ISSN 1064-2293, Eurasian Soil Science, 2009, Vol. 42, No. 11, pp. 1270–1281. © Pleiades Publishing, Ltd., 2009. Original Russian Text © N.O. Kovaleva, I.V. Kovalev, 2009, published in Pochvovedenie, 2009, No. 11, pp. 1362–1373.
SOIL BIOLOGY
Transformation of Lignin in Surface and Buried Soils of Mountainous Landscapes N. O. Kovalevaa and I. V. Kovalevb a
Institute of Ecological Soil Science, Moscow State University, Moscow, 119991 Russia b Faculty of Soil Science, Moscow State University, Moscow, 119991 Russia Received January 30, 2009
Abstract—The content and composition of the lignin phenols in plants and soils of vertical natural zones were studied in the Northern Caucasus region and Northwestern Tien Shan. Three types of lignin transformation were revealed: steppe, forest, and meadow ones. It was shown that the degree of oxidation of the biopolymer during the transformation of organic matter increased when going from the living plant tissues to humic acids in surface and buried soils. The portion of lignin fragments remained unchanged during the biopolymer transformation in the following series: plant tissues–falloff–litter–soil–humic acids–buried humic acids. It was also shown that the biochemical composition of the plants had a decisive effect on the structure of the humic acids in the soils. The quantitative analysis of the lignin phenols and the 13C NMR spectroscopy proved that the lignin in higher plants was involved in the formation of specific compounds of soil humus, including aliphatic and aromatic molecular fragments. The first analysis of the lignin content and composition in buried soils of different ages was performed, and an increase in the degree of oxidation of the lignin structures was revealed in the soil chronoseries. It was proposed to use the proportions of lignin phenols in surface and buried soils as diagnostic criteria of the vegetation types in different epochs. DOI: 10.1134/S1064229309110106
INTRODUCTION The development of quantum methods for studying soil organic matter [24] resuscitated interest in lignin as a precursor of humic acids. As far back as the early 20th century, Waksman [23] showed that the lignin arriving into the soil with plant falloff is resistant to microbial decomposition and can be considered as a prohumic substance. In the late 20th century, this hypothesis was again given consideration by the humus researchers in Germany and the United States [16, 19, 25] and then in Russia [4, 6–9, 14, 22, etc.]. It has been established by now that the rates and mechanisms of plant tissues' decomposition are determined by their biochemical composition, the hydrothermal environmental conditions, the presence of available nitrogen, and the nature of the microbial populations. Lignin is the most common natural phenolic compound of plant origin, and the lignification of cell walls (lignin fills the space between the cellulose fibers) is an essential evolution stage of the vegetable world, which ensures the functioning of the excretory and transport systems in vascular plants and their support in the soil. Lignocelluloses make up 70–90% of the dry weight of plant tissues. In terms of humification, lignin is interesting not only as a structural component of plant falloff relatively resistant to decomposition but also as an irregular trisubstituted biopolymer of large molecular weight composed of phenylpropane units and possessing col-
loidal properties. This is a compact microgel of a netlike structure whose particles are strongly branched and extremely polydispersed [1]. The relative proportions of lignin components and phenols are determined by the phylogenetic origin of plants and allow the formation of various low- and high-molecular-weight products of lignin decomposition in the soil, which are involved in humus formation and can act as agents of allelopathic interactions, inhibit soil enzymes, precipitate proteins, inhibit or catalyze biochemical reactions in soils, inactivate nitrification, and form coal and kerogen deposits [10]. We previously showed [4] that the complex aromatic structure, the colloid and hydrophobic properties, and the high biochemical stability of lignin suggest three most probable ways of lignin