II. Soil Organic Matter (SOM)

A LIFETIME PERSPECTIVE



5



lipids (alkanes, alkenes, saturated and unsaturated fatty acids, alkyl mono, di, and

tri esters) in SOM appear to be strongly retained by the aromatic SOM components and can only be separated from them with great difficulty. For example, even

after exhaustive extractions with n-hexane, followed by chloroform, Schnitzer and

Schuppli (1989) could remove only 10% of the total lipids from three agricultural soils sampled in western Canada. The separation of carbohydrates and proteinaceous materials from SOM requires prolonged hydrolyses with relatively

strong acids under reflux. Thus, the different chemical components of SOM are

closely associated to form a complex structure.



B. RELATIONSHIP AMONG SOM, HUMUS,

AND HUMIC SUBSTANCES

There is some confusion among soil chemists about the meanings of SOM, humus, and humic substances. Do these terms depict different materials? According

to Stevenson (1994), SOM is synonymous with humus. In my opinion, the term

total humic substance is also synonymous with SOM and humus as long as losses

occurring during the extraction and separation procedures are held to a minimum.

My definition of humic substances is that it is the sum of humic acid ϩ fulvic acid

ϩ humin. While essentially each of the three terms can be used, I personally, as a

SOM chemist, prefer use of the term SOM.



C. PROBLEMS ASSOCIATED WITH EXTRACTION

OF SOM FROM SOILS

The SOM content of agricultural soils usually ranges between 1 and 4% (w/w),

with most soils containing between 2 and 3% SOM. In the soil, because SOM and

inorganic soil constituents are closely associated, it is necessary to separate the two

before either can be examined in greater detail. This separation is usually achieved

by extracting the SOM with either dilute base (0.1–0.5 M NaOH solution) or by a

neutral salt solution such as aqueous 0.1 M Na4P2O7. Extraction of SOM with a

dilute base works reasonably well and was originated by Archard in 1786. Separation of the alkaline extract into HA, FA, and humin was first carried out by Sprengel (1826). The three fractions into which the alkaline SOM extract is partitioned

are (1) HA, which is that fraction of SOM that coagulates when the alkaline extract is acidified; (2) FA, which is the SOM fraction that remains in solution when

the extract is acidified, i.e., it is soluble in both acid and alkali; and (3) humin,

which is that SOM fraction that remains with the soil, i.e., it is insoluble in both

alkali and acid. Over the years, many objections have been raised against the use

of alkaline solutions, which are still the most efficient SOM extractants today.

Stevenson (1994) lists the following objections: (1) silica is dissolved from the



6



M. SCHNITZER



mineral matrix, which contaminates the SOM extract; (2) protoplasmic and structural components of fresh organic tissues are dissolved, and these mix with the

SOM extract; (3) autooxidation of some organic components occurs in contact

with air when the extracts are allowed to stand for extended periods of time; and

(4) other chemical changes can occur in the alkaline solutions, including condensation between amino acids and CuO groups of reducing sugars or quinones to

form Maillard reaction products. Some of these changes can be minimized by doing the extractions in the presence of an inert gas such as N2, but not all possible

changes can be excluded.

Another serious difficulty with the extraction of SOM from soils and the partitioning of the extract into HA, FA, and humin is that these are laborious and timeconsuming procedures that are not suitable for the analysis of large numbers of soil

samples. A new approach, not involving the use of wet chemical methods, is required to overcome these problems.



D. DIRECT ANALYSIS OF SOM IN WHOLE SOILS BY 13C

NUCLEAR MAGNETIC RESONANCE (NMR) AND PYROLYSIS –

FIELD IONIZATION MASS SPECTROMETRY (PY-FIMS)

In recent years we have witnessed the development of two analytical methods,

based on “high technology,” that appear to be suitable for the direct analysis of

SOM in situ, i.e., in whole soils. These methods are solid-state 13C NMR and PyFIMS. The solid-state 13C NMR analysis of whole soils has been described by Wilson (1987) and Arshad et al. (1988). This type of analysis requires that the soil contain at least 3% C and that the concentration of paramagnetic ions, e.g., Fe3+, in

the soil not be too high because paramagnetic ions interfere with the recording of

acceptable 13C spectra. According to Arshad et al. (1988), the C:Fe (w/w) ratio is

an important indicator for obtaining satisfactory solid-state 13C NMR spectra of

whole soils and particle-size fractions separated from them. If the C:Fe ratio is

