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V. Humic Acid-Like Materials from Geologic Deposits Not Classified as Soils
STRUCTURAL CHEMISTRY OF SOIL HUMIC SUBSTANCES
such time as their identity has been definitely established. Such is clearly
not the case at present.
The primary purpose of the present discussion of nonsoil sources
of humus-like substances is to review the techniques that have been
used recently and that may have applications to humic substances in
Recently Moschopedis et al. (1963) described the chlorination of a
humic acid from a subbituminous coal without apparent concomitant
oxidation. Gaseous Clz was bubbled through an aqueous suspension of
the humic acid both in the presence and in the absence of light. The
absorption of up to 25% C1 was reported as occurring in a 30-minute
period. IR studies of the products failed to reveal typical C-Cl band
absorptions but did reveal a market reduction in the 1600 cm.-' band
(carbonyl) with the appearance of new bands at 1640 cm.-l and in the
range of 2850 to 3000 cm.-'. No fractionations or presence of degradation
products were reported in this study.
B. ACID HYDROLYSIS
In a study of the ether oxygen in a kerogen concentrate from an
Esthonian bituminous shale, Aarna and Lippmaa (1957a) reacted the
material with anhydrous AlBr3 (1:5 w/w) at 100°C. under N2 for 1 to
3 hours. Over 80% of the product was soluble in pyridine, diphenylamine
( 5 5 O C . ), or aqueous diethylamine solution. The functional groups were
determined and the increase in phenolic hydroxyl was attributed to the
cleavage of aryl ether bonds. From this it was concluded that about 40%
of the 0 in kerogen concentrate existed as ether bond or about one ether
bond for each 20 C atoms in the original material. The total oxygen
content of the kerogen was 13.3%,whereas the typical 0 content in soil
humic acids ranges from 30 to 48% (Dubach and Mehta, 1963).
Degens et al. (1964) extracted the alkali-soluble material from the
surface layer of a marine sediment obtained off the coast of southern
California and hydrolyzed the acid-insoluble fraction with 6 N HCI under
NB at 105°C. Phenolic materials were extracted and chromatographed.
Some 13 phenolic compounds were detected, some of them being tentatively identified as p-hydroxybenzoic acid, syringic acid, vanillic acid,
salicylic acid, m-hydroxybenzoic acid, and phydroxyphenylacetic acid.
From these data it was suggested that the humic acids were perhaps
breakdown products of lignin material, and as such were derived from
terrestrial sources. Of interest is the observation that at least the first
three phenolic compounds listed were also observed in the free state in
G. T. FELBECK, JR.
If the 0 in humic acids exists as simple ether bonds it would appear
that refluxing with concentrated HI would tend to cleave these bonds
in the same manner as methoxyl bonds are cleaved in the Zeisel procedure (Fieser and Fieser, 1956, p. 137). However, the reviewer is aware
of only one report describing the application of this method to the
degradation of humus-like materials ( Raudsepp, 1954). In this study
kerogen was allowed to react with supersaturated HI (specific gravity
1.87 and 1.98) at 175-200°C. for 24 to 72 hours. The kerogen was completely broken down into ether-soluble or benzene-soluble products. When
15 ml. HI (specific gravity 1.98) was used per gram of shale during 48
hours' reaction time at temperature of 200"C.,93.1%of the kerogen was
rendered soluble in ether. The products were presumed to be aliphatic
and aromatic hydrocarbons, but no pure compounds were isolated. From
these observations it was concluded that the aromatic and aliphatic units
were connected by ether bridges, with about 1 aromatic nucleus for
each 13 to 17 C atoms in the original kerogen structure.
Extensive use has been made of hydrogenolysis and hydrogenation
in the study of the structure of lignin (Brauns, 1952) and coal (Van
Krevelen and Schuyer, 1957), but relatively few applications have been
made to polymeric substances of the nature of kerogen or soil humic
A comprehensive study of the hydrogenolysis of Colorado oil shale
kerogen was made by Hubbard and Fester ( 1959). They hydrogenolyzed
the kerogen at 355°C. for 4 hours using SnC12 as a catalyst. This process
rendered 80% of the kerogen C into a form soluble in benzene. The
soluble material was fractionated into straight-chain waxes (2% ), microcrystalline waxes ( 10%), straight-chain oil (3%), branched-chain and
cyclic oil (26%), and N- and O-containing material ( 36%) . From these
observations they suggested that kerogen was probably a complex polymeric material with portions being held together by comparatively weak
cross linkages that may include ,SyN, and 0. IBenzenoid struotures were
suspected of being either absent or present only in insignificant quantities. No observations were reported on the effect of SnC12 on materials of
known structure in order to estimate the amount of condensation or
rearrangement that might have occurred, if any. Therefore, the extent
to which this catalyst produces artifacts could n& be determined.
