Soil Organic Carbon Sequestration Mechanisms and the Chemical Nature of Soil Organic Matter—A Review

Abstract

This article presents a review of several non-exclusive pathways for the sequestration of soil organic carbon, which can be classified into two large classical groups: the modification of plant and microbial macromolecules and the abiotic and microbial neoformation of humic substances. Classical studies have established a causal relationship between aromatic structures and the stability of soil humus (traditional hypotheses regarding lignin and aromatic microbial metabolites as primary precursors for soil organic matter). However, further evidence has emerged that underscores the significance of humification mechanisms based solely on aliphatics. The precursors may be carbohydrates, which may be transformed by the effects of fire or catalytic dehydration reactions in soil. Furthermore, humic-type structures may be formed through the condensation of unsaturated fatty acids or the alteration of aliphatic biomacromolecules, such as cutins, suberins, and non-hydrolysable plant polyesters. In addition to the intrinsic value of understanding the potential for carbon sequestration in diverse soil types, biogeochemical models of the carbon cycle necessitate the assessment of the total quantity, nature, provenance, and resilience of the sequestered organic matter. This emphasises the necessity of applying specific techniques to gain insights into their molecular structures. The application of appropriate analytical techniques to soil organic matter, including sequential chemolysis or thermal degradation combined with isotopic analysis and high-resolution mass spectrometry, derivative spectroscopy (visible and infrared), or ¹³C magnetic resonance after selective degradation, enables the simultaneous assessment of the concurrent biophysicochemical stabilisation mechanisms of C in soils.

1. Introduction

1.1. Soil Processes and Carbon Sequestration

Carbon sequestration is the process by which carbon dioxide (CO₂) from the atmosphere is captured as accumulated C in soil and biomass, effectively removing it from biogeochemical circulation for extended periods. Carbon in the air is absorbed by photosynthetic plants and incorporated into complex organic macromolecules. Apart from CO₂, methane (at approximately 1.9 ppm (v/v)) is the second most notable species [1]. When plants die, their organic remains (leaves, stems, and roots) and microbial biomass undergo decomposition in the soil. A fraction of this C is incorporated as stable soil organic matter through humification processes, contributing to soil C sequestration. The remaining C is primarily released back into the atmosphere as CO₂ via mineralisation. Due to the relatively recalcitrant nature of soil C-forms, which may remain in the soil for hundreds to thousands of years, soil C sequestration plays a crucial role in controlling the concentration of CO₂ in the atmosphere [2,3].

Rough estimations suggest that the world soils represent the largest C reservoir on the planet, storing approximately 1500 to 2000 Pg (1 Pg = petagram = 10¹⁵ g) of soil organic C. This pool is more than twice the amount of C stored in living vegetation (around 560 Pg) and exceeds that in the atmosphere (750 Pg) [4,5]. As previously suggested, even small changes in soil C sequestration rates per unit area can significantly influence the global C balance [6]. In this balance, it should also be considered that an additional 800 to 1000 Pg are believed to represent the pool of soil inorganic C (primarily carbonate) [7,8,9,10].

Assuming that proper management practices can enhance soil carbon (C) sequestration, vast degraded and desertified semi-arid areas worldwide offer significant potential for storing atmospheric C [10,11]. This potential should be considered in transnational programs, especially if C storage is included in CO₂ credit markets. In this context, efficient and affordable environmental strategies and monitoring tools are essential to ensure effective stabilisation of C in soils. As a result, large-scale soil C sequestration initiatives present valuable scientific, social, and economic opportunities worth exploring [12,13].

The mechanisms responsible for the stabilisation of atmospheric CO₂ into biodegradation-resistant organic forms that accumulate in terrestrial ecosystems remain largely unknown. This is primarily because the chemical composition of soil organic matter and the key processes governing its formation are only broadly understood. This is particularly true for the so-called humic substances, which consist of complex, highly transformed macromolecular C-forms that constitute a significant portion of biodegradation-resistant materials in soils and sediments [14].

1.2. On the Variable Effectiveness of Soil C Sequestration

In general, C sequestration does not occur uniformly across all ecosystem compartments, and its efficiency varies depending on the interplay of abiotic (climatic, geologic, and edaphic factors) and biological constraints. Optimal conditions for C sequestration arise only under specific environmental scenarios in which these factors are favorably aligned. For instance, ecosystems with stable climatic conditions, fertile soils, and abundant vegetation tend to exhibit higher rates of sequestration. Conversely, regions with extreme temperatures, poor soil quality, and limited biological activity often show a reduced sequestration potential. This variability underscores the importance of understanding localised environmental conditions when designing strategies to enhance carbon storage [15,16]. Furthermore, the dynamic nature of these factors means that C sequestration is not a static process but one that can fluctuate over time in response to environmental changes, such as shifts in land use, climate patterns, and ecosystem management practices.

The role of soil as a C reservoir is still debated, given the varied results from studies across different climates and geological conditions [17,18]. Beyond climate, factors like vegetation type, soil texture, and mineralogical composition significantly influence the soil’s capacity to store C over the long term. Although agricultural sinks were recognised in the Kyoto Protocol (Article 3.4) [19], their inclusion was limited due to a lack of international consensus on the effectiveness of soil C sequestration during the first reporting period [20,21]. Consequently, the development of the Sink Table was restricted, as agricultural soils were not initially integrated into the agreement. This highlights the need for further research and international cooperation to better understand and harness the role of soil in storing atmospheric C.

