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This chapter was originally published in the book Advances in Agronomy, Vol. 113, published by Elsevier, and the attached copy is provided by Elsevier for the author's benefit and for the benefit of the author's institution, for non-commercial research and educational use including without limitation use in instruction at your institution, sending it to specific colleagues who know you, and providing a copy to your institutions administrator.

All other uses, reproduction and distribution, including without limitation commercial reprints, selling or licensing copies or access, or posting on open internet sites, your personal or institutions website or repository, are prohibited. For exceptions, permission may be sought for such use through Elsevier's permissions site at: http://www.elsevier.com/locate/permissionusematerial From: Morris Schnitzer and Carlos M. Monreal, Quo vadis Soil Organic Matter Research?: A Biological Link to the Chemistry of Humification. In Donald L. Sparks, editor: Advances in Agronomy, Vol. 113, Burlington: Academic Press, 2011, pp. 139-213. ISBN: 978-0-12-386473-4 Copyright 2011 Elsevier Inc. Academic Press.

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C H A P T E R

T H R E E

Quo vadis Soil Organic Matter Research?: A Biological Link to the Chemistry of Humification
Morris Schnitzer and Carlos M. Monreal Contents
1. Introduction 2. Criticism on Soil HS Research 3. Extraction of Soil Organic Matter 3.1. Extraction with dilute base 3.2. Other extractants 4. Analysis of SOM 4.1. In situ analysis of OM in whole soils 4.2. Analysis of OM in whole soils by 13C NMR 5. Analysis by Pyrolysis-Field Ionization Mass Spectrometry 5.1. Analysis of OM in soil extracts and whole soil by Py-FIMS 5.2. Py-FIMS analysis of HA 5.3. Py-FIMS analysis of FA 5.4. Py-FIMS analysis of humin 5.5. Py-FIMS analysis of whole soil 5.6. Summary of compound classes identified 6. Chemical Structure 6.1. The chemical structure of SOM 7. Chemical Characteristics of Humic Substances 7.1. Analytical characteristics of HAs and FAs 7.2. Infrared and Fourier transform infrared spectrophotometry 7.3. Oxidative degradation of HAs, FAs, and humins 7.4. Reductive degradation 8. Spectrometric and Spectroscopic Characteristics of HS 8.1. 13C NMR spectrometry of humic substances 8.2. Effect hot acid hydrolysis on the 13C NMR spectrum of HA 8.3. Curie-point pyrolysis-gas chromatography-mass spectrometry of HAs 8.4. X-ray analysis of FA 141 142 144 144 145 145 145 146 147 147 148 149 149 150 150 151 151 152 152 152 153 156 156 156 158 160 161

Agriculture and Agri-Food Canada, Eastern Cereal and Oilseed Research Center, Ottawa, Canada Advances in Agronomy, Volume 113 ISSN 0065-2113, DOI: 10.1016/B978-0-12-386473-4.00008-7
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2011 Elsevier Inc. All rights reserved.

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8.5. A 2D structure for HA 8.6. A 3D structure for HA 8.7. Relationships between HA and SOM 8.8. The corrected 2D HA model 8.9. A 3D model structure for SOM plus water 9. Effect of Time on the SOM Structure 9.1. Effect of long-term cultivation on the SOM structure 10. New Concepts on the Chemical and Microbial Synthesis of HAs and SOM 11. Microbial Humification of Small Organic Compounds into Soil Polyketides 11.1. A complex biological link to the chemistry of humification 11.2. Polyketides in nature 11.3. Some chemical and functional features of polyketides 11.4. Chemical analysis of polyketides in soils 11.5. Biosynthesis of polyketides 11.6. Ecological function and associated genetic evolution of polyketides 11.7. Plant polyketides 11.8. Microbial polyketides 11.9. Polyketides in soils 11.10. A microbial-PKSs model for studying biotic humification in soils 12. Thermodynamic, Energy, and Kinetic Considerations 13. Polyketides and the Central Structure of HS and SOM 13.1. Polyketides as a passive SOM pool 13.2. Biotic humification process forming the CUS of HS and SOM 14. Future Research References

167 169 171 172 174 175 175 176 178 178 179 180 181 181 185 187 189 190 192 194 198 198 200 201 203

Abstract
Soil organic matter (SOM) is the substrate and habitat of soil microorganisms and fauna. These biotic pools, along with inorganic soil components, contribute to the degradation and synthesis of humic acids (HAs) and SOM through chemical and biochemical reactions. During the past 225 years, most researches on the molecular structure and reaction of HA and SOM were done by soil chemists, with limited contribution from soil microbiologists. This may be one reason why progress in our knowledge of the molecular structure of SOM and HA has been slow. Closer cooperation between chemists and microbiologists would certainly have been beneficial. In this chapter, we summarize current knowledge of 2D and 3D molecular structures and propose new chemical reactions to synthesize HAs and SOM. We indicate how the application of recent advances in microbial biochemistry could assist soil chemists in their work on elucidating the molecular structures of HAs and SOM. We show that the continuous production of complex polyketides (PKs) from small soil oxoacids by soil microorganisms and

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associated polyketide synthases catalyzes the second biotic stage of humification in soils. The PKs involve complex alkylaromatic, aromatic, polyaromatic, phenolic, and polyphenolic structures. Due to their bioactivity, rapid adsorption to clay colloids and high energy content in their chemical bonds, biologically and chemically formed PK structures represent kinetically passive soil carbon pools, and so lend themselves as carbon skeletons that contribute to the formation of a stable central unit structure in humic substances and SOM.

1. Introduction
Soil organic matter (SOM) is a complex dynamic system whose chemical, microbial, and biochemical components change over time and space, depending on several abiotic and biotic factors, organic residue inputs, and on their degree of association with inorganic components. Functional outcomes of soil biotic and abiotic factor interactions may be diverse and represented simply as a response to altered nutrient availability for plant uptake, or as complex as the chemical communication between individual soil microbial cells and plants. These outcomes may also include the sequestration of atmospheric C in SOM as a result of anabolic reactions during microbial cell growth, or the microbial humification of simple soil organic molecules into alkylaromatic, polyaromatic, or polyphenolic structures and subsequent secretion into soil solutions to antagonize and compete with other species. Soil animals and microorganisms, intracellular and extracellular enzymes and inorganic surfaces are the catalysts that continuously process, modify, and bind residues into small molecules and metabolites of plants and microorganisms into humic substances (HSs) (Huang and Hardie, 2009). Over the years, there have been many definitions of HS. For example, Aiken et al. (1985) defined HS as a category of naturally occurring, biogenic, heterogeneous organic substances that can be generally characterized as being yellow-to-black in color, of high molecular weight, and refractory. An alternate definition indicates that HS are a category of naturally occurring materials found in or extracted from soils, sediments, and natural waters. They result from the decomposition of plant and animal residues (MacCarthy, 2001). In this chapter, we define HS as a portion of the total SOM that is extracted and solubilized with dilute alkali (0.10.5 M NaOH or KOH). The alkaline extract is usually partitioned into three fractions, which are HA, fulvic acid (FA), and humin. Detailed definitions of the three fractions are found in Section 3.1. Several biotic and abiotic factors and processes are involved in the formation of humus from soil organic residues. Briefly, humification of soil organic residues proceeds in two stages: the first stage involves physical fragmentation, microbial and enzymatic depolymerization, and decomposition of plant and microbial fragments and polymers into simpler structural

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or molecular units by microbes and enzymes (Kobayashi et al., 2001; Rehm, 2010). The second humification stage (i.e., synthesis) involves transformation of simple molecular units into large size molecules through processes catalyzed by biotic agents and mineral surfaces (Huang and Hardie, 2009). Conceptual precursors and ways of HS formation, including the modified lignin pathway, have been presented and discussed by several workers over many decades (Flaig, 1988; Huang and Hardie, 2009; Kononova, 1966; Stevenson, 1994; Waksman, 1936; Wershaw, 2004). Conversely, Piccolo (2001) proposed that HS are supramolecular associations of self-assembling heterogeneous and relatively small molecules, derived from the degradation and decomposition of dead biological material. In this supramolecular association model, weak dispersive forces instead of covalent linkages would stabilize the HS structure. The supramolecular model is proposed as a better representation of the actual state of SOM in soil extracts than the humic polymer models (Wershaw, 2004). Lately, Kleber and Johnson (2010) made an attempt to discredit the scientific concept of organic matter (OM) humification in soils, mostly through opinions and partial interpretations of scientific information on HS published during the past 20 decades. Over the past two decades, research on soil HS and SOM has focused to a large extent on scientific studies dealing with their chemical structure and the effects of inorganic colloids on the polymerization of single organic molecules (Liu and Huang, 2002; Schnitzer and Schulten, 1992, 1998). In spite of all scientific efforts conducted thus far, the basic chemical nature, biosynthetic pathways, and reactivity of HS and SOM are still poorly understood. Further, little information exists on the role played by soil microorganisms and associated enzymatic components and processes in the synthesis of humus and decomposition of humified soil materials. It appears in part that the limited existing knowledge on the biology (i.e., microorganisms, enzyme complexes, and gene expression) involved in humification processes of SOM has led some scientists to discredit the existing and past definitions and theoretical pathways of SOM humification (i.e., Kleber and Johnson, 2010). The aim of this review chapter is to demonstrate that: (a) the published information on the chemistry of HS is based on the use of rigorous scientific methods that helps elucidate 2D and 3D molecular structure of HS and SOM and (b) that biosynthetic processes controlling HS formation occur in soil ecosystems.

2. Criticism on Soil HS Research


In a recent review on SOM, Kleber and Johnson (2010) make two points on which we want to comment: (1) The authors severely criticize the extraction from soils of HS by dilute alkali, mainly 0.1 or 0.5 M NaOH solutions. They quote Waksman (1936) implying that HS obtained by

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alkaline extraction are chemically and physically different from organic materials actually occurring in soils (p. 92, lines 3537). On p. 93, they interpret Baldock and Nelson (2000), as well as Waksman(1936), as having postulated that the materials represented by alkaline extraction and commonly defined as HAs do not exist in nature. Over the years, a number of objections have been raised against the use of alkaline solutions. Stevenson (1994) lists the following: (1) silica is dissolved from the minerals; (2) protoplasmic and structural components of fresh organic tissues are dissolved and mixed with humic extracts; (3) auto-oxidation of some organic components may occur when extracts are allowed to stand in contact with air for extended periods of time; and (4) other chemical changes can occur in the alkaline solutions, including condensation between amino acids and C O groups of reducing sugars and quinones. Criticisms (1) and (2) are of minor importance with respect to the chemical structure of HS. Possible changes (3) and (4) can be minimized by doing the extractions under N2. On p. 93, 4th paragraph, Kleber and Johnson (2010) state that structural observations on alkaline extracts are not representative of SOM because they are chemically altered organic moieties and no longer in-situ components of SOM. On p. 120, line 33, they write: the products of the alkaline extraction procedures (HS) are different from natural organic matter and should not be used as proxies for organic matter dynamics. In spite of the objections raised by Kleber and Johnson (2010) and others against the use of alkaline solutions for the extraction of SOM from soils, dilute aqueous alkalis especially 0.10.5 M NaOH solutions, have been, by a wide margin, the most efficient extractants since Archard in 1786 until today. Over 95% of all SOM researchers have used and are still using dilute NaOH solutions for this purpose, and their work has produced several thousand scientific peer reviewed papers. Are we now going to throw rigorous scientific published articles into the waste paper basket and disregard this huge scientific literature? We hope not, because such action will leave us without any scientific facts and history, and may result in a new and false start for SOM research. One objective of our write-up is to show that there is no need for such drastic and unwarranted action. The second point which concerns us about the review by Kleber and Johnson (2010) is the following: these authors have understanding the molecular structure of SOM in the title of their review article but fail to show any molecular structure in the text although the literature contains a number of partial, 2D and 3D structures of HA and SOM (Burdon, 2001; Schnitzer, 2000; Schnitzer and Schulten, 1995; Stevenson, 1994). A chemical or molecular structure tells us more about the elemental composition of a compound, its molecular weight, functional groups, its aliphaticity, aromaticity, and its reactivity and possible origin and degradation and ecological functions than numerous pages of speculations and wishful thinking. Why are SOM specialists so reluctant to show and discuss chemical structures of HA and SOM? We plan to

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demonstrate in this write-up that chemical structures are important for a better understanding of HA and SOM chemistry. One of the major objectives of this chapter, however, is to summarize and interpret data obtained by analytical chemical methods, infrared (IR) spectrophotometry, chemical degradation, 13C NMR, chemical hydrolysis, X-ray analysis, and pyrolysis-soft-ionization mass spectrometry. With the aid of computational chemistry, the results of the analyses described above were converted to 2D and 3D HA and SOM structures, which are shown in this chapter. A second major objective is to demonstrate that HA, FA, and SOM have similar chemical compositions, therefore showing that the criticisms of chemical artifacts during SOM extractions are unjustified. During the past 200 and more years, most of the researches on soil HSs (HA, FA, humin) have been done by soil chemists. In spite of the fact that in soils, HS are substrates for microbial activity, soil microbiologists have contributed relatively little to structural studies on HSs. We believe that this relationship needs to change so that both soil chemists and soil microbiologists work side by side to resolve structural and other problems associated with SOM. It is true that it is often difficult to distinguish between chemical and microbial reactions. For example, the decarboxylation of fatty acids to alkanes may be brought about by chemical or by microbial reaction, and this applies to many other reactions. The input of soil microbiologists will certainly facilitate the work of soil chemists in structural studies on HSs.

3. Extraction of Soil Organic Matter


3.1. Extraction with dilute base
The SOM content of agricultural soils usually ranges between 1% and 5% (w/w). In the soil, OM and inorganic soil constituents are closely associated so that it is necessary to separate the two before either can be investigated in greater detail. Archard (1786) was the first scientist to use dilute base, either 0.10.5 M NaOH or KOH, for this purpose. Since that time, dilute NaOH has been found to be the most efficient extractant for OM from soils from all over the earths surface. The alkaline extract is usually partitioned into three fractions which are HA, FA, and humin. The definitions of the three humic fractions accepted by most soil scientists are: HA is that fraction of the alkaline extract which coagulates when the extract is acidified to low pH (< 2.0); FA is the OM fraction that remains in solution when the extract is acidified, that is, it is soluble in both base and acid; while humin is that fraction that remains with the soil, that is, it is insoluble in both base and acid. As mentioned in Section 1, a number of objections have been raised

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against dilute alkali as an extractant, but if the extractions are done under N2, some of these objections can be minimized.

3.2. Other extractants


Neutral salts of mineral and organic acids have been used for the extraction of SOM, but yields are usually low. Bremner and Lees (1949) proposed the use of 0.1 M Na4P2O7 (sodium pyrophosphate) solution at pH 7. The action of the neutral salt is thought to arise from the ability of the anion to interact with polyvalent cations bound to SOM to form a soluble salt of OM as illustrated by the following reaction: RCOO4 Ca2 Na4 P2 O7 ! RCOONa4 Ca2 P2 O7 1

where, R is the OM without COOH groups. Alexandrova (1960) found that Na4P2O7 solution, extracted not only HSs but also organo-mineral complexes without breaking up nonsilicate forms of sesquioxides. Kononova (1966) reports that the efficiency of extractions with Na4P2O7 solution can be improved by raising the pH from 7 to 9 and increasing the temperature. Schnitzer et al. (1958) demonstrated that pyrophosphate was difficult to remove from the humic material during the subsequent purification. Other approaches have been employed, without too much success, for the extraction of OM from soils. These were treatments with chelating resins (Levesque and Schnitzer, 1967; Ortiz de Sera and Schnitzer, 1972). Attempts have been made to extract SOM with organic solvents, Hays (1985) compared the extraction efficiency of 13 reagents which included dipolar aprotic solvents pyridine, ethylenediamine, organic chelating agents, ion exchange resins, Na4P2O7, and dilute NaOH solutions. The latter was found to be the most efficient and reproducible extractant. The danger of using organic solvents containing C and N for extracting SOM is that under these conditions C and N may be added irreversibly to the SOM, and so alter its composition and properties.

4. Analysis of SOM
4.1. In situ analysis of OM in whole soils
In recent years, we have witnessed the rapid development of two analytical methods based on state-of-the-art technologies, which appear to be suitable for the in situ analysis of OM in whole soils. These are solid-state 13 C NMR spectrometry and pyrolysis-field ionization mass spectrometry

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(Py-FIMS). It was hoped that the use of these methods would tell us whether extraction of soils with dilute alkali would damage or modify the extracted OM as claimed by Kleber and Johnson (2010).