ϾϾ1, the quality of the spectrum will be good; if the ratio is Ͼ1, a reasonable

spectrum will be obtained, but if the ratio is Ͻ1, the spectrum will be poor. The

quality of the spectrum can be improved by reducing the Fe3+ to Fe2+ by dithionite and then removing it. Another option is to separate the soil into particle-size

fractions and running 13C NMR spectra on the fractions (Wilson, 1987). Arshad et

al. (1988) report that SOM-rich soil and particle size fractions can be prepared by

flotation and that these yield well-defined 13C NMR spectra. Another approach that

can be used is to separate the soil on a magnetic separator into magnetic and nonmagnetic fractions, but this method requires specialized equipment and is too timeconsuming. Figure 1 shows solid-state 13C NMR spectra of particle-size fractions

separated from Culp and Rycroft soils from northwestern Alberta, enriched in

SOM by flotation (Arshad et al., 1988). Flotation increased the C content of the



A LIFETIME PERSPECTIVE



7



Figure 1 Solid-state 13C NMR spectra of (a) OM-enriched fine sand fraction isolated from Culp

soil, (b) OM-enriched sand fraction isolated from Rycroft soil, and (c) OM-enriched silt and clay fraction separated from Rycroft soil. From Arshad et al. (1988), with permission of the publisher.



sand fraction separated from the Culp soil from 2.05 to 20.15%, the C content of

a similar fraction separated from the Rycroft soil increased from 3.16 to 15.62%,

whereas the C content of the silt and clay fraction separated from the same soil increased from 3.08 to 8.59%. The three resulting spectra are well defined and are

characteristic of SOM or humic materials (Schnitzer and Preston, 1986). The major signals are at 30 ppm (paraffinic C), 73 and 102 ppm (carbohydrate C), 130

ppm (aromatic C) 150 ppm (phenolic C), and 173 ppm (C in CO2H groups). The

aromatic C content is lower than that of many soil HAs. It is hoped that with improvements in 13C NMR equipment and technology, it will be possible to analyze

soils that contain Ͻ3%C, which would include many agricultural soils.



8



M. SCHNITZER



Although 13C NMR spectroscopy provides information on the different types

of C in SOM, a method that yields data on SOM at the molecular level is Py-FIMS.

This method is more sensitive than 13C NMR and can also be used for the direct

analysis of SOM in soils.

The Py-FIMS spectrum of the whole Armadale soil (Schnitzer and Schulten,

1992), shown in Fig. 2, is dominated by carbohydrates (m/z 60, 72, 82, 84, 96, 98,

110, 112, 114, 126, 132, 144, and 162), phenols (m/z 94, 108, 110, 122, 124, 126,

138, and 154), monolignins (m/z 164, 166, 178, 180, 182, 194, 196, 208, 210, and

212), dilignins (m/z 246, 260, 270, 272, 274, 284, 286, 296, 298, 300, 310, 312,

314, 316, 326, 328, 330, 340, 342, and 356), and suberin derived esters (m/z 446,

474, 502, and 530). The signals at m/z 170 and 184 arise from tri- and tetramethylnaphthalene, respectively, whereas m/z 178, 192, 206, 220, and 234 are

due to phenanthrene, and methyl-, dimethyl-, trimethyl-, and tetramethylphenanthrene, respectively. Also, n-C10 to n-C18 alkyl diesters are present. Normal alkylbenzenes range from m/z 442 (C6H5иC26H53) to m/z 470 (C6H5иC28H57). The occurrence of N-containing compounds is indicated by m/z 59 (acetamide), 67

(pyrrole), 79 (pyridine), 81 (methylpyrrole), 93 (methylpyridine), 103 (benzonitrile), 117 (indole), 131 (methylindole), and 167 (not identified).

The two methods just described allow SOM chemists to obtain significant information on the chemical composition of SOM in whole soils, i.e., in situ. They

also make it possible to study the chemistry of SOM without extracting it from the

soil, without partitioning it into HA, FA, and humin, and without having to lower

the ash content of each of these fractions. It is noteworthy that while both 13C NMR

and Py-FIMS provide similar chemical information on SOM, the problem that

faces SOM chemists at this time is to decide whether the analysis of SOM in whole

soils by advanced methods is the path to follow or whether they want to continue

using the “classical” approach that involves the extraction and separation of HA,

FA, and humin. As shown in Section III,C, 13C NMR spectra of HA and FA demon-



Figure 2 Py-FI mass spectra of the whole Armadale soil. From Schnitzer and Schulten (1992),

with permission of the publisher.