Although progress has been made in studying the biogeochemical processes that regulate C sequestration, which is the core focus of this review, socioeconomic, cultural, and political factors that greatly affect its feasibility remain insufficiently explored [22].

1.3. Considerations on the Side-Effects of C Sequestration

Soil C sequestration, particularly in semi-arid and sub-humid regions, can not only offset C emissions but also contribute to restoring C in impoverished and degraded soils. This process provides a sustainable approach to combat land degradation and desertification while simultaneously enhancing agricultural productivity and promoting long-term soil conservation. From an agronomic perspective, the mechanisms driving C sequestration in soil include the humification of organic waste, formation of stable organomineral complexes that enhance soil aggregation, strategic placement of organic matter below the plough layer, promotion of deep-rooting, and calcification. These practices collectively represent environmentally friendly strategies for improving soil health and its resilience.

In summary, the indirect benefits of soil C sequestration are evident in the improvement of soil structural stability, water-holding capacity, nutrient availability, and microbial activity. These characteristics are also linked to the ability of organic matter to regulate the composition of the soil solution, thereby optimizing the uptake of macro- and microelements by plants. Furthermore, the increase in stabilised C, together with the soil mineral fraction, enhances its potential to act as an environmental filter for heavy metals. This includes reducing the leachability and bioavailability of organic and mineral pollutants, such as agrochemicals and heavy metals [23,24,25]. These combined effects underscore the multifaceted role of soil C sequestration in promoting soil health and environmental sustainability

From a biogeochemical perspective, humic substances, unlike unhumified biomass, play a central role in regulating nutrient transport mechanisms essential for sustaining soil life. Their remarkable ability to form complexes of varying stability with both elements and mineral compounds across scales, from free ions to mineral fragments, enables them to mediate the availability of bioelements within ecosystems.

Humic substances function as a dynamic interface between bioavailable and immobilised soil elements, bridging short-term nutrient cycling and long-term geochemical storage, including the formation of organic and inorganic soil minerals. The role of soil organic matter in mineral dynamics is dual: it enhances mineral weathering and contributes to biomineralisation processes. These processes are facilitated by humic-type organic matrices, which influence nucleation, crystal growth, and mineral morphology, resulting in the formation of biominerals such as carbonates, silicates, phosphates, sulfides, sulfates, and oxides [26,27,28].

In contrast, it should also be taken into consideration that, in addition to C, other essential elements, such as nitrogen (N) and phosphorus (P), are also sequestered in soil humus formations [29]. Therefore, in forest soils, effective C sequestration may often be associated with the accumulation of raw humus types (e.g., moder and mor), which are typically associated with slow biogeochemical cycling and limited primary productivity. In such humus formations, the interaction between organic matter and the mineral fraction is relatively weak compared to active humus types like mull. As a result, most pedogenic processes in such systems are less influenced by the formation of stable organo-mineral complexes but are instead characterised by the vertical redistribution of organic fractions along the soil profile and the generation of leachates with soluble organic fractions that, in some cases, may have adverse effects on water quality, causing the exportation of plant nutrients from the soil subsystem.

At this point, it is crucial to emphasise that in a global soil management strategy, monitoring the quality of organic matter sequestered in soils may be more significant than focusing solely on its total quantity. For instance, most semi-arid Mediterranean areas exhibit low concentrations of highly transformed soil organic matter but have high resilience to climate change. This stands in contrast to soil formation in subhumid areas, where large amounts of relatively less transformed organic matter are present [29]. In a possible future scenario of climate change with decreased humidity and increased temperature, these subhumid soils could become sources of CO₂ emissions due to low soil organic matter humification and the weak association between the organic and mineral fractions [30]. Therefore, the present article aims to highlight soil-specific factors that influence the effectiveness of C sequestration, independent of the variable and often contentious impacts of climate, such as annual fluctuations in temperature and moisture [31]. By focusing on these intrinsic soil properties, we can better understand and optimize C sequestration practices in diverse environments.

1.4. Basic Research on the C Sequestration in Soil

It should not be an oversimplification to consider that the long-term soil C sequestration process is fundamentally linked to the progressive formation of humic substances. These complex macromolecular compounds are ubiquitous and widely distributed in soils, water, and fossil organic deposits, representing the largest reservoir of organic C on the Earth’s surface [7]. Humic substances are composed of intricate structures derived from the alteration of biosynthetic materials, as well as newly formed macromolecules generated both abiotically and biosynthetically.

In addition to the complex functional relationships of humic substances in agroecological processes in soils, the scientific reappraisal of humification processes has acquired renewed interest in research on the environmental impact of the mechanisms involved in the carbon cycle [32]. This area of study is particularly challenging and complex due to the lack of a defined chemical structure for humic substances. Their chemical composition must be inferred using analytical descriptors, such as the extent of different structural domains, the presence of discrete macromolecular regions with selectively preserved entities resembling microbial or plant biomacromolecules, and the diagnostic presence of biomarker compounds with chemotaxonomic value [33,34]. Recent advancements in this field have been facilitated by the development of new analytical and instrumental tools [35,36,37,38,39,40]. Nevertheless, a more comprehensive understanding is required to elucidate the role of soil organic matter in terrestrial ecosystem conservation and productivity.