Takegami et al. (1963) and Landa and Eyem (1963) reported hydrogeno1ysi.s studies on humic acids from coal using Cu chromite and
WS2, respectively, as catalysts. However, it has been shown (Felbeck,
1965) that catalysts similar to these are very active in condensation
processes, producing under hydrogenolytic conditions yields of a ben-
STRUCTURAL CHEMISTRY OF SOIL HUMIC SUBSTANCES
zene-soluble tar, equivalent to 25% by weight from pure cellulose.
Therefore, the value of studies using these catalysts to elucidate structure appears to be quite limited,
As is the case in the study of soil humic substances, spectroscopic
methods are widely used in studies of other natural polymeric substances.
Wood et al. (1961) examined the IR spectra of humic substances isolated
from a North Dakota lignite and an Alberta subbituminous coal. When
the humic substances had been heated from 170" to 200°C. evidence for
the probable formation of 5-membered anhydrides was obtained. From
this they concluded that about 80% (3.5meq. COOH per gram out of
a total of 4.3 meq. COOH per gram) of the COOH groups existed in pairs
on adjacent C atoms. The remaining 0.8 meq./g. existed as isolated
COOH groups. Similar conclusions for a soil humic acid were reached
by Wagner and Stevenson (1965).
In further work on the same humic substances from the lignite and
subbituminous coal, Moschopedis ( 1962) obtained IR spectra on material
that had been methylated and then acetylated so as to remove any
intra- or intermolecular H bonding. The appearance of an absorption
band at 1660 cm.-' was cited as the first unequivocal evidence for nonH-bonded quinone carbonyl absorption in a humic substance. It might
be noted, however, that other carbonyl compounds have absorption
bands in the same region, e.g., the C=O group in a 4-pyrone (kojic
acid) absorbs from 1651 cm.-l to 1666 cm.-l, depending on the substituent side groups (Beklik, 1956).
In an interesting approach to the problem of estimating the degree of
aromaticity in a polymeric material, Aarna and Lippmaa (1957b) used
coupling with diazonium salts, chloromethylation, and addition of
mercuric acetate as indicators of aromaticity of kerogen from an Esthonian
shale. They estimated that 13 to 22% of the C atoms in the material
Occur in aromatic hydrocarbons. Although the possibilities of alternative
reactions are mentioned for each reagent, few data are given to support
the assumptions that these alternative reactions did not occur,
VI. Alternative Hypotheses for the Structure of Soil Humic Substances
Several schemes have been suggested for the molecular structure of
soil humic substances. Each of these has advantages and disadvantages,
depending upon one's viewpoint. Before discussing in detail the principal
hypotheses which, in the reviewer's opinion, most nearly meet the ob-
G. T. FELBECK, JR.
served characteristics, it might be appropriate to summarize those
observations on humic substances which seem to be reasonably well
established, even though in so doing there are the definite risks of oversimplification and of ignoring isolated data which may later turn out
to be pertinent.
ON SOIL HUMICSUBSTANCES
1. Elemental composition. The following ranges appear to include
most of the humic substances from a variety of soils (Dubach and Mehta,
1963),C, 45 to 65%; 0, 48 to 30%; N, 2 to 6%; H, about 5%. Humic
substances also usually contain 1-257 of methoxyl groups.
2. Molecular weights range from about 3,000 to over 300,000.As
the molecular weight increases, the C content also increases while the 0
content decreases. This shift in elemental composition has been ascribed
in part to decarboxylation (Martin et al., 1963).
3. A dark brown or black color is characteristic of the humic substances of higher molecular weights, whereas light brown or yellow
color is related to the lower molecular weight fractions. The chromophores responsible for the dark color are not known, but they may be
due to conjugation of quinonic type C=O with C=C bonds.
