzed via Diffuse Reflectance Infrared Fourier-Transform (DRIFT) spectroscopy to assess structural complexity. The results revealed that the chemical composition of the input materials significantly influenced the transformation dynamics, with orange by-products exhibiting a higher humification rate and degree. Vermicomposting emerged as the most efficient process, producing fertilizers with superior humic content, greater microbial biodiversity, and enhanced cation exchange capacity, thus markedly improving soil quality. Composting also contributed to the stabilization of organic matter, albeit less effectively than vermicomposting. Anaerobic digestion, by contrast, resulted in products with lower levels of HSs and reduced nutrient content. Aerobic processes, particularly vermicomposting, demonstrated the most rapid and effective transformation, producing structurally complex, stable humus-like substances with pronounced benefits for soil health. These findings underscore vermicomposting as the most sustainable and efficacious approach for generating HS-rich organic fertilizers, presenting a powerful alternative to synthetic fertilizers. Furthermore, this study highlights the potential of organic waste valorization to mitigate environmental pollution and foster circular economy practices in sustainable agriculture. Full article

(This article belongs to the Special Issue Humic Substances: Chemistry and Multidimensional Role in Agricultural Systems and Pollution Management)

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18 pages, 2764 KiB

Open AccessArticle

Effect of Vermicompost Application on the Soil Microbial Community Structure and Fruit Quality in Melon (Cucumis melo)

by Mei Tian, Rong Yu, Song Guo, Wanbang Yang, Shengfeng Liu, Huiying Du, Jinjin Liang and Xingxu Zhang

Agronomy 2024, 14(11), 2536; https://doi.org/10.3390/agronomy14112536 - 28 Oct 2024

Viewed by 1458

Abstract

Melon (Cucumas melon) is widely cultivated and popular because of its quality value and unique flavor. However, the continuous cropping of melons in greenhouses has various negative effects on the soil environment, melon growth, and quality. Recently, farmers have utilized organic fertilization, especially vermicompost, for melons to resist the harmful effects of continuous cropping. A field experiment was conducted to explore the effects of vermicompost on soil microbes and melon fruit quality via high throughput sequencing and chemical sequencing methods. The results showed that the application of vermicompost decreased (p < 0.05) soil pH and increased organic matter, available phosphorus, biomass, urease, catalase, peroxidase, and alkaline phosphatase. A total of 3447 bacterial and 718 fungal operational taxonomic units were identified in all soil samples. Application of vermicompost decreased (p < 0.05) the relative abundances of Acidobacteriota, Gemmatimonadota, Actinobacteriota, and unclassified and increased the relative abundance of Planctomycetota. Compared with the control soil, vermicompost application resulted in significantly higher bacterial Chao indices and a significantly lower Chao index under vermicompost of 60 t ha⁻¹ based on farmers’ normal fertilizer and significantly lower diversity under vermicompost of 90 t ha⁻¹. Otherwise, vermicompost application increased the photosynthetic rate and chlorophyll content of melon leaves and increased the total sugar, soluble solids, vitamin C, soluble protein, and organic acid contents of melon. The results of redundancy analysis indicated that Proteobacteria exhibited a positive correlation with soil ammonium nitrogen (AN) and pH, while showing a negative association with soil available phosphorus and organic matter. Spearman’s correlation analysis revealed that both total sugar content and central soluble solid content in melon had a significant positive correlation (p < 0.05) with Patescibacteria. This study demonstrates that the application of vermicompost alters the microbial community structure in melon cultivation, enhancing fruit quality; this not only promotes a healthier soil ecosystem but also contributes to sustainable and productive practices in melon farming. Full article

(This article belongs to the Special Issue Humic Substances: Chemistry and Multidimensional Role in Agricultural Systems and Pollution Management)

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20 pages, 1350 KiB

Open AccessArticle

The Role of Organic Matter in Phosphorus Retention in Eutrophic and Dystrophic Terrestrial Ecosystems

by Magdalena Debicka

Agronomy 2024, 14(8), 1688; https://doi.org/10.3390/agronomy14081688 - 31 Jul 2024

