Sen DOU,Jun SHAN,Xiangyun SONG,Rui CAO,Meng WU,Chenglin LI and Song GUANCollege of Resources and Environmental Sciences,Jilin Agricultural University,Changchun 308(China)

2State Key Laboratory of Soil and Sustainable Agriculture,Institute of Soil Science,Chinese Academy of Sciences,Nanjing 210008(China)

3College of Resources and Environment,Qingdao Agricultural University,Qingdao 266109(China)

4Jiangsu Key Laboratory for Eco-Agricultural Biotechnology around Hongze Lake,Huaiyin Normal University,Huaian 223300(China)

ABSTRACTHumic substances(HS),which are defined as a series of highly acidic,relatively high-molecular-weight,and yellow to black colored substances formed during the decay and transformation of plant and microbial remains,ubiquitously occur in nature.Humic substances represent the largest stable organic carbon pool in terrestrial environments and are the central characteristic of the soil.However,the validity of the HS concept and the justification of their extraction procedure have been recently debated.Here,we argue that the traditional humic paradigm is still relevant.Humic substances are distinctive and complex because the extracted HS formed during the humification are chemically distinct from their precursors and are heterogeneous among soils.By reviewing the concept,formation pathways,and stabilization of HS,we propose that the key question facing soil scientists is whether HS are soil microbial residues or unique synthesized compounds.Without revealing the distinctiveness of HS,it is impossible to address this question,as the structure,composition,and reactivity of HS are still poorly known owing to the heterogeneity and geographical variability of HS and the limits of the currently available analytical techniques.In our view,the distinctiveness of HS is fundamental to the soil,and thus further studies should be focused on revealing the distinctiveness of HS and explaining why HS hold this distinctiveness.

Key Words: heterogeneity,humic acid,humification,humus,microbial biomass,soil organic matter

INTRODUCTION

Soil organic matter(SOM)plays a vital role in global carbon(C)cycling as it holds more organic C than those found in the atmosphere and living plants (Schlesinger and Andrews,2000).It also has many ecological and environmental functions,such as maintaining soil fertility,sustaining plant growth,and controlling the fate of environmental contaminants(De Freitas Maia et al.,2013;Gerke,2018).Nowadays,the management of SOM is even more important than before due to high-intensity land use in terms of storing more C in the land and pursuing higher yields of crops.Since the early critique of“humus”was proposed by Waksman(1936),SOM has been generally used as a synonym of humus,which is comprised of non-humic substances(nHS)and humic substances(HS).The nHS belong to well-characterized classes of organic compounds,either simple or complex.However,HS are defined as a series of highly acidic,relatively highmolecular-weight,and yellow to black colored substances formed by biochemical and chemical reactions during the decay and transformation of plant and microbial remains(a process called humification)(IHSS,2007).

As the largest constituents of SOM,HS are ubiquitous in global environments and play a profound role in maintaining soil ecosystem services(Senesi,2010).Humic substances have a unique structural pattern that is different from that of any other group of soil compounds,and their chemical composition can well represent that of natural organic matter(García et al., 2019; Olk et al., 2019). Traditionally, HS are considered partially decomposed or microbially altered polymers from plant residues formed by polymerization reactions(Waksman,1936;Martin and Haider,1971;Flaig et al., 1975; Stevenson, 1994; Gerke, 2018). In the past decades,the understanding of the molecular composition of HS has changed significantly,resulting in HS being proposed as aggregates of macromolecules or supramolecular associations of self-assembling heterogeneous small molecules derived from defined plant,animal,and microbial materials and their decomposition products(Piccolo,2001;Sutton and Sposito,2005;Kelleher and Simpson,2006;Baigorri et al.,2007;Wells,2019).The supramolecular concept developed by Piccolo and coworkers (Piccolo, 2001, 2002; Nuzzo and Piccolo, 2013) and the concept of polymerized and polycondensed macromolecules(Stevenson,1994)may both be justified as extremes between the actual HS behavior(Baigorri et al., 2007). The chemically unique HS can be further divided into three major fractions,i.e.,humic acid(HA),fulvic acid(FA),and humin,which are products of humification and are defined operationally by their solubility under acidic and alkaline conditions(Stevenson,1994).The operational classification of HA,FA,and humin is not only a pH separation,but also a classification and isolation with a clear ecological and edaphic sense(Baigorri et al.,2009).Different pH and ionic strength conditions in soils constrain HS to adopt their own characteristics in each soil(Stevenson,1994;Baigorri et al.,2009).