1.5. Experimental Approaches to Monitor Soil C Sequestration Processes

There is compelling evidence that different simultaneous mechanisms may contribute to C sequestration in soil [41]. These mechanisms include the selective preservation of biomass fractions, diagenesis of biomacromolecules, and humification by neoformation sensu stricto. Neoformation pathways encompass the synthesis of humic-like substances by living organisms, in addition to enzyme-catalysed extracellular reactions in the soil, thereby promoting the condensation of humic acid precursors within the soil environment. This is also the case for humic substances formed via abiotic condensation, both spontaneous and catalysed by mineral oxides or clays. In the most favourable cases, the relative contributions of these mechanisms in different soils can be assessed using specific advanced techniques for molecular characterisation of humic substances. These techniques include the isolation and analysis of free biomarkers or signature compounds [42], selective chemical degradation followed by mass spectrometry, and non-destructive analytical methods such as visible, infrared, and nuclear magnetic resonance (NMR) spectroscopy [43,44,45,46].

Despite continuous advances in the chemical characterisation of soil organic matter, extraction remains a persistent limitation. It should be noted that protocols designed to isolate specific fractions, such as humic or fulvic acids, capture only a small proportion of the total soil carbon. Furthermore, these protocols may also introduce selective biases. This has prompted some authors to question whether the extracted humic substances accurately reflect the state of organic matter in situ [47].

Consequently, techniques that enable the study of soil organic matter without prior extraction have become increasingly relevant. Examples of such techniques include analytical pyrolysis and solid-state ¹³C NMR spectroscopy, which can be applied directly to whole soil samples. However, it should be noted that these techniques have several limitations [39]. Even when the most advanced molecular techniques are employed, a combination of techniques is required to produce a comprehensive overview, as each technique highlights different structural features while overlooking others. This is the case for techniques that detect only compounds in a narrow window of molecular weights. In other cases, the major structural units of humic substances may produce weak, overlapping, and non-diagnostic spectroscopic signals [34,37]. However, these limitations are not critical when standardised procedures are consistently applied across samples to ensure reliable comparisons.

1.6. The Soil Organic Matter Constituents: Biomacromolecules and Humic Substances

A portion of the total organic matter in soils, particularly that of recent origin, shows a chemical structure that can be considered analogous to that of individual or macromolecular constituents of the biomass, primarily derived from plant and microbial sources. Some of these organic substances in the soil, such as cellulose, hemicelluloses, lignins, cutins, suberins, and proteins, may undergo some degree of structural modification (diagenetic transformations), including depolymerisation, condensation, changes in the functional group composition, or the incorporation of products derived from microbial metabolism (e.g., lipid or protein). Although these plant constituents are readily metabolised and transformed mostly into CO₂ and H₂O through the mineralisation process, it is also possible that certain geological and climatic conditions can promote their long-term preservation in soils. In such cases, interactions with other soil constituents may render a significant proportion of these substances resistant to decomposition, allowing them to persist for extended periods [48,49].

In addition to the above-described soil organic constituents with more or less defined structures, the majority of the organic matter stabilised in soils, whether in particulate or colloidal forms, consists of a complex mixture of macromolecular structures based on three-dimensional networks of polyfunctional units, which form a series of structural domains of more or less condensed and chaotic structures [50]. These humic substances typically include aromatic (phenolic, alkylaromatic, and polycyclic) and aliphatic (O-alkyl and alkyl) domains, as well as oxygen-containing functional groups attached to surfaces with varying reactivity [35,51]. However, humic fractions extracted from soils may also contain recognisable biomolecular fragments that are intimately associated with and sometimes covalently bonded to the humic matrix. These fragments cannot be effectively separated [29], which further complicates their characterisation. The establishment of general structural models remains challenging for these substances [52].

Despite their extremely complex chemical structure, humic substances are extensively and widely studied due to their dual significance: they provide valuable information about the characteristics and function of the ecosystems, and they exert a key influence in shaping soil structure and influencing the mobility (and bioavailability) of almost all components—both organic and inorganic—in the soil solution [25,35,53]. In fact, owing to the long residence time of humic substances and the simultaneous occurrence in their structure of constituents derived from a variety of processes and synthetic pathways, the humic macromolecules can be viewed as biogeochemical records of past and present environmental processes in which soil organic matter has been formed. Nevertheless, the average residence time of humic substances remains controversial. It is evident that the data reported by various authors are challenging to compare due to the variability in the origin of the substances in question. It is acknowledged that residence times can vary considerably, ranging from decades to over 2000 years, depending on soil type, molecular size, depth within the soil profile, and environmental conditions [54,55,56].

2. Soil Carbon Stabilization Factors

This section reviews several well-established processes involved in humus formation, as well as more recent hypotheses concerning the origin of stable C forms in soils. The accumulation of stable C forms in soil depends on a variety of factors and processes, many of which involve the chemical composition of organic matter and its interactions with other soil constituents, often with similar efficiency [57]. Among the most relevant factors affecting the retention of C forms in soils, the following are particularly noteworthy (Figure 1).

2.1. Intrinsic Factors

This category encompasses factors related to the chemical nature of soil organic matter constituents and the different organizational levels of their macromolecular structures.

