INTRODUCTION

Humic substances:
Their definition. Humic substances have attracted the
attention of soil chemists for over two centuries; in spite of
intensive investigations, knowledge of the nature of these
materials is still limited, however.
Part of the difficulties experienced are caused by
inaccuracy and inconsistency of definitions used. Over the years
the terms humus, soil organic matter and humic substances have
been redefined continuously. The large number of definitions has
lead to many confusions and is partly caused by attempts of many
investigators to relate definitions of these materials with their
structural features. Because these materials have a very complex,
mainly macromolecular nature it is understandable that analytical
data of all kinds related to partial structures present in these
materials are interpreted in many different ways.
For a detailed historic review of these and other terms the
reader is referred to Waksman (1936), Kononova (1966), Stevenson
(1982) Aiken et al. (1985), Frimmel and Christman (1988). The
most recent definitions after Stevenson (1982) and Aiken et al.
(1985) are given in Tables 1 and 2.
Based on their solubility in alkali and acid, humic
substances are usually divided into three main fractions: humic
acids, which are soluble in dilute alkali but precipitate on
acidification of the alkaline extract; fulvic acids which
comprise that humic fraction which remains in solution when the
alkaline extract is acidified; humins, which comprises humic
fractions that cannot be extracted directly from the soil by
dilute alkali or acid but by alkali only after HF/HC1 treatment.
A certain number of subfractions can also be obtained by solvent
extraction or addition of electrolytes (Figure 1). Although the
definitions of these humic fractions have undergone changes over
the years as well they cause much less confusion because they are
operationally defined without reference to their structural

3
 HUMUS

extract with alkali

1
( insoluble ( soluble )

1 t r e a t with acid
HUMIN

( precipitated ) ( not precipitated

-
I
HUMIC ACID
1
FULVIC ACID

extract with alcohol redissolve in base

and add electrolyte

HYMATOMELANIC ACID
( precipitated ) ( not precipitated)
J I
GRAY HUMIC ACID BROWN HUMIC ACID

Figure 1. Scheme for the fractionation of humic substances, after

Stevenson (1982)

4
properties. Figure 2 shows a guide to nomenclature of humic
substances recently published by Thurman et al. (1988).
The classification schemes for defining components of humic
substances have been questioned from time to time. As early as
1936, Waksman stated that it is not justified to draw conclusions
on the chemical structures of various types of humic fractions
obtained by a separation based upon solubility. In other words,
humic acids, fulvic acids and humins are
operationally defined terms as already indicated above,
Humic and fulvic acids obtained by different procedures or
from different sources may differ significantly in their chemical
composition (Saiz-Jimenez et al., 1979; 1986). Therefore, these
fractions do not correspond to a unique chemical entity and they
cannot be described in terms of precise chemical structure. These
definitions have survived because of their practical utility, in
spite of striking facts, as for instance, the existence of a well
defined compound synthesized by Penicillium griseofulvum. P.
flexuosum and P. brefeldianum with the structural formula

called fulvic acid (Turner, 1971).

Because of this vagueness, many controversies about humic
fractions have originated over the years. However, whereas the
fractionation scheme is arbitrary to some extent it has
nonetheless • been widely accepted because the fractions are in
general more suitable for further analysis than unfractionated
humic substances.

5
Dissolved materials Solid materials

PRETREATMENT

e.g. filtration, e.g. drying, sieving,

lipid extraction, flotation,
etc. demineralization

EXTRACTION

e.g. adsorption (XAD e.g. NaOH, DMSO,

concentration, pyrophosphate,
reverse osmosis) etc.
soluble in alkali
I
ACIDIFICATION
Solution Precipitate

Fulvic acid fraction Humic acid fraction

■PURIFICATION
e.g. removal of inorganic and
identifiable organic
components by physical
methods I

VERIFICATION

FULVIC ACIDS HUMIC ACIDS

Figure 2. A guide to nomenclature of humic substances,

after Thurman et al. (1988)