4. Unsaturation is indicated by halogenation and, in a muck soil, by
the uptake of H in a hydrogenation reaction (Felbeck, 1965).The H uptake in the latter case specifically indicated the presence of one C=C
bond for each 4 C atoms, the possibility of H uptake by other structures
being essentially eliminated.
5. Acidity is due to O-containing groups, most probably COOH and
OH showing a phenol-like acidity function. The equivalent weight varies
from about 100 in lower molecular weight fractions to about 300 in higher
molecular weight fractions (Dubach and Mehta, 1963). If approximately
one-half of the acidity is due to COOH and the other half due to OH
(Broadbent and Bradford, 1952), then the COOH content varies from
about 2@4 in low molecular weight fractions to about 7% in higher
molecular weight fractions and the OH content varies similarly from 8
6. Except for certain Podzol B H preparations (Schnitzer and Desjardins, 1962) in which all of the 0 is accounted for as functional groups,
it appears that up to 50% of the 0 exists in nonreactive structural units.
These units most probably include ether bonds, heterocyclic 0, and unreactive C=O.
7. The presence of amino acids in acid hydrolyzates of humic substances has been widely observed ( Bremner, 1955;Kononova, 1961).
8. Approximately 30 to 50% of the N in the total organic matter of
STRUCTURAL CHEMISTRY OF SOIL HUMIC SUBSTANCES
soil is strongly resistant to acid hydrolysis. Most of this resistant N
appears to be associated with the soil humic substances ( Bremner, 1955).
9. Humic substances are quite sensitive to oxidation, large amounts
of C02, H20, acetic acid, and oxalic acid being produced by relatively
mild oxidizing agents. From this observation stable or fused ring aromatic compounds seem to be either absent altogether or present only in
negligible concentrations (Savage and Stevenson, 1961).
10. The carbon structures in the central parts of humic molecules
are resistant to both acid hydrolysis and microbial attack.
11. Carbohydrates do not seem to be an integral part of the humus
12. Phenolic compounds of both plant and microbial origin are released from humic substances in yields up to 25% by alkaline hydrolvsis
or by Na amalgam reduction. These phenolic compounds have also been
detected in alkaline permanganate and nitric acid oxidation products,
and, in lower concentrations, in acid hydrolyzates.
13. The fulvic acid fraction appears to be usually lower in C and
higher in 0 than the associated humic acid (Kononova, 1961, p. 90)
and therefore usually has a higher acidity. In many of its characteristics
the fulvic acid fraction of mineral soils resembles the organic fraction
extractable from the BH horizon of Podzol soils.
14. Lignoprotein in the sense used by Waksman (1936, pp. 188-190)
does not appear to exist in quantities greater than trace amounts in the
soil (Jenkinson and Tinsley, 1959, 1960).
B. HYPOTHESESON STRUCTURE
Depending on the nature of his observations, nearly every worker
in the field of molecular studies of soil humic substances has concluded
that certain structural features are present or absent. These conclusions
can be collected in various ways into several hypotheses on the nature
of humic structure. The particular hypothesis selected is related to the
relative degree of importance placed on different data and to which
sets of data are selected when there appear to be conflicts between two
or more sets.
Most of the hypotheses appear to have several aspects in common:
humic substances are amorphous, three-dimensional polymeric, acidic
substances of high molecular weight with a more or less aromatic
nature. Most hypotheses also agree that at least the humic acid and
humin fractions are chemically homogeneous but heterogeneous as to
It is also generally believed that no one specific structural formula
will adequately represent humic substances. Rather most hypotheses
suggest a “type” or “skeletal” structure in which only the general aspeots
are included, the details (e.g., specific location and number of functional
groups) being omitted. The hypotheses differ primarily in the nature of
the structural nucleus, i.e., whether it is primarily benzenoid, phenolic,
quinonic, or heterocyclic in nature, whether the N is a fundamental part
of such nucleus or is an accidental contaminant, and whether there is
a reasonable degree of uniformity in the nucleus or whether there is lack
of uniformity as reflected in a number of structural units randomly
distributed throughout the nucleus.