Cited by 1 | Viewed by 1166

Abstract

Phosphorus (P) retention in soils in the presence of organic matter (OM) has been, for years, a topic with no clear conclusions. Considering the important ecological functions of peatlands, the objective of this study is to examine the role of OM transformation in relation to P status in Histosols in the Oder Valley (Poland). Basic physical and chemical properties and the following P forms were determined in the organic horizons of 5 soil profiles from two habitats (eutrophic and dystrophic): total (Pt) and organic P (P_o), available P (P_M3), easily soluble P (P_CaCl₂), water-soluble P (P_W), and fraction of P_o in humic (P_o_HA) and fulvic (P_o_FA) acids after extraction with 0.5 mol L⁻¹ NaOH. The results were statistically verified in both examined habitat groups separately. The higher values of mobile P forms were found in the upper organic horizons released from OM constituents as a result of their decomposition. The role of OM in P retention was strongly related to the activity of humic substances (HS): a higher P_o percentage (6.9–99.4% of P_o) was observed in dystrophic, whereas a lower (9.3–28.6% of P_o) was observed in eutrophic Histosols. Humic acids played a dominant role in P retention compared to fulvic acids in most peat horizons, especially at pH < 5. The role of HA and FA in P retention was clearly dependent on forms found only in eutrophic Histosols. The important role of FA in P retention during OM transformation was confirmed by negative correlations between P_o_FA and macronutrient ratios in both soil groups. The results confirm the variable role of OM in P retention, depending on soil environmental conditions and OM type (peat and moorsh). This may have important applications not only in areas of natural importance, for which the release of mobile P forms may be a threat, but also in agricultural areas where, for a change, we struggle to increase P availability. Full article

(This article belongs to the Special Issue Humic Substances: Chemistry and Multidimensional Role in Agricultural Systems and Pollution Management)

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16 pages, 4248 KiB

Open AccessArticle

Molecular Composition of Humic Acids of Different Aged Fallow Lands and Soils of Different Types of Use in Northwest of Russia

by Vyacheslav Polyakov, Timur Nizamutdinov and Evgeny Abakumov

Agronomy 2024, 14(5), 996; https://doi.org/10.3390/agronomy14050996 - 9 May 2024

Cited by 1 | Viewed by 1126

Abstract

Post-agrogenic transformation of fallow soils leads to changes in soil carbon content, the molecular composition of humic substances, and rates of organic matter stabilization, which can affect climate change on the planet. In this regard, we analyzed the molecular composition of humic acids isolated from natural and fallow soils in the southern Taiga zone of northwest Russia. Different-aged soils on fallow lands represent a model of soil transformation in time, and data on the transformation of soil humic acid molecular composition make a significant contribution to the understanding of soil organic matter stabilization aspect issues. In this case, the molecular structure of humic acids isolated from natural and fallow soils in northwest Russia was analyzed. To study the molecular composition of HAs, the elemental composition was analyzed, and ¹³C (CP/MAS) NMR spectroscopy of HAs isolated from different aged abandoned soils and soils of different types of use was carried out. The obtained data showed that with the increasing age of soils in the fallow state, there is an increase in the carbon content of humic acids as well as a decrease in nitrogen content. As a result of the increasing age of soils in the fallow state, there are dynamics in the content of aromatic structural fragments in humic acids: 34% for 40 years old, 28% for 80 years old, and 31% for 120 years old. This is due to changes in the precursors of humification and the further transformation of plant residues in the soil. Re-involved fallow land soils lead to an increase in the content of aromatic structural fragments in the composition of HA in relation to HA extracted from mature soils. The lowest content of aromatic structural fragments was observed in the humic acids of 130-year-old agricultural soil, which is associated with the long-term application of organic fertilizers. Full article

(This article belongs to the Special Issue Humic Substances: Chemistry and Multidimensional Role in Agricultural Systems and Pollution Management)

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Review

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16 pages, 1283 KiB

Open AccessReview

The Formation, Stabilization Mechanism, and Environmental Impacts of Persistent Free Radicals in Soil Humic Substances

by Xuqin Wang, Yang Han, Yanwei Cao, Yiping Ni, Dengzhi Wang and Yaning Luan

Agronomy 2025, 15(3), 602; https://doi.org/10.3390/agronomy15030602 - 28 Feb 2025

Viewed by 693

Abstract

Persistent free radicals (PFRs) in humic substances (HSs) are relatively stable free radicals with a longer lifespan compared to transient free radicals. These PFRs are abundant in the ecological environment, such as in soil and water. The formation of PFRs in HSs occurs primarily through the humification of biological residues, electron transfer between quinone and phenolic groups in HSs, and interactions among microorganisms, HSs, and mineral particles. Mineral particles contribute significantly to the stability of these radicals. Conditions such as prolonged exposure to light and redox changes further influence their formation and stability. PFRs in HSs have been applied in the treatment and remediation of environmental pollution due to their ability to adsorb, degrade, and undergo redox reactions with soil pollutants. The formation mechanisms of PFRs (including semiquinone free radicals) in soil HSs are reviewed, and the factors affecting their stability are explored. Additionally, the interaction between PFRs and soil pollutants, and their effects on pollutant migration and transformation, and soil microbial communities, are discussed. Lastly, perspectives for future research aimed at improving innovation in understanding the impact of PFRs on pollutant migration and transformation, soil microbial communities, and the environmental behavior of PFRs are provided. Full article

(This article belongs to the Special Issue Humic Substances: Chemistry and Multidimensional Role in Agricultural Systems and Pollution Management)