A LIFETIME PERSPECTIVE



9



strate that the main structural features, as well as the aromaticity and aliphaticity

of the two humic fractions, are quite similar. Also, the 13C NMR spectrum of

humin, after deashing, is similar to that of HA (Preston et al., 1989). These findings do not support conclusions of earlier workers (e.g. Sprengel, 1826) that HA,

FA, and humin are different substances that are separated by the “classical” extraction procedure.

The terms HA, FA, and humin do not stand for distinct chemical substances.

Both 13C NMR and Py-FIMS show that the three are closely related materials and

that the separation scheme proposed by earlier workers has no chemical validity.

The obvious solution to the problem is the direct SOM analysis of the whole soil

or soil fractions by 13C NMR or Py-FIMS. Data so obtained tell us about the chemical composition of SOM in terms of aliphatics, proteins, carbohydrates, aromatics, phenols, heterocyclic N compounds, etc. These are chemical classes of compounds, and analytical data are readily understood by all chemists. It is essential

that we start to express ourselves in the language of chemistry and no longer use

terms that have no chemical meaning. I propose that SOM chemists use the term

SOM for all C-containing compounds in the soil, high molecular weight SOM for

HA, low molecular weight SOM for FA, and insoluble SOM for humin. Similarly, water chemists could use the term natural organic matter (NOM) for SOM, high

molecular weight NOM for HA, low molecular weight NOM for FA, and insoluble NOM for humin.

While it is true that enormous literature on the chemical and physical properties

of humic substances (HA, FA, humin), consisting of thousands of scientific papers,

has accumulated over the past 200 years, it is not necessary to abandon or disregard this huge literature. Older data can be reinterpreted and will help us to better

understand the information generated by the new analytical approaches.



E. HOW IS SOM AFFECTED BY LONG-TERM CULTIVATION?

Little is known about how SOM is affected by long-term cultivation. Schulten et

al. (1995) employed Py-FIMS of whole soils, a method described in the previous

section, to throw light on this problem. Soil samples originated from a Typic Haploboroll under a long-term crop rotation established in 1910 at Lethbridge, Alberta.

One soil sample, collected in 1910 from the Ahorizon after breaking the native grassland, had been stored. Another soil sample was collected in June 1990 from the A

horizon under a wheat-fallow, nonfertilized, rotation. This sample had been under

cultivation for 80 years. The native sample (collected in 1910) contained 3.03%

SOM but the cultivated sample (collected in 1990) contained only 2.23% SOM. Percentages of sand, silt, and clay and the exchange capacities of the two samples were,

however, almost identical. The aggregate stability of the native sample was 65%,

whereas that of the cultivated sample was only 42%. There were significant reduc-



10



M. SCHNITZER



tions in enzyme activities after 80 years of cultivation. The activity of dehydrogenase dropped by 60%, that of acid phosphatase by 77%, and that of urease by 82%.

While qualitatively the Py-FIMS spectra were similar, (total ion intensities) (TII),

which are related to SOM concentrations, were dramatically different for the two

samples. The TII value per milligram of soil for the native sample was 31.25 ϫ 104

counts compared to only 3.96 ϫ 104 counts for the cultivated sample. As illustrated

in Fig. 3, TII values of the summed signals characteristic of the major SOM components that are carbohydrates (carboh), phenols and lignin monomers (phenols),

alkylaromatics (alkylar), N-containing compounds (N-comp), peptides, lipids, and

lignin dimers (lignin) show quantitatively different compositions.

Despite similar SOM contents, the TII for the SOM in cultivated soil is only

one-sixth of that in the native soil. Signals in Py-FIMS spectra indicating the presence of carbohydrates, phenols and lignin monomers, alkylaromatics, and N-compounds in SOM of the cultivated sample are only between one-fifth and one-seventh of the intensities generated by the same compound classes in the SOM of the

native sample. TII values of the signals for peptides, lipids, and dimeric lignins in

the cultivated sample constitute even smaller proportions of similar components

of the SOM in the native sample.



Figure 3 Total ion intensity (TII) values of summed signals for major SOM components that are

carbohydrates (carboh), phenols and lignin monomers (phenols), alkylaromatics (alkylar), N-containing compounds (N-comp), peptides, lipids, (alkanes, alkenes, fatty acids, esters), and lignin dimers

(lignin). (a) Native sample, (b) cultivated sample. From Schulten et al. (1995), with permission of the

publisher.



