Evaluation of Conceptual Models of 

Natural Organic Matter (Humus) From a 

Consideration of the Chemical and 

Biochemical Processes of Humification

 By Robert L. Wershaw

 Scientific Investigations Report 2004-5121

 U.S. Department of the Interior

 U.S. Geological Survey 
 U.S. Department of the Interior

 Gale A. Norton, Secretary

 U.S. Geological Survey

 Charles G. Groat, Director

 U.S. Geological Survey, Reston, Virginia: 2004

 For sale by U.S. Geological Survey, Information Services

Box 25286, Denver Federal Center

Denver, CO 80225

 For more information about the USGS and its products:

Telephone: 1-888-ASK-USGS

World Wide Web: http://www.usgs.gov/

 Any use of trade, product, or firm names in this publication is for descriptive purposes only and does not imply 

endorsement by the U.S. Government.
iii

 Contents

Abstract.......................................................................................................................................................... 1

Introduction ................................................................................................................................................... 1

Purpose and scope.............................................................................................................................. 2

Degradation reactions of plant tissue ............................................................................................. 2

Degradation pathways of plant tissue components ............................................................................... 3

Enzymatic reactions ............................................................................................................................ 3

Primary metabolites ................................................................................................................... 4

Carbohydrates.................................................................................................................... 4

Lignin ................................................................................................................................. 5

Plant polyesters ................................................................................................................. 6

Aliphatic acids.................................................................................................................... 8

Plant waxes ........................................................................................................................ 8

Amino acids, proteins, and aminosugars .................................................................... 10

Secondary metabolites............................................................................................................ 10

Tannins............................................................................................................................... 10

Terpenes............................................................................................................................ 11

Pyrolytic reactions............................................................................................................................. 12

Cellulose and other polysaccharides.................................................................................... 14

Amino acids ............................................................................................................................... 14

Lignin and other phenolic compounds.................................................................................. 14

Terpenes..................................................................................................................................... 14

Other miscellaneous plant components ............................................................................... 16

Abiotic reactions................................................................................................................................ 16

Photochemical reactions ........................................................................................................ 16

Direct reactions ............................................................................................................... 16

Indirect reactions ............................................................................................................ 16

Radical oxidative degradation................................................................................................ 17

Browning reactions.................................................................................................................. 19

Interactions of degradation products ..................................................................................................... 29

Evaluation of NOM models........................................................................................................................ 31

Humic polymer model ....................................................................................................................... 31

Molecular aggregate models .......................................................................................................... 31

Compartmental model....................................................................................................................... 31

SOM compartments ................................................................................................................. 31

Partially degraded plant material.................................................................................. 32

Microbial biomass ........................................................................................................... 32

Organic coatings on mineral grains ............................................................................. 32

Pyrolytic carbon............................................................................................................... 33

Organic precipitates........................................................................................................ 33

DOM in soil water............................................................................................................ 34

DOM compartments ................................................................................................................. 34

Summary ...................................................................................................................................................... 35

References .................................................................................................................................................. 35
iv

 Figures

Figure 1. Lignin monomeric units. ........................................................................................................... 5

Figure 2. Lignin degradation pathways. ................................................................................................ 7

Figure 3. Cutin-forming acids. ................................................................................................................. 8

Figure 4. Model cutin monomeric unit according to Kolattukudy (2001). ........................................ 9

Figure 5. Hydrolysable tannin units. ..................................................................................................... 11

Figure 6. Ellagitannin formation. ........................................................................................................... 12

Figure 7. Nonhydrolysable tannins. ..................................................................................................... 13

Figure 8. Pathway for Rhodococcus erythropolis degration of (+)-(4R)-limonene according 

to van der Werf and others (1999). ............................................................................. 13

Figure 9. Formation of anhydro sugars. ............................................................................................... 15

Figure 10. Proposed mechanisms for photolysis of hydroquinone according to Joschek 

and Miller (1966) (Scheme I) and Richard and Grabner (1999) (Scheme II). ...... 18

Figure 11. Formation of benzotropolone rings according to Haslam (1998). ................................. 20

Figure 12. Auto-oxidation of pyrogallol (Haslam, 2003). .................................................................... 21

Figure 13. Possible oxidation reactions of thearubigins according to Haslam (2003). ................ 22

Figure 14. Thearubigin units according to Davies and others (1999). ............................................. 23

Figure 15. Formation of anthocyanin pigments (Salas and others, 2003). ...................................... 24

Figure 16. Proanthocyanidin oligomers............................................................................................... 25

Figure 17. Bicyclic and xythylium salt forms of flavene dimers. ..................................................... 26

Figure 18. Effect of pH on the structure anthocyanins. .................................................................... 27

Figure 19. First steps in browning reactions........................................................................................ 28
 Evaluation of Conceptual Models of Natural Organic 

Matter (Humus) From a Consideration of the Chemical and 

Biochemical Processes of Humification

 By Robert L. Wershaw

 Abstract

 Natural organic matter (NOM) has been studied for 

more than 200 years because of its importance in enhancing 

soil fertility, soil structure, and water-holding capacity and 

as a carbon sink in the global carbon cycle. Two different 

types of models have been proposed for NOM: (1) the humic 

polymer models and (2) the molecular aggregate models. In 

the humic polymer models, NOM molecules are depicted as 

large (humic) polymers that have unique chemical structures 

that are different from those of the precursor plant degradation 

products. In the molecular aggregate models, NOM is depicted 

as being composed of molecular aggregates (supramolecular 

aggregates) of plant degradation products held together by 

non-covalent bonds. The preponderance of evidence favors 

the supramolecular aggregate models. These models were 

developed by studying the properties of NOM extracted from 

soils and natural waters, and as such, they provide only a very 

generalized picture of the structure of NOM aggregates in 

soils and natural waters prior to extraction. A compartmental model, in which the structure of the NOM in each of the 

compartments is treated separately, should provide a more 

accurate representation of NOM in soil and sediment systems. 

The proposed NOM compartments are: (1) partially degraded 

plant tissue, (2) biomass from microorganisms, (3) organic 

coatings on mineral grains, (4) pyrolytic carbon, (5) organic 

precipitates, and (6) dissolved organic matter (DOM) in 

interstitial water. Within each of these compartments there 

are NOM supramolecular aggregates that will be dissolved by 

the solvent systems that are used by researchers for extraction 

of NOM from soils and sediments. In natural water systems 

DOM may be considered as existing in two subcompartments: 

(1) truly dissolved DOM and (2) colloidal DOM.

 Introduction

 Soils and sediments are composed of complex mixtures 

of inorganic and organic components. The organic components 

of soils (soil organic matter) constitute the largest pool of 

carbon on the surface of the Earth (González-Pérez and others, 

2004). This carbon pool is particularly important because soil 

properties such as buffering capacity, metal-binding capacity, 

stability of aggregates of soil particles, water-holding capacity, 

and sorption of hydrophobic organic compounds are dependent, to a large extent, on the amount of organic matter in a 

soil. All of these properties, with the exception of the last one, 

are important in controlling soil fertility.

 The maintenance of soil fertility is of paramount importance for the survival of human life on our planet. In the rich, 

industrialized countries of the world soil fertility is maintained 

by application of chemical fertilizers that are produced using 

large amounts of fossil fuels. The costs of chemicals fertilizers 

are expected to increase because of increases in costs of fossil 

fuels. The ready availability of the chemical fertilizers has 

encouraged farmers to employ intensive agricultural practices, 

such as growing a single crop year after year (monoculture), 

irrigation, and heavy use of pesticides. These practices oftentimes result in severe erosion and deterioration of soil texture 

and aggregation (Pimentel and others, 1995; Tilman, 1999).

 Mäder and others (2002) showed that organic farming 

systems are much more sustainable than conventional intensive agriculture. In addition, they found that organic farming 

reduced energy imput by 34 to 53 percent and pesticide imput 

by 97 percent. In organic farming practice, nutrients are supplied by the application of manure, green manure, and compost, and by growing legumes. Organic farming results in a 

marked increase in concentration of organic matter in an agricultural soil. The development of a more refined conceptual 

model of this organic matter will be the topic of this report. 

Such a model is necessary in order to be able to optimize 

sustainable agricultural practices and to better understand the 

chemical and biochemical reactions that take place in natural 

water systems.

 The pool of NOM in soils is not only an important factor 

in soil fertility, it also serves as a carbon sink. Increasing this 

pool not only improves soil fertility, it also reduces the amount 

of anthropogenic carbon dioxide that is added to the atmosphere. Thus, the active sequestration of carbon in soil can be 

an important tool in reducing climate change brought by the 

burning of fossil fuels and plant biomass.

 Naturally occurring organic compounds in soils have 

been studied for more than 200 years because agricultural 

scientists early recognized the importance of natural organics 

in enhancing soil fertility, soil structure, and water-holding 

capacity (Kononova, 1966). The most general term for the 

natural organic compounds in soils, sediments, and natural 

waters is natural organic matter (NOM); however, a number of 

other terms have been applied to different NOM fractions. 
2 Conceptual Models of Natural Organic Matter, Processes of Humification

The most commonly used of these are: humus, humic substances, humic acid, fulvic acid, and humin. The dissolved 

fraction of the NOM in natural waters is commonly called 

dissolved organic matter (DOM) or dissolved organic carbon 

(DOC). Historically, the term humus has been applied to the 

dark-colored, “rotted” organic matter in soils, and the terms 

humic acid, fulvic acid, and humin have been used to designate different fractions of humus (Kononova, 1966).

 Early workers (see Kononova, 1966, p. 13-45) understood 

that soil humus arises mainly from the degradation of the dead 

plant tissue with a lesser contribution from decaying animal 

remains. Many of these early workers assumed that humus was 

composed of the end products of synthetic reactions that alter 

the structures of plant degradation products. Other workers, 

however, maintained that humus is a complex mixture of plant 

degradation products. This controversy has persisted. Stevenson (1994) has provided a detailed exposition of the synthetic 

reaction postulate. He defined soil humic substances as “a 

series of relatively high-molecular weight, yellow to black 

colored substances formed by secondary synthesis reactions,” 

and humus as the “total of the organic compounds in soil 

exclusive of undecayed plant and animal tissues, their ‘partial 

decomposition’ products, and the soil biomass.” In contrast, a 

number of workers recently have proposed that humic substances consist mainly of the partial degradation products 

of plant polymers (Baldock and others, 1992; Hatcher and 

Spiker, 1988; Piccolo, 2001; Wershaw, 1986; Wershaw, 1994). 

These workers generally do not explicitly state, however, as 

Stevenson (1994) does, what NOM compounds are excluded 

from the category of humic substances; therefore, their definition of humic substances is necessarily incomplete. Most 

workers would agree that soil NOM consists of humified 

components (humus or humic substances) and the nonhumified components (unaltered plant components). Those workers 

who do not accept Stevenson’s definitions must, therefore, 

distinguish between those NOM components that have been 

altered enough to be called humic substances and those that 

have not. It is probably useful to exclude completely undecomposed plant and animal tissue and living microorganisms 

from the definition of humus; however, there is no practical 

way to exclude “partial decomposition” products from humus 

isolates, and indeed, it is likely that soil humus consists mainly 

of such products (Wershaw, 1994; Knicker and others, 1997).

 This controversy has led to the development of two 

different types of models for NOM: (1) the humic polymer 

model and (2) the molecular aggregate model. The belief that 

the component molecules of NOM are produced by secondary 

synthesis reactions from degradation products has led to models in which the NOM molecules are depicted as polymeric 

species that have distinctive chemical structures that are different from those of starting materials. These models, for want of 

a better designation, will be called humic polymer models in 

the following discussion. The proposal that NOM is composed of the partial degradation products of plant polymers 

has resulted in the development of models in which NOM is 

composed of molecular aggregates of the degradation products 

held together by non-covalent bonds (Wershaw, 1994; Piccolo, 

2001). Following the usage of Piccolo (2001), these models 

are designated as supramolecular aggregate models.

 In order to resolve this controversy Wershaw (2000) has 

proposed a paradigm shift in the study of NOM. He stated 

“* * *that a more fruitful approach to the study of NOM in 

soils and natural waters than that based on the humic substance paradigm is to study the chemical reactions that the 

chemical components of plant tissue undergo during and after 

senescence. That is to say, to concern oneself with the humification process rather than with ill-defined intermediates in 

the continuum from well-characterized plant components to 

carbon dioxide.” Humification may be considered as a threestep process of (1) degradation of components of plant tissue 

followed by (2) reassembly of the degradation products into 

NOM, and (3) the degradation of the NOM formed in step 2. 

The development, testing, and refinement of conceptual models are essential elements of scientific inquiry.

 A good model leads scientists to ask the right questions, 

that is to say, to design experiments that provide increased 

insight into the question at hand. At the present time, as 

pointed out above, there are two very different models for 

natural organic matter (NOM): (1) the humic polymer model 

and (2) the molecular aggregate model. Acceptance of one or 

the other of these models, either explicitly or implicitly, underlies the experimental design of most research in the field.

 Purpose and scope

 In this report the likely degradation pathways of the 

components of plant tissue will be discussed along with the 

likely mechanisms of interactions of the resulting degradation products in soils and natural waters. An analysis of these 

mechanisms will be used to develop a compartmental model 

of NOM. This model is a refinement of a previously proposed 

aggregate model for NOM (Wershaw, 1994). Techniques 

for the separation of the NOM compartments will also be 

described.