4.2. Analysis of OM in whole soils by 13C NMR


One of the first solid-state 13C NMR analyses of whole soils has been described by Wilson (1987). This type of analysis requires that the soil contains at least 3.0% C and that the concentration of paramagnetic ions, for example, Fe3 in the soil be relatively low as not to interfere with the recording of acceptable 13C NMR spectra. According to Arshad et al. (1988), the C/Fe3 (w/w) ratio is an important indicator for obtaining satisfactory solid-state 13C NMR spectra of whole soils and particle-size fractions separated from them. If the C/Fe3 (w/w) is ) 1, the quality of the spectrum will be good; if the ratio is > 1, a reasonable spectrum will be obtained, but if the ratio is < 1, the quality of the spectrum will be poor. The quality of the spectrum can be improved by reducing the Fe3 to Fe2 by dithionate and then removing it. Another option is to separate the soil into particle-size fractions and record 13C NMR spectra for each fraction. Another approach is to separate particle-size fractions by flotation or removal of paramagnet metal ions (Arshad et al., 1988). Figure 1 shows solid-state 13C NMR spectra of: (a) the whole Ap horizon of the Culp soil, a loamy sand from northwestern Alberta, classified as a Gray Luvisol; (b) the silt and clay fraction separated from the Ap horizon of the Culp soil; (c) the nonmagnetic silt and clay fraction separated from the Culp soil, and (d) the magnetic silt and clay fraction separated from the Culp soil. The 13C NMR spectrum of the whole Culp soil Fig. 1A shows relatively strong signals due to CH3 and (CH2)n between 0 and 40 ppm, small resonances due to C in methoxyls and in amino acids (4160 ppm), stronger signals arising from C in carbohydrates and in aliphatic structures bearing OH groups (61104 ppm), signals between 105 and 150 ppm (aromatic C), 151170 ppm (phenolic C), and a more prominent resonance between 171 and 190 ppm (C in CO2H groups) (Schnitzer and Preston, 1986). The 13C NMR spectrum of the silt and clay fraction separated from the Culp soil (Fig. 1B) is characterized by rounded broad signals although the different components produce resonances with similar chemical shifts as Fig. 1A. The spectrum of the demagnetized silt clay fraction (Fig. 1C) exhibits welldefined chemical shifts in the aliphatic, aromatic, and carboxylic regions. By contrast, the 13C NMR spectrum of the magnetic portion of the silt clay fraction (Fig. 1D) is very poorly resolved and illustrates the adverse effect of paramagnetic Fe3 on the development of the spectrum. Although at this time the use of solid-state 13C NMR for the in situ analysis of SOM is somewhat limited by the restrictions outlined above, we believe that with future improvements in 13C NMR technology, these difficulties will be overcome.

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73

30 A 173 153 129 73 32 173 127 102 135 165 73 B 129 84 110 61 30 173 24

158 C

200

100 d (ppm)

Figure 1 Solid-state 13C NMR spectra of: (A) whole Culp soil; (B) silt and clay fraction separated from the Culp soil; (C) nonmagnetic silt and clay fraction separated from the Culp soil; and (D) magnetic silt and clay fraction separated from the Culp soil. From Arshad et al. (1988), Fig. 1, p. 593. With permission of the publisher.

5. Analysis by Pyrolysis-Field Ionization Mass Spectrometry


5.1. Analysis of OM in soil extracts and whole soil by Py-FIMS
While 13C NMR spectrometry provides information on the types of C present in SOM, Py-FIMS tells us what individual chemical compounds are volatilized under these experimental conditions, that is, it yields data at the molecular level. Py-FIMS is more sensitive than 13C NMR and is not

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subject to interference by paramagnetic ions, and can be used for the in situ analysis of OM in whole soils. Schnitzer and Schulten (1992) investigated whether there were any significant differences in Py-FIMS spectra of OM extracted with 0.5 M NaOH solution and separated by the classical method into HA, FA, and humin and that of the untreated whole soil. Soil samples selected for this very purpose were taken from the Ap horizon (015 cm) of the Bainsville soil, a Haplaquoll, on the Central Experimental Farm in Ottawa. This soil had a pH (in water) of 6.3, and contained 2.43% (w/w) C and 0.21% (w/w) N.

5.2. Py-FIMS analysis of HA


The Py-FIMS spectrum of the HA (Fig. 2A) shows that this material is rich in carbohydrates, phenols, monomeric and dimeric lignins, n-fatty acids, and N-compounds. Prominent n-fatty acids, range from n-C14 to n-C34, with n-C24 and n-C26 fatty acids dominating. The presence of alkylbenzenes is indicated by m/z 540, 554, 568, 582, 596, 610, and 638
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Figure 2 Pyrolysis-field ionization mass spectrum of: (A) the Bainsville HA; (B) the Bainsville FA. From Schnitzer and Schulten (1992), Fig. 3, p. 1815. With permission of the publisher.

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(C6H5C33H67 to C6H5C40H81, respectively). Weak signals due to n-alkyl diesters, also shown in the Py-FIMS spectra of the whole soil and the humin, are present. The intense molecular ions at m/z 59, 81, 83, 97, and 99 are N-compounds.

5.3. Py-FIMS analysis of FA


The Py-FIMS spectrum of the FA (Fig. 2B) is also dominated by carbohydrates, phenols, and lignins. The signals at m/z 262, 276, and 290 appear to be due to hexa-, hepta-, and octa-methyl-phenanthrene. The presence of a series of n-fatty acids is indicated by m/z 382 (C26) extending to m/z 564 (C38). The FA is relatively rich in N compounds as shown by strong signals at m/z 59, 67, 83, 97, and 109.

5.4. Py-FIMS analysis of humin


The Py-FIMS spectrum of the humin (Fig. 3A) shows that the major components are carbohydrates, phenols, monomeric lignins, and alkyl esters. Other significant compounds are m/z 330, 344, 358, and 372
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Figure 3 Pyrolysis-field ionization spectrum of: (A) the Bainsville Humin. (B) the whole Bainsville soil. From Schnitzer and Schulten (1992), Fig. 4, p. 1815. With permission of the publisher.

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which are C6H5C18H37 to C6H5C22H45 n-alkylbenzenes. Signals at m/z 386, 400, and 414 are cholesterol, campesterol, and B-sitosterol, respectively. Signals ranging from m/z 202 to 342 appear to be due to n-C10 to n-C20 alkyl diesters, while m/z 242424 is due to n-C15 to n-C28 fatty acids. The spectrum of the humin also shows the presence of methyl napthalenes and a series starting with m/z 252 and extending to m/z 350 which we have so far not identified. The presence of N-compounds is indicated by m/z 59, 67, 81, and 93.

5.5. Py-FIMS analysis of whole soil


This Py-FIMS spectrum (Fig. 3B) is dominated by carbohydrates, phenols, monomeric and dimeric lignins, and alkyl esters. Molecular ions m/z 394 and 408 indicate the presence of small amounts of n-C28 and n-C29 alkanes, whereas the weak signals at m/z 442, 456, and 470 appear to be due to C6H5C26H53 to C6H5C28H57 n-alkylbenzenes, respectively. This whole soil contains suberin-derived aromatics esters at m/z 446, 474, 502, and 530 (Hempfling et al., 1985). The signals at m/z 170 and 184 arise most likely from tri- and tetra-methylnaphthalenes, respectively, while m/z 178, 192, 206, 220, and 234 are due to phenanthrene, methyl-, dimethyl-, trimethyl, and tetramethyl-phenanthene, respectively. The presence of N-compounds is indicated by m/z 59 (acetamide), 67 (pyrrole), 79 (pyridine), 81 (methylpyrrole), 93 (methylpyridine), 103(benzonitrile), 117 (indole), 131 (methylindole), and 167 (N-acetylglucosamine).

5.6. Summary of compound classes identified


Table 1 lists a summary of compound classes identified by Py-FIMS in the HA, FA, and humin, all extracted from the Bainsville soil, and in the whole Bainsville soil. The compound classes identified in the four materials are qualitatively identical, although there are some quantitative differences in the data presented in Table 1. The latter data obviate the need for laborious extractions, separations and purifications. At then same time, we can conclude from an inspection of the four mass spectra in Figs. 2 and 3 that extraction with 0.5 M NaOH solution has not caused any measurable losses nor alterations in the different molecular compound classes identified. Py-FIMS is possibly the first and so far the only procedure currently available which allow soil chemists to do comprehensive molecular analyses without any pretreatment, that is, in situ.

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Table 1 Compounds classes identied by Py-FIMS in the HA, FA, and humin fraction, all isolated from the Bainsville soil, and in the untreated whole Bainsville soil Compound classes identified HA FA
a

Humin

Soil

Carbohydrates Phenols Lignin monomers Lignin dimers n-Fatty acids n-Alkylbenzenes Methylnaphthalenes Methylphenanthenes N-Compounds n-Alkanes
a

Intensity of peak height: , 2040%; , < 20%.

6. Chemical Structure
6.1. The chemical structure of SOM
According to Hatcher and Spiker (1988), SOM is hypothesized to be formed by one of the following two pathways: (a) the degradation of plant and microbial biopolymers to form the central core or (b) condensationpolymerization reactions in which plant and microbial biopolymers are first degraded to small molecules which then repolymerize. Fisher and Schrader (1921) suggested that lignin was the mother substance of SOM. It soon became apparent, however, that microorganisms played an essential part in the synthesis of SOM because the lignin theory could not account for it is relatively high N content. To solve this problem, Waksman (1936) proposed that microbial-produced protein was chemically linked to microbial-modified lignin to form SOM. Another approach was advocated by Flaig (1964) who suggested that lignin was oxidatively degraded to simpler phenolic monomers, which then underwent oxidative polymerization to produce SOM. Along similar lines, oxidative polymerization of simple phenolic acids to SOM, catalyzed by enzymes, was proposed by Flaig et al. (1975), Sulflita and Bollag (1981), and by abiotic catalysis (Huang, 1990; Wang et al., 1986). Maillard (1913) suggested that SOM originated from interactions of reducing sugars with amino acids and amines, which produced brown to dark brown polymers rich in N. According to Maillard (1913), SOM was formed by purely chemical reactions in which microorganism did not play a direct role except to produce sugars from carbohydrates and amino acids from proteins. Burdon (2001) has criticized the Maillard reaction as a significant source of SOM because: (a) there are not

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sufficient concentrations of free monosaccharides and of proteins/peptides/ amino acids in soils for this reaction to proceed at any great extent; (b) since the Maillard reaction proceeds best at high pH, alkaline soils should contain much more SOM than neutral and acidic soils, but this is not the case; and (c) the Maillard reaction produces aromatic N-heterocyclic compounds but these compounds occur at only low concentrations in arctic soils compared to temperate and tropical soils. If humification proceeded via the Maillard reaction, there should be similar concentrations of aromatic compounds in all soils. Jokic et al. (2005), disagreeing with Burdon (2001), provide evidence that the Maillard reaction catalyzed by d-MnO2, which is common in soils and sediments, produces aromatic N-heterocyclic compounds which are the dominant N-structures in fossil fuels as well as amides, which are the dominant N-compounds in SOM and sediments. The role of dMnO2 as catalyst in the Maillard reaction is very important according to Jokic et al. (2005).

7. Chemical Characteristics of Humic Substances


7.1. Analytical characteristics of HAs and FAs
The elemental composition and functional group content of a typical HA extracted from the Ah horizon of a Haploboroll, and of a FA extracted from the Bh horizon of a Spodosol) are presented in Table 2. A more detailed analysis of the data shows that (1) the HA contains approximately 10% (w/w) more C, but 25% (w/w) less O than the FA; (2) there are quantitatively smaller differences between the two acids in H, N, and S contents; (3) the total acidity and the CO2H content of the FA are significantly higher than those of the HA; (4) the FA is per unit weight richer in phenolic and alcoholic OH as well as in ketonic C O groups than the HA but the latter is richer in quinonoid C O groups; (5) both materials contain relatively few OCH3 groups; and (6) the E4/E5 ratio of the FA is almost twice as high as that of the HA, indicating that the FA has a lower molecular weight than the HA.

7.2. Infrared and Fourier transform infrared spectrophotometry


IR and Fourier transform infrared (FTIR) spectra of HAs and FAs show bands at 3400 cm 1 (H-bonded OH), 2900 cm 1 (alphatic C H stretches), 1725 cm 1 (C O of CO2H, C O stretch of ketonic C O), 1630 cm 1 (COO 1, C O of carbonyl and quinone), 1450 cm 1 (aliphatic C H), 1 1400 cm (COO 1), 1200 cm 1 (C O stretch of OH deformation

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Table 2 Analytical characteristics of a Mollisol humic acid (HA) and a Haplaquod fulvic acid (FA) HA FA
1

C H N S O Total acidity COOH Phenolic OH Alcoholic OH Quinonoid CO Ketonic CO OCH3 E4/E6

Element (g kg ) 564 55 41 11 329 Functional groups (cmol kg 1) 660 450 210 280 250 190 30 4.3

509 33 7 3 448 1240 910 330 360 60 250 10 7.1

From Schnitzer and Schulten (1998), Table 8-1, p. 155. With permission of the publisher.

of CO2H), and 1050 (Si O of silicates). The bands are usually broad because of extensive overlapping of individual absorbencies. IR and FTIR spectra of HAs and FAs reflect the importance of oxygen-containing functional groups, that is, COOH, OH, and C O in these materials. The latter groups appear to indicate the presence of polyketides in the HA and FA fractions (see section 11 in this Chapter). IR and FTIR provide useful information on the outer surfaces of HA and FA molecules and on reactions that occur at these surfaces with metals, pesticides, and other organics, but they tell us little about the insides of these molecules.

7.3. Oxidative degradation of HAs, FAs, and humins


One of the most useful methods for obtaining information on the chemical structure of complex organic substances is oxidative degradation by a variety of methods. Oxidative degradations of methylated and unmethylated HSs with alkaline KMnO4 solution have been carried out by Schnitzer and coworkers and have been summarized by Schnitzer (1978). The somewhat milder oxidation with alkaline CuO as well the sequential oxidation with CuO NaOH KMnO4 and with CuO NaOH KMnO4 H2O2 solutions has also been employed. Humic substances have also been oxidized under acidic conditions with peracetic and nitric acids (Schnitzer, 1978).

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Other oxidants used included alkaline nitrobenzene, sodium hypochlorite, and H2O2 solutions. Degradations with Na2S and phenol have also been done (Hayes and OCallagham, 1989). Major components produced by the oxidation of methylated and unmethylated HSs from widely differing pedological and geographical origins under alkaline as well as under acidic conditions were aliphatic carboxylic, phenolic, and benzene-carboxylic acids (Figs. 46; Schnitzer, 1978). The most prominent oxidation products were aliphatic mono-, di-, tri-, and tetracarboxylic acids, phenolic acids with between 1 and 3 OH

CH3(CH2)14CO2H CH3(CH2)16CO2H CO2H (CH2)n, n = 08 CO2H CH2 CO2H CH CO2H

O R5

R2

R4

R3

CH2 CO2H

Figure 4 Major aliphatic oxidation products of humic substances. From Schnitzer (1978), Fig. 7, p. 28. With permission of the publisher.

CO2H CO2H

CO2H

CO2H CO2H

HO2C CO2H

CO2H CO2H

CO2H

CO2H CO2H

CO2H CO2H HO2C

CO2H CO2H

HO2C

CO2H

HO2C CO2H

CO2H

HO2C CO2H

CO2H

Figure 5 Major benzenecarboxylic acid oxidative degradation products of humic substances. From Schnitzer (1978), Fig. 8, p. 29. With permission of the publisher.

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groups and between 1 and 5 CO2H groups, and the tri-, tetra-, and pentabenzenecarboxylic acids. From the oxidation products identified, it appeared that in the initial HSs the aromatic rings were linked by paraffinic chains (Fig. 7). On oxidation, the aliphatic C closest to the aromatic rings were converted to Cs of the CO2H groups which remained bonded to the
CO2H CO2H

CO2H

CO2H

OH OH OH

HO OH

OH OH

CO2H

CO2H CO2H HO

CO2H CO2H HO2C

OH CO2H

OH CO2H CO2H

CO2H

HO2C CO2H

CO2H

Figure 6 Major phenolic oxidation products of humic substances. From Schnitzer (1978), Fig. 9, p. 29. With permission of the publisher.

OH HO

OH OH

COOH HOOC

OH COOH

COOH

HO

OH

COOH

OH OH OH

HOOC OH

COOH

Figure 7 Chemical structure for HS based on oxidative degradation products. From Schnitzer (2000), Fig. 12, p. 23. With permission of the publisher.

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rings, while the Cs in aliphatic chains were oxidized to aliphatic acids. The purpose of methylation prior to oxidation was to protect OH groups against attacks by electrophilic oxidants. This made it possible to isolate and identify phenolic- in addition to benzene-carboxylic and aliphatic acids. The oxidation products from soil HSs separated from soils from all the earths surface were remarkably similar. Two conclusions can be drawn from the oxidative degradation experiments: (1) isolated aromatic rings are important structural units in all HSs and in SOM and (2) aliphatic chains are linking aromatic rings to form an alkylaromatic network.