6
Humic substances:
Their origin. The term humus dates back to the time of the
Romans, when it was used to designate the soil as a whole.
Wallerius first defined humus, in 1761, in terms of
decomposed organic matter. The first historical reference to the
isolation of a compound similar to that which became later known
as humic acid dates to Achard, who, in 1786, extracted a brown
substance from soil and peat, by using alkaline solutions. Upon
adding sulphuric acid to the alkaline extract, he obtained a dark
brown to almost black precipitate.
In 1804, de Saussure introduced the term humus, the latin
equivalent of soil, to designate the dark-coloured organic matter
in soil.
The first comprehensive study on the origin and chemical
nature of humic substances was carried out by Sprengel (1837).
Many of the procedures he developed for the preparation of humic
acids became generally adopted, such as pretreatment of the soil
with dilute mineral acids prior to the extraction with alkali.
The modern foundations of humus chemistry are attributed to
Sven Oden (1919). Oden considered humic compounds as the light-
brown to dark-brown substances of unknown constitution which are
formed in nature by decomposition of organic matter through the
actions of atmospheric agencies or in the laboratory by chemical
reagents, and humic acids as those humic substances which show
acid properties and thus form salts with strong bases.
Years before, Maillard (1912) suggested that humus is the
product of a condensation, reaction between carbohydrates and
amino acids, in which microorganisms are not involved.
Eller (1921) stated that the oxidation of phenol, quinone
and hydroquinone in an alkaline solution yields compounds similar
to humic acids.
Beckley (1921) pointed out that the action of mineral acids
on carbohydrates results in the formation of hydroxymethyl
furfural, which on condensation gives rise to humus.
The most generally accepted theory is that humic substances
are derived from lignin. According to this theory, lignin is

7
 LIGNIN
attack by microorganisms
Lignin building units Residue
I
Further utilization by
demethylation, oxidation,
and condensation with N
compounds (e.g. proteins)
microorganisms
HUMIC ACID
fragmentation to smaller
molecules
FULVIC ACID

Figure 3. Schematic representation of the lignin theory of

humus formation, after Stevenson (1982)

LIGNIN
attack by microorganisms
cellulose and other non
Phenolic aldehydes lignin substrates
i
and acids utilization by
'microorganisms
I
further utilization polyphenols
by microorganisms and phenoloxidase
oxidation to C0„ enzymes
quinones
ami no compounds/ amino compounds
HUMIC ACID«- FULVIC ACID

Figure 4. Schematic representation of the polyphenol theory of

humus formation, after Stevenson (1982)

8
incompletely degraded by microorganisms and the residual moiety
becomes part of the soil humus (Figure 3).
Wehmer reported in 1915 that, during fungal degradation of
wood the lignocellulose is attacked, the cellulose is metabolized
and the lignin in converted into humic substances.
Waksman (1936) stated that the lignin theory is
substantiated by a number of facts, summarized as follows:
1. Decomposing wood and peat increase in lignin content with
time.
2. Lignin, as opposed to cellulose, gives rise to aromatic
transformation products. Humic acids are found to contain
aromatic moieties as well.
3. Both lignin and humic acids contain methoxyl groups; the
methoxyl content decreases with proceeding decomposition.
4. Both lignin and humic acids are acidic in nature; both can
react with bases and both are characterized by their capacity
of cation exchange, all be it to different degrees.
5. Both lignin and humic acids are insoluble in cold
concentrated acids, the degree of insolubility increasing
with proceeding decomposition.
6. When lignins are heated in aqueous alkaline solutions, they
are transformed into methoxyl-containing humic acids, which
does not hold for cellulose.
7. On oxidation under pressure, lignin gives rise to humic acids
and finally to aromatic carboxylic acids, but cellulose is
changed to other products.
8. Oxidation of brown coal under pressure gives
benzenecarboxylic acids and no furancarboxylic acids.
9. Both lignin and humic acids are oxidized by oxidizing agents,
such as permanganate and hydrogen peroxide.
10. Both lignin and humic acids are soluble in alkalis and
precipitated by acids.
11. Both lignin and humic acids are partly soluble in alcohol and
pyridine, depending also on the method of preparation; some
lignin and some humus preparations are completely soluble in
alcohol.