A LIFETIME PERSPECTIVE



11



A possible explanation for data obtained is that the components identified by

Py-FIMS in the cultivated sample originate from more thermally stable and higher molecular weight SOM than those present in the native sample. Thus, cultivation causes increased polymerization and cross-linking of the major SOM components, leading to the formation of larger molecules with higher molecular

weights, stability, and complexity (Schulten et al., 1995). Increases in molecular

size and chemical complexity of the SOM in the cultivated sample may explain

the observed decreases in enzyme activities involving the C, N, and P cycles.

Thus, anthropogenic disturbances through cultivation may induce significant

changes in the quality, chemical composition, and molecular size of SOM. While

these changes may help preserve and maintain SOM in agricultural soils, little is

known on how they affect soil biology.



III. HUMIC SUBSTANCES: ANALYTICAL

CHARACTERISTICS

A. CHEMICAL METHODS

Humic substances contain per unit weight relatively high concentrations of oxygen-containing functional groups (CO2H, OH, CuO). It is through these groups

that these materials are capable of attacking and degrading soil minerals by complexing and dissolving metals, transporting these throughout the soil, and making

them available to plant roots and microbes. Metal–humic complexes with widely

differing chemical and biological stabilities and characteristics are formed. Interactions between humic substances and metal ions have been described as ion

exchange, surface adsorption, chelation, peptization, and coagulation reactions

(Schnitzer, 1978). FA at any pH and HA at pH Ͼ 6.5 can form stable water-soluble metal complexes in competition with hydrolysis reactions. HA is water insoluble at pH Ͻ 6.5 but exhibits sorption properties that lead to the concentration of

metals, especially trace metals, and organics on its large surface.

The elemental composition and functional group content of a typical HA (extracted from the A horizon of a Haploboroll) and a FA (extracted from the Bh horizon of a Spodosol) are shown in Table I.

A more detailed analysis of data shows that (1) the HA contains approximately

10% more C, but 36% less O than the FA; (2) there are quantitatively smaller differences between the two materials in H, N, and S contents; (3) the total acidity

and CO2H content of the FA are significantly higher than those of the HA; (4) both

materials contain per unit weight significant concentrations of phenolic OH, total

CuO, and OCH3 groups, but the FA is richer in alcoholic OH groups than the HA;

(5) about 74% of the total O in the HA is accounted for in functional groups, but



12



M. SCHNITZER

Table I

Analytical Characteristics of a Haploboroll

HA and a Spodosol FA

HA

Element (g kgϪ1)

564

55

41

11

329

Functional groups (cmol kgϪ1)

Total acidity

660

COOH

450

Phenolic OH

210

Alcoholic OH

280

Quinonoid CuO

250

Ketonic CuO

190

OCH3

30

E4 /E6

4.3



C

H

N

S

O



FA



509

33

7

3

448

1240

910

330

360

60

250

10

7.1



all of the O in the FA is similarly distributed. The E4 /E6 ratio of the FA is almost

twice as high as that of the HA, which means that the FA has a lower particle or

molecular weight than the HA (Chen et al., 1976).



B. INFRARED (IR) AND FOURIER TRANSFORM

IR SPECTROSCOPY

IR and FTIR spectra of humic substances show bands at 3400 cmϪ1 (H-bonded

OH), 2900 cmϪ1 (aliphatic C–H stretch), 1725 cmϪ1 (CuO of CO2H, CuO stretch

of ketonic CuO), 1630 cmϪ1 (COOϪ, CuO of carbonyl and quinone), 1450 cmϪ1

(aliphatic C–H), 1400 cmϪ1 (COOϪ), 1200 cmϪ1 (C–O stretch or OH deformation

of CO2H), and 1050 cmϪ1 (Si–O of silicates). The bands are usually broad because

of the extensive overlapping of individual adsorbances. IR and FTIR spectra reflect

the preponderance of oxygen-containing functional groups, i.e., CO2H, OH, and

CuO in humic materials. While IR and FTIR spectra provide worthwhile information on the functional groups and their participation in metal–humic interactions,

they tell us little about the chemical structure of humic materials.

Celi et al. (1997b) applied FTIR to the analysis of CO2H groups in a number of

HAs. Concentrations of CO2H groups in HAs were determined directly from FTIR

spectra by totaling adsorbances at 1720–1710 cmϪ1 (CO2H) and 1620–1600

cmϪ1 (COOϪ). Good correlations were found between total carboxyl groups determined by FTIR, a wet chemical method, and by 13C NMR. Thus, depending on