7.4. Reductive degradation


Compared to oxidative degradation of HSs and SOM, reductive degradation has been less successful in obtaining structural information. The methods most widely used for this purpose were Na-amalgam reduction and Zn-dust distillation (Stevenson, 1994). Reduction of HSs with Na-amalgam produced phenols and phenolic acids. On the other hand, Zn-dust distillation and Zn-dust fusion (Hansen and Schnitzer, 1969) yielded small amounts of methyl-substituted naphthalene, anthracene, phenanthrene, pyrene, and perylene. The methyl groups on these polycyclic rings were probably remains of longer alkyl chains which linked the polycyclics in HSs and in SOM; or from microbial methylation during polyketide synthesis (see Section 11).

8. Spectrometric and Spectroscopic Characteristics of HS


8.1.
13

C NMR spectrometry of humic substances

One of the advantages of 13C NMR is that it indicates the presence in HSs a wide variety of C types whose determination by other methods would either be laborious and time consuming or not at all possible. In this sense, the use of 13C NMR offers unique possibilities. Of considerable interest is a comparison of solid-state or CP-MAS (cross polarization magic angle spinning) 13C NMR (nuclear magnetic resonance) spectra of HA (extracted with 0.5 M NaOH solution from the Ah horizon of a Haploboroll) and a FA (extracted with 0.5 M NaOH solution from the Bh horizon of a Spodosol). Both extracts were freeze-dried prior to NMR analysis. The CP-MAS 13C NMR spectrum of HA (see Fig. 8A) shows several distinct signals in the aliphatic (0105 ppm), aromatic (106150 ppm), phenolic (151170 ppm), and carboxyl (171185 ppm) C regions. The signals at 17,

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200 A

100

ppm
13

200

100

Figure 8 C NMR spectra of: (A) a Mollisol HA and (B) a Haplaquod Bh FA. From Schnitzer and Schulten (1998), Fig. 8-4, p. 158. With permission of the publisher.

21, 25, 27, and 31 ppm are due to alkyl C. The resonance at 17 ppm is characteristic of terminal CH3 groups and that at 31 ppm of (CH2)n in straight paraffinic chains. The peak at 40 ppm could include contributions from both alkyl and amino acid C. The broad signal at 53 ppm and the sharper one at 59 ppm may arise from C in OCH3 groups but amino acid C may also contribute to signals in this region (Breitmaier and Koelter, 1978). Carbohydrates in HA would be expected to produce signals in the 6065, 7080, and 90104 regions, although other types of aliphatic C bonded to O would also do so. The aromatic region exhibits a relatively sharp maximum near 130 ppm, due to alkylaromatics. The signal at 155 ppm shows the presence of O- and Nsubstituted aromatic C (phenolic OH and/or NH2 bonded to aromatic C). The broad signal near 180 ppm is due to C in CO2H groups, although amides and esters could also contribute to this resonance. The CP-MAS 13C NMR spectrum of the FA (Fig. 8B) consists of a number of aliphatic resonances in the 2050 ppm region, followed by signals from C in OCH3 groups and amino acids between 50 and 60 ppm and from carbohydrates between 61 and 105 ppm. The broad signal between 130 and 133 ppm indicates the presence of C in alkylaromatics. The strong signal between 170 and 180 ppm shows the presence of C in CO2H groups. In general, fewer sharper signals are observed in the spectrum of the FA than in that of the HA, possibly because of the occurrence of more H-bonding in the FA.

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The 13C NMR spectra in Fig. 8 show that the HA is slightly more aromatic than the FA, but the FA is significantly richer in CO2H groups, which appears to be the main difference between the two substances and accounts for the water-solubility of the FA in contrast to the HA which is insoluble in water. Other differences are that the HA is richer in paraffinic C but poorer in carbohydrate C than the FA. It is noteworthy that the main structural features such as aromaticity and aliphaticity are similar. Little is known about the chemical structure of humin, which is that portion of SOM which is left behind after extraction of the soil with dilute alkali. Preston et al. (1989) deashed a humin fraction separated from the surface horizon of the Bainsville soil and allowed it to stand with an aqueous solution of HClHF (1.16 M HCl 2.88 M HF) at room temperature for a prolonged period of time, changing the solvent at fixed intervals. With progressive deashing, the humin became more soluble in 0.5 M NaOH solution. After extensive deashing, the CP-MAS 13C NMR of the residue and its elemental composition were identical to those of the HA extracted with 0.5 M NaOH solution from the same soil. This indicates the humin is HA bound strongly to minerals and metal oxides and hydroxide in the soil, so that instead of three fractions, SOM contains only two fractions, that is, HA and FA. As has been mentioned before, since in most soils FA represents < 10% of the alkaline extract, and because FA is essentially a partial oxidation product of HA, it is clear that HA is the major component of SOM.

8.2. Effect hot acid hydrolysis on the 13C NMR spectrum of HA


Figure 9A shows the solution-state 13C NMR spectrum of a HA extracted with 0.5 M NaOH solution from the Ah horizon of a Cherozem from Central Alberta, Canada (Schnitzer and Preston, 1983). To facilitate the analysis of the 13C NMR data, the spectrum was divided into the following regions: 040 ppm (C in straight chain, branched and cyclic alkanes and alkanoic acids); 4160 ppm (C in branched aliphatics, amino acids, and OCH3 groups); 61105 ppm (C in carbohydrates and in aliphatics containing C bonded to OH, ether oxygens or occurring in five or six membered rings containing O; 106150 ppm (aromatic C); 151170 ppm (phenolic C); and 171190 ppm (C in CO2H groups). In Fig. 9B, the 13C NMR spectrum of the HA after hydrolysis with hot 6.0 M HCl for 24 h is shown. This spectrum is simpler than that in Fig. 9A, with distinct maxima remaining only at 18.7, 25.1, 31.3, 40.0, and 58.7 ppm in the aliphatic region and at 131 and 180 ppm in the aromatic region. The 13C NMR of the same HA, which had been hydrolyzed first by standing in contact with 12.0 M H2SO4 for 16 h at room temperature, and then refluxed with 0.5 M H2SO4 for 5 h, is shown in Fig. 9C. Note that curves 9B and 9C are very similar. In the aliphatic region of curve 9C, small signals can be seen at 16.5, 25.2, 31.4, 40.1, and 58.4 ppm. The persistence

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200
131.8

100

31.3 25.0

58.5

180.4 121.6 130.6

40.2

16.5

73.4

63.5

B
31.3 40.0 25.1

179.3 131.2

58.7

C
31.4 25.2

178.3 171.0 75.2 58.4

40.1 16.5

ppm
13

200

100

Figure 9 C NMR spectra of (A) Chermozem HA; (B) the same HA hydrolyzed with 6 M HCl; (C) the initial HA hydrolyzed first with 12 M H2SO4 and then with 0.5 M H2SO4. From Schnitzer and Preston (1983), Fig. 1, p. 204. With permission of the publisher.

of the distinct signal at 58.7 in Fig. 9B and at 58.4 ppm in Fig. 9C, as well as at 58.5 ppm in Fig. 9A, suggests that this signal is due to C in OCH3 groups. Resonances at 56.2, 63.5, and 73.4 ppm in Fig. 9A are no longer present in the spectra of the hydrolyzed HA (Fig. 9B and 9C). Chemical analysis for N-amino and carbohydrates indicated that the two acids had removed most

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of the proteinaceous compounds and carbohydrates from the acid-treated HAs. On the basis of these observations, and from data published in the literature, we could now assign the following bands in Fig. 9A; 56.2 ppm (C in amino acids), 63.5 ppm, and 73.4 ppm (C in carbohydrates). One striking observation was that the relative intensity of the aromatic regions in spectra Fig. 9B and 9C was greater than that in spectra Fig. 9A, although the position of the maximum remained near 130 ppm. For the initial HA, the HCl-hydrolyzed HA and for the H2SO4hydrolyzed HA (spectra Fig. 9AC) aromaticities were 41%, 62%, and 53%, respectively. Aromaticites were computed by expressing areas due to aromatic C (105165 ppm) as percentage of the total area (0165 ppm) but omitting contributions from carboxyl carbons (166185 ppm). Acid hydrolysis also reduces the intensity of the carboxyl region (170180 ppm). Area integration of the carboxyl regions (170180 ppm) in spectra Fig. 9AC showed CO2H contents of 4.5, 3.0, and 3.1 meq g 1, respectively. Thus, aside from lowering the CO2H by acid decarboxylation and by removing amino acids and carbohydrates, the hot acids also increase the aromaticity of the HA by removing aliphatics compounds such as amino acids and carbohydrates. To establish whether hot acid hydrolysis would affect other HAs and also FA in the same way as the Chenozemic HA, Schnitzer and Preston (1983) passed a HA extracted with 0.5 M NaOH solution from the Ah horizon of a Humic Gleysol and a FA extracted from the Bh horizon of Spodosol through the same procedure. The results obtained were identical to those described herein. Thus, hot acid hydrolysis removes proteinaceous materials and carbohydrates from HAs and FAs but leaves aliphatic and aromatic compounds intact. These findings may have important structural implications: (1) they suggest that proteinaceous compounds and carbohydrates are not structural compounds of HA, FA, or SOM but are adsorbed on their surfaces and in their voids. If they were structural components, they would have resisted dissolution by the hot acids; (2) the strong signal at 130 ppm in the 13C NMR spectra is due to C in aromatic rings not substituted by electrondonating O and N but by C, as in alkylaromatics, which indicates that the latter are very significant building blocks of HA, FA, and SOM.

8.3. Curie-point pyrolysis-gas chromatography-mass spectrometry of HAs


Curie-point pyrolysis-gas chromatography-mass spectrometry (Cp-GCMS) is a valuable method for structural studies on HSs (Schulten and Schnitzer, 1992) because the transfer of thermal energy from the wire to

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the sample is fast with temperature rises on the order of milliseconds. The resulting thermal shock produces small, stable organic molecules as pyrolysis products whose identification is based on two independent data sets: (1) gas chromatographic retention times and (2) computer assisted library searches of standard mass spectra libraries. Schulten and Schnitzer (1992) did Cp-GC-MS analyses of two HAs which had previously been examined by Py-FIMS. As shown in Table 3, the major compounds produced from the two HAs were benzene and alkylbenzenes, ranging from C6H6 to C6H5C22H45 (docosylbenzene). Other compounds produced were naphthalene and alkylated naphthalenes and phenanthrene and alkylated phenanthrenes. The alkylaromatics identified in Table 3 consisted of aromatic rings which were covalently linked to aliphatic groups or chains, and these building blocks were released during pyrolysis from an alkylaromatic structural network that was made up of the constituents listed in Table 3. A chemical structure for a HA skeleton, without functional groups, based on alkylbenzenes is shown in Fig. 10. This structure contains voids of varying dimensions which can trap and bind organic and inorganic components. An inspection of the data in Table 3 shows that Armadale HA produced a greater variety of alkylaromatics than the Bainsville HA. This may be related to differences in the origins of the two HAs. The Armadale HA was extracted from the Bh horizon of a Spodosol, about 25 cm below the soil surface while the Bainsville HA was extracted from the surface horizon of the Bainsville soil, a Haplaquoll. One of the striking features of a Spodosol Bh horizon is it is low microbial activity, which may have led to a better preservation of the alkylaromatics listed in Table 3.

8.4. X-ray analysis of FA


The X-ray diffraction pattern of a nonoriented flat powder specimen of a FA extracted with 0.5 M NaOH from the Bh horizon of a Spodol exhibited , accompanied by a few minor humps (Kodama a diffuse band at about 4.1 A and Schnitzer, 1967). The experimental intensity curve was corrected for polarization and absorption. Figure 11, curve A shows the corrected intensity curve as a function of over the range 0.020.5. Curve A was normalized to the total independent scattering curve B for FA in such a way that curves A and B approached each other. Curve B was obtained by adding the independent coherent scattering curve C to the incoherent scattering curve D. Calculations for the independent coherent scattering curve C and the incoherent scattering curve D were based on C28H23O19 (the molecular formula of FA), and included contributions from all atoms.

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Table 3 Building blocks of Bainsville and Armadale humic acids (HA) identied by Curie-point pyrolysis GCMS Intensity Armadale Bainsville Compounds

Benzene Toluene Ethylbenzene, xylenes Ethylmethylbenzene Propylbenzene Butylbenzene Methylpropylbenzene Tetramethylbenzene Pentylbenzene Hexylbenzene Octylbenzene Methyloctylbenzene Decylbenzene Methylnonylbenzene Undecylbenzene Methyldecylbenzene Dodecylbenzene Methylundecylbenzene Tridecylbenzene Tetradecylbenzene Pentadecylbenzene Hexadecylbenzene Heptadecylbenzene Octadecylbenzene Nonadecylbenzene Eicosylbenzene Hemicosylbenzene Decosylbenzene Styrene Methylstyrene Indene Indane Fluorene Naphthalene Methylnaphthalenes Dimethylnapthalenes Trimethylnaphthalenes Tetramethylnaphthalenes Pentamethylnaphthalene

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Table 3

(Continued ) Intensity

Armadale

Bainsville

Compounds

Phenanthrene Methylphenanthrene Dimethylphenanthrene Trimethylphenanthrene Tetramethylphenanthrene

Intensity of peak height: , 80100%; , 6080%; , 4060%; , 2040%; , observed. From Schulten et al., (1991), Table 1, p. 312. With permission of the publisher.

(CH3)04

(CH3)05

(CH3)05

Figure 10 Chemical structure of HA based on alkylaromatic building blocks without functional groups. From Schulten et al. (1991), Fig. 1, p. 311. With permission of the publisher.

8.4.1. Radial distribution analysis The radial distribution analysis of FA is based on the generalized Fourier method for polyatomic substances (Warren et al., 1936). For more details on how this method was used see Kodama and Schnitzer (1967). Figure 12 and two shows the existence of two peaks with maxima at 1.6 and 2.9 A . shoulders at 4.2 and 5.2 A

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4 103 3 a)

I (e.u.)

2 b)

c) 1

d) 0 0.2 (sin q)/l 0.4 0.6

Figure 11 X-ray intensity curve for FA in electron units per C20H12(CO2H)6(OH)5(CO)2 (the elemental and functional group composition of FA). (a) The corrected intensity curve; (b) the total independent scattering curve; (c) the independent coherent scattering curve; and (d) the incoherent scattering curve. From Kodama and Schnitzer (1967), Fig. 1, p. 90. With permission of the publisher.

As FA resembles low-rank coals in a number of properties, it seemed worthwhile to compare our results with those for carbon black as reported by Warren et al., (1936). His curve showed four distinct maxima at distances , indicating that carbon black was of separation at 1.5, 2.7, 4.05, and 5.15 A not amorphous but consisted of graphite-like layers. These four distances are in good agreement with the respective peak positions for FA. The broadness of the peaks and the similarity of their positions as compared to carbon black suggest that FA has a considerable random structure in which carbon atoms are arranged in a manner similar to that in carbon black. More dedicated analysis of each peak is complicated because in addition to carbon atoms, oxygen atoms also form part of the structure of FA. Contributions of hydrogen atoms can be neglected because they constitute less than 10% of the total number of electrons. Interatomic distances of bond pairs according to the Interatomic for a single bond or a partial Tables (1962) are 1.541.40 and 1.471.36 A double bond for carboncarbon and carbonoxygen, respectively. Assum may include contributions from both ing loose packing, the peak at 1.6 A atom pairs. The area under the peaks corresponds, therefore, to the sum of

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8 104 6
Km 4pr 2 rm (r )

16 700

6 000 2 r ()

Figure 12 Radial-distribution curve for FA. From Kodama and Schnitzer (1967), Fig. 2, p. 91. With permission of the publisher.

the electrons from carboncarbon and carbonoxygen bond pairs. One number-average molecular weight of FA contains 28 carbon atoms, 13 of which are either in functional groups or linked to the functional groups and may be diatomically bonded to 19 oxygen atoms (Table 2). Assuming that the effective number of electrons is 6 per carbon atom and 8 per oxygen atom, the average number of electrons due to carbonoxygen bond pairs will be 2(13 6 19/13 8) 1824. Subtracting this number from the area of the first peak ( 6000 electrons, see Fig. 12) leaves 4176 electrons which can only be due to carboncarbon bond pairs. The average number (n) of carbon neighbors nearest to each carbon atom can then be estimated from the relation 4176 2(28 6 n 6), so that n will be about 2.0. Contrary to carbon black, the number of first neighbors is smaller than 3, possibly indicating the presence of more non-aromatic carbon atoms. also agrees with this explanation. Analysis of the second peak at 2.9 A A distance of separation of 2.9 A may include oxygenoxygen bond pairs as well as second carbon neighbors. The area under the second peak ( 16,700 electrons) must arise from both contributions. Assuming for purposes of simplification that all consists of carbon atoms arranged as in carbon black, then the average number of second neighbors should be 9, and this relation should require 2(28 6 9 6) 18,144 electrons. This value is larger than the observed second area of 16,700, even including contributions from the other bond pairs. This means that the average number of second carbon

