Humic Matter in Soil and the Environment: Principles and Controversies - Chapter 4 pot

CHAPTER 4 GENESIS OF HUlMC MATTER 4.1 MAJOR PATHWAYS OF HUMIFICATION The process by which humic matter is formed has been called humification, which involves a number of biochemical reactions. It is closely connected to the organic and nitrogen cycles in the environ- ment. Though some people are of the opinion that the mechanisms for synthesis are not clear, a number of hypotheses have in fact been presented on how hurnic matter is formed. In general, these theories differ in the way the sources of original or raw materials are utilized in the synthesis of humic substances. Whereas one group of theories is based on depolymerization of biopolymers causing their direct transformation into humic substances, the other group envisages polymerization of small molecules, liberated by complete decomposition of the biopolymers, in the formation of humic matter. All agree that the materials for formation originate mostly from plant material, though in practice animal residue can also be transformed into humic matter. The depolymerization theory, called biopolymer degradation by Hedges (1988), assumes that the biopolymers in plants are gradually transformed into humin, which eventually will be degraded successively into humic acids and fulvic acids. The lignin theory of Waksman (1932) and its modern version are considered examples of MARCEL DEKKER, INC. 270 Madison Avenue, New York, New York 1001 6 TM Copyright n 2003 by Marcel Dekker, Inc. All Rights Reserved. 76 Chapter 4 the biopolymer degradation theory. In contrast, the polymerization theory claims that the plant biopolymers are decomposed first into their monomers or smaller organic components. Humic substances are then formed by interaction reactions between these small components. This theory assumes fulvic acid to be formed first, which by polymerization or condensation can be transformed into humic acids. The polyphenol or phenol, quinone, and sugar-amine condensation theories belong to the category of the polymerization theory. This second pathway of humification has recently also been called the abiotic condensation process (Hayes and Malcolm, 2001). The ligno- protein theory of Flaig et al. (1975; 19881, focusing on the breakdown of lignin and further oxidation of the degradation units into quinone derivatives, is an excellent example of the polymerization or abiotic condensation theory. Hayes and Malcolm (2001) believe that the rate of depolymerization depends on the oxygen content, and humification will be retarded in anaerobic conditions. It is true that a lot of oxygen is required for oxidation reactions, but the issue can be raised whether a lack of oxygen will severely inhibit the humification process. As discussed in Chapter 2, huge deposits of peat and bogs, rich in humic matter, are instead formed in wetlands, where anaerobic conditions prevail. Another important question is whether biopolymer degradation is really a humification process. Is humification a decomposition or a polymerization process? The present author would like to refrain from assessing judgment now and let the readers draw their own conclusion after reading the sections below on humic precursors and several theories on humification processes. 4.2 PRECURSORS OF HUMIC MATTER The plant biopolymers of importance in humic matter synthesis are for convenience called precursors of humic substances. The major components of higher plants, important as sources for formation of humic matter, are lignin, cellulose and hemicellulose, called poly- MARCEL DEKKER, INC. 270 Madison Avenue, New York, New York 1001 6 TM Copyright n 2003 by Marcel Dekker, Inc. All Rights Reserved. Genesis of Humic Matter 77 saccharides, and proteins. Phenols and amino sugars synthesized by microorganisms have recently been added as important raw materials for the synthesis of humic substances. Since degradation of lignin can also produce phenols, two sources of phenolic compounds can be distinguished in soils. All these compounds, present originally in the form of large molecules in the plant tissue and soils, will be discussed in more detail below in order to give a better picture of their characteristics and reactions related to the formation of humic substances. Moreover, many people are often confused about what the biopolymers are, what aromatics are and what the difference is between phenol and quinone. Even some hard-core scientists wonder about terms such as phenolic-OH and the like. It sounds like basic organic biochemistry, but it is not, though some of the basic definitions are needed to explain the chemical behavior of the compounds, which is necessary in understanding their interaction reactions in humic matter formation. 