2.1.1. The Resistance of Structural Units

The stability of organic matter chemical components against biological, chemical, and thermal degradation is determined to some extent by the relative abundance of specific structural units, such as aromatic, alkyl, N-, and O-containing groups. In particular, aromatic units have traditionally been considered more recalcitrant to degradation than aliphatic structures (e.g., O-alkyl and alkyl). Among aliphatic units, O-alkyl groups, primarily derived from carbohydrates, are thought to be preferentially used by soil microorganisms. In recent years, considerable progress has been made in understanding the structural and functional differences between soil organic matter fractions, particularly in terms of the proportions of their different constituents. These differences can be evidenced in the laboratory using mild degradation techniques, which yield variable amounts of non-degraded residues, or through multi-stage degradation approaches that progressively remove distinct macromolecular domains [58,59,60,61]. Furthermore, recent studies suggest that soils with varying C contents accumulate humic acids with distinct molecular compositions. This has enabled the identification of molecular proxies that correlate with the C sequestration potential of soils under different environmental conditions [62,63,64].

2.1.2. Intramolecular Bridging Factors

The chemical stability of soil organic matter against different types of degradation is closely linked to its degree of structural condensation. This, in turn, may be influenced by factors such as the number of intramacromolecular bridges or the abundance of polyfunctional “building blocks”. This can be illustrated by the differing susceptibility to enzymatic attack displayed by the syringyl- (3,5-dimethoxyphenyl) and guaiacyl- (3-methoxyphenyl)-type plant lignins. Syringyl-type lignins, which possess a higher number of methoxyl groups on their phenolic units, exhibit lower connectivity possibilities between structural units, resulting in reduced resistance to degradation compared to guaiacyl-type lignins [65]. The “potential resistance” of soil organic matter can be evaluated in the laboratory using appropriate techniques, such as selective methods of sequential degradation and approaches designed for the controlled cleavage of specific bond types [66,67,68].

2.1.3. Stability Induced by the Chaotic Structure

Evidence suggests that the high degree of structural heterogeneity in soil organic matter plays a substantial role in its resistance to biodegradation [69]. In the context of the biological evolution scenario, humification is a process that does not provide living organisms with a readily exploitable source of resources for their metabolism. During humification, the structural complexity of humic macromolecules progressively increases, rendering them less recognisable and accessible to microbial enzymes. This is attributed to the chaotic structure and fractal morphology [70] of humic colloids. Furthermore, successive perturbations of ecosystems, combined with a “long term memory” encoded in the humic structure [71,72], have recently been related to stabilisation of C.

2.2. Extrinsic Factors

In addition to climatic and local factors (waterlogging, temperature, topography, etc.), which are not soil-dependent processes and thus will not be described in this review, several extrinsic factors influence soil organic matter stabilisation independently of its chemical composition. Among these soil-related factors, the following can be emphasised [73].

2.2.1. Physical Protection

The importance of organo-mineral interactions in protecting organic matter against biodegradation has been well established since early research [74]. Recent studies have considered that, under certain conditions, this mechanism may be the primary driver of soil organic matter stabilisation and persistence [75,76]. It is widely accepted that microaggregates contain older organic carbon, including a significant proportion of extractable humic substances. In contrast, macroaggregates are primarily composed of younger organic material, predominantly in a particulate form [77].

There has been some debate regarding the extent to which the specific chemical composition of organic matter influences its recalcitrance and, consequently, its long-term storage in soil [78]. However, this should not be a contentious issue, as different processes operate in various soil types and mineral substrates, often occurring simultaneously and non-exclusively. Consequently, it is plausible that organic matter, regardless of its original chemical composition, can become resistant to degradation by being retained by minerals, subsequently acquiring a molecular composition that is characteristic of a higher degree of maturity due to its longer residence time in the soil. Conversely, it is equally plausible that certain biomass constituents and humic substances with specific characteristics—such as a chaotic structure, condensed three-dimensional configuration, high aromaticity, and a predominance of C–C bonds—are inherently resistant to biodegradation. The properties of these humic substances make them more suitable for forming various types of organomineral complexes. This is due to the higher proportion of reactive functional groups, which are capable of forming bridges with minerals. Alternatively, particulate organic matter may progressively fragment into small clay-sized particles (like “microplastics”), in such a way that, despite its initial recalcitrance to biodegradation (i.e., no additional protection required), they are also suitable for incorporation into the organomineral complex and microaggregate fraction.

Indeed, in the context of recently introduced organic matter to soil, a broad spectrum of factors is implicated in the soil microcompartmentation patterns of particulate fractions [79,80]. Soil horizons are constituted of aggregates of varying sizes, which function as their virtual structural units and engender a variety of microenvironments in which chemical and biological reactions proceed at different rates [81,82]. In general, the physical protection of otherwise readily biodegradable organic matter often involves selective preservation processes [48], where organic material is encapsulated within microaggregates, thus hampering the diffusion of microbial enzymes. The physical speciation of humins—humic fractions insoluble in alkaline extractants—is frequently associated with the accumulation patterns of recalcitrant alkyl C [67,83,84]. Recent studies suggest that the increase in sequestered C content in agricultural soils is driven by the accumulation of humins and humic acids, as well as an increase in the proportion of water-stable microaggregates [85].