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neighbors must be less than 9. All oxygen atoms in FA are in functional groups (Table 2). Assuming that each of these never meets more than two , the contributions due to carbon other oxygen atoms at a distance of 2.9 A carbon bond pairs would range between 11,500 and 14,200 electrons, corresponding to approximately 6 or 7 second carbon neighbors. These data suggest that the carbon skeleton of FA is a broken network rather than a continuous sheet as in graphite. Unlike coal, FA has no distinct 0.02 band which normally occurs in the region depending on the carbon content of the coal (Hirsch, 3.433.75 A is close to that of the g-band 1954). The position of the diffuse band at 4.1 A (Blayden et al., 1994). Thus, according to the interpretation of the g-band tells us that the by Curtz et al. (1956), the diffuse band of FA at 4.1 A structure of FA consists of irregularly packed poorly condensed aromatic layers and of appreciable numbers of disordered aliphatic chains or alicyclic rings at edges of the aromatic layers, in which a relatively uniform spacing of is maintained between layers of chains and rings. In general, this about 4 A interpretation is consistent with the structure of FA revealed by chemical and spectrometric studies reported in this chapter. 8.4.2. Proposed structures for FA on the basis of X-ray experiments As the number average molecular weight (Mn) and density (d) of FA are 670 and 1.61 cm3 g 1, respectively, the unit molecular volume (V) when Z 1 as calculated from the relation V ZM/dN (where Z the number of chemical formulae per unit cell, and N is Avogadros number 3. The spacing of 4 A observed in the X-ray (0.602 1024), is 690 A diffraction pattern of FA is thought to be equivalent to the average distance between aromatic layers, including aliphatic chains and/or alicyclic rings. Interlamellar adsorption studies of FA on montmorillonite showed that the corresponded to the thickness of a single layer of FA spacing of 4 A molecules (Schnitzer and Kodama, 1966). Consequently, the cross-sectional 3/4 A 172.5 A 2 (or area of one FA molecule was estimated to be 690 A 2 17.25 nm ). Possible models for a planar FA molecule must conform to the X-ray, chemical, and spectroscopic data. Thus, the model should contain two aromatic rings, six CO2H and two ketonic C O groups, two phenolic OH and three alcoholic OH groups. Allowances must also be made for the presence of aliphatic chains or alicyclic structures and for a limiting cross 2. Naturally, one can design a considersectional size of the order of 175 A able number of models that fit these requirements. Four of the more realistic models are shown in Fig. 13. These models only show carbon skeletons and maximum outlines of the modeled FA molecules. In diagram (A) in Fig. 13 two isolated aromatic rings are joined to each other via an acyclic fivemembered ring; in diagram (B) they are inked via two aliphatic chains; in diagram (C) one long aliphatic chain links two isolated aromatic rings,

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nm

0.5

Figure 13 Schematic representations of four possible structures for the planar FA molecule. From Kodama and Schnitzer (1967), Fig. 3, p. 93. With permission of the publisher.

whereas in diagram (D) the two aromatic rings are condensed and the aliphatic chains extend form one of the aromatic rings only. Although more detailed studies are needed for more definitive conclusions, it is noteworthy that the carbon skeletons of the FA models are in broad agreement with the results of physical and chemical studies that have so far been done on this FA and that the sizes of the models conform to the calculated cross-sectional area. It is also of interest that the model FA structures (B)(D) in Fig. 13 are alkylaromatics and that structures (B) and (D) contain voids.

8.5. A 2D structure for HA


With the aid of pyrolysis-soft ionization mass spectrometry, Schnitzer and Schulten (1992) demonstrated that carbohydrates, phenols, lignin monomers, lignin dimers, lipids (alkanes, alkenes, fatty acids, n-alkyl esters, and alkylaromatics), and N-containing compounds were the major HA components. Cp-GC-MS of HAs showed the presence of relatively large amounts of alkyl-substituted aromatic hydrocarbons (Schulten et al., 1991). Alkylaromatics consist of aromatic rings covalently linked to aliphatic groups and chains. In a first approach, Schulten et al., (1991) proposed that these

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HO O O (CH3)0-3 O HO

OH O

OH

O O

HO O

OH O OH (CH3)0-2

O OH OH OH O HO HO HO O OH O HO OH N H O OH O OH O OH OH O OH N O O OH HO OH O O OH (CH3)0-5 OH O O O O OH O OH O OH OH O
O OH

OH HO

OH O

(CH3)0-2 N H C N (CH3O)0-3 O

HO O OH

OH OH HO O O

OH O

(CH3)0-4 OH (CH3)0-4

(CH3)0-2

OH

OH HO O HO

(CH3)0-2

OCH3

C OH

N CH2OH

(CH3)0-5

Figure 14 A 2D chemical structure for HA. From Schulten and Schnitzer (1997), Fig. 1, p. 119. With permission of the publisher.

alkylaromatics were HA building blocks, released during pyrolysis from an alkylaromatic structural network. In the hand-drawn draft structure (Fig. 14), Schulten and Schnitzer (1993) assigned n-alkylaromatics a significant role. Oxygen was present in the form of carboxyls, phenolic, and alcoholic hydroxyls, esters, ethers, and ketones, whereas nitrogen occurred in nitriles and N-heterocyclic structures. The HA structure in Fig. 14 is in agreement with chemical (Schnitzer, 1978; Schnitzer and Khan, 1972), oxidative and reductive degradation (Schnitzer, 1978, Schnitzer and Khan, 1972), colloid-chemical (Ghosh and Schnitzer, 1980), electron microscopic (Stevenson and Schnitzer, 1982), and 13C NMR and X-ray data (Schnitzer, 1991) obtained over many years, as well as with exhaustive consultations of the voluminous literature on HSs. The elemental composition of the HA shown in Fig. 14 is C308H328O90N5, with a molecular weight of 5539.949 g mol 1 and an elemental analysis of 66.78% (w/w) C, 5.97% (w/w) H, 25.99% (w/w) O, and 1.26% (w/w) N. There are diverging views in the literature on SOM as to whether carbohydrates and proteinaceous materials are adsorbed on or loosely retained by HAs or whether they are bonded covalently by HAs (Sowden and Schnitzer, 1967). But regardless of the mechanisms considered,

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carbohydrates and proteinaceous materials are co-extracted with HAs, and their presence affects the elemental analysis and functional groups content of HAs. Carbohydrates have been reported to make up about 10% (w/w) of the HA weight (Lowe, 1978), a similar value has been suggested for proteinaceous materials in HAs (Khan and Sowden, 1971). Thus, Schulten and Schnitzer (1993) accepted these values and assumed that one molecular weight of HA interacted with 10% (w/w) carbohydrates and 10% (w/w) proteinaceous material. The resulting HA had an elemental composition of C342H388O124N12 with a molecular weight of 6650.831 g mol 1 and an elemental analysis of 61.67% (w/w) C, 5.88% (w/w) H, 29.83% (w/w) O, and 2.53% (w/w) N. When more carbohydrates and proteinaceous materials were added to the HA, the C content decreased but the O content increased. For the development of the HA structure (Fig. 14), Schulten and Schnitzer (1993) assumed that carbohydrates and proteinaceous materials were not integral structural HA components but were adsorbed electrostatically in internal voids and on external surfaces. One of the most interesting features of the proposed HA structure is the presence of voids of various dimensions that can trap and bind other organics such as carbohydrates and proteinaceous materials, lipids, and biocides as well as inorganics such as clay minerals and hydrous oxides.

8.6. A 3D structure for HA


Schulten and Schnitzer (1997) converted the 2D structure for HA (Fig. 14) to a 3D structure with the aid of HyperChem software, using the following four steps: (1) the C C skeleton was drawn by hand in the workspace and O and N were added; (2) H atoms were attached with accurate bond lengths and angles. In addition, seven H atoms were added to obtain a complete chemical compound with the composition C308H335O90N5; (3) the 3D HA structure was constructed with accurate bond distances, bond angles, torsion angles, van der Waals forces, and hydrogen bonds; and (4) the 3D stick structure of HA (C308H335O90N5) with 738 atoms is shown in Fig. 15. This structure has a molecular weight of 5547.004 g mol 1 and an elemental analysis of 66.69% (w/w) C, 6.09% (w/w) H, 25.96% (w/w) O, and 1.26% (w/w) N. Molecular mechanics calculations allowed the geometry optimization of this structure, which resulted in a favorable, energy-minimized conformation with a total energy of 710.70 kJ (0.1 nm) 1 mol 1 and a convergence gradient of 0.037 kJ (0.1 nm) 1 mol 1 (Schulten, 1995a,b). It was of interest to explore the capacity of the software (ChemPlus) to determine quantitative structureactivity relationship (QSAR) properties of HAs. Attempts were made, using QSAR calculations, to correlate the HA structure at a higher level of total energy and an optimal display of structural voids (Fig. 15) with properties that are of interest in soil- and

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636 637 95 157 165 195

248 642 586 282

344 747 638 746

Figure 15 Improved 2D model structure of HA. From Schulten and Schnitzer (1997), Fig. 3A, p. 122. With permission of the publisher.

nanochemistry. The investigated HA conformation had a total energy of 1696.02 kJ (0.1 nm) 1 mol 1 at a gradient of 0.83 kJ (nm) 1 mol 1, and the spatial dimensions (smallest rectangular box enclosing the molecule) were: height 2.81 nm, width 2.26 nm, and depth 4.93 nm. Utilizing the ChemPlus software, a (solvent accessible van der Waals) surface area of 44.08 nm2 and a volume of 11.50 nm3 were calculated. Also, an estimate for the octanolwater partition coefficient (log P) of 146.88, which is a measure of hydrophobicity, was obtained. In addition, data for molar refractivity (1.37 nm3) and polarizability (0.58 nm3) were also determined. To evaluate to what extent biological molecules could be inserted or trapped in the voids of the 3-D HA structure, Schulten (1995a) tested geometrically optimized 3D structures of a sugar and peptide. The sugar was a trisaccharide with 66 atoms, an elemental composition of C18H32O16

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with a molecular weight of 504.44 g mol 1 and an elemental analysis of 42.9% (w/w) C, 6.4% (w/w) H, and 50.8% (w/w) O. The hexapeptide tested was Asp-Gly-Arg-Glu-Al-Ly, which contained amino acids found in soils and which had an elemental composition of C26H46O10N10, a molecular weight of 658.71 g mol 1 and an elemental analysis of 47.4% (w/w) C, 7.0% (w/w) H, 24.3% (w/w) O, and 21.3% (w/w) N. Measurements of two voids in the 3D HA structure showed that the voids were of sufficient volume to retain the carbohydrate and the polypeptide. With the merger of the 3D HA with the structures of the two biological materials, the elemental composition of the humic complex (896 atoms) was C352H413O116N15 with an elemental analysis of 63.0% (w/w) C, 6.0% (w/w) H, 27.7% (w/ w) O, and 3.1% (w/w) N. The molecular weight was 6710.16 g mol 1. Compared with the elemental analysis of the 3D HA (Fig. 15), the C content of the complex dropped by 3.7% (w/w), but the H content increased by 0.1% (w/w), O by 1.8% (w/w), and N by 1.9% (w/w). The simulation experiments showed that trapping the biological molecules by the 3D HA alone was insufficient to account for the still existing oxygen deficiency. It appeared, therefore, that a significant portion of the biological molecules was either chemically or physically retained on the HA surface. The net effect of surface-retained biological molecules was to lower the C but to increase the O content of the HA.

8.7. Relationships between HA and SOM


In agricultural soils, the bulk of the SOM (about 70%, w/w) consists of HSs (HA, FA, humin). In addition, we find smaller amounts of carbohydrates (10%, w/w), N-compounds (10%, w/w), and lipids (10%, w/w) (Schnitzer, 1991). As has been mentioned earlier in this chapter, the analytical characteristics and chemical structure of humin are very similar to those of HA. Humin is so strongly complexed by clays and hydrous oxides that it can no longer be extracted by dilute base or acid. As far as FA is concerned, 13C NMR spectra of HA and FA show a similar contents of aliphatic and aromatic C. The major difference between HA and FA is that FA has a lower molecular weight and is richer in CO2H groups and carbohydrates than HA. Thus, FA is lower in C but richer in O than HA, which suggests that FA is partially oxidized HA. In many agricultural soils, except Spodosols, we find the following distribution of humic fractions: HA23%, FA 7%, humin70%). This indicates that FA is a relatively small humic fraction so that it may be realistic to model all HSs in terms of HA. Thus, an agricultural soil containing 3.0% SOM can be viewed as consisting of 2.5% HA 0.25% carbohydrates 0.25% proteinaceous materials, so that:

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SOM HA carbohydrates proteins

Another SOM component that needs to be considered is water. The water content of air-dry HA is of the order of 3.0% (Schnitzer, unpublished data). Schulten and Schnitzer (1997) further developed an improved SOM model structure to include 3.0% H2O, so that SOM HA carbohydrates proteins H2 O 3

8.8. The corrected 2D HA model


The 2D model shown in Fig. 14 needed to be corrected because it was too small to accommodate all oxygen-containing functional groups. Also, on the average, the aliphatic (CH2)n chains were too long because the proposed preliminary C C skeleton was based mainly on data obtained by Py-GC/ MS and Py-FIMS, which qualitatively indicated the presence of (CH2)n units ranging from n 1 to n 20. Essentially, however, the 2D structure shown in Fig. 14 was the basic structure which was adopted. The elemental composition and chemical analysis, as well as the molecular weight of the corrected HA, are listed in Table 4. The corrected 2D HA model structure
Table 4 Elemental composition, elemental analysis, molecular weight, and number of atoms in corrected 2D HA, water-free 3D SOM and 3D SOM 3% water Elemental composition Elemental analysis Molecular Number of atoms weight (g mol 1)

Corrected 2D HA

C305H299O134N16S

Water-free C349H377O161N26S 3-D SOM

3-D SOM 3% water

C349H401O173N26S

57.56% C 4.73% H 33.68% O 3.52% N 0.5% S 55.57% C 5.04% H 34.15% O 4.38% N 0.42% S 54.02% C 5.21% H 35.67% O 4.69% N 0.41% S

6364.81

755

7543.00

914

7760.15

950

From Schulten and Schnitzer (1997), Table 1, p. 121. With permission of the publisher.

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shown in Fig. 16 has an elemental analysis that is close to that of naturally occurring HAs (Schnitzer, 1978). The sizes of the structural voids are large enough to occlude peptides, polysaccharides, water, etc. The corrected 2D HA model structure shown as a simplified C, H, O, H, and S skeleton in Fig. 16 (sticks) contains 5 aliphatic and 21 aromatic CO2H groups, 17 phenolic OH, 17 alcoholic, 7 quinonoic and ketonic, 3 methoxyl groups, and 1 sulfur function. The aliphatic links between the aromatic rings have shortened to between 1 and 10 CH2 units, with an average of n 5. Hydrogen bonds are of particular importance in HAs. The use of HyperChem software can help to confirm favorable conditions for these bonds. Hydrogen bonds are formed if the hydrogen donor distance is less than 0.32 nm and the angle made by covalent bonds to the donor and acceptor atoms is less than 120 (HyperChem). In total, seven hydrogen bonds were calculated and displayed for the corrected HA (Fig. 15). The corresponding number of atoms (in brackets) and determined bond lengths (d) were: H (157) O (195) 0.298 nm, H (586) O (248), d 0.264 nm, H (746) O (344), d 0.240 nm, and H (747) N (638), d 0.256 nm if the acceptor H was covalently bound to O. For the three H-bonds for which N was the binding partner of the H,

Figure 16 Geometrically optimized 3D structure of HA. Element colours are: carbon (cyan); hydrogen (white); oxygen (red); and nitrogen (blue). From Schulten and Schnitzer (1997), Fig. 2, p. 120. The elements colors are: carbon (cyan); hydrogen (white); oxygen (red); and nitrogen (blue). With permission of the publisher.

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bond lengths increased slightly as might be expected: H (636) N (165), d 0.303 nm, H (642) N (282), d 0.310 nm, and H (637) N (95), d 0.391 nm. A preliminary check of the total energy by single point calculation (without changing the geometry) yielded a relatively high value of 4707.68 kJ (0.1 nm) 1 mol 1 at a gradient of 36.05 kJ (0.1 nm) 1 mol 1 and indicates a distorted sheet structure (height 4.2 nm, width 3.5 nm, depth 0.92 nm). Calculations of the QSAR properties gave a volume of 12.67 nm3 and a surface area (grid) of 51.04 nm2. The solvent accessible or van der Waals surface area was obtained by the grid method.

8.9. A 3D model structure for SOM plus water


The corrected 2D HA model was further developed into a 3D model of SOM that included a water content of 3% (Fig. 17), which is about the moisture content of an air-dried soil. The elemental composition and analysis as well as the molecular weight of the SOM H2O structure are listed in Table 4. Trapped in this structure are a typical soil hexapeptide (C26H46O11N10), a trisaccharide (C18H32O16), and 12 water molecules

Figure 17 Geometrically corrected 3D structure of SOM (HA carbohydrates proteins) (w/w) % water. From Schulten and Schnitzer (1997), Fig. 4B, p. 124. The element colors are: carbon (cyan), hydrogen (white), oxygen (red), and nitrogen (blue). With permission of the publisher.