transformation in soils depending on the thermodynamic environmental conditions: (1) the stabilization and conservation of lignin polymers as highly condensed polynuclear aromatic structures under reductive conditions; (2) the insignificant transformation of biopolymers because of an increase in its solubility under contrasting redox conditions at the preservation of the main lignin structures (precursors of humic acids); and (3) the degradation of lignins under oxidative conditions to simpler phenolic acids (agents of pedogenetic processes and biochemical transformations). However, it is still not clear how the properties of soil humus depend on the biochemical composition of the different falloff types, what are the specific forms
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and transformation mechanisms of aromatic phenols of plant origin into humic acids, and what is the role of separate phenolic compounds in the regulation of soil processes. Only foreign data are available on the content and transformation pathways of lignin in soils of different natural zones and ecosystems [15, 22, 25]. Problems related to the geographical features of the distribution of lignin in soils and sedimentary rocks and the profile distribution of phenols remain unsolved; no methods are available for the determination of the lignin pattern in soils. An idea has been formed that the results obtained by the conventional methods of lignin isolation (so-called residual lignin) are too approximate even for plant materials and extremely overestimated for litter and soil samples. Numerous authors [18] showed that the classical method is absolutely unsuitable for the determination of lignin because of the presence of humus and the formation of resinous condensation products during hydrolysis by strong acids. New methods have been searched for in different countries over more than 20 years; gas-liquid chromatography and high-performance liquid chromatography are the most common methods for the separation of lignin oxidation products obtained in oxidation reactions under mild conditions. A current idea of global science in the study of lignin is the reconstruction of the photosynthesis type on the basis of the 13C analysis of lignin preparations isolated from lacustrine or marine sediments [16]. However, no information on the structural composition and the amount of lignin phenols in buried horizons, soils, or cultural (anthropic) layers is available in Russian or foreign literature. Therefore, the immediate aim of this work was to study the transformation of aromatic lignin structures in the following series: plants–falloff–soils–humic acids–buried organic matter of different ecosystems. The studies were performed at the Institute of Soil Science and Soil Geography of the University of Bayreuth (Germany) with the hands-on assistance of Dr. I. Lobe, Dr. L. Haumaier, and Prof. W. Zech. EXPERIMENTAL Lignin preparations were isolated from different tissues of woody and herbaceous plants of the temperate and subtropical regions, litters, surface humus and mineral soil horizons, humic acids, buried humus horizons, and humic acid preparations from them. Studies were performed in the ecosystems of vertical natural zones under subboreal humid and subtropical continental climatic conditions. The soil–vegetation zones included, first, the mountainous biocenoses of the Northern and Western Tien-Shan (the Kyrgyz and Alai ranges): steppes on mountain chernozems, a thin juniper forest on cinnamonic mountain soils, a walnut forest on black-brown mountain soils, pine forest and subalpine meadow chernozem-like mountain and mountainmeadow subalpine soils, and an Alpine meadow on EURASIAN SOIL SCIENCE