9
12. Both lignin and huraic acids are decomposed with great
difficulty or not at all by the great majority of fungi and
bacteria.
Further, Waksman presumed that the nitrogen contained in
humic acids resulted from a condensation of modified lignin with
microbially derived proteins and other nitrogen-containing
compounds.

These days, the so called polyphenol theory as put forward

by Stevenson (1982) had become the more accepted one (Figure 4 ) .
It states that quinones of lignin origin, together with those
synthesized by microorganisms polymerize either autooxidatively
or by phenol oxidases in the presence of araino compounds to form
humic macromolecules. Stevenson mentioned that because lignins
are major plant constituents and relatively resistant to
microbial decomposition they are sometimes considered to be the
major, if not the primary, source of phenolic units in humic
substances.

Humic substances:
Their chemical structure. The study of the chemical nature
of humic substances has been hampered not only by their
insolubility and macromolecular structure but also by the fact
that they are a complex conglomerate of a variety of subunits and
do not constitute a uniform molecule.
Much work has been devoted to the elucidation of the
chemical structure of soil humic substances over a long period of
time. Because of the complexity mentioned, degradative methods
have been used with the aim of producing compounds that could be
identified and whose structures could be related to those of the
starting materials.
The degradative methods that have been used are mainly
oxidations, reductions and hydrolyses. The oxidative reagents are
often strong and could lead to significant alterations of the
original building blocks and to the formation of artifacts. These
artifacts are defined according to Norwood (1988) as identified

10
degradation product whose formation pathway is incorrectly
interpreted leading the investigator to false structural
inferences. This phenomenon has been demonstrated in the case of
permanganate oxidation (Hayes and Swift, 1978). Recently, Martin
et al. (1981) described persulfate as a mild oxidant which
degrades about 50X of soil humic acids, leaving a residue that
can easily be recovered and subjected to further analysis.
In general, much of the work on oxidative degradation is of
limited value, because the structures of the reaction products
only shed some light on those of the building blocks and not so
much on their interconnections. The interpretation can even be
incorrect because, in many instances, the naturally occurring
units could be altered before or after their release from the
macromolecular structure.
Reductive degradations e.g. by sodium amalgam have been
successfully applied by Martin et al. (1974) to a diversity of
soil humic substances and phenolic polymers. The method seems to
work with samples with a certain number of aromatic ether
linkages or with biphenyl structures where activating (hydroxy of
methyl) substituents are ortho and/or para to the connecting bond
(Hayes and Swift, 1978). Most of the compounds identified in
humic substances are similar to those obtained by degradation of
lignins or microbial phenolic polymers suggesting that these
types of materials are also present in soil humic substances.
Hydrolytic procedures are effective in removing protein and
polysaccharide constituents associated with humic substances.
However, the rest of the molecule is not affected (Riffaldi and
Schnitzer, 1973).
As a result of the identification of building blocks
obtained by various degradative and non-degradative methods some
model structures have been proposed for humic substances. Thus,
Haworth (1971) concluded that the e.s.r. signal of humic acids is
due to a complex aromatic core to which polysaccharides,
proteins, simple phenols and metals are chemically and/or
physically bound (Figure 5 ) .