 Degradation reactions of plant tissue 

 The components of plant tissue can undergo three possible types of degradation reactions in natural systems: 

(1) biotic (enzymatically catalyzed) reactions, (2) pyrolytic 

reactions, and (3) abiotic reactions exclusive of pyrolytic reactions. In most natural systems, it appears that the degradation 

of plant tissue is mainly the result of enzymatically catalyzed 

reactions or sporadic fires that burn plant material. Purely 

abiotic reactions appear to be less common; although plant 

components exposed to sunlight are subjected to abiotic photolytic degradation, this degradation often takes place in concert 

with biodegradation. Zepp and others (1995) pointed out that 

biodegradation may be accelerated or retarded by increases in 

solar ultraviolet radiation depending on the properties of each 

particular environmental system. Abiotic oxidative coupling 
Degradation pathways of plant tissue components 3

reactions may also be important in some environments; however, these reactions are often enzymatically catalyzed (Dec 

and others, 2003). Enzymatic reactions will be considered first 

followed by pyrolytic reactions and abiotic reactions.

 Enzymatic reactions are very substrate specific, and 

therefore, each chemical structural group of plant components 

must be dealt with separately. In our discussion of enzymatic 

degradation, NOM precursors will be divided in two categories: primary metabolites and secondary metabolites. According to Haslam (1995), primary metabolites are those which 

have 

“* * *a distinctive role in the life of all organisms* * *. 

* * *The pathways by which they are synthesised are similar 

if not identical in all organisms* * *.” Secondary metabolites 

on the other hand, “* * *occur sporadically throughout nature 

and the question of the role which they, or the processes by 

which they arise, may play in the economy and welfare of the 

producing organism is one which excites continued speculation.” Haslam (1998) has pointed out that secondary metabolites have three characteristics: (1) structural diversity of the 

metabolites, (2) accumulation of substantial quantities of the 

metabolites in plant tissue, and (3) limited taxonomic distribution. Thus the degradation products of primary metabolites 

will be found in practically all NOM mixtures, whereas those 

of secondary metabolites will be dependent on the source 

organisms for each particular NOM.

 Biodegradation of primary metabolites is carried out both 

by the organisms that originally produced the metabolites and 

by exogenous organisms. For example, carbohydrates and 

proteins that are produced in plant leaves are degraded during 

senescence by endogenous enzyme systems to monomeric 

species that can be stored for use in the following growing season. In addition, heterotrophic, exogenous microorganisms are 

dependent on primary metabolites produced by plants in their 

metabolic processes. The enzyme systems that catalyze these 

endogenous and exogenous reactions are often very similar. 

The biodegradation of primary metabolite polymers generally 

involves hydrolytic depolymerization reactions that ultimately 

produce monomeric species. As these reactions proceed, the 

average size of the molecules continually decreases until only 

monomers are left. Primary metabolites also undergo oxidative 

degradation during cell respiration to produce energy (catabolism). Organic acids produced during catabolism ultimately 

are utilized in one of the cell respiration cycles such as the 

citric acid cycle. Size reduction also occurs during oxidative 

degradation.

 Secondary metabolites are more specialized in function than primary metabolites and generally do not possess 

the structural regularity of primary metabolites. The lack of 

structural regularity of secondary metabolites causes their 

biodegradation pathways to be more complex than those of 

primary metabolites. The degradation of a secondary metabolite does not result in the release of a suite of well-defined 

monomeric units; in fact, it can lead to the formation of more 

complex structures. Of particular interest are the plant polyphenols (hydrolysable and nonhydrolysable tannins) and their 

degradation products that can form non-covalent bonds with 

proteins and carbohydrates. These are the types of compounds 

that promote the aggregation of the molecular fragments that 

constitute NOM.

 In addition to NOM components derived from plant 

biodegradation processes, black carbon (BC), which arises 

from the burning of plants, should be included as an important constituent of NOM (D.W. Rutherford, U.S. Geological 

Survey, written commun., 2004). BC provides ion-exchange 

sites for the binding of plant nutrients thereby improving soil 

fertility (Glaser and others, 2001; Lehmann and others, 2003). 

In addition to binding inorganic constituents, BC strongly 

binds hydrophobic organic compounds; BC has a much higher 

sorption capacity for these compounds than other NOM components (Karapanagiotti and others, 2000; Yang and Sheng, 

2003).

 Consideration of the formation processes of NOM in 

soil systems leads to a model in which NOM is represented as 

existing in separate interacting compartments. A tentative list 

of these compartments includes partially degraded plant tissue, 

biomass from microorganisms, organic coatings on mineral 

grains, pyrolytic carbon (charcoal), organic precipitates, and 

DOM in interstitial soil water. Within each of these compartments it is likely that there are degradation products that will 

form supramolecular aggregates in solution. These degradation products will be dissolved by the solvent systems that are 

used for extraction of NOM from soils and sediments. At the 

present time, methods do not exist for separating all of these 

compartments, but a start has been made. By studying each of 

these compartments separately it should be possible to develop 

a much more complete understanding of the properties of 

NOM in soils.

 Degradation pathways of plant tissue 

components

 The discussion in this section illustrates the degradation 

pathways and the processes that lead to the formation of NOM 

from degradation products of the organic components of plant 

tissue; it is not intended to be an exhaustive compendium of all 

possible groups of plant components. Only the most abundant 

or reactive groups of compounds will be discussed. It would 

be especially difficult to provide a comprehensive compendium of the reactions of all of the various types of secondary 

metabolites. This section is divided into subsections on enzymatic, pyrolytic, and abiotic reactions.

 Enzymatic reactions

 Reactions in biological systems are catalyzed by highly 

specialized proteins called enzymes. Enzymes are generally 

substrate-specific, and each enzyme normally catalyzes only a 

single type of reaction, such as hydrolysis.
4 Conceptual Models of Natural Organic Matter, Processes of Humification

 Primary metabolites

 Carbohydrates

 Carbohydrates are the most abundant components of 

plants. They act as both structural and energy-storage components of plants. Cellulose is the main structural component of 

vascular plants. It generally exists in the plants as crystalline 

or partially crystalline microfibrils of β-1-4-glucopyranose 

chains that are relatively resistant to biological degradation. In 

the native state the microfibrils are present in fiber-like aggregates (Atalla, 1999; Larsson and others, 1999). The microfibrils in these aggregates are bound together by hydrogen 

bonds between hydroxyl groups and van der Waals interactions between the hydrophobic surfaces of the pyranose rings. 

 Crystalline native cellulose (cellulose I) is a composite 

of two different crystalline allomorphs (Iα and Iβ) that have 

distinct 13C nuclear magnetic resonance (NMR) spectra (Kono 

and others, 2002). The preponderance of evidence indicates 

that the unit cell of the Iα allomorph consists of a single 

chain of anhydrocellobiose units, whereas the unit cell of 

th Iβ allomorph consists of two chains of anhydrocellobiose 

units (Atalla and VanderHart, 1999; Atalla, 1999). Typically, 

cellulose aggregates are composed of domains of the two 

allomorphs and noncrystalline domains. In lower organisms 

native cellulose fibers occur in a relatively pure state; however, 

in higher plants the cellulose fibers are generally found in intimate contact with the hemicellulose components of cell walls. 

 The hemicellulose components are mainly composed 

of β-1-4-linked pentoses and hexoses; however, some β-1-3-

gycosidic bonds also may be present. Some branching of the 

chains is present in most of the hemicellulose components. 

The monomeric units of hemicelluloses are: D-xylose, D-mannose, D-galactose, D-glucose, L-arabinose, 4-O-methylglucuronic acid, D-galacturonic acid, and D-glucuronic acid (Pérez 

and others, 2002). The relative concentrations of these monomers vary in different hemicelluloses. For example, hardwood 

hemicelluloses are composed mainly of glucuronic acid and 

xylose units, whereas softwood hemicelluloses are composed 

mainly of glucose and mannose units. In contrast to cellulose, 

hemicelluloses are soluble in dilute alkaline solutions. They 

may be isolated from plant tissue with dilute alkali after the 

plant pectins have been extracted with aqueous solvents and 

calcium chelating agents (Gregory and Bolwell, 1999).

 Pectins are polysaccharides that contain D-galacturonic 

acid residues in addition to other glycosyl residues (see Mohnen, 1999). Three groups of pectic polysaccharides have been 

extensively studied: rhamnogalacturonan I, rhamnogalacturonan II, and homogalacturonan (see Albersheim and others, 

1996). They are abundant in the primary walls of plant cells; 

much lower concentrations of pectins are found in the secondary walls. Albersheim and others (1996) have pointed out that 

the pectic polysaccharides form networks in the primary cells 

of plants that are intimately associated with the cellulose and 

hemicellulose networks in the cells. Pectins are soluble in 

aqueous solutions; if the pectins are present as calcium salts 

then chelation of the calcium ions may be necessary. Watersoluble pectin-like gums also are present in the seeds, roots, 

and bark of some plants.

 Pérez and others (2002) have reviewed the biodegradation 

of cellulose. Cellulose is degraded aerobically by eubacteria 

and fungi to carbon dioxide and water, and is degraded anaerobically by protozoa and slime molds to methane and water. 

These organisms secrete a variety of enzymes that attack cellulose in different ways. Cellulases hydrolyze β-1-4-gycosidic 

bonds; two different types of cellulases have been identified: 

endoglucanases and cellobiohydrolases (exoglucanases). The 

endoglucanases hydrolyze internal cellulose bonds; the most 

susceptible bonds are those in the noncrystalline regions of the 

cellulose fibers. The cellobiohydrolases attack the ends of the 

cellulose chains. Both types of enzymes ultimately reduce the 

cellulose chains to the disaccharide cellobiose. In order for the 

microorganisms to be able to use the hydrolysis products as an 

energy source, the cellobiose must be cleaved into two glucose 

molecules by β-glucosidases. Therefore, in order for microorganisms to effectively use cellulose as an energy source, they 

must secrete a mixture of endoglucanases, cellobiohydrolases, 

and β-glucosidases that are stable in the local environment.

 Anaerobic degradation has been studied less well than 

aerobic degradation. Pérez and others (2002) pointed out that 

cellulose degradation by the strict-anaerobic sporulated bacterium Clostridium thermocellum has been better studied than 

that caused by any other organism. In the Clostridium thermocellum system the enzymes are packaged in cellulosomes that 

are attached to the cell surfaces of the organisms. The cellulase 

enzymes are associated with the protein scafoldin that localizes the various enzymes so that they can more effectively act 

in concert (Atalla, 1999). The products of cellulolysis pass to 

the inside of the organisms through fibers that extend from the 

cellulose to the bacterial cells. Clostridium thermocellum is 

a thermophilic bacterium; mesophilic cellulolytic anaerobes 

have been isolated from soils, sediments, composts, and other 

anaerobic systems. Bacteria, fungi, and protozoa also anaerobically degrade cellulose in the rumina of ruminants.

 The hydrolytic degradation of hemicelluloses by hemicellulases produces monomeric saccharides and acetic acid 

(Pérez and others, 2002). Enzymes specific for the different 

saccharides and bonds in a given type of hemicellulose are 

required for the degradation of the particular hemicellulose. 

For example, if a hemicellulose is made up of chains of 

β-1-4-linked xylose units, then endo-1-4-β-xylanase is needed 

to cleave the chains into xylan oligosaccharides and xylan 

1-4-β-xylocidase to cleave the oligosaccharides into xylose 

monomers. Pérez and others (2002) have pointed out that four 

different enzymes are required to degrade the common hemicellulose O-acetyl-4-O-methylglucuronxylan: endo-1-4-β-xylanase, acetyl esterase, α-glucuronidase, and β-xylosidase. 

 The microbial degradation of pectic polysaccharides also 

is catalyzed by a group of specialized enzymes (pectinases) 

(Benen and others, 1996; Beldman and others, 1996; Bergmann and others, 1996; Hugouvieux-Cotte-Pattat and others, 
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 Figure 1. Lignin monomeric units. 

Degradation pathways of plant tissue components 5

1996). Pectinases may be divided according to their degradation mechanisms into polygalacturonases, pectin esterases, 

pectin lysases, and pectate lysases (Hoondal and others, 2002). 

As in the case of cellulose and hemicellulose, biological 

degradation systems ultimately depolymerize pectins to simple 

sugars.

 Lignin

 The lignin polymers in vascular plants are composed of 

varying amounts of p-hydroxyphenylpropanoid, guaiacylpropanoid and syringylpropanoid monomeric units (Douglas, 

1996; Saake and others, 1996). The most common linkages 

formed during lignin polymerization are β-O-4 ether linkages 

(fig. 1); however, Saake and others (1996) pointed out that 

other linkages such as α-O-4, β-β, β-5, and 5-5 also are present in lignins. Camarero and others (1994) have pointed out 

that, especially in grasses, the C-4 also may be attached to a 

hydroxyl group (C-4 phenol).

 In woody terrestrial plants lignin is imbedded in the 

hemicellulose matrix of the secondary cell walls. Lignin deposition is one of the final stages of xylem-cell differentiation 

(Donaldson, 2001). The deposition takes place in the interlamellar voids of the cell walls. During the deposition, chemical 

bonds form between the lignin and hemicellulose carbohydrates (Sun and others, 2000). Lam and others (1992) have 

shown that cinnamic acid groups in wheat lignin are bound to 

polysaccharides by ester linkages. Two different mechanisms 

have been proposed for the lignin polymerization. Lewis and 

his co-workers (see Burlat and others, 2001) have proposed 

that lignin deposition is initiated at specific dirigent binding 

sites in the cell wall. Monolignol-derived free radicals polymerize at these sites. Burlat and others (2001) pointed out that it 

has been proposed that “* * *lignification occurs through an 

iterative process of lignin assembly (following primary chain 

formation) in a template guided manner.” This process apparently favors β-O-4 linkages. Other workers, however, feel that 

lignin formation is a more “loosely ordered” process in which 

phenolic phenylpropanoid monomers other than p-hydroxyphenylpropanoid, guaiacylpropanoid, and syringylpropanoid 

monolignols can be incorporated into a lignin oligomer/

polymer by undirected free-radical coupling (see Ralph and 

others, 2001). In either scenario the lignin oligomers ultimately link to the hemicellulose chains of the matrix.