4.2.1 Lignin Lignin is a system of thermoplastic, highly aromatic polymers of the phenylpropane group. The name is derived from the Latin term lignum = wood. It is one of the three major components of wood, with the other two being cellulose and hemicellulose (Schubert, 1965). The bulk of lignin occurs in the secondary cell walls where it is associated with cellulose and hemicellulose. It is noted to coexist with the cellulosic plant components in such an intimate association that its isolation requires drastic chemical treatments that often alter the structure of the lignin itself. The latter raises questions about the assumption held by most biochemists that the libin is associated physically, rather than chemically, with the polysaccharides. The nature of the lignin-polysaccharide complex has still to be resolved and more definite data need to be presented refuting one or the other or supporting the presence of both physical and chemical interactions. The quantity of lignin increases with plant age and stem content. It is not only an important constituent of the woody tissue, but it contains the major portion of the methoxyl content of the wood. A MARCEL DEKKER, INC. 270 Madison Avenue, New York, New York 1001 6 TM Copyright n 2003 by Marcel Dekker, Inc. All Rights Reserved. 78 Chapter 4 large amount of lignin is also detected in the vascular bundles of plant tissue. The purpose is perhaps to strengthen and make the xylem vessels more water resistant. By virtue of the presence of larger amounts of vascular bundles, the lignin content of tropical grasses is considerably larger than that of temperate region grasses (Tan, 2000; Minson and Wilson, 1980). Consequently, soils under tropical grasses are expected to have higher lignin contents than soils under temperate region grasses. These differences may produce differences in the nature of humic substances formed. Lipnin Monomers The building stones of lignin are monomeric lignin possessing a basic phenylpropane carbon structure. Three types of lignin monomers can be distinguished on the basis of the type of wood or plant species, e.g., coniferyl, sinapyl, and p-coumaryl monomers (Figure 4.1). The coniferyl type characterizes lignin in softwood or co- niferous plants, and the sinapyl type represents lignin in hardwood, whereas the coumaryl type is typical of lignin in grasses and bamboos. Several of these monomers are linked together to form the total lignin polymer. The process, called polymerization, forms a very complex and long series of a lignin polymer structure (see Tan, 2000). Aromatization The ultimate source for formation of lignin is carbohydrates or intermediate products of photosynthesis related to carbohydrates. The process of conversion of the nonaromatic carbohydrates into substances containing phenolic groups characteristic of lignin is called aromatization. Enzymatic reactions are required to effect such a dras- tic transformation of nonaromatic carbohydrates into aromatic precur- sors of lignin. Several theories have been advanced on the aromati- zation process, e.g., aromatization of carbohydrates through a dehydra- tion process and the shikimic acid pathway. MARCEL DEKKER, INC. 270 Madison Avenue, New York, New York 1001 6 TM Copyright n 2003 by Marcel Dekker, Inc. All Rights Reserved. Genesis of Humic Matter SOFTWOOD Gymnosperm H Cloniferyl alcohol HARDWOOD Dicot. angiosperm Sinapyl alcohol GRASS-BAMBOO Monocotyledons p-Cournaryl alcohol Figure 4.1 Lignin monomers from softwood, hardwood, and grass or bamboo. In dehydration theory, carbohydrates, such as fructose, are releasing three water molecules, and with the assistance of enzymatic reactions, three possible aromatic end products are produced, e.g., pyrogallol, hydroxyhydroquinone, phloroglucinol, or a combination thereof (Figure 4.2). The shikimic acid pathway has been adopted from the theory for the biosynthesis of aromatic amino acids from carbohydrate precursors with the help of enzymes originating from Escherichia coli bacteria (Schubert, 1965). The end products, phenylpyruvic acid and p- hydroxyphenylperuvic acid, yield by transamination reactions phenyl- MARCEL DEKKER, INC. 270 Madison Avenue, New York, New York 1001 6 TM Copyright n 2003 by Marcel Dekker, Inc. All Rights Reserved. Chapter 4 0 H p yrogollol FRUC JOSE I I hydroxy hydroquinone p h loroglucinol Figure 4.2 Aromatization of fructose through a dehydration process. alanine and tyrosine, respectively. As illustrated in Figure 4.3, the chemical structures of these compounds show close similarities to those of the monomeric units of lignin. In particular, the structure of p- hydroxyperuvic acid is almost the same as that of p-coumaryl lignin, leading to the assumption that lignin monomers may have been formed through similar processes. In addition, the structures of phenylalanine and tyrosine are also very similar to those of ligno-protein compounds, the humic substances according to the ligno-protein theory. These find- MARCEL DEKKER, INC. 270 Madison Avenue, New York, New York 1001 6 TM Copyright n 2003 by Marcel Dekker, Inc. All Rights Reserved. Genesis of Humic Matter Carbohydrate COOH I HOOC 6' shikimic acid prephenic acid hy droxy pheny l p yruvic acid t yrosine COOH I FOOH \ transarnina tion f P=O Fl4~I-l~ COOH COOH p hen yl phen yl pyruvic acid alanine Figure 4.3 Bioformation of compounds in the shihmic acid pathway with molecular structures similar to lignin monomers. ings have an important bearing on the processes in the synthesis of humic substances, which will be discussed in more detail in one of the following sections. The similarities apparently support the hypothesis that plant biopolymers can be transformed into humic substances without drastic structural changes. MARCEL DEKKER, INC. 270 Madison Avenue, New York, New York 1001 6 TM Copyright n 2003 by Marcel Dekker, Inc. All Rights Reserved. 