The influence of carbonates in stabilising both particulate and soluble organic matter forms is also important in calcimorphic soils [74,86]. The additional contribution of soil lipids to the formation of waterproof soil aggregates, which effectively encapsulate organic matter, should not be overlooked [87,88]. Beyond aggregate size and stability, the depth of organic matter placement in the soil profile is a critical factor in determining its stability. Notably, subsoil layers below 1-m depth exhibit the greatest carbon sink capacity [89].

2.2.2. Matrix Interactions Between Organic and Mineral Surfaces

Interactions between organic and mineral colloids [90] enhance the resistance of organic matter to chemical and biological degradation [91,92]. From this viewpoint, there is a series of mineral surfaces that are particularly active. For instance, the stabilising effect of allophanic components on the accumulation of stable organic matter forms in soils developed on volcanic ashes has been well documented [93,94]. Various studies have shown that amorphous gels are among the most important factors contributing to the resistance of organic matter to biodegradation [95,96]. Furthermore, these gels influence the molecular composition of humic acids formed in volcanic soils with different geological substrates [97].

The extent to which colloidal humic substances are insolubilised within the mineral matrix can be evidenced in the laboratory by using strong demineralising treatments, followed by the extraction of the insolubilised organic matter. This protocol reveals the amount of C present in stable complexes with clays and sesquioxides, which is referred to as extractable humins [98].

2.2.3. Microbial Growth Inhibitors

Antimicrobial compounds (e.g., terpenes and fungal antibiotics) and enzyme inhibitors (e.g., certain phenols) released by plants or microorganisms can play a very effective role in controlling C recycling rates [99,100,101]. The accumulation of thick humiferous horizons under pine forests or ericaceous shrubs in temperate climates is often attributed to specific phenolic compounds [102] and diterpene structures released by the vegetation [42]. The biomass (wood and leaves) of several Mediterranean species also contains specific essential oils with allelopathic functions in living plants. These compounds remain active in the soil, leading to the non-selective preservation of many plant biomacromolecular constituents.

3. The Formation Processes of Humic Substances

Humic substances, comprising humic acids, fulvic acids, and humins, are widely regarded as the most characteristic organic fractions in soil [103]. These substances exhibit diverse molecular structures that vary across different soil types, reflecting the nature of the original material, the intensity of biogeochemical processes, and the effects of various environmental impacts on the ecosystem. Consequently, understanding the humification process is of paramount importance for both soil health and C sequestration mechanisms [104,105]. Among the processes contributing to the retention of organic matter in soil, the following section will focus on the mechanisms of humification sensu stricto—a series of non-exclusive pathways which have been postulated for the genesis of humic substances [106].

In principle, all biomass constituents can form humic substances, albeit through different mechanisms, which are summarised in the following sections. Some biomass constituents, such as lignins and aliphatic polymers (including cutins and suberins) found in plants, are intrinsically resistant to biodegradation. These constituents are progressively altered in the soil while retaining their primary skeletal structures. These constituents soon incorporate new functional groups, including oxygen-containing groups, alkyl groups, and nitrogen compounds.

Conversely, other constituents that are more easily biodegraded, such as proteins and many carbohydrates, are not considered macromolecular precursors of humic substances. Instead, they must first be biodegraded into their simplest units, which are then condensed through enzymatic or abiotic processes. Alternatively, they can be incorporated alongside the altered biomacromolecules mentioned above, thereby increasing molecular diversity and structural disorganisation (Figure 1).

3.1. Stabilization Mechanisms Involving Alteration of Pre-Existing Macromolecular Material (Inherited Organic Fractions) Not Requiring Complete Previous Degradation of the Starting Material

The accumulation of transformed particulate organic matter fractions (e.g., diagenetically altered macromolecules) contributes to the formation of particulate stable soil fractions, such as inherited humins [74]. These modifications are characteristic of humus types with low to moderate biological activity and primarily involve the selective preservation of plant-derived biomacromolecules (e.g., lignins). Over time, these compounds undergo progressive denaturalization involving a series of relatively simple reactions such as demethoxylation, carboxylation, incorporation of lipid and N-containing materials (likely of microbial origin), etc. [107,108]. These processes predominantly affect relatively recalcitrant plant and microbial biomacromolecules (such as lignins, cutins, and suberins), enhancing their resistance to microbial degradation and promoting long-term stabilisation in soils.

Microbial reworking, or structural reorganisation caused by microorganisms, is a crucial source of organic matter that is significantly more stable than the original material. This increase in the molecular complexity of lignin is often accompanied by the incorporation of nitrogen-containing products and alkyl structures [109]. These structures may originate from microbial metabolism during organic matter decomposition or from the inclusion of lipid compounds derived from higher plants.

In this section, it is also worth referring to the microbial processes that modify several imperfectly known aliphatic biomacromolecules, sometimes called lipid polymers. This is the case for biopolyesters, cutans, suberans, botryococcans, polyditerpenes, and recalcitrant biomacromolecules from vascular plants with unknown structures, likely “hybrid” substances containing lipid and carbohydrate domains [110,111]. Despite some of these substances being composed of cross-linked hydroxyfatty acid networks associated through ester bonds [112,113], their resistance to biodegradation is generally much higher than that expected from the labile nature of ester bonds, as indicated below. The presence of non-hydrolysable amides and non-hydrolysable esters is a characteristic feature often observed in complex, native, or transformed condensed three-dimensional networks in humic fractions with significant variability in structural units. In fact, non-destructive techniques (IR and NMR spectroscopies) indicate the existence of structures (e.g., sugars and fatty acids), which could a priori be considered easily removable by standard wet chemical methods; nevertheless, similar structures and bond types remain in the non-hydrolysed residue. It has been suggested that this stability may reflect the occurrence of steric impediments in organic matrices, molecular encapsulation, solid solution mechanisms, etc. [114].