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mountain-meadow Alpine soils. Studies were also conducted in the Northern Caucasus region: in the steppe zone of mountain chernozems; in the beech-hornbeamchestnut, fir, and spruce forests on brown forest soils; in the birch (with aspen) elfin forest and subalpine meadows on mountain-meadow subalpine soils; and on Alpine meadows with mountain-meadow Alpine soils (Karachai-Cherkessia). The determination of the lignin in the plants, soils, and humic acids involved the alkaline oxidation with copper oxide at 170°C under pressure in a nitrogen environment; the precipitation of humic acids; and the concentration of the phenolic products under pressure on compact disposable C18 columns. After the samples were passed through the columns, the columns were dried and the lignin was dissolved in ethyl acetate. The lignin preparations were isolated by the evaporation of ethyl acetate using a rotor evaporator [16]. The phenolic components of the lignin were separated using a gas-liquid chromatograph after preliminary derivatization and conversion into trimethylsilyl esters. A gas chromatograph with a mass spectrometer (Hewlett-Packard, Palo Alto, CA, United States) equipped with a flame-ionization detector and a capillary column was used. Nitrogen was the main and marking gas. The injector temperature was 250°C; the detector temperature was 300°C. The individual reaction products (vanillin; syringic aldehyde; and vanillic, syringic, p-coumaric, and ferulic acids) were identified by comparing the retention times (in minutes) and peaks with those of known components in known concentrations used as external standards (Fig. 1). The following standards were used: phenylacetic acid, 6.040 min; vanillin (4-hydroxy-3-methoxybenzaldehyde), 8.069 min; ethylvanillin, 8.518 min; syringic aldehyde (3,5-dimethoxy-4-benzaldehyde), 9.321 min; vanillic acid (4-hydroxy-3-methoxybenzoic acid), 9.626 min; syringic acid (3,5-dimethoxy-4-hydroxybenzoic acid), 10.538 min; p-coumaric acid (4-hydroxycinnamic acid), 10.840 min; and ferulic acid (4-hydroxy3-methoxycinnamic acid), 11.981 min. Although the method is highly sensitive and can determine trace amounts of phenols, the analysis was accompanied by significant losses of lignin decomposition products (up to 50%). To improve the reproducibility of the analytical results, glucose was added to the soil samples as an oxidation catalyst and ethylvanillin was added as an internal standard before the alkaline oxidation. Phenylacetic acid was added to the samples as the second internal standard before the derivatization. The reproducibility was thus increased to 95% [15]. The alkaline oxidation of vascular plant materials and residues with copper oxide in the soil yields 11 phenols [16], which can be grouped in accordance with their chemical nature into three structural families: vanillic (V), syringic (S), and cinnamilic (C) ones. The first two phenol types are found in mixed oxidation products of plant tissues as aldehydes (al), ketones, and acids
7
8
9 A a:
9 (d)
10 re
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10
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11.961
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12.259
a
re 41
11.965 12.090
A 61
. 17
11.012 11.108 11.181 11.228 11.358 11.423 11.482 11.541 11.720
10
11.858
6
97
10.837
9.573 9.631 9.713 9.780 9.743 9.842 9.878 9.913 9.971 10.072 10.132 10.218 10.385 10.333 10.438 10.542 10.590
8.638 8.666 8.709 8.745 8.794 8.900 9.089 9.165 9.193 9.3189.281
8.021
8.478
11
11.779
8 .4
1 :1
10.564
85
11.096 11.171 11.219 11.352 11.402 11.477 11.540
A r : ea
10
10.845
a:
10.540
01
. 18 .1
7 11
9.622
9
10.399
7 8.385
8.155
7.511 7.627 7.677 7.784 7.816
6.232
6.016
9
9.971
a
9.284
8.474
27
9.630 9.781
6 8
9.328
A re
8
9.085
7.
4 :1
8.633 8.719
8.485
7
8.023
7.780
6 7.215 7.239
6.062 6.180 6.321 6.451 6.494 6.605 6.730 6.802 6.904
7
8.062
6.031
pA 90 80 70 60 50 40 30 20
7.831
pA 55 50 45 40 35 30 25 20 15 6 6.027
7.520
8.378
10.259 10.430
9.970
12.253 12.417
12.051
11.781
10.913 10.955 11.013 11.088 11.167 11.219 11.351 11.473
10.128 10.221 10.340
9.190 9.339 9.501 9.571
8.634
8.113 8.152
7.681 7.821
7.235
6.769
6.323 6.491
9.627 9.769
9.283
8.671
8.481
8.024
6.025
11.970
11.541
10.539 10.714 10.838
9.081
8.764
6.226
pA 55 50 45 40 35 30 25 20 15 6 pA 90 80 70 60 50 40 30 20
6.223
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(b) 12
(c)
06
A re
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68
8 4.
A re
11 12
12 Retention time
Fig. 1. Chromatograms of phenolic compounds isolated from the (a) grass tissues, (b) surface horizon (chernozem), (c) buried horizon, and (d) humic acid preparation (chernozem).