11
 PEPTIDES CARBOHYDRATES
A
1 S
/ I
1 \
/ I
\
1 \
1
1 CORE 1
i

1 ,'
1 '
Ls
METALS PHENOLIC ACIDS

Figure 5. Diagramatic representation of a humic acid,

after Haworth (1971)

Figure 6. Structure of a fulvic acid, after Schnitzer (1978)

12
 Based on degradative and non degradative methods and
assuming that the major products thus obtained are building
blocks, Schnitzer (1978) proposed
a model structure (Figure 6) in which the phenolic and
benzenecarboxylic acids could be held together by hydrogen bonds,
Van der Waal's forces, and it-bonding.
According to Stevenson (1982) humic substances consist of a
heterogeneous mixture of compounds for which no single structural
formula will suffice. Each fraction must be regarded as
consisting of a series of molecules of different sizes, few
having precisely the same structural configuration or array of
reactive groups. As far as the structure of humic acids is
concerned, contemporary investigators favour a "model" consisting
of micelles of a polymeric nature, the basic structure of which
is an aromatic ring of the di- or trihydroxy-phenol type bound by
-0-, -CHj—NH-, -N=, -S- bridges and other groups. These
structures may contain attached proteinaceous and carbohydrate
residues.

Analytical pyrolysis.
Curie point pyrolysis in combination with either low voltage
electron impact mass spectrometry or gas chromatography -mass
spectrometry has been used for differentiating between
microorganisms and to study the chemical nature of synthetic
polymers, bio- and geopolymers (Meuzelaar et al., 1982).
Nagar et al. (1975) indicated that pyr o lysis-mass
spectrometry was promising for soil humus research. A more
extensive study was made by Meuzelaar et al. (1977) by comparing
humic acids from soils, peats and composted straw, with fungal
melanins and lignins. Humic acids from different soils and peats
and most of the fungal melanins gave similar pyrolysis mass
spectra with typical ion series, related to proteins,
polysaccharides and aromatic compounds. Furthermore, pyrolysis
mass spectra of humic acids showed ion series typical of lignin
(Haider et al., 1977).

13
 Saiz-Jimenez et al. (1979) studied the different organic
matter fractions present in a soil. The pyrolysis mass spectra of
humic acid fraction showed prominent mass peaks related to
polysaccharides, proteins and lignins. The spectrum of the humin
fraction resembled those of the humic acid ones although peaks
thought to originate from complex polysaccharides were more
evident. The pyrolysis-mass spectrum of the polysaccharide
fraction showed the characteristic pattern of the pyrolysate of a
complex polysaccharide together with fragments from polymers of
amino acids or amino sugars. The pyrolysis mass spectrum obtained
from the fulvic acid fraction showed clear dissimmilarities to
those of the humic acid fraction; signals from proteins as well
as those related to phenols were low. Depending upon the
isolation methods, pyrolysis mass spectra of fulvic acid
preparations showed different suites of peaks related to
polysaccharide and phenolic materials. Based on pyrolysis mass
spectrometry hymatomelanic acid fraction gave the impression that
it consisted of material rich in polysaccharides and lignins
removable from humic acids by extraction with ethanol. This
assumption was based on mass spectra with a limited mass range
(m/z below 180). However, upon pyrolysis-gas chromatography-mass
spectrometry analysis the hymatomelanic acid fraction was shown
to consist mainly of lipid materials (see chapter 3 ) .

Bracewell (1973) and Robertson (1977) have shown that

pyrolysis-mass spectrometry enables the recognition of variations
in humus type and differences of the genetic horizons in a soil
profile. Pyrolysis-mass spectrometry has shown great promise in
studies on the structures of soil organic matter and on the
humification processes involved. This analytical approach can be
used as a fingerprinting technique to clearly show similarities
and dissimilarities between different fractions and samples.
Preliminary tentative structural information is obtained as well.
To obtain more detailed insight into the structural
composition of soil humic substances firmer identifications of
pyrolysis products which reflect structural units present within
the macromolecular matrix, are required. This can be achieved by

14
application of pyrolysis-gas chromatography-mass spectrometry
(Py-GC-MS).
Previous Py-GC-MS studies (Martin et al., 1977, 1979) have
shown that common pyrolysis products of soil humic acids are
aliphatic hydrocarbons and compounds related to polysaccharides,
proteins, lignins. Further, Faix et al (1987) stated that
pyrolysis of lignins gives similar results to those obtained by
destructive (nitrobenzene oxidation) and non destructive (FTIR)
methods.