Therefore, the importance of aliphatic precursors in the formation processes of humic substances [115] is currently considered highly significant even in terrestrial soils, whereas classical studies have emphasised their importance only in the case of hydromorphic soils, which are traditionally known to be especially favourable for the selective preservation of aliphatic constituents (mainly protein and carbohydrate). In this sense, the comprehensive book by Stevenson [35] frequently refers to the fact that the resistance of lignin to microbial attack may have been overemphasised in classical literature, mainly based on the pioneering studies by Waksman [116] on peat soils. However, further studies have demonstrated that the enzymatic degradation of lignin [117] may occur at a rate similar to that of other leaf constituents [78,118,119]. Consequently, humification should not be regarded as a concentration of any specific constituent of plant biomass, which aligns with the fact that organic matter transformed into humic substances and associated with a mineral matrix is more resistant to decomposition than the original plant-derived material [103].

3.2. Neoformation Processes Based on the Condensation of Low Molecular Weight Precursors, or Structures Not Present in the Starting Material Derived Either from the Cleavage of Biomacromolecules or from Biological Synthesis

3.2.1. Biosynthetic Formation of Humic-like Substances

Microbial synthesis plays a relevant role in the accumulation of biodegradation-resistant black substances in soil [14]. The importance of the so-called fungal melanins was first highlighted in pioneering studies by Martin and Haider [120] and later reviewed by Bell and Wheeler [121]. In general, these melanins provide fungi with adaptive advantages, such as resistance to desiccation, solar irradiation, and enzymatic attack [122]. Over time, fungal melanins tend to accumulate in the soil, whereas other fungal constituents (e.g., chitin and proteins) are preferentially biodegraded.

Numerous studies—both classical, using ¹³C NMR [123] and more recent—have consistently demonstrated structural similarities between melanins and soil humic acids, regardless of the analytical techniques used [124,125,126].

In some cases, melanin is formed through well-defined biosynthetic pathways, such as those involving the condensation of binaphthyl derivatives. Certain fungi (e.g., Cenococcum, Alternaria, Aureobasidion, Ulocladium, and Hypoxylon) [127,128,129,130] produce a typical chromophor compound as a secondary metabolite, which appears green in dilute alkaline solutions. Therefore, these melanins can be readily identified using derivative spectroscopy in the visible range due to the presence of a 4,9-dihydroxyperylene-3,10-quinone unit [131,132,133], which exhibits distinct and well-defined absorption peaks at 455, 530, 570, and 620 nm [134].

Beyond their role as biomarkers for fungal contributions to stable soil organic matter [135], quinoid melanins may also influence soil properties. They tend to form highly stable mineral complexes [136] and exhibit greater resistance to biodegradation than humic substances derived from other humus-formation processes [137].

3.2.2. Extracellular Biochemical Processes

A large variety of reactive organic compounds from plants and microorganisms are released into the soil solution, where they coexist briefly before undergoing biodegradation or condensing into macromolecular structures. This spontaneous secondary synthesis, driven by abiotic and enzymatically catalysed reactions involving organic residues and their oxidative decomposition products, aligns with classical descriptions of humification [138]. For instance, such processes may involve the enzymatic condensation of products released after the biodegradation of lignin and other biomacromolecules [139].

Enzymatic browning, extensively studied in food technology, likely parallels soil reactions, where clays and colloidal oxides further catalyse condensation. Cell autolysis, triggered by the merging of lysosomal and vacuolar contents during secondary metabolism prior to cell death, also contributes to uncontrolled reactions, leading to free-radical-containing macromolecular material [140,141]. Continuous inputs from leaf leachates (abundant in some tropical soils) and root exudates provide reactive compounds that readily condense via soil enzymatic activity [142].

3.2.3. Abiotic Synthesis of Humic-like Substances from Aromatic or Aliphatic Precursors

Humification processes in the absence of microorganisms have mainly been demonstrated in model systems. Laboratory experiments have indicated that aging concentrated mixtures of reactive compounds, such as phenols or amino acids, produces brown-coloured condensation products. Over time, these products increase in molecular size through continuous incorporation of soluble units. For example, catechol-glycine-type substances form spontaneously within days under dark conditions at room temperature [143]. These classical reactions are greatly favoured by the presence of oxygen and metal oxides, particularly MnO₂ (birnessite, δ-MnO₂), which catalyzes the synthesis of humic acids from phenolic precursors [144].

Indeed, recent studies have exploited the catalytic role of metal oxides for their potential to accelerate the formation of humic-type substances in compost [145]. In general, several studies have shown that these reactions are greatly favoured by the presence of oxides, mainly MnO₂, Fe₂O₃, or Al₂O₃, as well as 2:1-layered clays [146].