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(H24O12). In order to find the best conformation for this SOM complex of 15 molecules and 950 atoms within a reasonable calculation time (600 calculation cycles, 1171 points, time about 2 days), the convergence limit was set by the determination gradient of 0.838 kJ (0.1 nm) 1 mol 1. Geometry optimization (and thus energy minimization) was performed with molecular mechanics calculations which yielded a total energy of 2453.99 kJ (0.1 nm) 1 mol 1. The C C skeleton containing H, O, N, S atoms of this geometrically optimized structure is shown in Figs. 16 and 17. Particularly interesting is that even after extensive calculations, and with progressing energy minimization, the SOM structure still showed mobility of the side chains and occluded molecules, changes in hydrogen bonding, and formation of voids. Thus, the dynamics and flexibility of the structure appeared to be a decisive structural property; it may be part of a novel definition of SOM. Figure 17 shows a 3D color plot for SOM containing 3% water in the disk version, which illustrates the high oxygen density of this macromolecule. At this stage of optimized SOM conformation, a total of 11 hydrogen bonds were determined. In the search for particular molecular properties, empirical calculations using ChemPlus software were employed. The resulting QSAR properties were: (1) surface area (approx.) 40.23 nm2; (2) more exactly but time consuming in calculation time, surface area (grid) 51.27 nm2; (3) volume 14.39 nm3; (4) Log P 124.80, log of the water-octanol partition coefficient as a measure of hydrophobicity; (5) refractivity 1.74 nm3; (6) polarizability 0.72 nm3; (7) mass 7760.15 g mol 1, which is consistent with the values listed in Table 4 obtained by HyperChem (Molinfo) directly; and (7) hydration energy 33.43 kJ mol 1 of the trapped hexapeptide. It is noteworthy that when comparing the structure of the corrected HA (Fig. 17), the surface area and the volume of the latter increased by approximately 25%, which is similar to increases in molecular weight and atom numbers (see Table 4). Of special interest are the 10 hydrogen bonds which are observed in the SOM H2O conformation obtained after geometry optimization (Figs. 15 to 17). The majority (six) of these hydrogen bonds were formed within the corrected HA.

9. Effect of Time on the SOM Structure


9.1. Effect of long-term cultivation on the SOM structure
The influence of long-term cultivation on the chemical quality of SOM was studied by Schulten et al. (1995). These authors employed Py-FIMS to analyze whole soil samples from a Typic Haploboroll under long-term

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cultivation established in 1910 at Lethbridge, Alberta. One soil sample was collected in 1910 from the Ah horizon after breaking the native grassland was stored. Another soil sample was collected in June 1990 from the A horizon under a wheat-fallow, nonfertilized rotation. This soil sample had been under cultivation for 80 years. The native sample (collected in 1910) contained 3.03% (w/w) SOM, but the cultivated sample (collected in 1990) contained only 2.23% (w/w) SOM. Percentages of sand, silt, and clay as well as the exchange capacities of the two samples were, however, almost identical. The aggregate stability of the native soil was 65%, whereas that of the cultivated soil was only 42%. There were significant reductions in enzyme activities after 80 years of cultivation. The activity of dehydrogenase dropped by 60%, that of acid phosphatase by 77%, and that of ureases by 82%. The total ion intensities (TIIs) of the Py-FIMS, which are related to carbon concentrations, were dramatically different for the two samples. The TII value per mg of soil for the native sample was 31.25 104 counts compared to 3.96 104 counts for the cultivated soil. The major signals in the Py-FIMS of the native sample were due to carbohydrates, phenols, lignin monomers, alkylaromatics, N-compounds, peptides, lipids, and lignin dimers but microbial biomarkers were present in the cultivated sample in much lower concentration, less than 1015% (w/w) or not present at all. The TII for the SOM in the cultivated soil was only about one-sixth of that in the SOM of the native soil. Thus, the SOM in the cultivated soil was significantly more thermostable than that of the native soil. Conversely the SOM in the native soil was more volatile and had a lower molecular weight than that in the cultivated soil. This means that cultivation depleted the easily metabolizable substrates and increased the resistance to pyrolysis of the major SOM components, leading to the formation of larger molecules with higher molecular weights and complexity. Increases in molecular weight, microbial biomarkers and size of the SOM in cultivated soils may also explain the observed decreases in enzyme activities, involving the C, N, and P cycles. While the effects of anthropogenic disturbances through cultivation to bring about significant changes in the quality, chemical composition, and molecular size of SOM have been discussed here in terms of 80 years of cultivation, one wonders how profound these effects would be after 300 or 500 years of continuous cultivation.

10. New Concepts on the Chemical and Microbial Synthesis of HAs and SOM
According to Burdon (2001), the following plant and microbial constituents in various stages of degradation contribute to the formation of HSs: plant carbohydrates and microbial carbohydrates, plant proteins and

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microbial proteins, plant lipids and microbial lipids, lignins and tannins, and microbial substances such as melanins and other polypeptides. This is an enormously complex mixture in which it is difficult to find well-defined chemical structures, and this has been the experience of many SOM chemists during the past 225 years since Archads first paper in 1786. We have now come to a point in time at which we have to further enhance structural research on HA and its formation process. In our opinion, it is possible and we are presenting a number of new ideas in order to demonstrate this point: (a) The experimental research on the chemistry of HS and SOM which we are presenting in this chapter shows that the complex mixture of plant and microbial products, degraded to small organic molecules which we have described above, is consistent with the first stage of HS formation. (b) The second stage of HS formation is through the selective chemical and microbial synthesis. (c) Chemical synthesis of HAs occurs from two major components of the plant and microbial polymer mixture, that is, lignins and lipids (n-fatty acids, n-alkyl mono- and diesters, and n-alkanes, see Table 1). Major HA building blocks are aromatic lignin degradation products such as benzene, naphthalene, and phenanthrene, while the major lipid degradation products participating in the synthesis are alkanes, which originate from decarboxylated fatty acids, released by lipids. The chemical covalent bonding of these two building blocks produces alkylaromatic structures. (d) Chemical synthesis of HA can also occur by the alkylation of the aromatic rings, which means that alkyl chains occupy positions on the aromatic rings, substituting hydrogens. This reaction may be brought about by free radicals, which are present in all HSs (Schnitzer, 1978). The alkylaromatic structures so formed by covalent bonds are chemically stable in that they resist degradation by strong hot acids. They can readily be seen in 13C NMR spectra of HAs sampled from soils from all over the earths surface. They also show in soft ionization mass spectra of all HAs and SOM samples so far analyzed. (e) Carbohydrates and proteinaceous materials in the mixture are not structural components of the chemically and microbially synthesized HA structures. Instead, they are physically adsorbed in the voids and electrostatically adsorbed on the surfaces of the alkylaromatic structural network, which now becomes SOM. They are the main sources of carbon, nitrogen, and energy for the microbes performing HA synthesis. (f) Finally, the microbial synthesis of HS occurs through the synthesis of complex and diverse polyketides from simple organic molecules residing in soil solutions. The sequence of events in the microbial

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synthesis of HAs, which we describe in the subsequent section, is based on published experimental data which we are presenting in this chapter.

11. Microbial Humification of Small Organic Compounds into Soil Polyketides


11.1. A complex biological link to the chemistry of humification
One important aspect of this review chapter deals with the microbial and enzymatic transformation (i.e., humification) of simple organic compounds (i.e., organic acids, amino acids) into large, complex, and diverse polyketide molecules (i.e., alkylaromatic, polyaromatic, and polyphenolic structures) in soils (Fig. 18). This biotic process is consistent with the second stage of OM humification (i.e., synthesis of complex soil organic molecules) as defined by Huang and Hardie (2009). Soil polyketides have their origin through the
Root exudates Plant tissues/ polymers Microbial tissues/ polymers Stage Ifragmentation depolymerization

Soil solutioni.e., organic acids

CO2 Synthesis
Soil microbes + PKSs I, II, III, Hyb + ACoA, MCoA

CO2

Humification

Stage IIstep 1 biosynthesis

Decomposition

Adsorbed/ fixed PKT


O

Stage IIstep 3 abiotic polymerization

H3 C

CH3 CH3

Polyketides (PKS)
O H 3C CH3
CH3 NO 2 CH3 O H O O

NO 2

CH3

Polymerization (inorganic colloids)

ea

Carbohydrates proteins, heterocyclic N

Macromolecules HS and SOM

ea

CUS-PKT in HS and SOM


O H3C CH3 CH3 CH3 O H O O

NO 2

Stage IIstep 2 Abiotic synthesis

Figure 18 A pathway involving biotic and abiotic processes for the humification of oxoacids (i.e., organic acids) into polyketides in soils. PKSs, polyketide synthases; MCoA, malonyl con-enzyme A; ACoA, acetyl co-enzyme A; ea, electrostatic attraction. From C. Monreal (2011, unpublished).

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excretion of secondary metabolites synthesized by crops and soil organisms (microorganisms and protozoa included) into their surrounding soil environment. The concept of microbial humification of soil oxoacids will be tested with published information on the synthesis of microbial polyketides from simple organic acids by complex and gigantic modules of polyketide synthases (PKSs). This chapter demonstrates that biotic humification occurs in soil, and that much remains to be investigated for establishing the most important biontic pathways of humification in soil-plant systems.

11.2. Polyketides in nature


This section reviews published information of polyketides, an important class of natural compounds that have been studied and reported in the scientific literature over the past four decades. Nature produces an amazing variety and number of biosynthesis products, especially secondary metabolites possessing diverse chemical structures. About 100,000 secondary metabolites of molecular weight less than 2500 have been characterized (Roessner and Scott, 1996); and some 50,000 are of microbial origin (Berdy, 1995; Fenical and Jensen, 1993). Figure 19 shows a few selected examples of polyketide compounds including simple and complex
A
O HO H3C HO H3C OH O OH O OH O O CH3 O OH OH CH3 O HO O

B
H N

CH3

Benastatin A C
O OH H3C

Fredericamycin A
Sug O

COOH O NO2 CH3 CH3 O O O OH OH OH O OH

OH

Candicidin/FR-008

Figure 19 Example of polyketides: (A) polyphenolic, (B) polyaromatic, and (C) alkylaromatics synthesized by polyketide synthases in microorganisms, plants, and other living organisms. Redrawn from Hertweck (2009), Scheme 8, p. 4694. Copyright Wiley-VCH Verlag GmbH & Co. KGaA; and from Das and Khosla (2009), Fig. 1, p. 632. Reproduced with permission from publishers.

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aromatic, polyaromatic, polyphenolic, and alkylaromatic compounds that are secondary metabolites synthesized by PKSs in microbial, protozoa, and plant cells. Polyketides are one of the largest and most structurally diverse classes of naturally occurring compounds which include many important compounds that have antihelmintic, insecticidal, antibiotic, and antienzymic properties even at low concentration (Demain, 1999; OHagan, 1991). The rich chemical diversity of polyketides results in part from an evolutionary process driven by selection for acquisition of improved defence against microbial attack or insect/animal predation (Dixon, 2001). Many important polyketide antibiotics are of medical or agricultural use, such as polyenoics, antibiotics, and macrolides. About 1% of all known organic molecules used in medicine are natural products, the rest are manmade, yet it has been recognized that more than a third of all drugs currently in use are at least in part derived from a natural source (von Nussbaum et al., 2006). Polyketides appear to play important functions in ecosystems, and comprise a significant fraction of the total number of microbial metabolites with secondary physiological activities (Cane, 1997). In this chapter and for simplicity, we use the terms alkylaromatics and polyketide alkylaromatics interchangeably.

11.3. Some chemical and functional features of polyketides


Many polyketides produced by living organisms are water soluble, may be synthesized in high yield, and are excreted into the growth media. From a chemical viewpoint, polyketides comprise aromatic, polyaromatic, alkylaromatic, and alkylated molecular structures such as polyphenols, macrolides, polyenes, enediynes, and polyethers (Hertweck, 2009). Plants, animals, and soil microorganisms synthesize polyphenols that include compounds having a moderate solubility in water, and a molecular weight of 5004000 Da, > 12 phenolic hydroxyl groups, and 57 aromatic rings per 1000 Da (Quideau et al., 2011). A macrolide is a large macrocyclic lactone ring to which one or more deoxy sugars, usually cladinose and desosamine, may be attached. The lactone rings are usually 14-, 15-, or 16-membered. Polyenes consist of long alkylated-aromatic moieties that contain one or more sequences of alternating double and single carboncarbon bonds. Polyenes and macrolides are biosynthesized by several strains of Streptomyces as antibiotics mostly targeting fungi but also bacteria (Dixon and Walsh, 1996). Enediynes have nine- and ten-membered rings containing two triple bonds separated by a double bond (Nicolaou et al., 1993). Much of the published information existing on polyketides relate to organic compounds that provide ecological protection and advantages to soil microorganisms and plants during periods of environmental and abiotic stress. From a functional viewpoint, polyketides include a large class of diverse compounds, including antibiotics, pigments, and regulatory substances

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associated with sporulation and cell differentiation processes. The biomolecular activity of polyketides may depend on the particular chemical structure rather than its concentration. For example, an alkylaromatic occurring in large amounts may not be as significant as an end product, but it might be important antagonistic intermediates during their decomposition (Firn and Jones, 2003). The latter needs special consideration when conducting ecological studies of the metabolism and dynamics of polyketides in soil systems. The energy content stored in the bonds of polyketides is high due to the aromatic and alkylaromatic nature of their molecular structures. Thermodynamically, excreted polyketides appear not to be the best substrate sources to provide carbon and energy during microbial growth in soils.

11.4. Chemical analysis of polyketides in soils


The chemical analysis of highly diverse soil alkylaromatic structures of microbial origin presents a significant challenge to soil and other scientists. Environmental chemists are continuously challenged in the analysis of bioactive substances in soils because these compounds present strong sorption to SOM and clay minerals, and the inherent complexities of soil ecosystem properties that vary from region to region (OConnor and Aga, 2007). Depending on the molecular properties of polyketides, such as solubility, polarity, or amphiphilicity, it is necessary to use different chemical extractants, and methods for sample clean up, separation and quantification (OConnor and Aga, 2007; Stoob et al., 2006; Turiel et al., 2006). For example, compound separation involves various techniques including liquid chromatography coupled with detection by UV, Diode Array Detection, and techniques of mass spectrometry (Hamscher et al., 2002). Thiele-Bruhn (2003) has reported and summarized analytical methods for analyzing soil antibiotics using various techniques of mass spectrometry, which were developed for natural and synthetic polyketide antibiotics. State-of-the-art Py-FIMS and field desorption mass spectrometry (FD-MS) have been used in pioneering the identification of several aromatic, phenolic, and alkylaromatics in HS and SOM, although the origin of these soil chemical structures remains unknown.