1. Hypothesis of Thiele and Kettner
On the basis of investigations into the characteristics of both natural
humic substances and model compounds, Thiele and Kettner (1953)
proposed a structure based on nuclei with associated reactive groups
combined by bridge elements. A diagram of the general structure they
FIG. 2. Diagrammatic scheme of relationship of nuclei ( N ) , reactive groups
( R G ) , and bridge units ( B ) in humic acid structure. (After Thiele and Kettner,
proposed is shown in Fig. 2. The specific structures included under
possible nuclei, reactive groups, and bridge units are listed in Table 11.
By selecting the specific structures involved in Thiele and Kettner’s
general scheme, it would be possible to devise a molecule that would
fulfill all the characteristics summarized in the previous section. In the
reviewer’s opinion, however, this universality is the greatest objection
to this hypothesis. In other words it would probably not be possible to
Possible Nuclei, Reactive Groups, and Bridge Units Suggested by Thiele
and Kettner (1953)
STRUCTURAL CHEMISTRY OF SOIL HUMIC SUBSTANCES
prove such a structure to be incorrect since it covers nearly all possible
feasible molecular arrangements. Therefore, it appears necessary to
consider hypotheses that are more specific.
2. Hypothesis of Kononova
A more specific hypothesis has been prepared by Kononova (1961)
after a review of current ideas on the nature and composition of soil
humic substances. She concludes that there exist in the soil only two
groups: humic acids and fulvic acids (designated as crenic or apocrenic
acids in her discussion). Each of these groups is thought to possess
a characteristic structural form, but substances extracted from different environments are not necessarily identical.
Thus humic acids, for example, are formed from two or possibly
three classes of organic compounds which condense in definite ratios into
high molecular weight units. The two main structural units are
thought to be compounds, either phenolic or quinoid in nature, and
compounds containing nitrogen (amino acids and peptides). It was
also suggested that carbohydrates may make up a third group in the
composition of the humic acid molecule, as indicated by the possible
presence of reducing substances. It was suggested that the alkaliinsoluble fraction, humin, was not different from humic acids, its insolubility being attributed to the h n e s s with which the material is
combined with the mineral part of the soil.
Fulvic acid is considered, in this view, to possess the same “structural
units” as humic acids, but the proportion of aromatic units is less, and
the peripheral aliphatic chain greater, than that occurring in humic
3. Hypothesis of Fluig
On the basis of results of comprehensive studies on the decomposition
of plants in the soil and model studies on postulated lignin breakdown
fractions, Flaig (1959, 196Oa, 1964) has proposed the hypothesis that
humic substances are the end product in the sequence starting with
degradation and demethylation of lignin to substituted polyphenols,
followed by oxidation to quinones and finally the condensation of these
quinones with amino acid units into humic acid or its immediate
precursors. A schematic diagram of the principal steps in this hypothesis
is presented in Fig. 3.
This hypothesis appears to require plant lignin as a source of the
elements that form the main structural units of humic acid. Several
observers ( Aleksandrova, 1962; Kang and Felbeck, 1965) have established that dark-colored products resembling humic acids in several
G. T. FELBECK, JR.
ways can be produced by a variety of microorganisms. Glycerol or
glucose are often used as the sole C source in these studies. Since many
of these organisms are ubiquitous soil inhabitants, it seems clear that
humic substances must at least partially be composed of these microbially
produced, dark-colored substances. These observations would appear to
rule out lignin as an essential source of the structural units of humic
substances, although the presence of lignin breakdown products in
FIG. 3. Principal steps in proposed scheme of production of humic acid from
lignin. (After Flaig, 1964.)
various hydrolyzates indicates that these products may be combined in
varying amounts with other sources of phenolic materials in humic
4. Hypothesis of Swaby and Ludd
Swaby and Ladd (1962,1965)examined a series of humic substances
extracted from Australian soils supporting a vegetation of highly lignified
plants and compared the results with studies on model compounds. Since
they were able to detect no peptide bonds and only trace amounts of
“lignin aldehydes,” they concluded that humic substances consist of
individual amino acids combined with phenols or quinones through the
amino group. Crosslinking was attributed ta the difunctional amino
acids lysine and cysteine.
Humic substances buried under volcanic ash deposits up to 3,000
years were found to differ little from recent humates. This resistance to
microbial attack could be explained if it were assumed that the humus
molecule consisted of many heterogeneous units cross-linked in an
irregular fashion by covalent bonds. Such a molecule could be attacked
STRUCTURAL CHEMISTRY OF SOIL, HUMIC SUBSTANCES
only by a variety of extracellular enzymes which would degrade the
molecule piece by piece from the outer surface. Under most soil conditions a process of this nature would be very slow.