A key challenge in composting lignocellulosic waste is the scarcity of a predominant mineral substrate, such as soil, despite the presence of nearly all the necessary components for humic substance formation. Consequently, even prolonged composting periods often fail to produce substances that are structurally similar to the humic acids found in soils. Instead, the resulting fractions typically exhibit the characteristics of oxidised lignoproteins [107,108]. However, despite these limitations, it is evident that the occurrence of thermophilic conditions during the composting process, which do not occur in natural soils, in conjunction with the addition of MnO₂, results in a decrease in NH₄⁺ losses and an acceleration of lignin degradation, promoting the recombination of breakdown products. This process facilitates the formation of humic-like substances with a structure resembling those found in soil [147].

Similarly, humic-type substances can form through abiotic pathways, such as the condensation of catechol with acetic acid at 25 °C [148]. Additionally, inorganic forms of nitrogen can be transformed into organic forms when combined with phenolic compounds in concentrated solutions, even in the absence of biological activity [149].

In contrast, short-chain water-soluble aliphatic compounds, particularly those containing unsaturated bonds, can undergo condensation to form resins or humic-like disordered substances that closely resemble soil fulvic acids. This phenomenon has been described in model products of the polymaleic acid type [150,151]. When the molecular complexity of such condensation products increases, so does their hydrophobicity. Consequently, long-chain compounds, such as fatty acids and alkanes, can be successfully entrapped into microporous, macromolecular networks or associated with surfaces through hydrophobic bonding. This could represent an alternative formation pathway that explains the presence of alkyl domains in humic substances [66,152,153].

3.2.4. Synthesis of Maillard’s Products

The condensation of amino acids and carbohydrates has long been studied in the context of the transformations occurring during food cooking [154,155]. These products, first described by Maillard [156] and generically referred to as melanoidins, are formed via a series of successive reactions at relatively high temperatures (typically > 80 °C) [157]. Nevertheless, evidence suggests that analogous reactions may occur over extended timescales in soils where organic matter is preserved from rapid biodegradation due to waterlogging, oligotrophic conditions, or the presence of antiseptic compounds.

Maillard’s reactions have often been invoked to explain the formation of aquatic humic substances in hydromorphic soils or dissolved organic matter [158]. However, numerous studies have shown that mineral surfaces, particularly clays and iron oxides, act as catalysts, accelerating melanoidin formation [159]. For instance, in more complex systems containing MnO₂ and catechol, Maillard darkening reactions are markedly enhanced, yielding humic-and fulvic-acid-like substances [160]. These findings suggest an interplay between the polyphenol and Maillard pathways, facilitated by MnO₂ under conditions representative of diverse soil environments [161]. Recent research supports the polyphenol-Maillard reaction [162]. While classical Maillard reactions require elevated temperatures (uncommon in soils), mineral phases such as gibbsite may lower the energy barriers, enabling these processes under ambient conditions.

Notably, several features are shared by Maillard products and macromolecular compounds formed abiotically through carbohydrate dehydration in nitrogen-deficient media (pseudomelanoidins). While N-containing compounds accelerate reaction rates and reduce the temperature required for condensation, simple sugars can also be transformed into a variety of reactive intermediates, including anhydrosugar derivatives (e.g., levoglucosenone), furans, and even benzenic compounds [163]. These reactions resemble the typical caramelisation or charring of carbohydrates [164,165] and may also occur in other oxygen-rich molecular systems [166], proceeding in both solution and solid-state environments [167,168].

Such humification pathways, driven primarily by typical carbohydrate reactions but facilitated by mineral catalysts and co-occurring reactive soil compounds, are most likely to occur in specific environmental settings. These include peatlands (where acid-catalysed carbohydrate dehydration dominates) and soils exposed to high temperatures, such as those during wildfires or controlled burns [169,170]. In extreme cases, like fire-affected soils, these processes yield charred organic particles and ultimately contribute to the formation of “black carbon” [171]. This topic is discussed in the following subsection.

3.2.5. Fire-Mediated Production of Pyogenic Organic Matter and Its Evolution into Humic-like Substances

The role of particulate carbon (C) forms in soil properties and C sequestration remains a topic of debate. For instance, black carbon (BC) has been widely regarded as highly recalcitrant, suggesting that historical fire practices (e.g., controlled burning of crop residues) could contribute to long-term C storage with greater residence times than humus formed through non-pyrogenic processes. However, studies have demonstrated significant biodegradation of these C forms in tropical soils, which challenges this assumption [172,173].

A major obstacle in assessing the environmental role of BC lies in accurately quantifying its concentration in soil. Current methods, such as wet chemical oxidation, are prone to substantial biases [6,174]. Furthermore, reliable surrogate indicators for charred materials are scarce. Although benzenecarboxylic acids produced during the oxidative degradation of soil organic matter have been proposed as markers of fire impact [175], their validity is limited. These acids can also form during the degradation of unburned lignin, and their yields vary with the experimental conditions (e.g., temperature) [176].

Despite these uncertainties, evidence consistently shows that fire enhances the stability of soil organic C against thermal, chemical, and biological degradation under natural and controlled heating conditions in the laboratory [177]. Controlled laboratory incubations have revealed reduced mineralisation rates (CO₂ release per unit of soil C) in heated soils. Nevertheless, no single descriptor can fully capture the impact of fire on organic matter, as discussed below.