Recently,the validity of the concept HS,the existence of HS in nature,and the justification of HS extraction procedure were questioned(Schmidt et al.,2011;Lehmann and Kleber,2015;Kleber and Lehmann,2019).The core issues are that:i)no HS exist in nature;ii)the alkaline extraction process for extracting HS is problematic,and iii)the“humic”paradigm should be set aside. In terms of refuting the existence of HS, Schmidt (2011) considered the stability of HS/SOM as an ecosystem property and Lehmann and Kleber(2015)and Kleber and Lehmann(2019)stated that SOM is a continuum of progressively decomposing organic compounds(soil continuum model (SCM)) and no HS exist in soil.While they proposed many arguments to support their view of SCM, they did not provide any arguments for refuting the existence of HS. The academic community reached a consensus in the middle of the 20th century that HS exist in soil as a natural body(Hayes and Swift,2018).The HS chemistry implies the transformation of biological sources(plant decomposition,microbial decomposition,plant decay, macro/micro fauna decay, biochemical re-synthesis,and microbial re-synthesis)to chemical structures(aliphatic chains,aryl structure,O-alkyl structure,carboxylic groups,amine groups,and sulfhydryl groups),and this inhomogeneity may be a source of confusion for pure chemists.In fact,the existence of HS makes SOM distinctive,which is also one of the most important reasons for soil science to be an independent scientific discipline. Thus, before substantial scientific evidence emerges, refuting the existence of HS is unconvincing and unacceptable.The concerns regarding the extraction of HS by alkaline solutions(commonly 0.1 mol L-1anoxic NaOH) have already been discussed and addressed several decades ago.One of the problems of alkaline extraction is that most of the mineral components and the original colloidal structure are lost during the acid precipitation of HA. However, the main criticisms raised by Lehmann and Kleber(2015)are the possible chemical alteration of the structure of HS,owing to the formation of laboratory artifacts and the inability to distinguish HS from nHS during the alkaline extraction process.Generally,the problems related to potential HS structure changes can be minimized by performing the extraction process under inert gas(such as diatomic nitrogen(N2)),which has been widely proved and used in the literature(Stevenson,1994;Olk et al.,2019).Thus,the claims of alteration during extraction with alkali are exaggerated.Moreover,there are few reports that show any structural alterations to HS extracted from alkaline substances(Olk et al.,2019),mainly because there are no records of any structure that functions as a control(García et al.,2019).In fact,the functions and reactions of HS do not necessarily rely on the extraction methods.Some studies have compared the13C-nuclear magnetic resonance(NMR) spectra of extracted material with the spectra of the original soil materials demonstrating that the extraction process does not change the structure of the HS(Fernandes et al.,2010;Weber et al.,2018).In addition,nHS such as sugars and amino acids can be extracted and separated after the alkaline extraction process.It is not justified to ignore the scientific progress based on the structural characterization of HS extracted from soil.By simply criticizing the limitations of the alkaline extraction methods, Lehmann and Kleber(2015)claimed that SOM or HS should no longer be seen as large and persistent,chemically unique substances,but as unremarkable intermediates in a continuum of degrading organic compounds. Actually, the concept of SCM is not novel.In 1981,Jenkinson already pointed out that“in the long run, no fraction of organic matter in plants and animals can withstand decomposition to carbon dioxide and water...”(Jenkinson,1981;Hayes and Swift,2018).Indeed,the traditional view considers the formation of HS(humification) as a progressive and continuous transformation process where soil organisms play a vital role(Baveye and Wander,2019).Four years have passed since the novel SCM concept was proposed and it seems that the recommendation of Lehmann and Kleber is not accepted by soil scientists,as the number of published articles related to the humic paradigm,including“humus”,“humic substances”,“humic acid”,or“humification”,continuously increased year by year based on the dataset of Web of Science(Fig.1).