Framework of the thesis.

In this thesis several soil organic matter fractions and
composted humic acids are investigated in order to elucidate
their composition and origin. Moreover, different types of
lignins and woods and some of their degraded counterparts have
been studied in an attempt to understand the complex structural
changes occurring to woods and lignins by biotic and abiotic
diagenesis.
In Chapter 2 the results of investigations of soil fulvic
acids, a soil polysaccharide and polymaleic acid are presented.
The soil fractions and the synthetic polymer were analysed by
pyrolysis-gas chromatography-mass spectrometry in order to
determine whether or not fulvic acids are mainly composed of
polysaccharides and to what extent polymaleic acid-like
structures occur in this humic fraction.
A detailed analysis of the soil organic matter fractions
obtained from a representative soil, following the classical
method of fractionation of humic substances, is summarized in
Chapter 3. The data allow for an easy discrimination of these
soil fractions based on the major characteristic series of
pyrolysis products of each fraction.
Because lignin appears to be an important contributor to
some of the soil humic fractions, a description of the pyrolysis
products obtained from a spruce milled wood lignin, before and
after fungal degradation, is presented in Chapter 4. The data

15
were also compared to those of a synthetic lignin and an
industrial lignin.
Chapter 5 stresses the significance of lignin pyrolysis
products as biomarkers. Pyrolysis permits a differentiation of
the three types of lignins based on the presence or absence of
characteristic phenols.
In Chapter 6 wet chemical and spectroscopie data of buried
and present-day woods are compared with analytical pyrolysis
data. The results obtained indicate that the pyrolysis data are
in good agreement and are also complementary with the other data.
Because soil humic acids are complex mixtures of a broad
variety of materials, acid hydrolysis was applied to remove the
polysaccharide and proteinaceous moieties followed by solvent
extraction of lipids. The residual humic acid fractions were
pyrolysed and the significance of the greater part of thé
pyrolysis mixture, consisting of homologous series of straight
chain alkanes, alk-1-enes and a, w-alkadienes is discussed in
Chapter 7. A more detailed study of the most resistant part of
soil humic acids is described in Chapter 8. The residues obtained
after acid hydrolysis and persulfate oxidation gave similar
chromatograms of pyrolysis products, dominated by homologous
series of aliphatic hydrocarbons, which might be related to the
highly aliphatic biopolymers encountered in plant cuticles and
suberins (Nip et al. 1986).

The chemical characterization and the pyrolysis data of the

humic acid fraction of a sludge obtained from waste water of
olive mills after disposal in lagoons is presented in Chapter 9.
This and other wastes or composted materials are being used to
fertilize agricultural soils. Chapter 10 describes some of the
chemical and pyrolysis data of humic acids extracted from
vennicomposts (manure composted by earthworms). The results show
that lignin constitutes an important part of the organic matter
present in the composted manures and can be isolated in the humic
acid fraction.

16
 TABLE 1

Glossary of terms after Stevenson (1982)

Terms

Humus Total of the organic compounds in soil

exclusive of undecayed plant and animal
tissues, their partial decomposition
products, and the soil biomass

Soil organic matter Same as humus

Humic substances A series of relatively high-molecular-

weight, brown to black coloured substances
formed by secondary synthesis reactions.
This term is used as a generic name to
describe the coloured material or its
fractions obtained. on the basis of
solubility characteristics. These materials
are distinctive to the soil (or sediment)
environment in that they are dissimilar to
the biopolymers of microorganisms and higher
plant (including lignin)

Nonhumic substances Compounds belonging to known classes of

biochemistry, such as amino acids,
carbohydrates, fats, waxes, resins, organic
acids, etc. Humus probably contains most, if
not all, of the biochemical compounds
synthesized by living organisms

Humic acid The dark-coloured organic material which can

be extracted from soil by various reagents
and which is insoluble in dilute acid

17
Fulvic acid The coloured material which remains in
solution after removal of humic acid by
acidification

Humin The alkali insoluble fraction of soil

organic matter or humus

Hymatomelanic acid Alcohol soluble portion of humic acid.