Fire-induced stabilisation of soil organic matter is characterised by decarboxylation, demethoxylation, reduced colloidal properties, heterocyclic N-forms, and increased aromaticity. These changes result not only from the selective degradation of aliphatic moieties in humic substances but also from the thermal condensation of newly formed cyclic and polycyclic structures [178]. A notable consequence of soil heating is the accumulation of heterocyclic nitrogen compounds, which are typically absent in unburned soils [179]. These thermal transformations are accompanied by quantitative shifts in carbon fractions, conversion of soluble fractions and fulvic acids into humic acid-like substances, further transformation of humic acids into humin, and eventual conversion of humin into black carbon [177]. In addition, fire induces soil water repellency, which can significantly alter physical and microbiological soil processes [180,181,182,183].

The role of black carbon (derived from carbohydrates, nitrogenous compounds, and lignins) in humic acid formation has been widely studied. Early research on dark volcanic soils in Japan suggested that charred residues of Miscanthus sinensis (Japanese pampas grass) substantially contribute to humic acid formation [184]. Subsequent studies have explored BC as a key component of the aromatic domain in humic acids [185] and references therein.

However, given the transformative effects of fire on organic matter, it is unlikely that BC integrates directly into humic acids without oxidation and functional group incorporation. This also applies to biochar, which is often used as a soil amendment. Instead, pyrogenic carbon is more likely to be incorporated into the humin fraction due to thermal alteration, which removes oxygen-containing functional groups, increases hydrophobicity [186], reduces solubility, and promotes the conversion of fulvic acids to humic acids and, ultimately, to humins. Notably, these humins contain most of the pyrogenic carbon, which remains insoluble across all pH levels.

3.2.6. Abiotic Condensations of Long-Chain Alkyl Compounds

The reliability of abiotic condensation involving unsaturated lipids, leading to the formation of macromolecular materials not only in dissolved organic matter (e.g., marine humic acids [187]) but also in terrestrial soils, has gained increasing recognition in recent years. The traditional view that alkyl molecules act as “peripheral constituents” of humic macromolecules, which are presumed to possess an “aromatic core”, largely stems from indirect evidence. For instance, the preferential release of aliphatic compounds during mild laboratory degradation experiments [58] has been cited to support this model. However, alternative pathways merit further consideration. While free fatty acids and glycerides are often assumed to be rapidly metabolised in incubation experiments (as evidenced by their swift disappearance from solvent-extractable pools), this observation does not solely reflect their biodegradation. Although microbial consumption accounts for much of the alkyl material, unsaturated lipids can also undergo rapid condensation reactions, yielding polyalkyl networks via mid-chain bridging, followed by cyclohexane formation and even aromatic rings [188]. Such abiotic processes are particularly relevant in environments where lignin-derived precursors are scarce, such as marine systems. For example, photo-oxidation has been proposed as the primary humification mechanism for plankton-derived organic matter in open sea environments [189].

In terrestrial systems, the presence of oxides, clays, and other mineral catalysts significantly accelerates the condensation of unsaturated fatty acid chains, especially when these acids remain esterified as triglycerides. Consequently, the rapid decline in solvent-extractable fatty acids during soil incubation may reflect not only microbial degradation but also their incorporation into stable, non-extractable polyalkyl structures [42].

It has been hypothesised that mechanisms of humification, based on precursors derived from alkyl compounds, such as waxes, cutins, suberins, and microbial biopolymers, may be more relevant in the case of certain humin fractions described as predominantly aliphatic [114,190] than in humic acids. Polyalkyl structures are not particularly susceptible to supporting a large number of hydrophilic oxygen-containing functional groups that confer characteristic solubility properties to humic acids. Considering that these aliphatic acids could also condense with pre-existing humic substances after a previous weak association through hydrophobic interactions, we consider that the role of non-aromatic humic precursors should be emphasised in most soil types under a variety of bioclimatic conditions.

4. Concluding Remarks

The processes that govern carbon storage in the soil are highly complex and interrelated, making it difficult to establish the specific contribution of each mechanism (inheritance, neoformation, etc.).

When defining “sequestered soil organic carbon”, it is essential to emphasise the importance of organic matter quality or maturity, which is understood as the degree to which the molecular structure of soil organic matter diverges from that of the original biomass compounds. This quality dimension often outweighs the total C quantity, as it determines the long-term stability of C under future environmental changes.

Several studies have highlighted that carbon sequestration through humification is a complex process that varies across ecosystems. While aromatic compounds from lignin and microbial activity are often key to forming mature humic substances, humification can also occur from aliphatic precursors like sugars and lipids, especially where biodegradation is limited, such as in waterlogged or fire-affected environments.

Similarly, although organo-mineral interactions are widely regarded as essential for organic matter stabilisation, they may play a minimal role in ecosystems such as peatlands or mor-type forest humus, where clay-humus complexes are absent. Nevertheless, these systems are the most effective terrestrial C sinks, due to the transformation of biomacromolecules into highly recalcitrant organic matter.

All extant evidence points to the diverse sequestration mechanisms functioning concurrently, rather than exclusively. The sequestration of carbon in soil should be regarded as a multitude of scale-dependent processes inherent to distinct soil types. This perspective would facilitate the comprehension of biogeochemical processes and recommended ecosystem management practices.