Overall,it is normal and reasonable to discuss the core contents of SOM or the boundary concepts of HS such as composition,formation,transformation,function,and influencing factors,but it is not necessary to deny the existence and even change the name of HS unless the secrets hidden behind SOM or HS are unveiled.To make our view clear,the concept,formation,and stabilization of HS deserve to be reviewed.

THE CONCEPTS OF SOM AND HS

Fig.1 Number of papers on soil organic matter published annually from 2015 to 2019. All the papers were collected from the Web of Science database on June 20,2019 after the novel concept“soil continuum model”was proposed by Lehmann and Kleber in 2015.The following search items were used to collect the relevant papers: “humus”, “humic substances”,“humic acid”,or“humification”.

The terms SOM and HS are widely used in soil science and deeply rooted among scientific communities.Although the term SOM is popular,there is currently no single definition.For example,Hayes and Swift(1978)regarded SOM as the non-living components of soil that represent a heterogeneous mixture composed largely of products resulting from microbial and chemical transformations of organic debris.However, Huang and Hardie (2009) defined SOM as the total of the diverse organic components in the environment exclusive of un-decayed plant and animal tissues,their“partial decomposition”products,and the soil biomass.When defining SOM,the main difficulties include:whether or not SOM contains living life? Does SOM contain plant and animal residues,or mainly plant residues(PRe)? Whether the anthropogenic organic contaminants in the agricultural soil,which were usually called“non-natural organic matter”(nNOM),should be included? We view SOM as all the“fine”(invisible size)carbonaceous organic matter in the soil.The definition of SOM in some textbooks did not have“fine”as a limit,which is not correct(Dou,2001,2010).Therefore,SOM should include:i)fine living organisms(LivOM);ii)fine animal and plant residues(mainly PRe)that have not been decomposed or have been partially decomposed;and iii)decayed organic matter or its re-synthesized products,which are often called humus(Eq.1).

Soil microbes only account for a very small volume,commonly less than 0.001% of soil volume, and the C in living microbial biomass is less than 4%of SOM(Bae et al.,1972;Sparling,1985;Liang and Balser,2011;Jiang et al.,2013).In addition,the non-decayed fine PRe accounts for a small proportion and cannot be separated from existing organic matter. In some situations, SOM and humus are almost synonymous(Stevenson,1982,1994;Senesi et al.,

2009).In a narrow sense,SOM mainly represents humus,but it is necessary to distinguish between SOM and humus for soils that contain more PRe. The HS can be extracted according to the“humus composition modification method”by a mixed alkaline solution of 0.1 mol L-1Na4P2O7and 0.1 mol L-1NaOH(Dou et al.,2007).Humic substances are divided into water floating humic substances (WFS),water-soluble humic substances(WSS),extractable humic substances (EHS), and residual humin (Dou et al., 2003;Dou,2010).Considering this,WFS and WSS are SOM,but strictly,they are not humus.Humus is humified SOM whose main body is HS,including HA,FA,and humin.

SOM=WFS+WSS+EHS+humin (2)

Humus=WSS(part)+EHS+humin (

EHS=HA+FA (

HS=FA+HA+humin (?3)4)5)

Heterogeneity is the main characteristic of HS,just like leaves have similar characteristics on trees but are apparently different from each other in detail(Dou,2010).If soils are regarded as the“skin”of the Earth,HS should be the“protein”component of the skin.The formation and molecular structure of such an important“protein”remains a mystery(Dou, 2011). The heterogeneity of HS essentially reflects the complexity of HS in terms of its formation pathways and stabilization mechanisms.In other words,the composition and structure of HS are extremely complex because of the heterogeneity of HS,which is also the major reason why studying HS is difficult.No matter the complexity,HS are, after all, a class of naturally occurring chemical substances that can be extracted and have general characteristics(such as alkali solubility and dark color).Humic substances are called a class of substances because they are different from their precursors in terms of chemical composition and structural characteristics.During the humification process,new biochemical reactions occur among different precursor materials,which are derived from animals,plants,and microorganisms,resulting in secondary synthesis.Kleber and Lehmann (2019) considered that the secondary synthesis is not provable because alkaline extraction could not separate materials created by secondary synthesis from other ionizable organic compounds. However, special chemical combinations or special proportion of chemical combinations, as well as a certain organic chemical structure of unknown compounds,are formed in the secondary synthesis,which is the so-called “structural distinctiveness”of HS.The heterogeneity is only a part of this“structural distinctiveness”.Scientists have been trying to find proof of this“structural distinctiveness”for decades, but they have not succeeded due to technical constraints and the heterogeneity of HS. In fact, there is no single piece of scientific work with sufficient molecular resolution that can directly prove what exactly exists in the traditional alkaline solution of soil(Piccolo,2016).To make comprehensive advances in the understanding of the“structural distinctiveness”of HS,state of the art instrumentation and close cooperation among excellently-equipped laboratories are required.