It was suggested in this hypothesis that humic substances are formed
by rapid condensation or polymerization of free radicals formed enzymatically in plant and microbial cells shortly after death, when the
autolytic enzymes are active but the cell has not yet been attacked by
other microbes. The actual synthesis of humic substances is thought to
be the result of heterogeneous chemical catalysis rather than of directed
enzymatic activity, as indicated by the poor crystallinity of humic substances.
5. Hypothesis Based on 4-Pyrone Units
In a study of the hydrogenolysis of the nonhydrolyzable fraction of
a muck soil (Felbeck, 1965), it was shown by means of nuclear magnetic
resonance spectroscopy that the hydrogenation products consisted primarily of long chains of methylene units with occasional branching.
The only pure product isolated and identified was an n-CZ5or an n-CZ6
hydrocarbon. From these observations, in addition to data on elemental
analyses, number of C=C double bonds per carbon atom, and the
known ability of humic substances to form chelates and to fix NH3, it
was suggested that humic substances consisted of a microbially produced
central unit of 4-pyrone units linked together in a chain by methylene
bridges at the 2,Spositions. Phenolic and amino acid units are thought
to be attached to the central structure as shown in a schematic form
in Fig. 4. This hypothesis is similar to that of Swaby and Ladd (1962,
1965), but with a modification of the structure of the central unit.
Schnitzer and Desjardins (1962) observed that Podzol BH preparations were aromatic in nature, having no 0 in the central structure. If
fulvic acids are structurally related to Podzol BH organic matter, then
they could be derived primarily from oxidized plant and microbial
phenols attached to the central pyrone structure by ester linkages (COOH
from the phenolic acid and OH from the pyrone) as was suggested in
Section 11, B, 3.
It was observed that NH3 and glycine condensed rather easily with
a 5-hydroxy-4-pyrone (kojic acid) ( BeBlik, 1956) forming 4( IH)-pyridones. On the basis of this observation it was suggested that this mechanism could account for the observed NHBfixation by humic substances
(Burge and Broadbent, 1961) and also might account for that part of
the N in soil humic substances that is resistant to acid hydrolysis.
The sources of such N most probably are amino acids produced by the
G. T. FELBECK, JR.
The principal objection to this hypothesis is that a polymer consisting of 4-pyrone units has never been observed, as far as the reviewer
is aware, among microbially synthesized substances, even though kojic
acid is a common fungal metabolite. In addition, the only pure compound
isolated from the hydrogenation products represented less than 2% of
the C in the nonhydrolyzable organic fraction, and it is entirely possible
FIG.4. General relationship ( I ) and a specific example of possible units (11)
contained in a proposed scheme for the molecular structure of humic acid and fulvic
that other substances present, but as yet unidentified, could lead to
equally plausible alternative structures.
The last decade has witnessed a substantial increase in the knowledge
of the structure of soil humic substances. This advance has been due
largely to the application of newly developed chromatography techniques
to the problems of separation and characterization of degradation products. Much less progress has been made in the actual degradation process
and in the analysis of functional groups. Probably the most important
technique still remaining to be developed is a degradation process that
will produce high yields of monomeric materials which are simple
enough to be characterized completely, while still retaining sufficient
STRUCTURAL CHEMISTRY OF SOIL HUMIC SUBSTANCES
structure to be informative. Allied with this problem is the need for more
carefully purified starting materials. Since it seems to be clear that at
least part of humic materials is the result of microbial activity independent of plant compounds except as a source of C and energy, a
possible approach to “purer” humic substances might be the examination
of such compounds as are produced by a single microbial species grown
on a fully defined medium.
It is hoped that intensive application of new techniques, combined
with a critical point of view such as described by Platt ( 1964), will
lead to further progress in the coming decade.
The writer wishes to thank Drs. P. Dubach, J. W. Parsons, M. Salomon, M.
Schnitzer, C. Steelink, R. J. Swaby, and G. H. Wagner for unpublished reports of their
research work and for helpful advice. Sincere appreciation is extended to the %ode
Island Agricultural Experiment Station for the opportunity to prepare this review
and to Miss Gladys Coggeshall and her assistants for deciphering and correcting the
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