 Terashima and others (2002) have differentiated between 

lignin isolates and lignin as it exists in plant cells. They call 

the lignin that is chemically and physically bound to hemicellulose in plant cell walls protolignin. 13C NMR analysis 

indicates that protolignin is a heterogeneous polymer. For 

example, Terashima and others (2002) found that β-O-4 linkages are the most abundant bonds in wheat straw protolignin, 

but that β-5, β-1, and β-β linkages also are present. The 

protolignin forms a three-dimensional network in the hemicellulose matrix that apparently provides increased compressional 

strength to the aerial organs of land plants (Donaldson, 2001). 

The hemicellulose polymers are commonly linked to lignin 

polymers by benzyl ester, benzyl ether, and phenyl glycosidic 

linkages (Lawoko and others, 2003; Sun and others, 2000).

 In as much as lignin occurs as hemicellulose-lignin complexes, lignin degradation cannot be considered independently 

of the hemicellulose degradation. For example, Wershaw and 

others (2003) isolated intact hemicellulose-lignin complexes 

from a natural black water derived from degradation of wheat 

straw. The complexes occur in the black water as soluble, high 

molecular weight molecules. The more hydrophilic hemicellulose chains probably constitute the exterior surfaces of the 

particles with the lignin chains in the interior portions of the 
6 Conceptual Models of Natural Organic Matter, Processes of Humification

particles. Therefore, degradation of the lignin will require 

prior degradation of the hemicellulose.

 Lignin degradation by specialized microorganisms is 

relatively rapid. Ulmer and others (1983) found that lignin 

isolates were reduced to low molecular weight soluble products by the white-rot fungus Phanerochaete chrysosporium

within 6 to 8 days. The enzymatic degradation of lignin by 

microorganisms generally involves both depolymerization 

and aromatic-ring cleavage (Crestini and others, 1998). This 

degradation of lignin is brought about by extra-cellular oxidative enzymes. The action of these enzymes generally has been 

studied using lignin model compounds and lignin isolates that 

have been mechanically and chemically freed from the surrounding hemicellulose matrix.

 Crestini and others (1996) studied the degradation of 

lignin model compounds by the white-rot fungus Lentinus 

edodes. β–O-4 linkages were oxidized to arylglycerol compounds, and aromatic rings were cleaved. Cleavage of aromatic rings coupled with β–O-4 oxidation can lead to the formation of cyclic carbonate structures. The aromatic rings are 

cleaved by enzymatic systems that mostly follow the β–ketoadipate pathway. Harwood and Parales (1996) have reviewed 

the enzymatic reactions that take place in the β–ketoadipate 

pathway. A wide variety of aerobic soil bacteria and fungi 

secrete enzymes that convert aromatic compounds to aliphatic 

carboxylic acids. The first step of the conversion involves 

mono- or di-oxygenation of the aromatic ring to produce a 

dihydroxylated phenyl ring. Fission of the ring can take place 

either between two adjacent hydroxyl groups (ortho-fission) 

or adjacent to a hydroxyl group (meta-fission). Examples of 

these reactions are shown in figure 2. Schemes I and II (orthofission) in figure 2 lead to the production of β–ketoadipate. 

Scheme III is the first step of meta-fission, and Scheme IV is 

the first step in the gentisate pathway in which fission takes 

place between the carbon atom that is attached to a carboxylate group and an adjacent phenolic carbon. Schemes III and IV 

do not lead to the formation of β–ketoadipate (Fairley and others, 2002; Mohamed and others, 2001; Zaar and others, 2001).

 In addition to the degradation of lignin polymeric units, 

the microbial degradation of other phenolic compounds, aromatic hydrocarbons, aminoaromatic compounds, and chlorinated aromatic compounds often follow the β–ketoadipate 

pathway in soils. Fused ring aromatic compounds also can 

undergo degradation by the β–ketoadipate pathway (Hinter 

and others, 2001). Similarly, carboxylate-substituted aromatic 

compounds other than lignin units may be degraded by the 

gentisate pathway (Fairley and others, 2002; Mohamed and 

others, 2001; Zaar and others, 2001).

 Plant polyesters

 There are two types of polyester structural components 

in plants: cutin and suberin (Kolattukudy, 2001). The outer 

covering (cuticle) of the aerial parts of plants is composed of 

cutin. Suberin is a component of cork cells in the bark of vascular plants and in the epidermis, endodermis, exodermis, and 

phellem of roots; it also is deposited in wounds to seal them 

(Bernards, 2002).

 Cutin is a heterogeneous polyester composed to a 

large extent of hydoxy-substituted, saturated C16 acids and 

unsaturated C18 acids (Kolattukudy, 2001). Some of the more 

common cutin-forming acids are shown in figure 3; a model 

of a cutin monomeric unit is given in figure 4. The cuticles of 

plants consist of the cutin polymer embedded in a matrix of 

soluble plant waxes.

 Both bacteria and fungi secrete enzymes that degrade 

cutin. Cutin-degrading fungal enzymes have been studied 

more extensively than bacterial enzymes; however a bacterial 

cutinase has been isolated that hydrolyzes the ester linkages of 

cutin (Berto and others, 1999; Kolattukudy, 2001). The fungal 

enzymes have received more attention because of the damage 

caused by plant fungal diseases. Kolattukudy (2001) pointed 

out that a large number of phytopathogenic fungi secrete 

enzymes that hydrolyze cutin polyesters, thereby allowing the 

fungi to penetrate plant cuticles and harm the internal plant tissue. Cutin oligomers and monomeric fatty acids are produced 

during enzymatic cutin hydrolysis.

 Whereas cutin is composed mainly of straight-chain fatty 

acids, suberin is made up of aromatic and aliphatic monomeric 

units. The suberin polymer appears to consist of polyaromatic 

and polyaliphatic domains (Bernards, 2002; Bernards and 

Lewis, 1998). Bernards and Lewis (1998) pointed out that 

“While it is clear that suberized tissues contain both polyaromatic and polyaliphatic domains, and that each of these has 

its own unique characteristics, whether there is a contiguous 

macromolecule that can be called suberin is an open question.” 

They stated further that “* * *the notion of a heteropolymer, 

called suberin, must be questioned and it is perhaps more prudent to describe tissues as suberized, rather than as containing 

suberin per se.”

 Kolattukudy (2001) has reviewed the literature on the 

biodegradation of suberized tissue. He pointed out that suberized tissue is very resistant to degradation. Parameswaran and 

Wilhelm (1979) found in their study of the degradation of 

beech and spruce bark that suberized tissue was more resistant to degradation by soil microorganisms than other bark 

components. However, fungi can penetrate suberized tissue 

and degrade it (Kolattukudy and Espelie, 1989). This degradation leaves holes in the suberized tissue of bark and roots. 

Kolattukudy and Espelie (1989) pointed out that a number 

of fungal species produce enzymes that degrade suberized 

tissue. There are two different types of enzymes: those that 

degrade the aromatic domains and those that degrade the 

aliphatic domains. The esterase that catalyzed the hydrolysis 

of esterfied aliphatic acids of suberized tissue was shown to 

have identical properties to a cutinase produced by the same 

fungal species. All types of monomeric aliphatic acids were 

released from the suberized tissue by the enzyme. The enzyme 

or enzymes that degrade the aromatic domains have not been 

isolated. It appears that they mainly hydrolyze ester linkages; 

ether linkages appear to be resistant to attack.
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Degradation pathways of plant tissue components 7
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8 Conceptual Models of Natural Organic Matter, Processes of Humification

 Aliphatic acids

 Two groups of aliphatic acids will be covered in this 

section: (1) The low molecular weight acids and (2) the 

straight-chain fatty acids. Ohta (1989a) has reviewed the low 

molecular weight aliphatic acids. Some of these acids serve as 

metabolic intermediates in the citric acid and glyoxylate cycles 

of plants, and therefore, they are transient components of plant 

tissue. Their concentrations in a tissue will be dependent on 

the organs from which the tissue comes and on the time in 

the life cycle of the plant when a tissue sample is isolated. 

Examples of these acids are citric acid, succinic acid, fumaric 

acid, malic acid, pyruvic acid, and malonic acid. In addition to 

those acids that are constantly being cycled by plant metabolic 

processes, there are other low molecular weight acids that are 

end products of plant metabolism that generally accumulate in 

cell vacuoles. Examples of these acids are lactic acid, oxalic 

acid, and tartaric acid. These acids are commonly found in 

high concentrations in some plant species and in much lower 

concentrations in other species (see Ohta, 1989a, p. 267–268). 

Low molecular weight lactones and other alicyclic compounds 

also are found in some plant species (Ohta, 1989b).

 The fatty acids accumulate in plant tissue as energy 

reserves. They commonly occur as triglyceride esters; however, free fatty acids are also found in plant tissue (Zinkel, 

1989). Fatty acids are readily degraded by soil microorganisms. The degradation pathways of fatty acids are similar in all 

organisms and will not be described in detail here. In general, 

fatty acids undergo β-oxidation in which two carbon atoms are 

successively removed from the ends of the chains until the last 

two carbon atoms are left as acetyl-CoA. The acetyl residue 

then undergoes oxidation to carbon dioxide. Fatty acids also 

can undergo ω-oxidation in which a terminal methyl group is 

oxidized by monoxygenase enzymes to a carboxylate group, 

thereby yielding an α,ω-dicarboxylic acid (Eschenfeldt and 

others, 2003).

 Plant waxes

 Kolattukudy and Espelie (1989) have reviewed the plant 

waxes that are associated with cutin and suberin. The most 

common components of plant waxes are long-chain alkanes, 

long-chain primary and secondary alcohols, fatty aldehydes 

and ketones, fatty acids, and monoesters of fatty acids and 

long-chain alcohols. So-called very-long-chain fatty acids 

(greater than 18 carbon atoms in length) are the precursors of 

most components of waxes (Mariani and Wolters-Arts, 2000). 

The relative proportions of the wax compounds vary from 

plant species to plant species and from one type of organ to 

another. Plant waxes are present both as layers covering plant 

cuticles and imbedded in the cutin of the cuticles (Heredia, 

2003).

 Plant waxes are relatively resistant to microbial degradation; however, there are some microorganisms that produce 

enzymes that can degrade them. Some groups of microorganisms, such as those belonging to the genus Acinetobacter, produce wax esters for energy storage (Ishige and others, 2003; 

Rontani and others, 2003). These microorganisms, therefore, 

must possess enzyme systems that can degrade wax components. Berto and others (1999) have shown that the spores of 

the phytophathogenic fungus Alternaria brassicicola contain a 

lipase that can hydrolyze cuticle waxes. McKenna and others 
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Degradation pathways of plant tissue components 9
10 Conceptual Models of Natural Organic Matter, Processes of Humification

(2002) have isolated strains of non-streptomycete actinomycetes from soils that produce enzyme systems that can degrade 

plant waxes. They have used these organisms to ameliorate 

water repellency in a soil.

 Amino acids, proteins, and aminosugars

 Proteins are the most abundant polymeric components 

in living cells. With the exception of some bacterial peptides 

and some peptide antibiotics, all proteins are composed of 

the same set of twenty L-amino acids. The amino acids of 

peptides and proteins are linked together by peptide bonds 

formed by the condensation of the α-carboxyl group of one 

amino acid with the α-amino group of another amino acid. 

Because peptide bonds are formed by the elimination of water, 

they are susceptible to hydrolysis; however, the bonds are 

stable because their activation energies of hydrolysis are high. 

Hydrolysis of peptide bonds requires strongly acidic or basic 

conditions or enzymatic catalysis.

 The enzymes that hydrolyze peptide bonds are called 

proteases. Soil microorganisms secrete proteases to hydrolyze 

proteins into their constituent amino acids (Martens and Loeffelmann, 2003). The amino acids so produced then are utilized 

by the microorganisms as sources of carbon and nitrogen 

(Martens, 2001, Reitzer, 1996a,b; McFall and Newman, 1996). 

Microorganisms can utilize the amino acids in two possible 

ways. They can reduce them to ammonia (or ammonium ion) 

and hydrocarbon compounds and then use the ammonia to 

produce new amino acids and aminosugars for incorporation 

into larger polymers, or they can assimilate the existing amino 

acids and use them as building blocks for proteins and other 

polymeric species (Reitzer, 1996b). Assimilation of amino 

acids can involve partial degradation; for example, members of 

Enterobacteriaceae convert L-arginine, L-glutamine, L-histidine, and L-proline to L-glutamate (McFall and Newman, 

1996). The L-glutamate can be utilized as an energy source via 

the α-ketoglutamate pathway while still maintaining much of 

the carbon skeleton and the C-N bond intact.

 Microbial biomass also contributes to NOM. The chemical composition of microorganisms is much different from that 

of plants; and therefore, the composition of the soil microbial biomass will be different from that of plant-derived soil 

organic matter (SOM). For example, bacteria are composed of 

proteins, RNA and DNA, lipids, lipopolysaccharides, peptidoglycans, glycogen, polyamines, various metabolites, cofactors, 

and inorganic ions (Neidhardt and Umbarger, 1996). Because 

microbial biomass constitutes only about 1 to 5 percent of the 

total carbon in most soils (Angers and others, 1993; Anderson 

and Domsch, 1993; Jenkinson and Ladd, 1981) one might not 

expect it to greatly alter the composition of SOM. However, 

the presence of certain constituents, such as N-acetylglucosamine and N-acetylgalactosamine, are diagnostic of microorganisms. In contrast to soils, microbial biomass can be a 

major component of dissolved and suspended NOM in natural 

waters.