THE POSSIBLE PATHWAYS OF HS FORMATION AND STABILIZATION

Several pathways have been proposed for the HS formation during the decay of plant,animal,and microbial residues in soil(Fig.2)(Stevenson,1994;Dou,2001).Since Achard(1786)first extracted HS from peat using a KOH solution in 1786,HS are primarily defined as decayed substances during plant decomposition,which are not distinguished from products of microbial decomposition or re-synthesis.In the first decade of the 20th century,Tpycov(1914)considered that HS were derived from the cellulose of the plants,which is easily used by soil microbes as indirect sources of HS.Cellulose participated in the synthesis of HS by pre-transforming into the cytoplasm of microorganisms,which is called the microbial synthesis theory of HS formation(pathway 1 in Fig. 2). Conversely, other organic materials (e.g., lignin)were considered as direct resources for HS formation.Aromatic compounds were primarily formed by hydrolysis of lignin and then were oxidized to quinone by oxidases(Senesi et al.,2009).Finally,HS formed during the condensation,which is the lignin polyphenol theory(pathway 2 in Fig.2).Waksman(1936)proposed another view,which represents the first critique of the humification process.He considered that the compounds that are easily used by microorganisms have limited effect on HS formation and proposed that the main sources for HS formation are lignin and proteins,which are both hard to be used by microorganisms and are synthesized by the cytoplasm of microbes.Lignin and proteins can combine and form lignin-protein complexes,which are the core of HS.This process represents the lignin-protein theory for HS formation(pathway 3 in Fig.2).Until the middle and late 20th century,scientists found that HS could be formed by polyphenols derived from soil microbes even without lignin(Kononova,1966)(pathway 4 in Fig.2).Finally,there are two additional theories, namely, the autolysis theory(Schnitzer and Khan,1972)(pathway 5 in Fig.2)and carbohydrate amine condensation theory,which is based on the Maillard reaction(Stevenson,1982)(pathway 6 in Fig.2).The former states that HS are formed from dying plant and microbial cell tissues undergoing enzyme autolysis,random condensation, and free radical polymerization. The latter considers that without the presence of lignin derivatives and enzymatic reactions,sugars and amides can be condensed into glucosylamine or glycocyamine and finally polymerized to HS(Aiken et al.,1985;Huang and Hardie,2009).

Fig.2 Possible pathways for formation of humic substances(HS)(adapted from Dou(2001)):microbial synthesis theory(1);lignin polyphenol theory(2);lignin protein theory(3);microbial polyphenol theory(4);autolysis theory(5);and sugar-amide condensation theory(6).The question marks indicate whether quinones are a required intermediate step for the polymerization of HS is a matter of debate.