11.5. Biosynthesis of polyketides


The large chemical diversity of polyketides is derived from the biotic polymerization of some of the simplest building blocks available in nature, such as acetic acid or propionic acid, and occasionally, butyric acid (Hertweck, 2009). Many organisms including among other, protists, plants, and bacteria produce polyketides by PKSs. A set of b-ketoacylsynthase (KS), acyltransferase (AT), and acyl carrier protein (ACP) domains, as well as optional b-keto processing domains constitute a PKS module, and generally each module is responsible

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Fatty acid (FAS) or polyketide synthase (PKS) CO2H O MT SCoA R O SCoA AT KS = Ketosynthase KR = Ketoreductase DH = Dehydratase ER = Enoyl reductase ACP = Acyl carrier protein AT = Acyl transferase MT = Malonyl transferase

CO2H O

SACP R -CO2

O SKS

Acyl transfer

Condensation
KS

O R

Reduction
SACP KR R

OH

Dehydration
SACP DH R

Reduction
ER SACP R

O SACP

B A
O H3C O O SX R O O OH

C D
O SX R n O OH

Polyketide

Reduced polyketide

Fatty acid

Figure 20 Basic mechanisms involved in polyketide and fatty acid synthesis by PKSs and FASs, respectively. (A)(D) represent the alternative versions of the reductive cycle that lead to keto, hydroxy enoyl, or methylene functionality, respectively, at specific b-carbons during the assembly of reduced polyketides. Note that the starter unit may be acetyl coenzyme A (CoA) (where R CH3) or an alternative CoA ester for all classes of synthases, while the chain extender unit is malonyl CoA for the synthesis of fatty acids and aromatic polyketides, but varies for reduced polyketides. From Hopwood (1997), Fig. 13, p. 2465. Reproduced with permission of the American Chemical Society.

for only one elongation cycle (Fig. 20) (Hopwood, 1997). The domains of a complete PKS include the starting or loading module: AT-ACP-; the elongation or extending modules: -KS-AT-[DH-ER-KR]-ACP-; and the termination or releasing module: -TE, where DH, dehydratase; ER, enoyl reductase; KR, keto reductase. The polyketide chain and the starter groups are bound with their carboxy group to the SH groups of the ACP and the KS domain through a thioester linkage: R C( O)OH HS protein , R C ( O)Sprotein H2O. The growing chain is handed over from one SH group to the next by trans-acylations and is released at the end by hydrolysis or by cyclization (http://www.chemistrydaily.com/chemistry/Polyketide). The chemical steps of chain extension and corresponding enzymatic catalysis are strikingly similar to those of fatty acid synthases (FASs; Cane et al., 1998; Hopwood and Sherman, 1990). All polyketides are the result of successive rounds of PKS activities catalyzing the Claisen condensation of a starter coenzyme A (CoA) ester, such as acetyl-CoA, with extender CoA esters, such as malonyl-CoA (Fig. 21) (Hopwood, 1997). The unraveling of the biosynthetic origins of polyketides started when radioactive isotopes of C and H and 13C NMR

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Loading Module 1
KR AT ACP KS AT

Module 2 Module 3

Module 4 Module 5
ER DH KR ACP KS AT KR ACP

Module 6 TE
KR KS AT ACP TE

KR ACP KS AT ACP KS AT ACP KS

AT

S O O HO

S O HO HO mMCoA mMCoA

S O O HO HO mMCoA

S O

S O HO

S O HO HO O HO HO mMCoA O HO HO mMCoA

S O

O HO HO mMCoA

OH O O OH OH

6-dEB (1)

Figure 21 The deoxyerithronolide-B-synthase, giant multimodular megasynthases, required for erythromycin biosynthesis as an example of a modular type I PKSs in prokariots. From Hertweck (2009). Scheme 2, p. 4691. Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission from the publisher.

became widely available and were used to conduct the first microbial in vitro studies in the early 1970s. In the 1980s and 1990s, there were many published studies resulting in the identification of the fundamental precursors of these secondary metabolites and the first detailed information on the manner in which the individual building blocks were assembled and subsequently modified inside microbial cells (Hopwood, 1997; Robinson, 1991). The PKS catalyzed reactions involve chain elongation or b-ketoacyl synthesis, followed by some or all of a sequence of ketoreduction, dehydration, and reduction (Thomas, 2001), although the reduction steps are optional (Hopwood, 1997). The PKSs can use a broad range of building blocks and are also involved in the formation of various chain lengths (Hertweck, 2009). Most aromatic polyketide backbones are synthesized by the minimal PKS involving four dissociated enzymes acting mainly through decarboxylation of a malonyl building block, although other polyketides may be derived from nonacetate sources (Tang et al., 2004). Different types of multifunctional PKSs are classified as type I, type II, type III, and hybrid modules. These PKSs produce the complex and diverse polyketide molecules based on their protein architecture (Chopra et al., 2008; Hertweck, 2009). Type I PKSs are multifunctional enzymes that provide a construction plan for the assembly of complex structures from simple carbon building blocks (i.e., acetic acid). A minimal PKS module contains at least KS, AT, and ACP domain and b-keto acyl synthase (b-KS) activities. Type I PKSs are giant megasynthases subgrouped as iterative or modular; found mostly in bacteria but also in protozoa and fungi (Hertweck, 2009; Moore and Hopke, 2001; Moss et al., 2004). The active sites of type I PKSs are organized linearly into modules, such that each module catalyzes one cycle in the assembly and

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modification of polyketide elongation. Once a module has acted on a nascent acyl chain (i.e., removed one or more OH groups from an oxoacid) it is passed to the next downstream module. Type II PKSs are a system of individual enzymes that carry a single set of iteratively acting activities to synthesize aromatic polyketides (Olano, 2011). A minimal set consists of two ketosynthase units (a- and b-KS) and an ACP, which serves as an anchor for the growing polyketide chain. Additional PKS subunits such as ketoreductases (KRs), cyclases, or aromatases define the folding pattern of the polyketo intermediate and further post-PKS modifications, such as oxidations, reductions, or glycosylations are added to the polyketide (Hertweck et al., 2007; Rix et al., 2002). The only known group of organism that employs type II PKS systems for polyketide biosynthesis nchez and Verpoorte, 2009; Hertweck, 2009; Seow is bacteria (Flores-Sa et al., 1997). Type III PKSs are the condensing enzymes that catalyze the synthesis of aromatic polyketides mainly in plants, but also in fungi and bacteria (Austin and Noel, 2003; Austin et al., 2004; Funa et al., 2007; Seshime et al., 2005). Type III PKSs are condensing enzymes that lack ACP and act directly on acyl-CoA substrates. The specificity of AT for malonyl-CoA, methylmalonyl-CoA, or other a-alkylmalonyl-CoAs determines which carbon extender is used. Since the latter two substrates have a chiral center, their incorporation gives different stereoisomers of the prolonged polyketide chain. After condensation of an activated acyl starter unit with malonylCoA-derived extender units, the oxidation state of the b-carbon is either kept as a keto group or modified to a hydroxyl, methine, or methylene group by the optional activity of KR, DH, and ER domains ( JenkeKodama et al., 2006). Further, molecular variability of polyketides comes from the existence of two types of KR domains that create different stereoisomers regarding the chiral b-carbon (Caffrey, 2003). So far, most of the type III PKSs has not been characterized fully. Hybrid PKSs has also been identified as one of the types of PKSs, where modules from type I PKSs are linked, for example, to nonribosomal peptide synthetase (NRPS) modules, which result in the production of polyketide peptide hybrid metabolites, such as phenolic lipids that appear to play an important role as minor components in the biological membrane in various bacteria. Other hybrid systems include type III/type I, type I/type II, and FAS/PKS hybrids (Hertweck, 2009). For example, the aromatic moieties of the phenolic lipids in Azotobacter vinelandii are synthesized by two type III PKSs, ArsB and ArsC, which are encoded by the ars operon. In turn, the aliphatic moieties arise from C22C26 fatty acids that are synthesized from malonyl-CoA by two type I FASs, ArsA and ArsD, which are members of the ars operon. Miyanaga et al. (2008) showed through in vivo and in vitro reconstitution of phenolic lipid synthesis systems with the Ars enzymes that the C22C26 fatty acids produced by ArsA and ArsD remained attached to the

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ACP domain of ArsA and were transferred hand-to-hand to the active-site cysteine residues of ArsB and ArsC. The type III PKSs then used the fatty acids as starter substrates and carried out two or three extensions with malonyl-CoA to yield the phenolic lipids. The phenolic lipids in A. vinelandii were found to be synthesized solely from malonyl-CoA by the four members of the ars operon. Miyanaga et al., (2008) were the first to demonstrate that a type I FAS interacts directly with a type III PKS through substrate transfer. In some cases, the hybrid enzymes could be either inactive or catalytically inefficient (Kennedy and Hutchinson, 1999). The genes encoding for the biologically active polyketides are important because their presence express the capacity of various soil microorganisms to synthesize polyketides that are excreted into the surrounding soil, thus contributing to the structure of SOM. The encoding genes for active polyketides are located within DNA stretches of 20 kb to more than 100 kb. The genes for antibiotic biosynthesis are frequently located near one or more genes mediating resistance to the corresponding antibiotic (Dworkin, 2006). It has been revealed that genes ArsB and ArsC express type III PKSs in A. vinelandii, capable of catalyzing the synthesis of alkylresorcinols and alkylpyrones, respectively, which are essential for encystment as the major lipids in the bacterial cyst membrane (Funa et al., 2006). In Streptomyces griseus, the srs operon consisting of srsA, srsB, and srsC is responsible for the synthesis of methylated phenolic lipids derived from alkylresorcinols and alkylpyrones (Funabashi et al., 2008). The srsAB- and srsABC-like operons are distributed in both Gram-positive and -negative bacteria (Nakano et al., 2009). The phenolic lipids synthesized by the complex of srs enzymes confer resistance to b-lactam antibiotics (Funabashi et al., 2008). The microbial alkylaromatic structures have longer alkyl chains than those in Fig. 4. Noteworthy, Mu ller (2004) indicated that the classification of PKSs into the four types is too limiting because a growing body of evidence showing nature realized limitless transitional stages between differently classified biosynthetic systems during evolution. Although there are only four known module architectures, which are classified as type A, B, C, and D (Fig. 22), the potential permutation of combining the different structural variants gives an enormous diversity of polyketide molecular structures (Fig. 23). Theoretically, a PKS system comprising six elongation modules could produce more than 100,000 possible polyketide chemical structures (GonzalezLergier et al., 2005).

11.6. Ecological function and associated genetic evolution of polyketides


The diversity of plants and microorganisms in soil ecosystems is enormous, however, only a minor proportion of soil bacteria, fungi, and actinomycetes have thus far been cultured and examined for secondary metabolite

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Module type

KS

AT

DH

320

ER

KR

ACP

KS

AT

70

DH

320

KR

ACP

KS

AT

KR

ACP

KS

110

AT

90

ACP
R R R R

determines

OH (L or D configuration)

Figure 22 Different module types of modular PKSs and their influence on the structure of the polyketide backbone. The numbers written between domains give the typical length of the respective interdomain region in terms of amino acid residues. Adapted from Jenke-Kodama et al., (2006), Fig. 1, p. 1211. No permission required to reproduce, www.ploscompbiol.org.
OH
CH3

CH3 O Sug

H O O

CH3 CH3 H CH3


H3C CH3

CH3

CH3

CH3 OH
O OH

OH CH3 CH3

H3C O HO O H

OH
H

O O

H3C
OH OH

H3C

O O

NH OH

H3C

N
O H OH CH3

H COOH

HO

OH

O H3C CH3 CH3

OH H3C

OH OH O CH3
O Cl Cl Cl

CH3 O
O

O O
CH3 CH3

NO2

CH3

(CH2)8COOH

HO
O O H3C O O CH3 OH OH O

H3C

O O

C CH3 O H3 O CH3

OH

H3CO

CH3 CH3

CH3

CH3

CH3

CH3

H3C CH3 H3C

CH3 CH3 OH O O

CH3 H3C HO O H3C NH H3C


CH3

H3C HO COOH

NH O H3C
H3C CH2 S N H3C CH3 O CH3

H3C CH3 H3C O

Figure 23 A few selected examples of the chemical diversity of polyketides. Redrawn from Hertweck (2009). Cover page, p. 4688. Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Adapted with permission from the publisher.

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production. Only 5% of the total number of fungal species has been described, and of those described (69,000), only 16% (11,500) have been cultured (Hawksworth, 1991). Differences of opinions have existed with respect to the potential ecological functions of secondary metabolites in living organisms. Some scientists have been of the opinion that most microbial natural products had no function in enhancing the fitness of the producer organism and they were considered as waste products or accidents of metabolism (Bennett, 1995). Other chemical ecologists adhere to the view that natural product diversity is associated with processes of genetic selection allowing chemical protection against the attack by other living organisms (Fraenkel, 1959). Fungi, in particular, but also actinomycetes, have successfully used their chemical arsenal against bacterial competitors for millions of years (Firn and Jones, 2003). As cited by Hartman (2008), Ernst Stahl was an early pioneer of chemical ecology, who in 1888, suggested that plants use chemical protective means to control competitors and aggressors. Lately, it has been reported that bacteria can differentiate between surrounding microorganisms and tailor their responses to the presence of competing microorganisms, as well as a variety of responses targeted to specific bacteria (van Ooij, 2011). Molecules that kill their living neighbors or prohibit their replication have also coevolved with their producers and their resistance strategies. Based on the warfare view for the ecological role of natural products, diverse soil polyketides represent key weapons of living organisms in the continuous struggle for ensuring vital space, and access to carbon, nutrients, and energy resources in terrestrial ecosystems. From an evolutionary view point of the molecular diversity of polyketides and SOM, it is important to know whether repetitive PKS modules within a single genome has an impact on the diversification of the polyketide products synthesized by microorganisms and other living cells in soil ecosystems. Data obtained from phylogenetic studies of Streptomyces avermitilis shows that homologous recombination of DNA has led to exchange, loss, and gain of domains and domain fragments and hence to a natural reprogramming of the PKS assembly lines ( Jenke-Kodama et al., 2006). It is also plausible to suggest that the chemical arsenal of synthesized polyketides excreted by soil microbial cells and other living organisms into their surrounding milieu has had an important effect on the humification processes and chemical quality of SOM during the thousands of years of pedogenesis.

11.7. Plant polyketides


Plants have evolved adaptive processes that involve the production and use of many secondary metabolites to perceive and translate that perception into an adaptive response to repel attacks from fungi, bacteria, nematodes, and insects (Dangl and Jones, 2001; Dixon, 2001). Plants produce five groups of

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secondary metabolites: polyketides, isoprenoids (e.g., terpenoids), alkaloids, phenylpropanoids, and flavonoids, many in response to environmental stress nchez and Verpoorte, 2009; Pankewitz and Hilker, 2008). Rice, (Flores-Sa corn, soya bean, Arabidopsis, and the model legume Medicago truncatula are rich sources of antimicrobial indole, terpenoid, benzoxazinone, and flavonoid and isoflavonoids (Dangl and Jones, 2001). Flavonoids, which are polyphenols, are synthesized in plants by the combination of polyketides and phenylpropanoids (Verpoorte and Memelink, 2002). The compounds of the phenylpropanoids family are synthesized predominantly by plants but also by endophytic Penicillum brasilianum (Pacheco et al., 2010). Chalcone synthase (CHS) catalyzes the first step in the synthesis of flavonoids which uses malonyl CoA and 4-coumaroyl CoA as substrates. CHS is a family of plant PKSs that accumulates transcripts and produces chalcones (i.e., aromatic ketones) in response to stimuli such as pathogen attack (Arias et al., 1993; Meier et al., 1993), light and UV light (Batschauer et al., 1991; Knogge et al., 1986), wounding (Lawson et al., 1994), elicitor treatment ( Junghans et al., 1993), and symbionts (Harrison and Dixon, 1993). A few polyketides have physiological functions in plants (i.e., spatial pattern of cell development in plant embryos) (Seigler, 1998). Attack by pathogens to plants causes accumulation of flavonoids and isoflavonoids, and their importance as antibiotic phytoalexins is well established (van Etten et al., 1989; van Etten and Pueppke, 1976). The flavonoid phytoalexins, known to be produced in response to invasion by fungi and bacteria, have been found and described predominantly in legumes, but have also been identified as major phytoalexins in cereals like wheat, sorghum, and rice (Feng and McDonald, 1989; Hipskind et al., 1990; Ioset et al., 2007; Kodama et al., 1992; Seigler, 1998). In barley, flavonoids are present at constitutive levels in the epidermis and mesophyll of leaves (Fro st et al., 1977; Seikel et al., 1962), and, as seen in other species, illumination with UV light prompts the accumulation of flavonoids in barley and rye leaves (Christensen et al., 1998; Haussuehl et al., 1996; Lanz et al., 1991; Reuber et al., 1996). The presence of gchs2 indicated that CHSrelated enzymes are involved in the biosynthesis of pyrone (a heterocyclic with five carbon and one O atom and two double bonds), that confer plants resistance to insects and pathogens (Eckermann et al., 1998). Pankewitz and Hilker (2008) published and extensive review article on the ecological role and evolutionary aspects of polyketide biogenesis in insects. The chemical structures of plant polyketides and flavonoids have structural and bond similarities with respect to their aromatic and alkyl moieties, and bioactivities against other living organisms. The input of the latter two types of natural products into soils may contribute with basic carbon skeletons to the formation of a central unit structure (CUS) in HS and SOM. Little is known, however, about the ecological effects on soil biota by plant secondary metabolites, especially once plant residues containing

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polyketides on their tissue surfaces are incorporated into the soil. The potential microbial utilization or decomposition of plant polyketides in soils is also unknown, as well as pathways for incorporating plant polyketides into soil HS and SOM (Fig. 18).