 Secondary metabolites

 Tannins

 Tannins are commonly divided into two groups: hydrolysable and nonhydrolysable tannins. The hydrolysable tannins 

contain gallic acid moieties (fig. 5). Gallic acid apparently is 

produced in plants by degradation reactions (Gross, 1999); 

Haslam (1998) concluded that gallic acid is formed mainly 

from the dehydrogenation of 3-dehydroshikimate. It then 

apparently undergoes esterification to form β-1,2,3,4,6-pentagalloyl-D-glucose, which appears to be an important precursor 

in the formation of the majority of gallotannins and ellagitannins in plant tissue. Ellagitannins are formed by oxidative 

coupling that results in the formation of C–C bonds between 

adjacent galloyl groups as shown in figure 6 (Gross, 1999). 

Hydroxyl groups adjacent to acid groups in these dimers can 

undergo esterification to form internal esters (lactones), as 

shown in figure 6. The dimer shown in figure 6 can undergo 

further polymerization to form larger oligomers. The esterification of two gallic molecules produces a compound called a 

depside.

 The nonhydrolysable or condensed tannins are oligomers 

of phenolic flavan-3-ol (catechin) units as shown in figure 7 

(Haslam, 1998). The biosynthesis of condensed tannins starts 

with the conversion of L-phenylalanine to cinnamate; further 

steps in the biosynthesis are outlined by Haslam (1998). As 

shown in figure 7, the flavan-3-ol units generally are linked to 

each other at the 4 and 8 positions to form proanthocyanidin 

oligomers and polymers; however, other linkages such as 4–6 

also exist (see Ferreira and others, 1999). Oligomers of 2 to 

6 units generally are soluble; larger oligomers are insoluble; 

the monomeric units of these oligomers are called anthocyanidins. Anthocyanidins often exist as glycosides in plants; these 

glycosides are called anthocyanins. The polymeric species 

are called proanthocyanins. The larger insoluble oligomers 

and polymers are generally in higher concentration in plant 

tissue than the soluble oligomers. Flavan-3-ol units also can be 

linked to gallotannins or ellagitannins to form “complex tannins” (Ferreira and others, 1999).

 It has been shown that hydrolysable tannins are degraded 

by some filamentous fungi. These fungi produce enzymes 

called tannases that catalyze the hydrolysis of ester bonds 

between phenolic acid moieties and alcohols or polyols 

(Scalbert, 1991; Gross, 1999). Depside bonds also are cleaved 

by tannases. Some fungi also secrete polyphenoloxidases that 

appear to oxidize tannins. The biodegradation of nonhydrolysable tannins has been much less well studied than that of the 

hydrolysable tannins; however, it appears that some yeasts can 

depolymerize them (Scalbert, 1991).

 Tannins exhibit antimicrobial activity towards many types 

of fungi, bacteria, and yeasts. Scalbert (1991) has reviewed the 

antimicrobial properties of tannins. Four different mechanisms 

have been proposed to explain these properties: (1) inhibition of extracellular microbial enzymes, (2) deprivation of 
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Degradation pathways of plant tissue components 11

substrates required for microbial growth, (3) inhibition of 

oxidative phosphorylation in the metabolic processes of the 

organisms, and (4) deprivation of iron and other metals to the 

organisms by metal binding. The inhibition of some enzymes 

by tannins may be due to binding of the enzymes to the tannins. It is well known that tannins precipitate proteins and 

polysaccharides by forming complexes with them.

 Terpenes

 Terpenes are organic compounds that are composed of 

isoprene units arranged either in open chains or cyclic structures. They generally exist as monoterpenes (two isoprene 

units), sesquiterpenes (three isoprene units), diterpenes (four 

isoprene units), triterpenes (six isoprene units), or tetraterpenes 

(eight isoprene units). Wagner and Elmadfa (2003) pointed out 

that most naturally occurring terpenes are cyclic unsaturated 

hydrocarbons with attached oxygen-containing functional 

groups. At least 15,000 terpenes have been identified. Wagner 

and Elmadfa (2003) stated that “In nature terpenoid molecules 

are implicated in almost every interaction between plant and 

animal, plant and plant, or plant and microorganisms as phytoalexins, insect antifeedants, defense agents, pheromones or 

signal molecules.” Consumption of terpenes in fresh fruits and 

vegetables is purported to have important health benefits.

 Isoprene, the simplest terpene, constitutes about 50 percent of the biogenic non-methane hydrocarbons in the atmosphere (Claeys and others, 2004). Isoprene and some of the 

monoterpenes constitute the most abundant group of reactive 

biogenic compounds in the atmosphere (Limbeck and others, 

2003; Simpson and others, 1999; Guenther and others, 1995). 

Limbeck and others (2003) have shown that “humic-like 

substances” form when isoprene reacts with sulfuric acid in 

aerosols. These substances will precipitate from the atmosphere and contribute to the NOM pool.
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12 Conceptual Models of Natural Organic Matter, Processes of Humification

 Resin acids from trees constitute a major source of terpenes in the environment (Yu and others, 2000). Resin acids 

are composed of tricyclic diterpenoid carboxylic acids. Yu 

and others (2000) isolated a number of bacterial strains from 

hydrocarbon-contaminated Arctic tundra soils that degrade 

resin acids.

 Leenheer and others (2003) have identified degradation products of terpenes in landfill leachates, and surface 

and ground waters. They pointed out that alicyclic terpenoid 

structures are more resistant to bacterial degradation than are 

open-chained or alkene terpenoid structures. In addition, structures containing aliphatic quaternary carbons and branched 

methyl groups are resistant to microbial degradation. Leenheer 

and others (2003) have shown that bands characteristic of 

quaternary aliphatic carbon atoms and branched methyl groups 

in the 13C NMR spectra of NOM isolates are indicative of the 

presence of resistant terpenoid structures in the isolates.

 Some organisms, however, produce enzymes that degrade 

cyclic terpenes. Van der Werf and others (1999) found that 

ring scission is one of the pathways of limonene degradation brought about by a strain of the gram-positive bacterium 

Rhodococcus erythropolis isolated from a ditch sediment. 

Limonene is a very abundant monocyclic monoterpene that is 

found in more than 300 plant species. Other studies (see references cited in van der Werf and others, 1999) also have shown 

that enzymatically catalyzed β-oxidation of the alicyclic ring 

of limonene takes place. Other oxidation products also are produced during microbial degradation of limonene. The pathway 

for Rhodococcus erythropolis degradation of (+)-(4R)-limonene according to van der Werf and others (1999) is shown in 

figure 8.

 Harder and Probian (1995) found that anaerobic bacterial 

cultures can oxidize some monoterpenes in the presence of 

nitrate as an electron acceptor. Acyclic, monocyclic, and bicyclic monoterpenes were degraded. Resin acids also undergo 

anaerobic degradation (Martin and others, 1999). Molecules 

of resin acids generally are composed mainly of hydrophobic 

groups, and they readily sorb to suspended mineral particles. 

When the mineral particles settle out of suspension to form 

sediments, the sorbed resin acids are transported to an anoxic 

environment. The anaerobic biodegradation of the resin acids 

is poorly understood; however, it appears that some resin acids 

undergo aromatization and decarboxylation to form alkylated 

polycyclic aromatic hydrocarbons (Martin and others, 1999). 

Complete mineralization of resin acids by nitrate reducing 

bacteria in anaerobic environments also may take place. 

 Pyrolytic reactions

 Pyrolytic reactions lead to the formation of the so-called 

black carbon (BC) fraction in soils. BC is the least studied component of soil organic carbon (SOC). BC has been 

detected in many soils and sediments (Cambardella and Elliot, 

1992; Karapanagiotti and others, 2000; Schmidt and others, 

1999; Skjemstad and others, 1996; Skjemstad and others, 

2002) and is probably present in most soils. Gélinas and others 

(2001) estimated that BC accounts for as much as 55 percent 

of SOC. They divided BC into two components: char/charcoal 

BC (CBC) and soot/graphitic BC (GBC). The CBC fraction 

is formed by the charring biomass mainly of plant origin, 

whereas the GBC fraction is formed by vapor-phase reactions. 

Gélinas and others (2001) pointed out that GBC particles are 

much smaller than CBC particles, and that they are 

“* * *rapidly dispersed through aerosol formation and atmospheric transport and deposition.” The CBC particles, on the 

other hand, generally will remain in the soil near where they 

are produced. The GBC particles may be transferred long 

distances before they precipitate out of the atmosphere and 

become incorporated into soils and natural waters.

 Simoneit (2002) has reviewed the literature on the 

organic compounds that are associated with GBC particles 

produced by the burning of wood and other biomass. 
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Degradation pathways of plant tissue components 13
14 Conceptual Models of Natural Organic Matter, Processes of Humification

Simoneit (2002) pointed out that during the heating of wood, 

the sequence of decomposition with increasing temperature is 

hydrolysis, oxidation, dehydration, and finally pyrolysis. During the heating process the constituents of wood are converted 

into combustible volatile compounds, tars, and char. When 

the ignition temperature of the volatiles or the tars is reached, 

spontaneous exothermic oxidation (combustion) begins. During combustion, resins, the thermal decomposition products of 

cellulose, hemicellulose, and lignin, and water are volatilized. 

These vapors then undergo either partial or complete combustion. Some of the products of partial combustion are deposited 

on soot particles.

 Cellulose and other polysaccharides

 The pyrolysis of cellulose, the most abundant constituent biomass, has been studied more extensively than that of 

any other biomass constituent. Shafizadeh (1982, 1984) has 

delineated two different pathways for cellulose pyrolysis. 

At temperatures below about 300oC bond scission, formation of free radicals, dehydration, and formation of carbonyl, 

carboxyl, and hydroperoxide groups predominate. These 

reactions are followed by production of carbon monoxide, 

carbon dioxide, and char. The carbon monoxide is apparently the result of decarbonylation and the carbon dioxide of 

decarboxylation. At temperatures above 300oC bond cleavage 

by transglycosylation, bond fission, and disproportionation 

reactions take place. This second pathway (fig. 9) leads to the 

formation of anhydro sugars such as levoglucosan by dehydration. (See Nimlos and others, 2003, for a detailed discussion of 

dehydration of carbohydrates.) Sanders and others (2003) have 

developed a more detailed model of the saccharides. They 

found that at temperatures between 300 and 600oC low-molecular weight oxygenated compounds such as anhydro sugars 

and simple aromatic compounds are formed. At higher temperatures polynuclear aromatic hydrocarbons are produced. 

However, McGrath and others (2003) found that polynuclear 

aromatic hydrocarbons form between 300 and 650oC. Polynuclear aromatic hydrocarbons also are formed from pyrolysis of 

lignin (Sharma and Hajaligol, 2003) and amino acids (Sharma 

and others, 2003).

 Shafizadeh (1984) pointed out: “The production of volatiles leaves a solid residue that is neither intact substrate nor 

pure carbon, but a different material at various stages of charring and carbonization. The intermediate chars are characterized by the functional groups present (including aromatic and 

olefinic structures); a high concentration of free spins trapped 

in a rigid structure or stabilized by aromatic and olefinic 

structures; a large surface area; and high degree of reactivity.” 

Fused ring aromatic structures develop during the charring 

process.

 Amino acids

 Sharma and others (2003) studied the pyrolysis of 

α-amino acids in an inert atmosphere. They identified polycyclic aromatic hydrocarbons and nitrogen-containing polycyclic 

aromatic compounds as products. At low temperatures (300oC) 

single-ring heterocyclic compounds also were formed. Further 

research will be necessary to determine the applicability of 

these results to pyrolysis in air.

 Lignin and other phenolic compounds

 Simoneit (2002) pointed out that “Lignin pyrolysis 

products are major components in the fine aerosol fraction 

of smoke samples from fires burning wood* * *.” Rogge and 

others (1998) analyzed the smoke from burning pine and oak 

wood in residential fireplaces. They identified approximately 

200 different organic compounds including n-alkanes, cyclohexylalkanes, n-alkanals, n-alkanoic acids, alkenoic acids, 

dicarboxylic acids, resin acids, hydroxylated and methoxylated 

phenols, lignans, substituted benzenes and benzaldehydes, 

phytosterols, polycyclic aromatic hydrocarbons, and oxy-polycyclic aromatic hydrocarbons. They attributed the hydroxylated and methoxylated phenols and the substituted benzenes 

and benzaldehydes to pyrolysis of lignin. The lignin-derived 

compounds were the most abundant products in the wood 

smoke. Resin acids (terpenes) also were abundant in the pine 

smoke.

 Terpenes

 Terpenes undergo dehydrogenation reactions to form 

products such as dehydroabietal and dehydroabietic acid during burning of biomass (Simoneit, 2002). Further heating of 

diterpenes with the abietane skeleton leads to decarboxylation 

to form dehydroabietin, and finally to aromatization to form 

retene (Oros and others, 2002). Simoneit and others (1993) 

found that dehydroabietic acid is the dominant compound 

in pine smoke. Many terpenes are volatile and are driven off 

during burning along with water vapor. Depending on the 

temperature of the fire, some or all of the terpene vapors will 

completely burn. If the fire temperature is not too high, some 

of the terpenes will remain intact and be transported along 

with other combustion products (Rogge and others, 1998).

 Oros and others (2002) have analyzed dichloromethane/

methanol-soluble fractions from soils and litters from forests, 

grasslands, and chaparral landscapes before and after burning. 

