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V. Humic Acid-Like Materials from Geologic Deposits Not Classified as Soils

V. Humic Acid-Like Materials from Geologic Deposits Not Classified as Soils

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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.


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

sea water.



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-



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-



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.




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

to 3%.

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



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).



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

molecular weight.

It is also generally believed that no one specific structural formula

will adequately represent humic substances. Rather most hypotheses


G. T.


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)











Reactive groups




Bridge units











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



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



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

soil microflora.



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.

VII. Conclusions

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



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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Beblik, A. 1956. Aduan. Carbohydrate Chem. 11, 145-183.

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Brauns, F. E. 1952. “Chemistry of Lignin.” Academic Press, New York.

Bremner, J. M. 1950. J . Soil Sci. 1, 198-204.

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