According to the above-mentioned pathways,the formation of HS is a chronosequence.For instance,HA is formed earlier in the lignin protein theory and microbial synthesis theory than FA, but FA is formed earlier in the microbial polyphenol theory and the lignin polyphenol theory(Stevenson,1982).It is hard to say which pathway is correct as the origin of HS is quite different in diverse environments,and a completely satisfactory scheme for explaining the nature and occurrence of HA and FA has not yet been established.All the above-mentioned pathways may be involved in HS formation in soil,but to different extents in different soils or with different importance(Stevenson and Cole,1999).The relative importance of these pathways would depend on vegetation,topography,mineralogical composition and surface chemistry of soil particles, cropping practices, microbial population and activity,and environmental conditions(Dou,2010).Although the hypotheses regarding the formation of HS are different,it is well accepted that the formation of HS cannot be separated from the effects of microorganisms and enzymes.Previously,the contribution of microbial biomass residues to SOM formation was considered less than 6%(Insam,1996;Dalal,1998).However,increasing evidence showed that microbial biomass residues at various stages of decay and transformation constitute a large percentage of SOM (Kögel-Knabner et al., 2008; Liang et al., 2011;Miltner et al., 2012; Kögel-Knabner, 2017). It has been reported that dead microbial biomass could contribute more than 50%to the extractable SOM and account for 80%of soil N in certain soils(Simpson et al.,2007).Based on modeling approaches and the concept of“microbial biosynthesis acceleration”,it is estimated that the contribution of microbial biomass to SOM at an equilibrium stage ranges 47%-80%,depending on the quality and quantity of substrates(Fan and Liang,2015).Besides the contribution to the formation of SOM,soil microbes also play crucial roles in promoting plant growth, nutrient turnover, and tolerance to drought (East,2013).The stimulated plant growth will offer more substrates(e.g.,C and N)to soil microbes,which will,in turn,enhance their microbial activities and even their contribution to SOM formation.

The chemical recalcitrance of SOM is only meaningful at its initial formation stage,while physical protection may exert profound effects on the stabilization of SOM(Blanco-Canqui and Lal,2004;Von Lützow et al.,2006).The inherited humin and HA are often physically protected in macroaggregates of SOM,and the soil fraction richest in micropores exerts better protection on organic matter(Agnelli et al.,2008).Humic substances can also be considered the residual garbage eaten by microbes and may be re-synthesized by microorganisms in the clay-protected SOM of microaggregates(Dou,2001).An incubation experiment was conducted with14C-labelled wheat straw in black soil and showed that HA was formed primarily in macroaggregates and transformed into FA with time, while HA14C/FA14C decreased (Guan et al., 2015)(Fig.3).However,FA was formed primarily in microaggregates or via the decomposition of HA in macroaggregates.Another incubation experiment showed that the addition of organic molecules with a hydrophobic and hydrophilic structure character,such as HA,is able to improve aggregation in soils with a high weathering degree(Bastos et al.,2005).These results indicated that selective formation of HA and FA exists between macroaggregates and microaggregates.The HS formation in macroaggregates and microaggregates can be explained by Waksman’s lignin protein theory and Kononova’s polyphenol theory,respectively.The selective formation and preservation of HS between soil macroaggregates and microaggregates would play a more important role in poorly drained soils and lake sediments,compared with more aerated environmental conditions during humification processes(Senesi et al.,2009).DEBATES ON THE HETEROGENEITY OR STRUCTURAL DISTINCTIVENESS OF HS

The heterogeneity of HS is strongly determined by the origin,age,and decomposition processes of parent organic materials(Muscolo and Sidari,2009).During the formation of HS,special chemical combinations or special proportion of chemical combinations,as well as certain organic chemical structures of unknown compounds, are formed, i.e., the“structural distinctiveness”of HS.Actually,the“structural distinctiveness”of HS is intrinsic, inherent, and innate to their nature because HS are the result of chemical and biochemical actions of variable agents (microorganisms and chemical reactions)on variable substrates(vegetation,micro/meso/macro fauna)in variable conditions(moisture,mineralogy,temperature,insolation,etc.)in soils from all over world(geographically variable).These variables will certainly result in a unique HS chemical structure.However,the distinctiveness of HS has been questioned by several authors(Kelleher and Simpson,2006;Lehmann and Kleber,2015). They considered HS as a continuum of degrading biomolecules or mechanically mixed intact and degrading biopolymers of the categorized compounds,both of which are against the traditional views on the structure of HS.

Fig.3 Changes in the ratio of newly formed humic acid carbon(HA14C)to newly formed fulvic acid carbon (FA14C) in different aggregate size fractions of a black soil with 14C-labelled wheat straw added during a one-year incubation(adapted from Guan et al.(2015)and reproduced by permission of Elsevier).Different lower-case letters for a same sampling time indicate significant differences(P ＜0.05)between aggregate size fractions.Different upper-case letters for a same aggregate fraction indicate significant differences(P ＜0.05)between sampling times.Bars represent standard deviations.