82 Chapter 4 Lignification In the growth of woody plants, carbohydrates are synthesized first. The formation of lignin then begins, and the spaces between the cellulose fibers are gradually filled with lignified carbohydrates. This process is called lignification and serves several functions: 1. It cements and anchors the fibers together 2. It increases the resistance of the fibers to physical and chemical breakdown 3. It increases rigidity and strength of cell walls. In the process, the lignin monomers are bonded together, by a process called polymerization, to form a complex chain of large lignin molecules (Figure 4.4). It is believed that after lignification, the lignified tissue then no longer plays an active role in the life of plants, but serves only as a supporting structure. Nonlignified plant parts contain more moisture, are soft and break more easily. Decoml~osition of Lignin Lignin is insoluble in water, in most organic solvents, and in strong sulfuric acid. It has a characteristic W absorption spectrum and gives characteristic color reactions upon staining with phenols and aromatic amines. It hydrolyzes into simple products as do the complex carbohydrates and protein. When oxidized with alkaline benzene, it produces up to 25% vanillin. Lignin is considered an important source for the formation of humus, and especially humic matter. The high resistance of lignin to microbial decomposition is perhaps the reason why it accumulates in soils. It is believed that, depending upon the condition, this could result in the formation of peat, which in time can be converted into kerogen, coal and ultimately oil (fossil fuel) deposits. Nevertheless, lignin can be attacked by very specific microorganisms in the group of Basidiomy- MARCEL DEKKER, INC. 270 Madison Avenue, New York, New York 1001 6 TM Copyright n 2003 by Marcel Dekker, Inc. All Rights Reserved. Genesis of Humic Matter Figure 4.4 A hypothesis for a softwood lignin structure by a systematic linkage of coniferyl alcohol monomers. cetes (Schubert, 1965; Paul and Clark, 1989). Several forms of these so- called lignolitic fungi have been reported as the major organisms responsible for the partial decomposition of lignin, e.g., white-rot, brown-rot, and soft-rot fungi. In well-aerated soils, the white-rot fungi are reported to decompose wood containing lignin into CO, and H,O. Patches of a white substance are often formed in the residue, hence the name white-rot. These white patches have been identified as pure forms of cellulose. According to Paul and Clark (1989), the brown-rot fungi are useful for the removal of the methoxyl, -OCH,, group from lignin, leaving the hydroxyphenols behind, which upon oxidation in the air produce brown colors. However, Schubert (1965) believes that the cellulose and other associated carbohydrates are attacked MARCEL DEKKER, INC. 270 Madison Avenue, New York, New York 1001 6 TM Copyright n 2003 by Marcel Dekker, Inc. All Rights Reserved. 84 Chapter 4 preferentially, leaving the lignin behind, which turns the residue brown in color. The soft-rot fungi are most active in wet soils and are specifically adapted to decomposing hardwood lignin. The hydroxyphenol units resulting from demethylation of lignin by white-rot fungi can be oxidized to form quinones. The latter are believed to be capable of reacting with amino acids to form humic substances (Flaig et ai., 1975). Lignin itself has the capacity to react with NH,. The process, called ammonia fixation, has been applied in industry for the production of nitrogen fertilizers by treatment of lignin and other materials rich in lignin, e.g., sawdust, and peat, with NH, gas. The exact mechanism of fixation is still not known, but it is believed that the NH, reacts with the phenolic functional groups in lignin. 4.2.2 Phenols and Polyphenols Phenols are aromatic carbon compounds with a general formula of C6H,0H. They are derived from benzene, C,H,, by replacing one or more of the hydrogens with OH. Benzene, a flammable colorless compound, is called aromatic because of its characteristic structure marked by six carbon atoms linked by alternate single and double bonds in a symmetrical hexagonal configuration. The C6H, group in phenol is called the phenyl group, from the Latin termphene = shining, because burning benzene produces a very bright light. By linking several monomeric phenols together polyphenols are produced. As indicated earlier, the phenols and polyphenols can be derived from two sources, from the decomposition of lignin and from the synthesis by microorganisms. Stevenson (1994) believes that uncombined phenols are present in higher plants in the form of glucosides and tannins. Limin Derived Phenols and Polvphenols Biodegradation of lignin has been implicated in producing polyphenols and phenols. Specific types of fungi have been discovered MARCEL DEKKER, INC. 270 Madison Avenue, New York, New York 1001 6 TM Copyright n 2003 by Marcel Dekker, Inc. All Rights Reserved. [...]