They identified a number of diterpenes and triterpenes in the 

dichloromethane/methanol extracts of some of their samples. 

In soils from some of the burned areas, they also detected 

combustion products such as dehydroabietic acid and retene.
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Degradation pathways of plant tissue components 15
16 Conceptual Models of Natural Organic Matter, Processes of Humification

 Other miscellaneous plant components

 Oros and others (2002) detected n-alkanes, n-alkanoic 

acids, n-alkanols, and phytosterols in all of the extracts they 

analyzed. The first three of these groups of compounds are 

components of epicuticular plant waxes. The phytosterols are 

components of plant lipid membranes and plant waxes. Biomarkers for specific plant species or types of plants are found 

within all of these categories of compounds.

 Oros and others (2002) detected specific alterations in all 

of the categories of compounds brought about by fire. They 

pointed out, for example, that the n-alkanes of the epicuticular 

waxes of Rye grass are composed of odd numbers of carbon atoms from 25 to 33, with a strong odd carbon number 

predominance. In unburned soil covered by Rye grass, the 

range of carbon atoms was from 15 to 31 with an odd carbon 

number predominance. After burning, the n-alkanes of the 

grassland soil had a range of carbon atoms from 15 to 33 and 

an even stronger odd carbon number predominance than in the 

unburned soil. However, the fatty acids in the unburned grassland soils had a strong even carbon number predominance 

with carbon numbers between 16 and 24. After burning the 

carbon numbers ranged between 14 and 32, and even carbon 

numbers were still predominant, although to a lesser degree 

than in the unburned soil.

 Abiotic reactions

 Photochemical reactions

 Photochemical reactions are classified as being either 

direct or indirect. Direct reactions are those in which chromophores in the molecule being degraded absorb electromagnetic 

radiation (light) that promotes the degradation. Chromophores 

can absorb radiation in the visible or ultraviolet range, or both. 

The added electromagnetic energy causes a chromophoric 

group in a molecule to go from its ground state to an excited 

state. The lower wavelength limit for solar radiation reaching 

the surface of the earth is approximately 300 nanometers (nm), 

and therefore, only chromophores that absorb radiation of 

300 nm or greater will undergo transitions to excited states. 

Aromatic and heteroaromatic functional groups, and conjugated polyenes and carbonyl groups undergo π-π* and n-π* 

transitions at these wavelengths; most other functional groups 

commonly present in NOM absorb at wavelengths shorter 

than 300 nm and are, therefore, inactive (Brown, 1999). The 

energy changes for these transitions can be of the order of the 

energy involved in forming a covalent chemical bond (Pine 

and others, 1980). The work of Osburn and others (2001), 

who studied the photodegradation of DOM from bog water, 

provides an example of direct degradation. They found that 

aromatic functional groups were preferentially degraded by 

solar radiation. Aliphatic functional groups that do not absorb 

solar ultraviolet radiation were more stable than the aromatic 

groups when exposed to solar radiation.

 NOM also may undergo photochemical reactions by indirect processes in which photo-activated organic or inorganic 

species react with unactivated molecules (Goldstone and others, 2002; Faust, 1999; Larson and Marley, 1999; Zafiriou and 

others, 1984). Zafiriou and others (1984) pointed out that there 

is evidence that manganese, iron, copper, mercury, tantalum, 

carbon, nitrogen, oxygen, sulfur, chlorine, bromine, and iodine 

species enter into photochemical reactions in surface waters. 

Thus, for example, photoactivated oxygen-containing species 

could react with NOM. 

 A brief overview of direct and indirection photochemical reactions that take place in surface waters will be given in 

this section. In addition, the contribution to the NOM pool of 

chemical compounds produced by solar irradiation of plant 

components in the atmosphere will be discussed. A further 

complication arises because photodegradation often takes 

place in concert with biodegradation. Zepp and others (1995) 

pointed out that biodegradation may be accelerated or retarded 

by increases in solar ultraviolet radiation depending on the 

properties of the particular system undergoing solar irradiation.

 Direct reactions

 Aromatic functional groups are plentiful in NOM. Richard and Grabner (1999) state that hydroquinone is particularly 

relevant environmentally because it absorbs radiation in the 

ultraviolet portion of the solar spectrum, and it has long-lived 

triplet states that are efficiently populated. Joschek and Miller 

(1966) found that irradiation of hydroquinone both in the 

presence and absence of oxygen in water produced similar 

products. Oxidation and hydroxylation products were formed 

along with a C–C dimer, 2,2’,6,6’-tetrahydroxybiphenyl. 

They proposed the reaction mechanisms shown in figure 10 

(Scheme I) to explain the products they detected. Richard and 

Grabner (1999) proposed a slightly different mechanism for 

the formation of the tetrahydroxybiphenyl product in which a 

triplet-state hydroquinone intermediate reacts with p-benzoquinone that also is produced during the photolysis as shown 

in figure 10 (Scheme II). Polycyclic aromatic hydrocarbons 

undergo similar types of direct photochemical reactions in 

water (Pagni and Sigman, 1999).

 Indirect reactions

 Indirect photochemical reactions are brought about by 

both organic and inorganic species. The most abundant group 

of organic compounds in natural waters is DOM. The colored 

constituents of DOM (humic substances) in natural waters 

participate in indirect photochemical reactions (Zafiriou and 

others, 1984). Although all the chromophores in DOM have 

not been well characterized, it is likely that they are mainly 

aromatic moieties. Zepp and Schlotzhauer (1983) have shown 

that the presence of algal material in fresh water accelerates 

the photodecomposition of nonionic organic compounds. 
Degradation pathways of plant tissue components 17

The authors found in their study of the photo-degradation of 

aniline, m-toluidine, p-chloroaniline, parathion, and methyl 

parathion that these compounds were reversibly sorbed by the 

algal cells and that the degradation took place on the surfaces 

of the cells. The photosensitizing agents on the cell surfaces 

were not identified.

 Photolysis of DOM produces hydroxyl radicals (OH•). 

Vaughan and Blough (1998) photolyzed Suwannee River fulvic acid (SRFA), both aerobically and anaerobically, in water. 

They found that under anaerobic conditions hydroxyl radicals 

were produced in proportion to the amount of SRFA present. 

They concluded from their observations that the OH• radicals 

were produced by direct photolysis of SRFA “* * *through 

a dioxygen-independent pathway* * *”. They proposed that 

a possible mechanism for production of OH• radicals in the 

absence of dioxygen is through the abstraction of hydrogen 

from water by photo-activated (triplet state) benzoquinone 

groups in the SRFA.

 Goldstone and others (2002) showed that OH• radicals 

react with SRFA to produce low-molecular weight organic 

acids (for example, formic, acetic, malonic, and oxalic acids). 

The photo-bleaching rates (reaction rates) they observed for 

SRFA were relatively slow, and they concluded that the OH•

radical reaction with DOM only is able to account for a small 

part of observed photo-bleaching rates in natural water systems. The only exceptions would be in waters with high iron 

concentrations where the photo-Fenton reaction 

(Fe(II) + H2O2 → Fe(III) + OH• + OH-) would produce large 

concentrations of OH•. Brinkmann and others (2003) irradiated 

a bog-lake water with simulated solar ultraviolet radiation. 

They proposed that the observed production of formic, acetic, 

pyruvic, oxalic, malonic, and succinic acids was the result of 

a combination of photochemical and biochemical degradation 

reactions.

 In addition to Fe(II), many other inorganic species produce reactive free radicals when irradiated with solar ultraviolet radiation in water (Faust, 1999). The most important of 

these are dioxygen (O2), nitrate ions, and Fe(III) and Cu(II) 

ions. Inorganic surfaces also catalyze the photodecomposition 

of organic compounds (Ollis, 1985; Ollis and others, 1991; 

Pagni and Sigman, 1999). Solar illumination of an oxide semiconductor surface produces excited electrons (e-

) and holes 

(h+); thus, the excited electrons are reducing agents and the 

holes are oxidants. Concerning the excited electrons and the 

holes, Ollis and others (1991) pointed out “These can migrate 

to the oxide surface and participate in half-cell reactions that 

are part of a closed, catalytic cycle.” In natural waters the photodecomposition of organic compounds, no doubt, is brought 

about by a combination of reactions, some of which take place 

in solution and some on surfaces.

 Precipitation of organic compounds produced by photooxidation in atmospheric aerosols also can contribute to DOM. 

Isoprene is the most abundant biogenic nonmethane hydrocarbon in the atmosphere. Claeys and others (2004) identified two 

diastereoisomers of 2-methyltetrol in natural aerosols from the 

Amazonian rain forest. They attributed these compounds to 

photo-oxidation of isoprene by OH• radicals in the aerosols.

 Radical oxidative degradation

 Free radical oxidative degradation processes take place 

during the later stages of leaf senescence. Leaf senescence is 

one of the major processes leading to the formation of DOM. 

In living plant tissue the reactions between the chemical components in plant cells are tightly controlled. The early stages of 

senescence also are highly regulated with specialized metabolic pathways being activated (Dangl and others, 2000). The 

enzyme systems active in these metabolic pathways degrade 

plant polymers to monomeric units that are then transported 

to storage organs for reuse during the next growing season. 

For example, carbohydrates and proteins are depolymerized to 

monosaccharides and amino acids respectively. Phospholipid 

cell-membrane components are degraded to saturated and 

unsaturated fatty acids. Nucleic acids and plant pigments also 

are degraded.

 During the later phases of senescence, death of the leaf 

takes place, and the various compartments in the plant cells 

rupture and their contents mix together. After the cellular and 

subcellular walls rupture, chemical species produced in one 

subcellular compartment will be exposed to all of the chemical components in the leaf tissue. Oxygen-containing free 

radical species such as superoxide, hydroxyl, peroxyl, alkoxyl, 

polyunsaturated fatty acid, and semiquinone radicals are abundant in the cytoplasmic mixtures formed as cell walls rupture 

(Leshem, 1988; Philosoph-Hadas and others, 1994). These 

free radical species depolymerize plant biopolymers that have 

survived the earlier enzymatically-mediated depolymerization 

reactions. The amino acids, peptide, simple sugars, fragments 

of polyphenolic polymers, and other biopolymers produced 

are then leached from the dead leaves to become components 

of DOM (Wershaw and others, 1998a,b).

 In addition to depolymerization reactions, plant phenolic 

compounds such as hydrolysable and nonhydrolysable tannins 

can undergo oxidative coupling reactions. The most extensively studied sequence of coupling reactions is that which 

takes place during tea fermentation. The processing of tea is 

initiated by withering and mechanically macerating the tea 

shoots to rupture the cell walls; this is followed by fermentation in a high-humidity environment where oxidation reactions 

catalyzed by polyphenol oxidases and peroxidases take place 

(Baruah and Mahanta, 2003). Haslam (2003) has reviewed 

the reactions that take place during tea fermentation. Two 

major groups of phenolic compounds have been identified in 

fully fermented tea (black tea): theaflavins and thearubigins. 

Theaflavins constitute as much as 6 percent and thearubigins 

as much as 75 percent of the solids in brewed tea. Theaflavins 

are characterized by the presence of the seven-carbon atom 

benzotropolone ring (B′ in fig. 11). Much less is known about 

the structures of thearubigins.

 Haslam (1998) has pointed out that the benzotropolone 

ring arises from oxidative condensation of the otho-dihydroxy-
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and Richard and Grabner (1999) (Scheme II). 

18 Conceptual Models of Natural Organic Matter, Processes of Humification
Degradation pathways of plant tissue components 19

phenyl B ring of an epicatechin with the otho-trihydroxyphenyl B ring of an epigallocatechin (fig. 11). In the mechanism 

given by Haslam (1998), two of the three hydroxyl groups of 

the epigallocatechin B ring are oxidized to carbonyl groups 

before ring opening takes place. Davies and others (1999) 

show a slightly different mechanism in which all three of the 

hydroxyls are oxidized to carbonyls prior to ring opening.

 Haslam (2003) speculated that thearubigins have structures similar to those that have been proposed for the products 

of the auto-oxidation of pyrogallol in basic solutions (fig. 12). 

The pyrogallol products result from nonezymatically catalyzed 

oxidative coupling reactions. A sequence of possible oxidation reactions for thearubigins proposed by Haslam (2003) is 

shown in figure 13. Davies and others (1999) suggested that 

thearubigins are composed of units such as those shown in figure 14. All of these proposed structures are highly speculative; 

at the present time there are no definitive data on thearubigin 

structures.

 Coupling reactions of phenolic compounds also also have 

been extensively studied in the aging of wines (Salas and others, 2003). The reactions in red wines have been of particular 

interest because the red pigments are flavonol glycosides 

(anthocyanin pigments shown in fig. 15). Proanthocyanidins 

(fig. 16), condensed oligomers of two to six anthocyanidin 

(aglycone) units linked through the C-4 and C-8, also are present in red wines. The proanthocyanidins undergo cleavage of 

the 4–8 bond forming flavanols in the process. These flavanols, in turn, react with anthocyanin flavylium cations to form 

a colorless flavene dimer. The dimer, in turn, can undergo 

oxidation to the red flavylium cation (fig. 15) or conversion to 

a xanthylium salt or a bicyclic structure (fig. 17).

 The flavylium cation is the dominant form for anthocyanins in the pH range between 1 and 3 (Kähkönen and Heinonen, 2003); at higher pH values the quinonoidal anhydrobases and anionic quinonoidal anhydrobases form as shown 

in figure 18 (Haslam, 1998). In aqueous solution flavylium 

cations are converted to carbinol bases with time or dilution 

at pH values between 4 and 5 (Jurd, 1963; Kähkönen and 

Heinonen, 2003).