Kelleher and Simpson(2006)studied the structural characteristics of four common biopolymers,namely,proteins(albumin),polysaccharides(amylopectin),lipids(cuticle),and lignin, as well as their mixtures, by two-dimensional(2D) NMR techniques-heteronuclear single quantum coherence(HSQC)(Fig.4).By comparing the signals of the above-mentioned plant and microbial compounds with those of IHSS peat HA,they found that most signals overlapped.Therefore,they suggested that the vast majority of HS is a complex mixture of microbial and plant biopolymers and their degradation products,without a distinct chemical category(Kelleher and Simpson,2006).This study did not rule out the existence of a distinct category of HS,as claimed in the text by the authors themselves.However,this study was improperly interpreted by some scientists, leading to the trend of denying the distinctiveness of HS.For example,Lehmann and Kleber(2015)argued that SOM/HS do not represent a distinct chemical category but a very complex mixture of microbial residues and plant biopolymers with their decomposition products. Our viewpoint on Kelleher and Simpson’s study is as follows.First,the HSQC technique can be used to investigate the differences in sample composition by detecting functional groups of large compounds or mixtures.However,it cannot be conversely used.If the signals of two different samples in the HSQC spectra are not different,it just shows that the chemical environment of the C functional groups is the same,but does not mean that the whole molecular or compound structures are the same.Second,in their study,the signals of proteins,carbohydrates,aliphatic biopolymers,and lignin do not completely overlap with those of IHSS peat HA,which means that the chemical structures of these microbial and plant biopolymers are different from the IHSS peat HA.Third,the IHSS peat HA may be different from HA extracted from other soils and cannot completely represent HS,as HS is widely heterogeneous in nature.Fourth,in terms of investigating or elucidating the structure of HS,any single technique is currently imperfect and has limited capability.For example,the1hydrogen(1H)NMR-based HSQC technique used by Kelleher and Simpson(2006)can only detect C bonded to H in the abundant small molecules because these fractions yielded the sharpest NMR lines and strongest surviving signals in the 2D solution NMR

Fig.4 Two-dimensional nuclear magnetic resonance heteronuclear single quantum coherence(2D-NMR-HSQC)of:four common biopolymers illustrated in different colors(i.e.,albumin(blue),amylopectin(red),cuticle(green),and lignin(grey))(a),the four biopolymers illustrated in black(b),International Humic Substances Society(IHSS)humic acid extracted from peat(IHSS peat HA)(c),and the biopolymers(black)overlaid on IHSS peat HA(green)(d)(adapted from Kelleher and Simpson(2006)and reproduced by permission of American Chemical Society).

spectra. In fact, using quantitative solid-state13C NMR methods, which can detect all C regardless of solubility and molar mass,Cao and Schmidt-Rohr(2018)investigated the structure of IHSS peat HA and compared the IHSS spectra with that of microbial and plant biopolymers.Their results showed that the ISSH peat HA are clearly distinct from plant and microbial biopolymers in terms of abundant condensed aromatics,nonprotonated aromatic carbons near oxygen,oxygen-bonded nonprotonated carbons such as aryl ketone,and abundant carboxylic acid groups.The distinctive structural differences between HS and biopolymers clearly refute the SCM concept proposed by Lehmann and Kleber (2015). In addition, there was another misconception regarding the concept of HS when the polycyclic aromatic C within SOM/HS was attributed to black (pyrogenic) C(BC)(Gerke,2018).Although BC represents a particularly stable fraction of SOM and accounts for more than 25%of the total organic C in certain soils(Mao et al., 2012), the three most widely used methods for determining BC (the benzene polycarboxylic acid, ultraviolet (UV)/NMR, and13C-NMR methods)strongly overestimate the BC content in soils(Gerke,2019).The reason why these three methods overestimate BC in soils lies in that the polycyclic aromatic C in HS or that formed during lignin degradation is determined as BC(Simpson and Hatcher,2004;Waggoner et al.,2015;Gerke,2019).