18
 TABLE 2

Glossary of terms after Aiken et al. (1985)

Terms Definition

Humus The organic portion of soil, brown or black

in colour, consisting of partially or wholly
decayed plant and animal matter, that
provides nutrients to plants and increases
the ability of soil to retain water. This
term is not entirely synonymous with humic
substances, although it is often used as a
synonym.

Humic substances A general category of naturally occurring,

biogenic heterogeneous organic substances
that can generally be characterized as being
yellow to black in colour of high molecular
weight, and refractory.

Humification The process of fermentation of humic

substances; generally the decomposition of
organic material.

Humic acid That fraction of humic substances that is

not soluble in water under acid conditions
(below pH 2 ) , but becomes soluble at greater
pH.

Fulvic acid That fraction of humic substances that is

soluble under all pH conditions.

Humin That fraction of humic substances that is

not soluble in water at any pH value.

19
 REFERENCES

Achard, F.K. (1786) Chemische Untersuchung des Torf. Crell's

Chem. Ann. 2, 391-403.
Aiken, G.R., McKnight, D.M., Wershaw, R.L. and MacCarthy, P.
(1985) Humic Substances in Soil, Sediment and Water.
Geochemistry, Isolation and Characterization. Wiley, New
York.
Beckley, V.A. (1921) The preparation and fractionation of humic
acid. J. Agric. Sci. 11, 66-68.
Bracewell, J.M. and Robertson, G.W. (1973) Humus type
discrimination using pattern recognition of the mass spectra
of volatile pyrolysis products. J. Soil Sci. 24, 421-428.
Bracewell, J.M. and Robertson, G.W. (1977) Pyrolysis studies on
humus in freely drained Scottish soils. In Analytical
Pyrolysis, C.R.C. Jones and C.A. Cramers (eds.), pp. 167-178,
Elsevier, Amsterdam.
Eller, W. (1921) Kunstliche und natürliche Huminsauren.
Brennstoff. Chem. 2, 129-133.
Faix, 0., Meier, D. and Grobe, I. (1987). Studies on isolated
lignins and lignins in woody materials by pyrolysis-gas
chromatography-mass spectrometry and off-line pyrolysis-gas
chromatography with flame ionization detection. J. Anal.
Appl. Pyrol. 11, 403-416.
Frimmel, F.H. and Christman, R.F. eds. (1988) Humic Substances
and their Role in the Environment. Wiley, Chichester.
Haider, K., Nagar, B.R., Saiz, C., Meuzelaar, H.L.C. and Martin,
J.P. (1977) Studies on soil humic compounds, fungal melanins
and model polymers by pyrolysis mass spectrometry. In Soil
Organic Matter Studies, vol. 2, pp. 213-220, I.A.E.A.,
Vienna.
Haworth, R.D. (1971) The chemical nature of humic acid. Soil Sci.
Ill, 71-79.