 The oxidative reactions outlined above illustrate that the 

types of abiotic reactions that the polyphenolic components 

of NOM can undergo. It is important to point out, however, 

that enzymatic systems also are capable of catalyzing many 

of these reactions, and therefore, in environmental systems it 

often is not possible to distinguish between the results of biotic 

and abiotic oxidative mechanisms.

 Browning reactions

 Another possible non-enzymatic reaction pathway is the 

browning reaction that commonly takes place in foods. In this 

type of reaction, reducing sugars react with free amino groups 

in compounds such as amino acids and proteins to form the 

so-called Maillard products. Amino acids may have only a products. Amino acids may have only a 

single α amino group or more than one amino group as in the 

case of lycine with an additional ε amino group. Both primary 

and secondary amines are susceptible to reaction with sugars 

(Friedman, 1996; Ledl and Schleicher, 1990). The first step in 

the reaction is the formation of a Schiff base followed by re- base followed by rearrangement to an Amadori product (figure 19). Further reaction leads to the formation of heterocyclic nitrogen compounds 

(see Friedman, 1996; Ledl and Schleicher, 1990). The distribution of reaction products will be dependent on relative rates of 

the different steps in the reaction sequence. Unfortunately, the 

kinetics of the different reaction steps are complex and much 

work remains before definitive predictions on relative concentrations of reaction products can be made (van Boekel, 2001).

 Browning reaction products are most commonly formed 

during the cooking of foods. Many of these products are 

important in improving the taste, aroma, and appearance of 

cooked foods. However, mutagenic, carcinogenic, and other 

types of harmful browning products also can be formed during 

the cooking process (Friedman, 1996). In addition, Tauer and 

others (2004) found that increased amounts of Maillard products inhibited enzymatic fermentation of carbohydrate-amino 

acid mixtures.

 The importance of non-enzymatic browning reactions 

has not been demonstrated in humification; however, in some 

special circumstances browning reactions may be important. 

González-Pérez and others (2004) pointed out that Maillard

reactions may take place in soils heated by fires. It should be 

noted, however, that simple sugars would be expected to be 

rapidly consumed by microorganisms in natural systems and, 

therefore, would not be available to participate in browning 

reactions.
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20 Conceptual Models of Natural Organic Matter, Processes of Humification
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Degradation pathways of plant tissue components 21
 Figure 13. Possible oxidation reactions of thearubigins according to Haslam (2003). 

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Degradation pathways of plant tissue components 23
 Figure 15. Formation of anthocyanin pigments (Salas and others, 2003). 

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Degradation pathways of plant tissue components 25
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26 Conceptual Models of Natural Organic Matter, Processes of Humification
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Degradation pathways of plant tissue components 27
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28 Conceptual Models of Natural Organic Matter, Processes of Humification
Interactions of degradation products 29

 Interactions of degradation products

 Wershaw (1994) proposed that NOM is composed of 

molecular aggregates of the degradation products of plant 

tissue. That is to say, humification can be considered as a 

three-step process of (1) degradation, (2) aggregation of the 

degradation products, and (3) degradation of the aggregation 

products. All living organisms are made of molecules that 

form non-covalently bonded molecular aggregates (supramolecular aggregates). In solution supramolecular aggregation 

is a reversible process in which the molecules are constantly 

combining and breaking apart (Lou and others, 2004). In 

living organisms the various supramolecular aggregates are 

segregated in a hierarchy of compartments such as organelles, 

cells, tissues, and so on. However, when an organism dies 

the barriers between the compartments break down, and their 

contents are mixed together and degraded. As outlined in the 

Degradation Pathways of Plant Tissue Components Section, 

the products of biodegradation of the major components of 

plant tissue are monomeric or oligomeric units of the polymers 

that existed in the original organism, and therefore, they can be 

expected to undergo the same types of non-covalent interactions as the precursor polymers. The polyphenolic compounds 

(tannins) constitute a notable exception to this rule. They 

undergo oxidative coupling reactions that produce new types 

of structures.

 During the mixing of the biodegradation products, new 

supramolecular aggregates form. The process of building these 

new supramolecular aggregates results in the formation of 

NOM. In general, with the exception of the polyphenolic compounds, one need not invoke the formation of new covalent 

bonds in the humification process that leads to the production 

of NOM.

 Wershaw (1994) reviewed observed properties of NOM 

isolates, and from these data proposed that “* * *the depolymerization and oxidation reactions that take place during the 

enzymatic degradation of biopolymers produce amphiphiles–

molecules that have a polar (hydrophilic) part and a nonpolar 

(hydrophobic) part. These amphiphiles that result from the 

partial oxidative degradation of dead biomass assemble spontaneously into ordered aggregates in which the hydrophobic 

parts of the molecules form the interiors and the hydrophilic 

parts of the molecules make up the exterior surfaces of the 

aggregates. These ordered aggregates constitute the humus in 

soils and sediments. Humus ordered aggregates most likely 

exist as bilayer membranes coating mineral grains and as 

micelles in solution.” Piccolo (2001) proposed a similar model 

for humic substances in which they are composed of 

“* * *supramolecular associations of self-assembling heterogeneous and relatively small molecules derived from the 

degradation and decomposition of dead biological material.” 

Piccolo (2001) has proposed that supramolecular aggregates 

are “* * *held together mainly by hydrophobic dispersive 

forces.” 

 More recent observations of the properties of leaf leachates (Wershaw and others, 1998a,b, and Wershaw and Kennedy, 1998) indicate that hydrolysable and nonhydrolysable 

tannins are important sources of NOM that were not considered explicitly in the model of Wershaw (1994). However, it 

now appears that these compounds are of particular importance in understanding the aggregation of NOM because they 

form strong non-covalent bonds with a variety of different 

types of compounds and, in particular, with carbohydrates and 

proteins. Haslam (1998) has reviewed “non-covalent inter- and 

intra-molecular interactions” of phenolic compounds. Haslam 

has called these types of interactions “molecular recognition” 

because of their importance in biochemical processes. Noncovalent interactions in aqueous solutions may be classified 

into three categories: Lifshitz-van der Waals forces, Lewis 

acid-base (electron-donor/electron-acceptor) forces, and 

electrical double-layer forces (van Oss, 2003). The electron 

donor-acceptor interactions are by far the strongest of these 

interactions. Included within this category are hydrogen 

bonds, hydrophobic interactions, ionic interactions, and other 

polar interactions. 

 Haslam (1998) pointed out that phenolic compounds are 

by their very nature amphiphiles because phenolic hydroxyl 

groups are hydrophilic and aromatic rings are hydrophobic. 

The amphiphilic character of phenols enhances their interaction with carbohydrates and proteins because the phenolic 

hydroxyls form hydrogen bonds with the polar groups of carbohydrates and proteins, and the aromatic rings interact with 

the more hydrophobic parts of the carbohydrate and protein 

molecules.

 Electron-donating or electron-withdrawing substituents 

on an aromatic ring will polarize the aromatic π electrons. 

Thus, for example, an alkoxy substituent will cause the ring to 

exhibit a net negative charge, and conversely, a carbonyl group 

will cause the ring to exhibit a net positive charge (Cubberley 

and Iverson, 2001). Oppositely charged rings, therefore, will 

be attracted both by hydrophobic and electrostatic effects. If a 

single molecule is composed of aromatic rings that are linked 

by a flexible polymeric backbone, then hydrophobic interactions will cause the rings to tend to stack one on top of another 

to form a more compact conformation (foldamer) in solution 

(Gellman, 1998). If the rings are oppositely charged, then the 

stacking tendency will be enhanced.

 Intermolecular stacked aromatic complexes also form 

in solution (Haslam, 1998). The association of caffeine with 

polyphenols has been extensively studied. Cai and others 

(1990) showed that caffeine molecules are sandwiched 

between the gallate rings of theaflavin digallate, thereby bridging between theaflavin digallate molecules in solution. Haslam 

(1998) proposed that the precipitation of some proteins by tannin polyphenols involves “multi-dentate” binding of a protein 

molecule to a polyphenolic molecule. He pointed out that an 

average tannin polyphenolic molecule of molecular weight 

of 1,000 has between 12 and 16 phenolic hydroxyl groups. 

If both the tannin and protein molecules possess significant 

conformational mobility then it should be possible to form 
30 Conceptual Models of Natural Organic Matter, Processes of Humification

hydrogen-bonded complexes with them (see Haslam, 1998, 

p. 143-145) and hydrophobic-interaction complexes between 

the hydrophobic regions of the polyphenols and the proteins. 

The bound polyphenols would shield the proteins (or peptides) 

from the action of proteases. Zang and others (2001) found 

that proteinaceous material from a green alga is preserved 

from microbial degradation by a highly refractory cell wall 

component called algaenan.

 Carbohydrates also form weak-bonded complexes with 

polyphenols and proteins. Cai and others (1990) found that 

hydrogen bonding and hydrophobic interactions are important 

in the complexation. The sorption of polyphenols by cyclodextrins allows one to study these two types of interactions. The 

hydrophobic interiors of the cyclodextrins will sorb phenolic 

groups that are small enough to penetrate the cyclodextrin 

cavities; phenolic hydroxyl groups interact with the exterior 

hydroxyl groups of the cyclodextrins (Cai and others, 1990; 

Sojo and others, 1999). Many polysaccharides such as starch 

(amylose) exist in coiled conformations that can form hostguest complexes with polyphenols (Cai and others, 1990). 

Many carbohydrates form hydrogen-bonded complexes with 

enzymes and other proteins. This binding is often stereospecific, resulting from an intricate network of hydrogen bonds at 

specific binding sites (Quiocho, 1986).

 Etter (1990) has attempted to devise a method for predicting the patterns (morphologies) of hydrogen bonds between 

proton-donor and proton-acceptor functional groups by studying hydrogen-bond patterns in crystalline solids. She proposed 

a set of three general rules for hydrogen-bond formation 

between organic compounds in solution or in the solid state:

 1. All good proton donors and acceptors are used in 

hydrogen bonding.

 2. Six-membered-ring intramolecular hydrogen bonds 

form in preference to intermolecular hydrogen bonds.

 3. The best proton donors and acceptors remaining after 

intramolecular hydrogen-bond formation form intermolecular hydrogen bonds to one another.

 With regard to the third rule, many authors have attempted 

to develop scales that rank compounds with respect to their 

hydrogen-bond acceptor basicities or hydrogen-bond donor 

acidities (Haslam, 1998). These scales are not universally 

applicable because they normally are developed by measuring 

hydrogen-bond formation constants of a group of proton-donor 

compounds with an arbitrarily chosen strong proton-acceptor standard or the formation constants of a group of protonacceptor compounds with an arbitrarily chosen strong protondonor standard. The relative positions of the donor or acceptor 

compounds in the scales will be to some extent dependent on 

the choice of standards and solvents used for the measurements. Anions in general are the strongest proton acceptors 

and proton-containing cations are the strongest donors (Abraham and others, 1989). Thus, carboxylates, which are the most 

common anionic functional groups in NOM, can be expected 

multiple hydrogen bonds between the phenolic molecules and 

the proteins molecules. In this scenario, proteins with loose, 

random conformations in which large numbers of binding 

sites are exposed would interact more strongly with polyphenols than would compact globular proteins with few exposed 

binding sites, and this is indeed the case. A similar mechanism 

would explain the interaction of polyphenols with polysaccharides.

 The work of McIntosh and others (1999) provides 

another example of promotion of aggregation by tannins; 

they found that hydrolysable tannins (so-called tannic acid) 

strongly interact with phospholipids in bilayer membranes 

to bring about aggregation of the bilayer structures. They 

argue that the tannic acid molecules act as molecular bridges 

between adjacent bilayers.

 The ability of tannin polyphenols to form non-covalent 

bonds provides an explanation for the preservation of proteins 

and peptides in soils. NMR studies of soils have demonstrated 

that almost of all of the nitrogen is in the form of amides (see 

Knicker, 2002). These findings are supported by the work of 

Martens and Loeffelmann (2003) who found, using a methanesulfonic acid hydrolysis procedure they developed, that more 

than 85 percent of the total nitrogen in the soils they analyzed 

was recovered as amino acids, aminosugars, and ammonia. 

They concluded that these hydrolysis products are most likely 

derived from peptides and aminosugars in the soils. Larger 

polypeptides (proteins) also may be present. The aminosugars are probably in the form of bacterial peptidoglycans. The 

preservation of those peptides that contain free amino groups 

is probably due to the formation of protein or peptide-clay 

complexes (see Wershaw and Pinckney, 1980). Wershaw and 

Pinckney (1980) showed that deamination of clay-humic 

complexes released a nitrogen-containing organic phase from 

the clays. Deamination will eliminate the free amino groups 

of basic amino acids in proteins and peptides. Laird and others 

(2001) found that 50 to 66 percent of the total amino acids 

associated with the fine clay fractions of the agricultural soils 

they analyzed was arginine. Arginine is the most basic amino 

acid found in proteins; the guanidinium group of arginine will 

be positively charged to pH values in excess of 12. Proteins 

containing arginine or free arginine, therefore, can bond to 

negatively charged sites of clay surfaces (Laird and others, 

2001).

 This mechanism cannot be invoked if free amino groups 

are not present. Zang and others (2000), however, found that 

humic acid isolates also protect proteins from degradation. 