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33 pages, 1473 KiB

Open AccessReview

Humic Substances: Bridging Ecology and Agriculture for a Greener Future

by Angela Maffia, Mariateresa Oliva, Federica Marra, Carmelo Mallamaci, Serenella Nardi and Adele Muscolo

Agronomy 2025, 15(2), 410; https://doi.org/10.3390/agronomy15020410 - 6 Feb 2025

Cited by 1 | Viewed by 2802

Abstract

Humic substances (HSs) are emerging as multifunctional natural catalysts in sustainable agriculture, offering novel opportunities to enhance soil health, plant productivity, and environmental resilience. This review synthesizes recent insights into the chemical diversity, biological mechanisms, and ecological impacts of HSs, presenting a new perspective on their role as dynamic agents in agroecosystems. Derived from decomposed organic matter, HSs regulate critical processes such as nutrient cycling, carbon sequestration, and pollutant detoxification. Unlike plant and microbial biomass, which undergo rapid mineralization due to their active dynamism, HSs exhibit significant resistance to biodegradation, leading to a prolonged residence time in soil that spans years or even centuries. This stability allows HSs to maintain their functional roles over extended periods, contributing to long-term soil health and ecosystem sustainability. Their integration into agricultural systems has demonstrated profound effects, including improved soil structure, increased water retention, and the stimulation of microbial activity, which collectively bolster plant stress tolerance and yield. Notably, it has been proposed that HSs exhibit hormone-like properties, influencing plant signaling pathways to enhance root architecture and nutrient acquisition. Moreover, HSs contribute to environmental remediation by regulating the leaching of heavy metals, mitigating nutrient runoff, and fostering climate resilience. This review highlights the synergistic potential of combining HSs with organic amendments like compost and biochar, positioning HSs as a cornerstone of regenerative farming practices. Addressing challenges such as variability in composition and application methods, the discussion underscores the urgency of developing standardized approaches to harness their full potential. By framing HSs as versatile and adaptive tools, this review paves the way for advancing sustainable agricultural systems while addressing global challenges like food security and climate change. Full article

(This article belongs to the Special Issue Humic Substances: Chemistry and Multidimensional Role in Agricultural Systems and Pollution Management)

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23 pages, 3853 KiB

Open AccessReview

The Use of Spectroscopic Methods to Study Organic Matter in Virgin and Arable Soils: A Scoping Review

by Evgeny Lodygin and Evgeny Abakumov

Agronomy 2024, 14(5), 1003; https://doi.org/10.3390/agronomy14051003 - 9 May 2024

Cited by 6 | Viewed by 2264

Abstract

The use of modern spectroscopic methods of analysis, which provide extensive information on the chemical nature of substances, significantly expands our understanding of the molecular composition and properties of soil organic matter (SOM) and its transformation and stabilization processes in various ecosystems and geochemical conditions. The aim of this review is to identify and analyze studies related to the application of nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy techniques to study the molecular composition and transformation of organic matter in virgin and arable soils. This article is mainly based on three research questions: (1) Which NMR spectroscopy techniques are used to study SOM, and what are their disadvantages and advantages? (2) How is the NMR spectroscopy technique used to study the molecular structure of different pools of SOM? (3) How is ESR spectroscopy used in SOM chemistry, and what are its advantages and limitations? Relevant studies published between 1996 and 2024 were searched in four databases: eLIBRARY, MDPI, ScienceDirect and Springer. We excluded non-English-language articles, review articles, non-peer-reviewed articles and other non-article publications, as well as publications that were not available according to the search protocols. Exclusion criteria for articles were studies that used NMR and EPR techniques to study non-SOM and where these techniques were not the primary methods. Our scoping review found that both solid-state and solution-state NMR spectroscopy are commonly used to study the structure of soil organic matter (SOM). Solution-phase NMR is particularly useful for studying soluble SOM components of a low molecular weight, whereas solid-phase NMR offers advantages such as higher ¹³C atom concentration for stronger signals and faster analysis time. However, solution-phase NMR has limitations including sample insolubility, potential signal aggregation and reduced sensitivity and resolution. Solid-state NMR is better at detecting non-protonated carbon atoms and identifying heterogeneous regions within structures. EPR spectroscopy, on the other hand, offers significant advantages in experimental biochemistry due to its high sensitivity and ability to provide detailed information about substances containing free radicals (FRs), aiding in the assessment of their reactivity and transformations. Understanding the FR structure in biopolymers can help to study the formation and transformation of SOM. The integration of two- and three-dimensional NMR spectroscopy with other analytical methods, such as chromatography, mass spectrometry, etc., provides a more comprehensive approach to deciphering the complex composition of SOM than one-dimensional techniques alone. Full article

(This article belongs to the Special Issue Humic Substances: Chemistry and Multidimensional Role in Agricultural Systems and Pollution Management)

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