... by the linkage of amino acid molecules through the carboxyl and amino groups: Ha-C-C-OH I Glycine + H-N-C-C-OH I Glycine + H,N-C-C-N-C-C-OH I I + H,O Dipeptide The bond linking the two groups is called the peptide bond, and the compound formed is called a peptide, or protein Under refluxing with 6 N HCl for 1 8-2 4 hours, the protein may be hydrolyzed into its constituent amino acids Twenty-one amino... of Humic Matter 103 purine bases adenine and guanine, and by the pyrimidine bases thymine and cytosine For the basics of nucleic acids in soils, see Tan (1998) Upon decomposition of plants, these nucleic acids will be released into the soil Though, they are one of the organic compounds from living cells that are apparently broken down rapidly in soils, they are also potentially capable of being incorporated... C=O I H-C-OH I OH-C- H I H-C-OH - H-C- OH OH-C-H 0 I I I H-C-OH I i H- H-C- OH o ," I .dOCH \H !H20H Glucose Open chain A HCOt HO/ C OH C H Cyclic structure Ring FH20H HOCH NH I Glucosomine (Chitin monomer) glucosa mine FHflH Chitosan ( glucosamine polymer) 9 Molecular structures of glucose, glucosamine, chitin, and chitosan (some of the H and OH are not drawn due to space limitations) Figure 4. 8 TM... orcinol and benzoic acid, respectively,yielding quinone (After Schubert, 1965; Flaig et al., 1975; Stevenson, 19 94. ) 4. 2 .4 Protein and Amino Acids In the early days, protein and amino acids were not considered compounds making up humic matter Many scientists believed humic acid to be a plain hydrocarbon substance and information has been presented off and on providing the argument for humic acid-like... hormonal effect of humic matter The molecular structure of auxin (Figure 4. 10) shows the presence of carboxyl and hydroxyl groups for easy hook-up to phenols, quinones, lignin, and other humic acid precursors by chelation or other interaction processes Vitamins .- These compounds have hitherto been considered only of importance in animal and human nutrition The term vitamine,from Auxin- b Figure 4. 10 Molecular... Humic Matter 93 The main reaction process for the decomposition of protein and amino acids is hydrolysis Hydrolysis of protein, brought about byithe enzymes proteinase and peptidase of soil microorganisms, results in cleavage of the peptide bonds, releasing in this way the amino acid constituents The latter substances are broken down further into NH, by the enzymes called amino acid dehydrogenase and. .. Humic Matter 109 Xenobiotics The name xenobiotic refers to foreign organic substances, here meaning foreign to the soil They are introduced into soils by the modern and extensive agricultural and industrial operations of today The major xenobiotics expected to participate in the synthesis of humic matter include pesticides and their degradation products, e.g., the triazines, substituted ureas, and phenylcarbamates... experiments in growing plant cells, conducted in 1910 and the following years by Boysen-Jensen (1936) Since then a large number of investigations have been carried out on the subject of growth promoting substances in plants, which finally resulted in the term hormone being adopted for compounds showing the capacity to stimulate the growth of plants The name auxin from the Greek term to increase was... NaNO,, asparagine, and peptone (Filip et al., 19 74; 1976;Saiz-Jiminez et al., 1975) .The substances formed are identified by chemical analysis to be composed of phenols, orsellinic , p-hydroxybenzoic, p-hydroxycinnamic acids, anthraquinones and melanins Their appearance as dark-colored microbial products in the culture media is the reason for associating them with humic acids, since phenols and their derivatives... physiology and humic acid chemistry It is called the B complex, since it is composed of several types of vitamin Bs, each responsible for specific functions in the plant body Thiamin, riboflavin, nicotinic acid or niacin, vitamin B,, biotin and pantothenic acid are examples of vitamin B This B complex also includes aminobenzoic acid and inositol Since these vitamins are produced by plants, they are suspected . the carboxyl and amino groups: Ha-C-C-OH + H-N-C-C-OH + H,N-C-C-N-C-C-OH + H,O I I I I Glycine Glycine Dipeptide The bond linking the two groups is called the peptide bond, and. In addition, the structures of phenylalanine and tyrosine are also very similar to those of ligno-protein compounds, the humic substances according to the ligno-protein theory. These find-. Stevenson, 19 94. ) 4. 2 .4 Protein and Amino Acids In the early days, protein and amino acids were not considered compounds making up humic matter. Many scientists believed humic acid to be a plain hydrocarbon