In our previous work(Wang et al.,2014),using different precursors such as glucose, cellulose, lignin, straw,and sawdust as substrates, we studied the formation of humus-like substances by several fungi,i.e.,Trichoderma viride,Aspergillus niger,Penicillium,and their mixed strains,showing that the molar ratio values H/C of the newly formed HA (HLA) were all higher than 1.5, being different from that(1.0)of soil extracted HA(Fig.5).Thus,it cannot be concluded that the precursor materials of HS or the simple cultured products really have the characteristics of soil HS.Besides, many studies are still devoted to elucidating the heterogeneities of SOM/HS using sophisticated analytic tools such as solid-state NMR,electrospray ionization mass spectrometry(ESI-MS),and 2D electrophoresis techniques(Skokanová and Dercová,2008;Nebbioso et al.,2010;Mao and Cao,2011;Karim et al.,2013;Tahiri et al.,2016b).A recent study also indicated that different soil humic acids could exert different ecological functions due to their different chemical structures (Wu et al., 2019), which also reflects the heterogeneity of HS.Taken together,there is not enough evidence for denying the heterogeneity and distinctiveness of HS until now.

Although the HS concept is based on solubility characteristics, and minor alteration exists during the extraction processes,HS are heterogeneous and distinctive(Aoyama,2015;Tahiri et al.,2016a;Ghio and Madden,2018).Elucidating the distinctiveness of HS will be helpful for understanding the roles of HS in relation to the improvement of soil structure,stimulation of plant growth,C fixation,and interactions with xenobiotics(Hayes and Clapp,2001).We agree that it is crucial to study the composition and structure of HS in situ,but before state-of-the art technique emerges,it is still necessary to rely on the alkaline extraction method to characterize the elemental composition,building blocks,and heterogeneity of HS, as the isolation and fractionation of the components of SOM/HS represent a prerequisite for studying them.The distinctiveness of HS will only be revealed when the chemical properties, such as elemental composition,building blocks,reactivity,and heterogeneity,of HS become known. Furthermore, the ability to isolate and characterize the fractions of HS will not only improve our understanding of the distinctiveness of HS but will also allow us to gain a deep insight into the mechanisms of interactions between specific components of HS with organic and inorganic contaminants.

CONCLUSIONS

Based on our view, HS are not just an operationally defined fraction of SOM. Instead, they do exist in nature and possess a distinctive structure. It is unreasonable and unacceptable to set aside the concept of HS.Our knowledge of the intrinsic mechanisms of the genesis of HS in nature still needs to be advanced.What the distinctiveness of HS is and why HS hold this distinctiveness will shed light on the mystery of HS.Humic substances research on the alkaline extraction procedure and the subsequent fractionation(i.e.,HA,FA,and humin)is still relevant and is currently the most effective way to explore and elaborate the crucial and complex role of HS in terrestrial ecosystems. To comprehensively understand the chemistry of HS and enhance our recognition of the relationship between HS structure and reactivity,multiple sophisticated(non-invasive and in situ)techniques should be encouraged in the future.

ACKNOWLEDGEMENTS

This study is supported by the National Natural Science Foundation of China(Nos.41571231 and 41201221),the National Key Research and Development Program of China(No.2016YFD0200304),the Scientific Instrument and Equipment Development Project of Chinese Academy Sciences(CAS)(No.YJKYYQ20170058),the Natural Science Foundation of Jiangsu Province,China(No.BK2012496),the Youth Innovation Promotion Association, CAS (No.2017362),and the Natural Science Foundation of the Jiangsu Higher Education Institutions of China(No.19KJB180010).Dr.Jun Shan is grateful to the China Scholarship Council(No.201804910070)for supporting his study abroad.We thank the two anonymous reviewers for their valuable and constructive comments and suggestions.

Fig.5 Van-Krevelen distributions of glucose,cellulose,lignin,and components extracted from microbial residues(MR)and soil for assessing the origin of humic substances(adapted from Wang et al.(2014)).FA=fulvic acids;HA=humic acids.

CONTRIBUTION OF AUTHORS

Sen Dou and Jun Shan contributed equally to this work.