20
Hayes, M.H.B, and Swift, R.S. (1978) The chemistry of soil
organic colloids. In The Chemistry of Soil Components, D.J.
Greenland and M.H.B. Hayes (eds.), pp. 179-230, Wiley,
Chichester.
Kononova, M.M. (1966) Soil Organic Matter, Pergamon Press,
Oxford.
Maillard, L.C. (1912) Formation d'humus et de combustibles
mineraux sans intervention de 1'oxygene atmospherique, des
microorganismes, de hautes temperatures, ou de forte
pressions. C.R. Acad. Sci. 155, 1554-1556.
Martin, F., Saiz-Jimenez, C. and Cert, A. (1977) Pyrolysis-gas
chromatography-mass spectrometry of soil humic acids. I. The
low boiling point compounds. Soil Sci. Soc. Amer. J. 41,
1114-1118.
Martin, F., Saiz-Jimenez, C. and Cert, A. (1979) Pyrolysis gas-
chromatography-mass spectrometry of soil humic acids. II. The
high boiling point compounds. Soil Sci. Soc. Amer. J. 43,
309-312.
Martin, F., Saiz-Jimenez, C. and Gonzalez-Vila, F.J. (1981) The
persulfate oxidation of a soil humic acid. Soil Sci. 132,
200-203.
Martin, J.P., Haider, K. and Saiz-Jimenez, C. (1974) Sodium
amalgam reductive degradation of fungal and model phenolic
polymers, soil humic acids and simple phenolic compounds.
Soil Sci. Soc. Amer. Proc. 38, 760-765.
Meuzelaar, H.L.C., Haverkamp, J. and Hileman, F.D. (1982)
Pyrolysis Mass Spectrometry of Recent and Fossil
Biomaterials. Compendium and Atlas. Elsevier, Amsterdam.
Nagar, B.R., Waight, E.S., Meuzelaar, H.L.C. and Kistemaker, P.G.
(1975) Studies of the structure and origin of soil humic acid
by Curie-point pyrolysis in direct combination with low
voltage-ionization mass spectrometry. Plant Soil 43, 681-685.
Nip, M., Tegelaar, E.W., de Leeuw, J.W., Schenck, P.A. and
Holloway P.J. (1986) A new non-saponifiable highly aliphatic
and resistant biopolymer in plant cuticles.
Naturwissenschaften 73, 579-585.

21
Norwood, D.L. (1988) Critical comparison of structural
implications from degradative and nondegradative approaches.
In Humic Substances and their Role in the Environment, F.H.
Frimmel and R.F. Christman (eds) pp. 133-148, Wiley,
Chichester.
Oden, S. (1919) Die Huminsauren. Kolloidchem. Beihefte. 11, 75-
260.
Riffaldi, R. and Schnitzer, M. (1973) Effects of 6 N HCl
hydrolysis on the analytical characteristics and chemical
structure of humic acids. Soil Sci. 115, 349-356.
Saiz-Jimenez, C, Haider, K. and Meuzelaar, H.L.C. (1979)
Comparisons of soil organic matter and its fractions by
pyrolysis-mass spectrometry. Geoderma 22, 25-37.
Saiz-Jimenez, C, Hawkins, B.L. and Maciel, G.E. (1986) Cross
13
polarization, magic angle spinning C nuclear magnetic
resonance spectroscopy of soil humic fractions. Org. Geochem.
9, 277-286.
Saussure, Th. de (1804) Recherches chimiques sur la vegetation.
Paris. As quoted by Waksman (1936).
Schnitzer, M. (1978) Humic substances: chemistry and reactions.
In Soil Organic Matter, M. Schnitzer and S.U. Khan (eds.),
pp. 1-64, Elsevier, Amsterdam.
Sprengel, C. (1837) Die Bodenkunde oder die Lehre vom Boden.
Muller, Leipzig.
Stevenson, F.J. (1982) Humus Chemistry, Wiley, New York.
Thurman, E.M., Aiken, G.R., Ewald, M., Fischer, W.R., Förstner,
U., Hack, A.H., Mantoura, R.F.C., Parsons, J.W., Pocklington,
R., Stevenson, F.J., Swift, R.S. and Szpakowska, B. (1988).
Isolation of soil and aquatic humic substances. Group Report.
In Humic Substances and their Role in the Environment, F.H.
Frimmel and R.F. Christman (eds) pp. 31-43, Wiley,
Chichester.
Turner, W.B. (1971) Fungal Metabolites, Academic Press, London.
Waksman, S.A. (1936) Humus, Williams & Wilkins, Baltimore.
Wallerius, J.G. (1761) Agriculturae fundamenta chemica spez. De
Humo. Diss. Upsala. As quoted by Waksman (1936).

22
Wehmer, C. (1915) Zum Abbau der Holzsubstanz durch Pilze. Ber.
Deut. Chem. Gesell. 48, 130-134.