THE SUPRAMOLECULAR STRUCTURE OF HUMIC Science 2002... · THE SUPRAMOLECULAR STRUCTURE OF HUMIC SUBSTANCES…

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    0038-075C/01/16611-810832 November 2001Soil Science Vol. 166, No. 11Copyright 2001 by Lippincott Williams & Wilkins, Inc. Printed in U.S.A.

    HUMIC substances (HS), natural organic sub-stances that are ubiquitous in water, soil, andsediments, are of paramount importance in sus-taining plant growth and controlling both thefate of environmental pollutants and the biogeo-chemistry of organic carbon (OC) in the globalecosystem (Piccolo, 1996). Despite their role inthe sustainability of life, the basic chemical natureand the reactivities of HS are still poorly under-stood. The scientific community of humic scien-tists has so far failed to provide a unified under-standing of this field of science, and there is still,therefore, a poor awareness of fundamental as-pects of humic structures and reactivities. Never-theless, the implications of the relevance ofawareness of HA structure should extend far be-yond the interests of a few chemists; HA struc-tures affect the ways that the soil ecosystem work,as well as the bioavailabilty of organic substances

    (including potential pollutants) in the soil envi-ronment (Tate, 1999).

    Most of the difficulties encountered in chem-ically defining the structures and reactivities of HSderive from their large chemical heterogeneityand geographical variability. The substances areundoubtedly mixtures that develop randomlyfrom the decay of plant tissues, from microbialmetabolism-catabolism, or from both. Thus, thechemistry is not only highly complex,but it is alsoa function of the different general properties ofthe ecosystem in which it is formed, such as veg-etation, climate, topography, etc. It is not surpris-ing that, despite the efforts of many excellent sci-entists in the distant and recent past (Kononova,1961; Stevenson, 1994), there is still much to bedone to achieve an appropriate awareness of hu-mic chemistry.

    The objective of this contribution is to reporton recent experimental findings leading to a newunderstanding of the conformational nature ofHS and to mention the profound implicationsthat this may have on our understanding of soilorganic matter (SOM) functions and reactivities.

    THE SUPRAMOLECULAR STRUCTURE OF HUMIC SUBSTANCES

    Alessandro Piccolo

    Dipartimento di Scienze Chimico-Agrarie, Universit di Napoli Federico II 80055,Portici, Italy. E-mail: alpiccol@unina.it.

    Received June 4, 2001; accepted Aug. 21, 2001.

    The scientific understanding of the molecular size and shape of humicsubstances (HS) is critically reviewed. The traditional view that HS arepolymers in soil is not substantiated by any direct evidence but is as-sumed only on the basis of laboratory experiments with model moleculesand unwarranted results produced by incorrectly applying either analyt-ical procedures or mathematical treatments developed for purified andundisputed biopolymers. A large body of evidence shows an alternativeunderstanding of the conformational nature of HS, which should be re-garded as supramolecular associations of self-assembling heterogeneousand relatively small molecules deriving from the degradation and de-composition of dead biological material. A major aspect of the humicsupramolecular conformation is that it is stabilized predominantly byweak dispersive forces instead of covalent linkages. Hydrophobic (vander Waals, -, CH-) and hydrogen bonds are responsible for the ap-parent large molecular size of HS, the former becoming more importantwith the increase of pH. This innovative understanding of the nature ofHS implies a further development of the science and technology for thecontrol of the chemistry and reactivity of natural organic matter in thesoil and the environment. (Soil Science 2001; 166:810832)

    Key words: Humic substance structure, size exclusion chromatogra-phy, supramolecular associations.

  • Traditional Paradigms in Humus ChemistryThe amount of HS in soils is several times

    greater than the amount in waters.Up to 7080%of the OC in mineral soils may be composed ofhumic material, with recognizable plant remainsconstituting a small percentage of the organicmatter (OM) of mineral soils. Even though theabundance of OC in HS is from 2 to 3 timesgreater than that in the terrestrial biomass, the lat-ter greatly influences the OM dynamics in soils(Insam, 1996). Biological activity rapidly decom-poses labile plant materials on entering aerobicsoil environments with adequate water supplies,but more resistant components transform slowlyin the same environment. The compositional di-versities and the differences in the modes oftransformation of the components make it ex-tremely difficult to define accurately the grossmixtures that compose SOM, or the dissolved(DOM),or the particulate organic matter (POM)of waters.