11.8. Microbial polyketides


Prokaryotic microorganisms are the largest reservoir of genetic diversity on earth, yet to date only a small fraction (may be 0.11%) of all microbes have been cultured and rigorously described (Wawrik et al., 2005). Of the 12,000-polyketide antibiotics compounds known in 1995, 55% were produced by soil actinomycetes of the genus Streptomyces, 12% from nonfilamentous bacteria, 11% from other actinomycetes, and 22% from fungi (Berdy, 1995; Strohl, 1997). In general, the accumulation of inducible antagonistic microbial compounds is often orchestrated through signal transduction pathways linked to perception of the pathogen by receptors encoded by host resistance genes (Dangl and Jones, 2001). To exert an effect, most of the antimicrobial compounds must penetrate cell membranes and attack at least one adversarial molecular target (Nikaido, 2003). Most polyketides when excreted into their surrounding disrupt protein synthesis, DNA replication, and the integrity cell membranes of microorganisms lacking resistance (Hertweck, 2009). Microbial polyketides including several polyaromatic compounds such as anthracyclines, with medicinal application, are synthesized primarily by Streptomyces peucetius, a soil actinomycete (Alekhova and Novozhilova, 2001; Das and Khosla, 2009). Polyketides are also synthesized by fungal and bacteria species commonly found in soil systems (i.e., Penicillium sp., Aspergillus sp., Mycobacterium sp.). The capacity to produce polyketides in vitro varies and depends on the species, strain type, and other abiotic factors, and may range from a few mg ml 1 to thousands mg ml 1. For example, Streptomyces globisporus may produce from 1.3 to 14 mg ml 1 of a nine-member ring enediyene over 5 days of growth (Horsman et al., 2010). Relative to other Streptomyces species and strains, the strain N2 of Streptomyces chrysomallus displayed an increased capacity to biosynthesize polyketide antibiotics during several days of growth in vitro. S. chrysomallus was able to produce 730 mg ml 1 of actinomycin after 5 days of growth; and the production of actinomycin by several producing streptomyces strains, their mutants and genetically transformed strains ranged from 25 to 1120 mg ml 1. The minimum inhibiting concentration of actinomycin was 5000 mg ml 1 for the same species. Other polyketide compounds, however, inhibit the growth of S. chrysomallus at concentrations ranging from 15 to 50 mg ml 1 (Alekhova and Novozhilova, 2001). Microbial synthesized alkylaromatics are also named phenolic lipids in the published literature. For example, alkylresorcinols and alkylpyrones,

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which have a polar aromatic ring and a hydrophobic alkyl chain, are found in fungi, bacteria, and plants (Miyanaga et al., 2008). Su et al. (1981) identified the synthesis of 10 phenolic lipids in A. vinelandii during encystment because of carbon substrate induced stress. Encystment and sporulation is a frequent process carried out by bacteria and other microorganisms under adverse nutrient and environmental conditions in soils. During encystment, the phospholipids of cell membranes are replaced by a family of 5-n-alkylresorcinols and 6-n-alkylpyrones (Reusch and Sadoff, 1983). The encystment of A. vinelandii, a Gram-negative, free-living nitrogenfixing soil inhabitant, can be induced by transferring exponentially growing cells from glucose to 0.2% b-hydroxybutyrate. The encystment is completed within 46 days and results in the conversion of more than 90% of the vegetative cells into cysts (Lin and Sadoff, 1968). Lipid phenols are synthesized at a time in the differentiation process when lipids are turning over (Reusch and Sadoff, 1979, 1981). Su et al., (1981) identified various phenolic lipids in A. vinelandii including 6-n-eneicosylresorcylic acid methyl ester and 6-n-tricosylresorcylic acid methyl ester, 5-n-(2-hydroxy) heneicosylresorcinol and 5-n-(2-hydroxy)tricosylresorcinol, 5-n-heneicosyl-4-acetylresorcinol and 5-n-tricosyl-4-acetylresorcinol, 6-n-heneicosyl4-hydroxypyran-2-one and 6-n-tricosyl-4-hydroxypyran-2-one, and 6-(2oxotricosyl)-4-hydroxy-pyran-2-one and 6-(2-oxopentacosyl)-4-hydroxypyran-2-one. The polymethylene chains of the cyst lipids average 2.7 nm in length and 57 nm3 in volume, which are larger in size than the phospholipids of vegetative cells (1.9 nm length and 40 nm3 volume); and the proportion of lipids allocated into the cyst membrane was equivalent to 95% of the total lipids found in cysts (Reusch and Sadoff, 1983).

11.9. Polyketides in soils


This section presents and discusses published information on the production of soil polyketides, the effects of antibiotics on soil microbial activity, and the effect of soil management and the importance of adsorption reactions on soil polyketides. In spite of their natural occurrence, the biochemistry and chemistry of microbial polyketides in vivo soils has received little attention. Most research on polyketides and the microorganisms responsible for their syntheses has been conducted in vitro in the laboratory. Soils are a source of many and diverse bioactive compounds involving polyketide structures that are produced by indigenous microorganisms (Gottlieb, 1976; Thomashow et al., 1997). About 3050% of actinomycetes isolated from soil are capable of synthesizing antimicrobial compounds (Topp, 1981). In soil-plant ecosystems, the interactions of living organisms with their surroundings are complex and involve plants, microorganisms, insects, animals, and inorganic colloids. In soils, the microbial synthesized polyketide antibiotics help the microbial producer to gain access to nutrients and the suppression of plant

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root pathogens, among other benefits. For example, isoepoxydon is an alkylaromatic compound synthesized by fungi and actinomycetes that causes growth interference between Poronia puntata, a late colonizer of animal dung and earlier colonizer species of Ascobolus furfuraceous and Sordaria fimicola (Gloer and Truckenbrod, 1988). Harris and Woodbine (1967) examined a total of 560 bacterial isolates from four rhizosphere and eight nonrhizosphere soils and tested them for resistance to seven different antibiotics. They found marked differences between the overall levels of antibiotic resistance in the different soils. The bacteria isolates inhabiting the rhizosphere were more susceptible to antibiotics than the corresponding bacteria from nonrhizosphere soils. The latter authors also showed that only 210% of the total soil bacterial isolates showed resistance to the seven antibiotics. In comparison, the bacteria populations in the rhizosphere of 10 plant species were shown to have a greater resistance to streptomycin than the comparable bacteria from nonrhizosphere soil when assessed by a plate count technique Brown (1961). Penicillin activity added to unsterilized soil at 1000 mg g 1 completely disappeared after 2 weeks (Pramer and Starkey, 1951). The latter study, however, did not determine the potential effects of biotic decomposition and adsorption reactions by soil colloids on the disappearance of streptomycin. Other studies show that microbial populations in various soils differ in their reactivity to antimicrobial compounds (Harris and Woodbine, 1967; Williams and Davies, 1965); and there is no universal antibiotic or set of antibiotics capable of inhibiting all the soil heterotrophic activity. The optimum concentration of polyketide antibiotics and their degradation susceptibility need to be investigated before conducting CO2 soil respiration studies (Anderson and Domsch, 1973). Soil respiration studies show a dose response to added antibiotics after 24 and 48 h of incubation in selected soil samples (Thiele and Beck, 2001). There is little and inconclusive evidence demonstrating the proportion and type of soil microorganisms that are susceptible or resistant to microbial polyketides in situ. Published information clearly indicates, however, that some soil organisms and bacteria isolates have resistance genes to protect themselves from polyketides secreted by other living organisms in soils. The synthesis and stabilization of various polyketide antibiotics were studied in sterilized and unsterilized soil samples amended with organic residues and inoculated with Streptomyces aureofaciens and Streptomyces rimosus. The maximum amount of aureomycin found in the soybean amended sterilized soil samples was 0.15 mg g 1 soil, and that in unsterile amended soil samples was 0.03 mg g 1 soil after 16 days of incubation. In comparison, the amount of terramycin in the same soil treatments was about six times greater. After 30 days of incubation, the amount of aureomycin was reduced by about 50% in sterile soil and by nearly 90% in unsterile soil (Soulides, 1965). The antibiotic glycotoxin produced by the soil saprophyte fungus,

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Gliocladium virens, to control soil borne pathogens was detected at concentrations between 0.2 and 0.37 mg cm 3 soil in clay and composted mineral soil (Lumsden et al., 1992). Several antibiotic compounds persist in the environment and are not transformed (Winkler and Grafe, 2001). Antibiotic residues given to animals for treatment of illness or growth promotion are found in soils, after manure application, at concentrations of 0.02 mg g 1 soil (Aga et al., 2005; De Liguoro et al., 2003; Hamscher et al., 2002). In a different study, the concentrations of residual tetracyclines in soils ranged from 0.025 to 0.105 mg g 1, but sulfonamide antibiotics were not detected (Cengiz et al., 2010). Polyketides are strongly adsorbed to soils organic and inorganic particles. Strongly adsorbed antibiotics to soil colloids may be leached from soils through preferential transport in macropores (Thiele-Bruhn, 2003). The strong adsorption of polyketide antibiotics to OM and clay colloids in soils is associated with charge transfer and ionic interactions and not to hydrophobic partitioning. The distribution coefficient for the adsorption of antibiotics to soil materials varies widely depending on soil type and on the chemical structure and amount of the compound, and as expected, sorption is stronger in clay than in sand particle size fraction (Thiele et al. 2002; Thiele-Bruhn, 2003). Aminoglycosides, tetracyclines, and tylosin are more strongly adsorbed to expandable than to nonexpandable clay minerals while desorption of aminoglycosides was not observed (Bewick, 1979; Pinck et al., 1961a,b). Effects of antibiotic addition on soil organisms include shifts in microbial communities and development of resistance under frequent and high addition of these compounds to soils via animal manures. The reviewed published information clearly indicates that indigenous polyketide antibiotics and those of added to soil through manures or other animal residues results in rapid adsorption and fixation by soil organic and inorganic components, and thus their biological utilization and decomposition is largely prevented in soil ecosystems.

11.10. A microbial-PKSs model for studying biotic humification in soils


The soil solution in native and cultivated amended and unamended soils is a key central and fast cycling SOM pool (Monreal and McGill 1989a,b). In the path for enhancing our knowledge on the biotic humification of soil organic molecules residing in soil solution, it will be extremely useful to identify and establish a model or a few models of representative soil microorganisms and PKSs. Actinomycetes and fungi in general, and the genera Streptomyces in particular, are active in synthesizing polyketides in soils compounds that also serve as biocontrol agents against fungi in crop rhizospheres (Blaak and Schrempf, 1995; Yuan and Crawford, 1995). Streptomyces constitute the most abundant (> 90%) soil streptomycete, and soils rich in

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SOM such as grasslands contain the largest number of these filamentous bacteria that live under various soil conditions including different pH (Schrempf, 2006). Soil streptomycetes produce compounds with antibacterial and antifungal activities and are active during the initial decomposition of OM (Kutzner, 1981; Dworkin, 2006). Soil is the most important habitat for streptomycetes, and most soils contain 104107 colony forming unit g 1 soil, representing 120% or more of the total viable counts. Over 20 genera of soil actinomycetes were isolated and reported by Lechevalier and Lechevalier (1967). Other important genera of soil actinomycetes appearing in culture media include Micromonospora, Rhodococcus, and Streptosporangium (Williams and Wellington, 1982). The rhizosphere of soil-crop systems is a location with high microbial diversity and activity, and carbon, energy, and nutrient fluxes, and it is an ideal place to study the increased synthesis of polyketides by Streptomyces and PKSs. The scientific community has been improving the standard cultivation techniques of soil microbes by developing better methods for the efficient identification and screening of pure culture actinomycetes, and also of soils containing PKSs and secondary metabolites potentially useful for industrial application. For example, molecular methods using DNA tools are useful to detect Streptomyces strains colonizing and producing streptomycin in the rhizosphere of soybeans (Huddleston et al., 1997). The streptomycin biosynthetic gene cluster of two species, S. griseus and S. glaucescens, has been well studied (Distler et al., 1992). Genes for resistance and biosynthesis have also been cloned and characterized (Piepersberg, 1995). For streptomycin resistance, the gene cluster is comprised of over 30 genes, including an aminoglycoside phosphotransferase gene, strA (Huddleston et al., 1997). Metsa -Ketela et al. (1999) obtained the partial sequences of PKS genes from six known and 29 unidentified soil actinomycete isolates using degenerate PCR primers. Pigment- and antibiotic-producing PKS gene clusters could be clearly distinguished based on their KSa sequences, indicating that functional information may be derived from PKS sequence data. A different study by Metsa -Ketela et al., (2002) found that the phylogenies of 16S rRNA and PKS genes in actinomycete soil isolates were not congruent, indicating that the phylogenetic grouping of actinomycetes is an inadequate predictor for the type of secondary metabolites these strains produce. A study conducted to directly access PKS gene soil diversity by terminal restriction fragment length polymorphism (TRFLP) patterns found that some soil samples contained a particularly rich and unique actinomycetes community; and that the same soil samples also contained the greatest diversity of KSa genes as determined by TRFLP analysis of KSa PCR products (Wawrik et al., 2005). These workers identified seven novel clades of KSa genes by vector cloning. Greatest sequence of diversity was observed in a sample containing a moderate number of peaks in its KSa TRFLP.

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Cluster of sequences were most similar to the KSa involved in the production of several antibiotics such as ardacin, angucycline, simocyclinone, pradimicin, and jasomycin. Work conducted by several scientists on phenolic lipids has involved characterization of phenol lipids and PKSs associated in cysts of A. vinelandii (Miyanaga et al., 2008). A molecular technique approach that helps confirm the occurrence of given soil biotic process is the characterization of soil mRNA to measure the gene expression controlling the synthesis of specific proteins (Nannipieri et al., 2010). The confirmation of the humification pathway for the biontic production of specific soil polyketides as intermediate or end products may be confirmed by characterizing the expression of genes associated with the complex PKS systems in model soil fungi or actinomycetes. Important progress has been made on the development of methods for the characterization of mRNA and so detecting the expression of gene sequences in soil (Krsek et al., 2006). Accordingly, A. vinelandii, Streptomyces sp., associated PKSs complexes, and polyketide products, such as phenol lipids, may be used as microbial-gene-enzyme-product models to elucidate the role of PKSs in the formation of alkylaromatic or polyaromatic structures from simple soil oxoacids (first step of humification synthesis, Fig. 18), and to elucidate their role as basic backbone structures contributing to the formation of the CUS of HS and SOM, and complementary physicochemical reactions between polyketides with colloidal surfaces and other organic molecules (second step of humification synthesis, Fig. 18).

12. Thermodynamic, Energy, and Kinetic Considerations


Determining the energy, kinetic, and thermodynamic relationships of soil microbial metabolism is basic to the understanding of interactions between the activities of microbial communities, available substrates, and the humification processes associated with polyketide synthesis and decomposition in their geochemical environments. Microbial intracellular enzymes capture the free energy in chemical bonds of substrates to conduct work for the maintenance, growth, and transport of organic and inorganic species across the cell membrane during anabolic and catabolic processes (Russell and Cook, 1995). Adenosine triphosphate (ATP) is the cells energy currency to conduct work, and it is mostly produced through oxidative phosphorylation during catabolic cell respiration processes in aerobic and anaerobic cells. To a lesser extent, ATP is also produced by substrate-level phosphorylation occurring during glycolysis and the Krebs cycle. Part of the energy produced is conserved by translocating protons across the membrane and synthesizing ATP from adenosine diphosphate

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and inorganic phosphorus (Senez, 1962). The complete microbial oxidation of glucose in vitro produces 38 ATP mol per mol (Brock and Madigan, 1991) and releases 2881 kJ mol 1 of free energy. Conversely, the anaerobic fermentation of glucose produces 2 molecules of ATP and releases 180 kJ mol 1. Bacteria require about 70 kJ mol 1 of free energy to conserve 1 mol of ATP under the physiological conditions of a cell ( Jackson and McInemey, 2002). Depending on the intracellular pH, between 10 and 12 kcal ( 41.8 and 50.2 kJ) is required for the synthesis of 1 mol of ATP from ADP and Pi in anaerobic bacteria (Thauer et al., 1977). The average heat of combustion (energy content) of whole Pseudomonas fluorescens cells was 22.3 kcal (93.2 kJ) g 1; and this average energy content is similar to values obtained for other heterotrophs (Powers et al., 1973). The biontic humification pathways for soil polyketide synthesis from simple oxoacids residing in soil solution require consistency with the laws of thermodynamics and kinetic principles. In general, soils are characterized by having small amounts of available carbon sources, and thus the production of chemical free energy for biological work is limited, thus deviating little from thermodynamic equilibrium ( Jin and Bethke, 2007). A minimum quantum of free energy (12.7 2 kJ mol 1) sustains microbial metabolism of acetate and butyrate by the synthropic fermentative bacteria Synthrophomonas wolfei and Desulfovibrio strain G11; and surprisingly, this free energy value was lower than the 20 kJ mol 1 for thermodynamic limits predicted by Jackson and McInemey (2002). The latter authors concluded that substrate and bacterial metabolism can proceed near thermodynamic equilibriuma condition that is often thought to be a biological improbability. Under conditions of limited free energy, metabolic reactions must proceed in the forward and reverse (or branched) directions to have consistency with thermodynamics, such as data reported from physicochemical studies , 2005; Wolfe, 2005). Most microbial respiration studies conducted (Pekar in vitro only show forward directions with high rate of reactions because abundant substrate is used to produce high levels of chemical free energy (Monod, 1949). With low free energy levels, however, the rate of reaction for soil microbial metabolism is defined by the difference between the forward and reverse rates ( Jin and Bethke, 2007). As a contribution to the study of evolution of SOM, Tardy et al. (2005) indicated that the thermodynamic properties and stability of soil HSs may be delineated as a function of polymerization indexes. Others have examined the thermodynamic and free energy relations for the sorption onto clay colloids of aromatic, soil HSs, and contaminants (Chin and Weber, 1989; Faria and Young, 2010; Ghabbour et al., 2004); but no work has been associated with the thermodynamics of secondary metabolites in soils. There are several articles and books published with information on thermodynamic properties including enthalpies of reaction and formation

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for single organic compounds (Rossini et al., 1952; Wagman et al., 1982; Yaws, 1999), but not for natural compounds such as polyketides. The authors of this chapter used the modeling approach of Banfalvi (1999) to estimate the relative energy contents (kJ mol 1) for two polyketides, tetracenomycin and a triketide pyrone, both biosynthesized from acetic acid. Tetracenomycin (C20H12O8; MW 366 g mol 1) is a four-member polyaromatic ring structure with eight double bonds synthesized by S. glaucescens, a Gram-positive soil bacterium (Tang et al., 2004). The triketide pyrone is an alkylaromatic polyketide (C21H43; MW 295 g mol 1), produced in vegetative cells and cysts of A. vinelandii (Miyanaga et al., 2008). The estimated energy content for tetracenomycin is 7420 kJ mol 1, and for the triketide pyrone is 15,240 kJ mol 1. Acetic acid has a MW 60.05 g mol 1, and an energy content of 869.4 kJ mol 1 (Fig. 24). Thus, the humification of acetic acid by PKSs modules in S. glaucescens produced tetracenomycin, a humified compound whose molecular weight is five times larger than acetic acid, and contains 8.5 times more energy than that stored in the chemical bonds of the parent compound. The biotic production of the triketide pyrone resulted in a humified polyketide that is six times larger but contains 16.5 times more energy than that stored in the chemical bonds of acetic acid. In comparison, the 3D modeling work done by Schulten and Schnitzer (1997) indicates that the total energy stored in the chemical bonds of a thermodynamically stable HA molecule (31.3 nm3) is 169,600 kJ mol 1 (see Section 8.6), an energy content that is 1123 times greater than the energy stored in the bonds of the previous two polyketide
OH O OH CH3 COOH

PKS1
O H 3C OH

HO

OH

Tetracenomycin (6,100 kJ mol-1)

Acetic acid (869.4 kJ mol-1)

PKS2

R = C21H43
OH

Triketide pyrone (15,240 kJ mol-1)

Figure 24 The estimated change in molecular bond energy content during the biotic humification of acetic acid to tetracenomycin and a triketide pyrone in soils. From C. Monreal (2011, unpublished).