They proposed that this protection is brought about by “encapsulation into hydrophobic domains of humic acids.” A purely 

hydrophobic mechanism has been used to explain the sorption 

of hydrophobic organic compounds in soils and sediments 

(see Xing, 1998, and references therein); however, proteins are 

characterized by both polar and nonpolar parts. A more likely 

encapsulation mechanism would be by hydrogen bonding and 

hydrophobic interactions of the proteins (or peptides) with the 

tannin polyphenolic components of NOM. The polyphenols 

would protect the proteins (peptides) by forming multi-dentate 
Evaluation of NOM models 31

to form especially strong hydrogen-bonded complexes with 

strong proton donors such as phenols.

 Evaluation of NOM models

 Humic polymer model

 As pointed out in the Introduction Section of this report, 

in the humic polymer model it is postulated that the component molecules of NOM are polymeric species that have been 

synthesized by secondary synthesis reactions from plant degradation products. It is further postulated that they have unique 

chemical structures that are different from precursor plant 

polymers. A large number of these models have been proposed 

(see Stevenson, 1994, p. 287-294). The models generally have 

been developed for soil humic acid isolates by taking into 

account the chemical and physical data that been determined 

for a particular isolate or group of isolates. The data commonly include functional group distributions, elemental compositions, and number- or weight-average molecular weights.

 With the advent of computer modeling (Bruccoleri and 

others, 2001; Diallo and others, 2001; Schulten and Schnitzer, 

1995; Sein and others, 1999), modeling of NOM has reached 

new levels of mathematical sophistication. The real problem in 

using these modeling programs is determination of the input 

data to be used by the program. For example, in the computational scheme used by Diallo and others (2001) the required 

input data “* * *include: (i) atomic ratios, (ii) functional group 

ratios, (iii) structural groups centered on carbon atoms and 

hydrogen atoms, (iv) the nature and amount of molecular fragments and the interfragment bonds, and the number average 

molecular weight.”

 In both the manual and computer-aided modeling efforts 

for soil humic acids, the input data have been obtained by 

making measurements on humic acid isolates. These isolates 

are commonly obtained by extraction of soils with 0.1 Molar 

(M) NaOH and precipitation of the humic acid by adjusting 

the solution pH to between 1 and 2. Different purification 

steps to remove minerals matter also may be used. The resulting isolates are complex mixtures of different molecular species (Wershaw and Pinckney, 1973; Wershaw, 1994). This is 

not at all surprising in light of the fact that soil organic matter 

is composed of partially degraded plant tissue, biomass from 

microorganisms, coatings on mineral grains, pyrolytic carbon, 

organic precipitates, and DOM in soil water (Cambardella and 

Elliott, 1992 and 1994; Six and others, 2001; Sohi and others, 

2001; Wershaw, 2000). Simpson and others (2002) demonstrated by multi-dimensional NMR analysis of reverse-phase 

chromatographic fractions that NaOH extracts of soils are 

composed of mixtures of “* * *aliphatic acids, ethers, esters 

and alcohols; aromatic lignin derived fragments; polysaccharides and polypeptides.”

 Sein and others (1999) have attempted to model a “humic 

acid building block” and the stereochemistry of this “building block.” The evidence cited in the previous paragraph that 

humic acids are complex mixtures precludes the existence of 

such a “building block.” That is to say, humic acid is not a 

single, identifiable entity. Similar objections can be made to 

all of the other polymer models that have been proposed.

 Molecular aggregate models

 The molecular aggregate models that have been proposed 

by Wershaw (1994) and Piccolo (2001) are better representations of the actual state of NOM in soil extracts than the 

humic polymer models; however, they provide only a very 

generalized idea of the structure of NOM aggregates in soils 

prior to extraction. As pointed out in the previous section, soil 

NOM consists of partially degraded plant tissue, biomass from 

microorganisms, organic coatings on mineral grains, pyrolytic carbon, organic precipitates, and DOM in soil water; for 

want of a better term, each of these will be called a separate 

NOM compartment. A similar set of compartments exists in 

sediments. The NOM in each one of these compartments has 

a different composition. Unfortunately, the usual practice of 

extracting NOM from a whole soil yields a solution in which 

the components of NOM from all of these compartments that 

are soluble in the extracting solvent system are mixed together. 

 Compartmental model

 The concept of NOM compartments described above is 

somewhat analogous to the phase concept in thermodynamic. 

A phase is a homogeneous region in a thermodynamic system. 

Only the DOM compartment is truly a phase. The other compartments are not homogeneous regions; however, they are 

identifiable regions that can be separated from soils or sediments. Each of these compartments is composed of a set of 

components (chemical substances); some of these components 

are inorganic and some organic. The organic components 

constitute the NOM in the compartment. These components 

will, in general, interact with each other and the inorganic 

components in the compartment. As in thermodynamics, one 

must identify the components and their interactions in each 

phase or compartment before attempting to study the entire 

system, whether it be a soil or a well-defined system of two or 

three components.

 SOM compartments

 Separation and characterization of soil or sediment NOM 

(SOM) from each of the compartments (partially degraded 

plant tissue, biomass from microorganisms, organic coatings 

on mineral grains, pyrolytic carbon, organic precipitates, and 

DOM in soil water) should allow one to develop more representative models for NOM supramolecular aggregates in soil 

and sediment systems. This is especially important because the 
32 Conceptual Models of Natural Organic Matter, Processes of Humification

hydrate bands are the dominant ones in these spectra. In the 

occluded d < 1.6 fractions and the more dense fractions, the 

relative intensities of the carbohydrate bands were reduced in 

comparison to bands attributable to alkyl hydrocarbons and 

aromatic moieties. A similar reduction in the relative intensities of carbohydrate bands was observed in the solid-state 

13C NMR of litter samples that had undergone weathering for 

different lengths of time on the floor of a pine forest (Qualls 

and others, 2003); Qualls and others (2003) observed that the 

longer the weathering period, the greater the loss of carbohydrates from the litter. Preston and others (1998) also observed 

a loss of carbohydrates brought about by decay in coarse 

woody debris in coastal forests of British Columbia. Nierop 

and others (2001) found lower concentrations of carbohydrates 

in the NOM in soils than in the overlying litter or fresh plant 

material from a primary vegetation succession (algae, moss, 

heather, and pine) in the central Netherlands. Accumulation 

of resistant aliphatic polymers derived from mosses also was 

detected in the soils. They found that lignin degraded more 

rapidly than tannins.

 Microbial biomass

 Soil microbial biomass is difficult to isolate and characterize; however, Golchin and others (1996) and Baldock and 

others (1990) have used an indirect method to characterize 

soil biomass. Baldock and others (1990) isolated bacteria and 

fungi from a sandy loam soil. The isolates were amended with 

glucose and the solid-state 13C NMR spectra of the cultures 

were measured. They found that the spectra from the two 

materials were distinctive. The bacterial material contained 

more alkyl, aromatic, and carboxyl groups and less O-alkyl 

and acetal groups than the fungal material. They compared 

these NMR spectra with spectra of soil fractions incubated 

with 13C-enriched glucose and concluded that “* * *the soil 

microbial population was dominated by fungi.” Golchin and 

others (1996) extended this work by inoculating artificial 

soils with soil microorganisms and incubating the artificial 

soils with unlabelled and 13C-labelled glucose. They found 

that 24 to 25 percent of the carbon was in the form of alkyl 

groups, 56 to 63 percent in the form of O-alkyl groups, 11 to 

15 percent in the form of carbonyl groups, and a small amount 

in aromatic and olefinic groups. By using proton-spin relaxation editing techniques they were able to distinguish between 

slowly relaxing (long T1(H)) functional groups and rapidly 

relaxing (short T1(H)) functional groups. Alkyl carbons in 

long- and short-chain structures had long T1(H) values as did 

N-alkyl groups. Carbonyl carbon atoms and -alkyl groups. Carbonyl carbon atoms and O-alkyl groups 

had short T1(H) values. Golchin and others (1996) concluded 

that the alkyl carbon groups were attached to clay mineral surfaces by polysaccharide mucilages and that the O-alkyl groups 

represented neutral and acidic extracellular saccharides. Lichtfouse and others (1998) also found evidence for straight-chain 

aliphatic biopolymers that are resistant to degradation in soils. 

This resistance to degradation may be due to attachment of the 

aliphatic biopolymers to clay minerals.

NOM in each of these compartments can be expected to have 

different sorption, ion exchange, and buffering properties.

 A start has been made on techniques to achieve these 

separations. A number of groups have fractionated soils by 

particle size and density, and have characterized the NOM in 

these fractions with solid-state 13C NMR (Baldock and others, 

1992; Chen and Chiu, 2003; Golchin and others, 1997; Kahle 

and others, 2003; Laird and others, 2001; Preston and others, 2000; Schmidt and Kögel-Knabner, 2002; Six and others, 

2001; Sohi and others, 2001). Solid-state 13C NMR provides a 

nondestructive means of characterizing the NOM in soil fractions without alteration.

 The physical separation techniques used by the various 

groups were designed to isolate the size and density fractions with minimal alteration of the NOM. The procedure 

used by Golchin and others (1997) is a typical density fractionation procedure. In this procedure 20-gram (g) samples 

from Japanese volcanic ash soils (andosols) were suspended 

in 200 milliliters (mL) of sodium polytungstate solution of 

density 1.6 grams/cubic centimeter (g/cc) in capped 250 mL 

glass centrifuge tubes by inverting the tube gently five times 

by hand. The tubes were allowed to stand for 30 minutes 

(min) and was then centrifuged for 30 min at 2,000 revolutions 

per minute (rpm). The floating particles and the supernatant 

solution were decanted, and the particles were isolated by 

filtration and washed to remove residual polytungstate. The 

particles were dried and weighed; this fraction was designated 

the free light fraction with a density (d) less than (<) 1.6 g/cc. 

The residual soil from the first floatation was resuspended in 

80 mL of 1.6 g/cc solution and sonicated in an ice bath for 5 

min. The volume was then adjusted to 200 mL with polytungstate solution and centrifuged, filtered, and washed as before. 

The fraction isolated was called the occluded 

d < 1.6 g/cc fraction. The residual soil was further fractionated 

by sequential suspension in 1.8 g/cc and 2.0 g/cc solutions. 

In this way, fractions with densities between 1.6–1.8 g/cc and 

1.8–2.0 g/cc and greater than (>) 2 g/cc were isolated. A free 

fraction isolated by floatation with pure water also was isolated; it was designated as the free d < 1.0 g/cc fraction.

 Size fractionation by dry sieving also has been used on 

soil samples; the dry sieving may be followed by wet sieving to isolate smaller particle-size fractions. Clay fractions 

are generally isolated by dispersing sieved soils in water and 

allowing the different clay size particles to settle from suspension. Kahle and others (2003) isolated coarse and fine clay 

fractions from illitic soils (soils in which illite was the major 

clay mineral in the soils). NOM was in the form of coatings on 

the clay surfaces.

 Partially degraded plant material

 The different soil fractions of Golchin and others (1997) 

represent different stages in the diagenesis of NOM. The least 

degraded material was isolated in the free d < 1.0 g/cc and the 

free d < 1.6 g/cc fractions; they yielded NMR spectra very 

similar to those of fresh plant litter on soil surfaces. Carbo-
Evaluation of NOM models 33

 Pyrolytic carbon

 Techniques for the isolation and characterization of pyrolytic carbon (so-called black carbon) in soils and sediments 

have not been well developed. Golchin and others (1997) 

found by light microscopy that some of the density fractions 

they isolated were enriched in black carbon; however, other 

soil components also were present in the fractions. Skjemstad and others (2002) used a series of physical and chemical 

treatments to isolate charcoal from agricultural soils. Unfortunately, a high-energy photo-oxidation step was included in 

their procedure to remove “more labile fractions of organic 

matter”; this step could very well alter existing charcoal or 

produce black carbon from the so-called labile organic matter. Another highly aggressive chemical oxidation procedure 

using dichromate and sulfuric acid has been used to isolate 

black carbon from sediments (Masiello and Druffel, 1998). 

This procedure can be expected to markedly alter the black 

carbon. Recently, a group of scientists convened a meeting 

at the Friday Harbor Laboratories of the University of Washington called “New Approaches in Marine Organic Biogeochemistry”. Part of the meeting was devoted to developing 

black-carbon reference materials (Schmidt and others, 2003). 

Hopefully, efforts of this nature should lead to better isolation 

and characterization techniques.

 Rutherford (D.W. Rutherford, U.S. Geological Survey, 

written commun., 2004) has prepared wood charcoals under 

conditions similar to those that prevail during forest fires. 

He measured the surface areas, elemental compositions, and 

NMR and IR spectra of charcoals prepared by heating pine 

and poplar wood at different temperatures for different time 

durations. Future studies are planned to compare the charcoals 

prepared under controlled conditions with charcoals collected 

from areas that have undergone forest fires.

 Organic precipitates

 Precipitation of NOM in soils can take place during 

evaporation of soil solutions. Metal ion bridging between 

NOM molecules also will bring about precipitation of NOM. 

At the present time (2004), no techniques exist for the isolation of organic precipitates from soils. Natural precipitates, 

however, can be simulated by adding selected metal ions to 

interstitial solutions collected from soils or by evaporating 

these solutions.

 Merdy and others (2000) and Guillon and others (2001) 

attempted to simulate the interaction of metal ions with NOM 

by studying the binding of Cu (II), Mn(II), and Fe(III) with 

lignin monomeric, dimeric, and polymeric model compounds. 

These metals formed strong complexes with the lignin model 

compounds. Guillon and others (2001) proposed that part of 

the Fe(III) in soils will be immobilized by complexation with 

insoluble lignin components, and that part of Fe(III) will be 

mobilized by complexation with soluble lignin components. 