    Because of the heterogeneity of humic mix-tures, simplification and reductionism must beadopted. Stevenson (1994), in summarizing pre-vious reports and definitions, stated that humusincludes a broad spectrum of organic con-stituents, many of which have their counterpartsin biological tissues. He distinguished betweennon-HS and HS, the former of which consists ofcompounds belonging to the well known classesof organic chemistry such as amino acids, carbo-hydrates, lipids, lignin, and nucleic acids, whereasthe latter are unspecified, transformed, dark col-ored, heterogeneous, amorphous and high mole-cular weight (MW) materials.

    The classical definitions of HS are opera-tional only and are based on solubility propertiesin the aqueous solutions used as soil extractants.The generalized terms humic acids (HAs), fulvicacids (FAs), and humins cover the major fractionsstill used to describe HS components, but theboundaries between these fractions have notbeen yet clarified in chemical terms. Becausemodern analytical techniques for organic com-pounds were not available during the first half ofthe 20th century, the data that were availablefrom elemental analyses (C, H, O) and determi-nations of acidic functional groups suggested thatHAs and FAs from many different soils were rel-atively similar (Kononova, 1961). Such simplecorrelations, although not based on any molecu-lar understanding, encouraged scientists to con-sider humic fractions to be chemical entities hav-ing specific properties rather than complexmixtures of nonspecific compounds.

    Many of the modern concepts of HS derivefrom theories illustrated in the book of Ko-nonova (1961). In a review of hypotheses ad-vanced predominantly by Russian scientists,Kononova introduced the concept of HS as a sys-tem of polymers, based on the observation thatelemental composition, optical properties, ex-change acidities, electrophoretic properties, andMW characteristics varied consistently with soilclasses. Using this concept, the various fractionsof HS obtained on the basis of solubility charac-teristics are imagined to be part of a heteroge-neous mixture of molecules, which, in any givensoil, range in MW from as low as several hundredsto perhaps over 300,000 Da, and exhibit a con-tinuum of any given chemical property (Steven-son, 1994).

    It must be noted that despite the existence ofdata showing that the molecular dimensions (asmeasured by osmometry, viscometry, and diffu-sion) of some HS were scarcely beyond one ortwo thousand Da (Scheffer and Ulrich, 1960;Schnitzer and Khan, 1972), more reliance wasplaced on the early work of Flaig and Beu-telspacher (1958) who showed, using the ultra-centrifuge, that MW values were in the range of30,000 to 50,000 Da for HAs and about 10,000Da for FAs. One reason for such bias towardshigh molecular weight (HMW) structures maybe found in the historical hypotheses that consid-ered HS to be products of biologically-assistedsyntheses from compounds derived from degra-dations of lignin, polyphenols, cellulose, andamino acids. Evidence for this polymeric as-sumption was researched in many classical labo-ratory experiments that indicated possibilities foreither abiotic or biotic condensations of simplemolecules into humic-like materials (Kononova,1961). Some of these early laboratory studieshave been researched in more carefully definedconditions in recent times (Haider and Martin,1967; Martin and Haider, 1969; Flaig et al., 1975;Flaig, 1988; Hedges, 1988). However, no directevidence has ever been provided for the occur-rence of such polymer build-up processes in nat-ural soil systems.

    The polymeric view of HS has included thegeneral concept of polydispersity (Dubach andMetha, 1963), in which the HS are made of poly-mers with different MW values, similar to thatwhich applies for other natural biological macro-molecules such as proteins, polysaccharides, andlignin. The experimental difficulties encounteredin isolating chemically homogeneous fractions ofHS was compared with those observed for bio-

    VOL. 166 ~ NO. 11 SUPRAMOLECULAR STRUCTURE OF HS 811

  • polymers with varying molecular dimensionssynthesized in the living cell. This unwarrantedsimilarity supported the polydisperse polymericconcept of HS and justified the observation thatthese are mixtures of compounds.The concept ofHMW and polydisperse polymers became a par-adigmatic part of the descriptive definitions ofHS proposed thereafter (Schnitzer and Khan,1972; Aiken et al., 1985; Malcolm, 1990; Steven-son, 1994).

    The assumption that HS are polymers, whichhas no sound molecular basis, has promulgated theuse of simple physical-chemical measurements tocharacterize HS. An example is the E4/E6 index,the ratio of the absorbance of HS at 465 nm to thatat 665 nm, introduced by Welte (1955) and repro-posed by Kononova (1961). This ratio supposedlyindicates a reverse relationship with progressivehumification as well as increased condensation as-sumed to produce large amounts of polyconden-sated aromatic-ring structures. Despite its wide-spread and continued application, the E4/E6 ratiohas repeatedly been shown not to hol

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