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compounds. The difference in energy content and molecular size of biosynthesized polyketides and HAs may be explained by either the small size of polyketides used in the previous example or by additional bond energy attained by polyketides during polymerization catalyzed by the surface of inorganic soil colloids. These estimated energy and molecular size data are consistent with the conventional definition for humification of SOM. The latter data also suggest that during the humification synthesis of simple soil organic compounds, soil microorganisms have to produce the additional energy stored in the chemical bonds of polyketides through branched metabolic pathways and are likely working near the thermodynamic equilibrium in non-rhizosphere soils. In other cases of soil polyketides enedynes or polyphenol biosynthesis, the differences in molecular size and bond energy content between the resulting humified products and the parent soil oxoacids will be much greater. The aromatic, polyaromatic, alkylaromatic, and polyphenolic biosynthesized structures are also characterized by high chemical stability (Krygowski and Cyranski, 2001). The biosynthesis of soil polyketides is associated with the production of free energy by soil microorganisms, which in turn, depends on the availability and chemical quality of soil substrates available for metabolism. The half saturation value for limiting microbial growth on glucose and several other C sources is between 10 and 100 mM or 10100 mg C L 1 (assuming 1000 g C mol 1) (Pirt, 1975). Soil microorganisms can use a multicity of simple and complex organic and inorganic substrates that may also serve as electron acceptors or donors in the cells metabolic network. In order to satisfy their energy needs, soil microorganisms can also switch between pathways in the metabolic network when the free energy content of certain metabolites in a given pathway is not favorable (Banfalvi, 1994; Wolfe, 2005), and thus, the magnitude of the reverse (or branched) metabolic processes may be quite significant for soil microorganisms due to limited capacity of a soil to supply C. For example, the microbial mineralization of SOM was limited in an Andept of Chile supplying 38 mg C L 1; but it was not limited in a Mollisol of Canada supplying 220 mg C L 1 (Monreal et al., 1981). In soil rhizospheres, the production of polyketides will be associated with conditions of substrate availability, especially when crop photosynthesis and root exudation supplies adequate amounts of organic substrates for microorganisms during 38 weeks of crop growth after seeding (Fig. 25); or after the incorporation of crop residues into soils at harvest. Supply and availability of soluble C sources also occurs through cell excretion and the lysis of soil microbial cells. Research is warranted to conduct studies for establishing the relations between free energy production by soil microorganisms and soil C supply in relation to the synthesis and decomposition of soil polyketides. Thus far, the question on how the energy, kinetic, and thermodynamic relationships operate in soil systems, especially those relations associated with the biontic humification of simple organic compounds into polyketides, has

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1400
Organic-C in soil solution (mg C ml-1)

1200 1000 800 600 400 200 0


16 24 35 42 Time (days after seeding) 56

24 19 14 9 4 -1 -6 -11 -16 -21 -26

Figure 25 The dynamics of soluble organic carbon (dark cyan) and the d 13C in the soil solution (light yellow) of an in vivo soil-canola system labeled for 2 hours with 13CO2 in a closed chamber under continuous flow at different growth stages during the growing season of canola (Brassica napus L.). The d 13C values in soil solution indicate significant production and supply of small organic compounds through root exudation, such as oxoacids, for the potential biosynthetic humification of soil polyketides. From C. Monreal (2011, unpublished experimental data).

not been answered. Within this context, a thermodynamic analysis based on the modeling approach reported by Jin and Bethke (2007) may be used to examine the relationships associated with the biotic humification of simple oxoacids into polyketides in soils. The latter authors used a new and rigorous way to explain how the rate law can account quantitatively for the thermodynamic driving force of kinetic reactions in oligotrophic and eutrophic conditions. An understanding of the energy, kinetic, and the thermodynamic relationships of biotic humification will also benefit from the following additional information: (a) the intramolecular energy of chemical bonds in polyketides; (b) the intermolecular energy between polyketide moieties and other classes of soil organic compounds, and the surface of soil inorganic colloids; (c) characterize the free energy, kinetic, and thermodynamics for all the biosynthesis pathways and decomposition of excreted microbial polyketides, and their incorporation into the CUS of HS and SOM, as shown in Fig. 18.

13. Polyketides and the Central Structure of HS and SOM


13.1. Polyketides as a passive SOM pool
Published information shows that polyketides are adsorbed strongly and rapidly by soil inorganic colloids once released into soils. The latter abiotic process together with the antimicrobial properties of the biosynthesized

d 13C ()

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polyketides and the high-energy content in their bonds appear to prevent their metabolism by many soil microorganisms, protozoa, nematodes, and fauna. These biotic and abiotic factors and processes would favor the longterm stabilization of polyketides in soils, contributing to the large turnover time of soil carbon (> 1000 year) in soil HS and microaggregates (> 265 year) (Campbell et al., 1967; Monreal et al., 1997). The chemical analysis of SOM by Py-FIMS provides indirect information of soil putative polyketides as represented by alkylaromatic, aromatic, phenolic, and lipid pyrolytic products during soil sample analyses. Recently, the analysis of SOM by Py-FIMS showed that putative polyketides represented by alkylaromatic, aromatic, phenolic, and lipid structures make up the great majority of SOM residing in the clay and nanosize soil fractions where the age of carbon is > 1000 years (Monreal et al., 2010). Diverse studies conducted in microorganisms have shown that the amounts of native and residual polyketide antibiotics in soil samples obtained from incubation or field trials range from as little 0.01 to about 0.1 mg g 1 (or 0.050.5 mg ml 1 soil solution, assuming a soil moisture content of 20%, w/w, at field capacity). S. globisporus produced even higher amounts, from 1.3 to 14 mg ml 1, of a nine-member ring enediyene produced in a fermenter during 5 days of growth (Horsman et al., 2010). The amounts polyketide antibiotics produced in vitro studies are somewhat similar and within an order of magnitude of those amounts reported for indigenous and residual polyketides measured in soils (Aga et al., 2005; Cengiz et al., 2010; Soulides, 1965). The authors of this chapter used selected published data to make some general inferences about the net flow of polyketide through soils during pedogenesis. The production and accumulation of polyketides in soilplant systems appears to have occurred continuously during pedogenesis. On a long-term basis, a glaciated Chernozemic soil with a content of 2% organic C (48,000 kg SOM ha 1 20 cm 1, assuming a bulk density of 1.2 g cm 3) would need a net accretion of 0.1 mg g 1 wk 1 of polyketides for 16 wk yr 1 (or 1.92 kg polyketide ha 1 yr 1), to produce a kinetic passive and chemically diverse soil putative polyketides pool of 19,200 kg ha 1 during 10,000 yrs of soil formation. This passive putative polyketide pool would represent 40% of the total SOM content; and its chemical composition is similar to the relative proportion of lignin monomers, phenols, alkylaromatics, lipids (i.e., n-alkanes, alkenes, and n-alkylesters), and fatty acids, as determined by Py-FIMS analysis of the SOM residing in a clay and nanosize fraction of a Chernozemic soil having a carbon turnover time > 1000 yrs (Monreal et al., 2010). Using the specified SOM content at the end of pedogenesis (i.e., 48,000 Mg ha 1 20 cm 1), the accumulation of putative polyketides in A horizons during pedogenesis was represented by the exponential function of Fig. 26 after using the Century model of Parton et al. (1987) to describe the dynamics of the passive SOM pool. It appears that soil microbial polyketides constitute an important chemical pool of SOM with turnover time > 1000 yrs.

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50,000 Soil organic matter (Mg/ha/20 cm)


Total SOM

40,000

30,000

20,000
Putative polyketides
y = 21,228.34 (1-e-0.000378t )

10,000

0 0 2000 4000 6000 8000 10,000 Time (years)

Figure 26 An exponential model for the hypothetical accumulation of putative soil polyketides in the 020 cm depth of a Black Chernozem growing a G3 grass in Canada during 10,000 years of pedogenesis. The exponential function describing the accumulation of putative polyketides was derived from data obtained for a passive SOM pool after a simulation run with the Century model. From C. Monreal (2011, unpublished data).

13.2. Biotic humification process forming the CUS of HS and SOM


Figure 18 shows that humification consists of stages I (i.e., depolymerization) and II (i.e., synthesis). Stage II in turn involves three steps, step 1, which is a biocatalytic step, and steps 2 and 3, which are abiotic catalytic steps. The first step in stage II humification is a key and complex biosynthetic process catalyzed by soil microorganisms having PKS module types I, II, III and hybrids, and malonyl-CoA and acetyl-CoA to synthesize polyketides (PKs). The synthesized PKs are adsorbed rapidly and strongly onto inorganic soil colloidal surfaces, minimizing thereby their potential decomposition by resistant soil organisms. The biosynthesized PKs, such as alkylaromatics, may contribute thus to the formation of the central unit structure of HS and SOM. The CUS derived through microbial biosynthesis reacts electrostatically with carbohydrates, proteins, and N-heterocyclic to produce macromolecular assemblies (stage IIstep 2 of humification; Fig. 18). In parallel, a third abiotic process (step 3) involves the polymerization of adsorbed PKs and macromolecules of HS and SOM by catalytic surfaces of soil inorganic colloids into even larger size humified macromolecules in soil (step 3 in stage II of humification; Fig. 18). Steps 2 and 3 in Fig. 18 appear to be the abiotic processes contributing to a pool containing large and highly diverse soil chemical macromolecules such as those HS structures modeled by Schnitzer and Schulten (1998), and those described in Section 1.

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In order to act as a central architectural unit in HS and SOM, the CUS needs to have the following associated characteristics: (1) is a microbial synthesized polyketide core involving polyphenolic, alkylaromatic, polyaromatic, and aromatic rings bonded to alkyl moieties of different C lengths; (2) is kinetically stable due to the high energy content in chemical bonds with predominance of covalent bonds in and between adjacent aromatic rings and between aromatic rings and aliphatic moieties; (3) is devoid of N-protein and carbohydrates; (4) has moieties allowing H bonding, electrostatic attractions to other macromolecules such as carbohydrate and proteins; (5) continuously generates a random molecular structure variance to HS and SOM by binding with other molecules. The net accumulation of soil polyketides appears to be a function of the rates of adsorption and desorption, the prevalence of microbial populations with the ability of PKSs expression and polyketide resistance, type of polyketide, and associated pedogenetic processes. The biotic and abiotic soil factors that influence the residence time of soil microbial polyketides, and the amounts and frequency of synthesis that occurs at an ecological significant level under field conditions in soilplant systems during humification of soil oxoacids awaits further elucidation.

14. Future Research


As we move forward, further work is warranted to better understand the synthesis and decomposition of soil polyketides in relation to soil microorganisms, PKSs and catalytic inorganic surfaces in vivo soilplant systems. Within this context, it will be necessary to demonstrate the consistency of biohumification processes with the rate laws of thermodynamics. Information from future studies will be obtained from soil microcosm studies involving microbial-gene-enzyme-product models to determine the factors controlling the humification of small soil organic molecules into polyketides, and their subsequent incorporation into the CUS in HS and SOM. Such work may also result in the discovery of new soil polyketides, PKSs, and their decomposition metabolites. Future studies on the structure of SOM will benefit from including sensitive biosynthetic DNA and RNA molecular approaches that may detect either the potential for soil polyketide biosynthesis as inferred from the presence or expression of biosynthetic genes, and/or an activity attributable to gene expression through the direct presence of the metabolite of interest. The biosynthetic molecular techniques are important and complementary tools to the use of isotopes and direct techniques of mass spectrometry to allow for the elucidation and quantification of humification via secondary polyketide pathways for the formation of the CUS of SOM in situ. New specific and

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standardized tests of soil microbiology, chemistry, and biochemistry are needed to further clarify and characterize the exact role of polyketides and PKSs in soil environments under the auspices of multidisciplinary scientific research teams. Modern tools of chemistry, thermodynamics, molecular biology, nanoscience and nanotechnology, simulation modeling, and microscopy will enable soil scientists and others to further and better characterize natural occurring substances produced by microbial humification processes in vivo soilplant systems. These tools include, among other, DNA and RNA transcription and expression for proteins, Py-FIMS, Py-FDMS, ESI-MS, LC-ESI-MS/MS, electrophoresis-MS, 1H, 15N, and 31P NMR, solid 13CCP/MAS NMR. SOM chemists should benefit from the rapid advances in these fields, which should enable them to develop more and more precise knowledge of the chemical structure of HAs. Some of these tools will support carrying out kinetic studies to characterize the microbial and enzymatic transformation of isotopically labeled substrates into intermediates and end products to better determine the turnover of soil polyketides. The chemical techniques need to be complemented by studies involving atomic force microscopy (AFM), transmission electron microscopy (TEM), scanning electron microscopy (SEM), NanoSIMS (secondary ion mass spectrometry) and Raman confocal microscopy to better characterize the spatial disposition of polyketides, trace elements and isotopes in particle size fractions, soil organic domains, and in OM-mineral complexes at the nanoand microscales. Such interdisciplinary studies will provide scientific information on: (a) the humification of small organic molecules, and on the soil ecological significance of microbial and plant synthesized/excreted polyketides in soils, (b) the formation of a CUS in HS and SOM, (c) the chemical diversity and dynamics of polyketides at soil nano- and microscales, and (d) the chemical structure of SOM and HS. For many decades, research on the molecular structures of SOM and HS has taken place in almost entirely different scientific worlds, with little crossfertilization between laboratories or individual researchers working on the chemistry, microbiology, and biochemistry. We hope this chapter will stimulate the exchange of such disciplines, ideas, and concepts, and result in effective collaboration and healthy competition for enhancing human knowledge. In the future, multidisciplinary approaches will be required to continue making progress on elucidating the chemical structure of SOM. But the most important recommendation for the future is closer cooperation between soil chemists, soil microbiologists, and soil biochemists in research on HS and SOM. We have provided scientific evidence showing that polyketides are a category of some 100,000 secondary plant and microbial metabolites of molecular weight less than 2500, of which some 50,000 have origin in soil microorganisms. Polyketides are produced by complex PKSs modules and

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are expressed as active compounds against other living organisms even at low concentrations in soilplant systems. Theoretically, the potential permutation of the presently known four existing PKSs modules could produce more than 100,000 possible alkylaromatic, polyaromatic, and polyphenolic structures from simple soil oxoacids. The synthesis and excretion processes of soil microbial polyketides constitute an example of the first step of HS synthesis, being consistent with the scientific definition already established for the second stage of SOM humification. Due to their bioactivity, strong and rapid adsorption to clay colloids and high energy content of their bonds, the microbial excreted polyketide structures represent kinetically passive carbon pools, and so lend themselves as basic carbon skeletons that contribute to the formation of a CUS in HS and SOM. Moieties of the CUS would then adsorb carbohydrates, proteins, lipids, and N-heterocyclic, molecular associations that can be further modified and polymerized by catalytic soil inorganic surfaces to form large soil humified macromolecules of diverse chemical composition.

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