They further proposed that sorbed Fe (III) will promote oxidation of the NOM. The studies of Merdy and others (2000) 

and Guillon and others (2001) could easily be extended to the 

 Organic coatings on mineral grains

 Kahle and others (2003) measured the solid-state 13C NMR spectra of fine and coarse clay fractions from soils 

from seven different sites. They found that the fine clay fractions had more amide carbonyl carbon and less carbohydrate 

carbon than the corresponding coarse clay fractions. Kahle 

and others (2003) pointed out that NOM may also be associated with iron oxides in the fine clay fractions.

 The presence of iron oxides or other paramagnetic materials in a sample presents special problems for NMR analysis (Smernik and Oades, 2002). If the concentration of the 

paramagnetic species is similar to that of the carbon content 

of the sample, then severe broadening of the 13C NMR bands 

will occur. Gonçalves and others (2003) have developed a 

procedure using hydrofluoric acid to remove iron oxides from 

soil samples prior to NMR analysis. Carbon functional group 

distributions do not appear to be altered significantly by their 

procedure.

 Wershaw and others (1996) showed that compost leachate DOM formed bilayer membrane-like coatings on alumina surfaces. The experiments conducted by Wershaw and 

others (1996) were designed to simulate what happens when 

leachate from decomposing plant litter is sorbed by hydrous 

metal oxides in soils. Alumina was chosen as a representative hydrous metal oxide because it is not paramagnetic, and 

therefore, NMR can be used to characterize the sorbed organic 

material. In addition, alumina surfaces are present in clay minerals. Hydrous metal oxide surfaces are composed of hydroxyl 

ions bonded to metal ions. Wershaw and others (1996) showed 

that organic anionic species from the compost leachate 

replaced hydroxyl ions to form inner sphere complexes with 

aluminum ions. Some of the anions from the compost leachate 

adsorbed to the alumina surface were amphiphiles. Wershaw 

and others (1996) proposed that the amphiphile molecules 

(such as long-chain aliphatic acids) “* * *are adsorbed on 

metal oxide surfaces through a combination of polar and 

hydrophobic interaction mechanisms. This combination of 

polar and hydrophobic mechanisms will lead to the formation 

of bilayer coatings of the leachate amphiphilic molecules on 

the oxide surfaces.”

 Sorption of NOM also takes place on clay mineral 

surfaces. These surfaces oftentimes are negatively charged 

because of isomorphous cation substitution (Sposito and 

others, 1999). As pointed out above, basic amino acids and 

peptides, proteins, and amino sugars containing basic amino 

acids are bound to negatively charged clay mineral surfaces. 

Wershaw and Pinckney (1980) characterized a colloidal clayhumic complex that had been isolated as a fraction of a soil 

humic acid. Wershaw and others (1988 and 1990) used liquidstate 13C NMR to characterize colloidal clay-humic complexes 

isolated from different soils and sediments. They found that 

NMR spectra of many of these clay-complexes consisted of 

very sharp carbohydrate bands. These bands are most likely 

due to the presence of amino sugars or peptidoglycans that are 

bound to the clay surfaces by basic amino acid moieties.
34 Conceptual Models of Natural Organic Matter, Processes of Humification

that “As the hydrophilic character of the fraction increases, 

carbon percentage decreases and nitrogen percentage 

increases.” Barber and others (2001) have applied this procedure with some modifications to characterization of DOM in 

water-treatment wetlands. This procedure can be used for the 

characterization of soil-water DOM.

 DOM compartments

 The work of Leenheer and others (2000) indicates that 

DOM operationally defined by passage through a 0.4 micrometer filter actually consists of two compartments: (1) truly dissolved DOM and (2) colloidal DOM. The colloid DOM compartment would include not only the high molecular weight 

organic colloids that Leenheer and others (2000) isolated, but 

also NOM adsorbed to colloidal mineral particles.

 Evidence exists that DOM in natural waters also forms 

supramolecular aggregates. Using polarography Hunter and 

Lee (1986) found that humic acid isolated from a natural 

water was four times more surface active than any of the other 

organic components in the water. When the humic acid and 

the other organic components were mixed at natural concentrations the surface activity of the mixture was only about 

50 percent of the sum of the surface activities of the individual 

components. They interpreted the results of the work as indicating that the DOM components form aggregates in solution. 

Wershaw and Hayes (2001) have reviewed the literature on 

solubilization of anthropogenic compounds by DOM. They 

concluded that the enhanced solubilities of hydrophobic 

organic compounds that have been observed in natural waters 

are due to partitioning of the molecules of the hydrophobic 

compounds into premicellar DOM aggregates. Premicellar 

aggregates are those that form at concentrations lower than the 

critical micelle concentration (CMC) for the amphiphilic molecules under consideration. For example, Zimmels and Metzer 

(1976) found evidence for the formation of premicellar oleate 

aggregates at concentrations as low as 0.01 CMC. Aggregation of amphiphilic molecules (molecules that have separate 

hydrophilic and hydrophobic parts) is caused by hydrophobic 

interactions between the hydrophobic parts of the molecules. 

Polyvalent cations also can bring about intermolecular 

aggregation by bridging between polar groups of two or more 

molecules (Gamble and others, 1984). Another possible aggregation mechanism for DOM is by interaction of tannins with 

protein and carbohydrate components of DOM as discussed in 

the Interactions of Degradation Products Section. Pacheco and 

others (2003) studied the interactions of solutions of soil, peat, 

Leonardite, and coal humic acids with a variety of different 

organic and inorganic xenobiotic compounds. They concluded 

that some of these compounds form supramolecular complexes 

with the humic acid molecules. They proposed that ion binding, hydrogen bonding, van der Waals forces, ligand exchange, 

hydrophobic and hydrophilic adsorption, charge-transfer 

complexation, and sequestration accounted for the aggregation 

they observed.

interaction of metal ions with DOM components isolated from 

soil interstitial solutions in order to provide a more realistic 

simulation of the actual interactions in soil-water systems.

 DOM in soil water

 Soil solutions may be readily collected using different 

types of lysimeters (see Charbeneau and Daniel, 1992, and 

the references therein). The pressure-vacuum lysimeter is the 

most commonly used type. It consists of a porous cup that is 

placed in a borehole; the space between the cup and the inside 

of the borehole is filled with a fine silica powder. The cup is 

evacuated with a vacuum pump to draw water from the soil 

into the cup. Qualls and Haines (1992) and Hagedorn and others (2004) used these devices to collect soil solutions. Qualls 

and Haines (1992) found that in oak and hickory forests of 

the southern Appalachian Mountains large quantities of DOM 

are generated on the forest floors. They have interpreted their 

data as indicating that most of the DOM is rapidly adsorbed 

in the A horizons of the forest soils; a small amount of DOM 

remains in solution. It appears that biodegradation in the solution phase is too slow to remove much DOM from the soil 

solutions; however, the increased concentration of NOM on 

the soil particles appears to favor biodegradation. Hagedorn 

and others (2004) have interpreted the data they collected from 

13C tracer studies in model beech and spruce forest ecosystems in a somewhat different way. Their results suggest that 

organic carbon compounds freshly leached from forest floor 

litter are rapidly degraded to carbon dioxide, and that only 

a small amount remains in solution. The DOM in the soil 

solutions collected at 5-10 cm depth “* * *originated mainly 

from native soil C and pools of humified C smaller than 50 

μm.” They further point out “* * *that dissolved organic C is 

produced during incomplete decomposition of recalcitrant soil 

C, while easily degradable components are rapidly consumed 

by soil microbes and thus make minor contributions to the 

dissolved organic C.” Neither Qualls and Haines (1992) nor 

Hagedorn and others (2004) have characterized the DOM in 

their soil solutions. Nierop and Buurman (1999) used solidstate 13C NMR to characterize the total DOM in water extracts 

of podzol soils; however, they did not fractionate their extracts. 

Fractionation would provide a more detailed characterization 

of the soil-water composition.

 Leenheer and others (2000) have developed the most 

comprehensive fractionation and characterization procedure 

for DOM that has yet been published. This procedure yields 

five different fractions: (1) a colloidal fraction, (2) a hydrophobic NOM fraction, (3) a fraction of intermediate polarity 

(called transphilic NOM), (4) a hydrophilic acids plus neutrals 

NOM fraction, and (5) a hydrophilic base NOM fraction. 

Leenheer and others (2000) used this procedure to characterize the DOM in several drinking-water supplies. They found 

that the colloidal fractions isolated from these waters consisted 

mainly of N-acetyl amino sugars; the hydrophobic fractions -acetyl amino sugars; the hydrophobic fractions 

were similar to the fulvic acids that are commonly isolated 

from natural waters by resin adsorption. They further found 
References 35

weight organic colloids and NOM adsorbed to colloidal 

mineral particles. There is evidence that DOM molecules also 

form supramolecular aggregates in natural waters.

 References 

 Abraham, M.C., Duce, P.P., Prior, D.V., Barratt, D.G., Morris, 

J.J., and Taylor, P.J., 1989, Hydrogen bonding. Part 9. Solute proton donor and acceptor scales for use in drug design: 

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 Albersheim, P., Darvill, A.G., O’Neill, M.A., Schols, H.A., 

and Voragen, A.G.J., 1996, An Hypothesis: The same six 

polysaccharides are components of the primary cell walls 

of all higher plants, in Visser, J. and Voragens, A.G.J., eds., 

Progress in biotechnology 14—Pectins and pectinases: 

Amsterdam, Elsevier, p. 47–55.

 Anderson, Traute-Heidi, and Domsch, K.H., 1993, The metabolic quotient for CO2

 (qCO2) as a specific activity parameter to assess the effects of environmental conditions, such 

as pH, on the microbial biomass of forest soils: Soil Biology 

and Biochemistry, v. 25, p. 393–395.

 Angers, D.A., N’dayegamiye, A., and Côté, D., 1993, Tillage-induced differences in organic matter of particle-size 

fractions and microbial biomass: Soil Science Society of 

America Journal, v. 57, p. 512–516.

 Atalla, R.H., 1999, Celluloses, in Pinto, B.M., ed., Comprehensive natural products chemistry, vol. 3, Carbohydrates 

and their derivatives including tannins, cellulose, and related 

lignins: Amsterdam, Elsevier, p. 530–598.

 Atalla, R.H. and VanderHart, D.L., 1999, The role of solid 

state 13C NMR spectroscopy in studies of the nature of 

native celluloses: Solid State Nuclear Magnetic Resonance, 

v. 15, p.1–19.

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A.M., and Wilson, M.A., 1992, Aspects of the chemical 

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13C NMR spectroscopy: Biogeochemistry, v. 16, p. 1–42.

 Baldock, J.A., Oades, J.M., Vassallo, A.M., and Wilson, M.A., 

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 Summary

 Agricultural scientists have recognized the importance of 

natural organic matter (NOM) in enhancing soil fertility, soil 

structure, and water-holding capacity for more than 200 years. 

More recently, its importance as a carbon sink in the global 

carbon cycle has been recognized. NOM is generally divided 

into two different pools: (1) soil organic matter (SOM) and 

dissolved organic matter (DOM) with the SOM pool being 

the larger of the two. Early workers understood that SOM is 

formed mainly from the degradation products of dead plant 

tissue with a much lesser contribution from decaying animal 

remains. Most of these early workers assumed that SOM was 

composed of the end products of synthetic reactions that alter 

the structures of plant degradation products. Other workers, 

however, maintained that SOM is a complex mixture of plant 

degradation products. This controversy has persisted to the 

present day, and a similar controversy exists with regard to 

DOM.

 This controversy has led to the development of two different types of conceptual models for NOM: (1) the humic 

polymer model and (2) the molecular aggregate model. The 

belief that the component molecules of NOM are produced by 

secondary synthesis reactions from degradation products has 

led to models in which the NOM molecules are depicted as 

large (humic) polymers with unique chemical structures that 

are different from those of starting materials. The proposal that 

NOM is composed of the partial degradation products of plant 

polymers has resulted in the development of models in which 

NOM is composed of molecular aggregates (supramolecular 

aggregates) of the degradation products held together by noncovalent bonds.

 The preponderance of evidence favors the molecularaggregate models. The molecular-aggregate models were 

developed by studying the properties of NOM extracted from 

soils and natural waters, and, as such, they provide only a very 

generalized picture of the structure of NOM aggregates in 

soils, sediments, and natural waters prior to extraction. 

A compartmental model would be more representative of 

NOM in soils and sediments. In this model NOM is divided 

into the following compartments: (1) partially degraded plant 

tissue, (2) biomass from microorganisms, (3) organic coatings 

on mineral grains, (4) pyrolytic carbon, (5) organic precipitates, and (6) DOM in water. Within each of these compartments there are NOM supramolecular aggregates that will be 

dissolved by the solvent systems that are used for extraction of 

NOM from soils and sediments. In order to better understand 

the interactions of NOM in soils and sediments, the NOM in 

each of these components must be studied separately. This 

is especially important because the NOM in each of these 

compartments can be expected to have different sorption, ion 

exchange, and buffering properties.

 DOM as normally isolated actually consists of two compartments: (1) truly dissolved DOM and (2) colloidal DOM. 

The colloid DOM compartment consists of high molecular 
36 Conceptual Models of Natural Organic Matter, Processes of Humification

 Cai, Ya; Gafney, S.H., Lilley, T.H., Magnolato, Daniele; 

Martin, Russell; Spencer, C.M., and Haslam, Edwin, 1990, 

Polyphenol interactions. Part 4. Model studies with caffeine 

and cyclodextrins: Journal of the Chemical Society, Perkin 

Transactions 2, 1990, p. 2197–2208.

 Camarero, Susana; Galletti, G.C., and Martínez, A.T., 1994, 

Preferential degradation of phenolic lignin units by two 

white rot fungi: Applied and Environmental Microbiology, 

v. 60, p. 4509–4516.

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