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(ac); the latter include only acid forms: ferulic (F) and p-coumaric (K) acids. The phenol mixture obtained after the oxidation of lignin is best described by the parameters characterizing the weight combinations of its components. The sum of the oxidation products is thus indicative of the total lignin content in the sample. In the plant tissue and soil samples from the above objects of study, the carbon and nitrogen were determined using a Vario El CNS analyzer (Elementar GmbH, Hanau); the humic acid preparations were isolated [12], and their 13C NMR spectra were recorded [24]. The sampling procedure of the plant tissues and the values of the plant productivity were reported earlier [3]. RESULTS AND DISCUSSION The lignin in plant tissues. The rate of lignin decomposition in soils is largely determined by the source of this biopolymer: the type of plant tissues and their anatomic structure. Although insufficient data are available on the biochemical composition of the different organisms involved in humification [11], it is considered established [1] that different types of plant tissues (gymnosperms and angiosperms, woody and nonwoody, and aboveground and underground) have contrasting lignin parameters. The data in Table 1 show that we confirmed the existing tendencies and found three known lignin types in our samples. First, the lignin of coniferous plants (soft wood lignin) contains vanillin phenols as major structural units: up to 60 mg/g Corg in juniper roots and up to 80 mg/g Corg in the fir falloff. The content of cinnamic alcohols in the needles, wood, and especially roots of pine and juniper is low; syringic acids and aldehydes are almost absent in the needles, and S/V = 0. Second, the lignin of deciduous trees (hardwood lignin) predominantly consists of similar amounts of vanillic and syringic structures. The lignin in deciduous species of Caucasian mountain forests (birch, beech, and chestnut) hardly differs from that in the tree leaves of the temperate zone [7] and the walnut mountain forest of the Tien-Shan and contains similar amounts of vanillic and syringic phenols. The S/V and C/V ratios are higher than 0 but lower than 1. The content of cinnamic phenols is close to 0. The third lignin type is the lignin of herbaceous plants, which contain the largest amounts of cinnamic structural units: their content in steppe and meadow grasses and herbs increases to 20–30 mg/g C, which is higher than in woody plants by 4–6 times. The content of syringic phenols in herbs is similar to that in angiosperm wood but exceeds that in leaves by 5–6 times. Ferulic acids are mainly associated with hemicelluloses in the cell walls of grass fibers. Their content reaches 15–20 mg/g Corg in the aboveground phytomass and roots of grasses. EURASIAN SOIL SCIENCE
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It is also noteworthy that the largest amount of lignin phenols is in the underground tissues of plants. Both in the tree and herbaceous plant covers, the root lignin is predominant: up to 66 mg/g C in juniper roots, as well as up to 51 mg/g C in grass roots at a depth of 0–30 cm and 49 mg/g C at a depth of 30–60 cm. The coefficient of correlation between the lignin content in the soil and root biomass is 0.92 (ê = 0.95) for subalpine meadow ecosystems, 0.93 (ê = 0.95) for alpine ecosystems, and 0.99 (ê = 0.95) for thin juniper forests. Detailed information on the biomass structure of the plant communities considered was reported earlier [3]. This fact can be indicative of a more important contribution of the root biomass to the formation of soil humus than is generally considered. Thus, the oxidation and chromatographic separation of lignin biopolymers into simpler phenols provide information about the types of plant tissues. In addition, the S/V ratio can be used to discriminate between the tissues of gymnosperms and angiosperms, and the C/V ratio can be used to separate the organic matter of wood and nonwood origin. The type of plant tissues primordially creates unequal conditions for the transformation of aromatic components of plant origin into aromatic compounds of soils. The comparative study of lignin accumulation in the living phytomass and falloff of plants allows the conclusion to be drawn that the amount of lignin arriving onto the soil surface in the ecosystems of coniferous forests is double that in hardwood forests. The highest content of lignin was found in the mats of subalpine (26.47 mg/g Corg) and Alpine (81.68 mg/g Corg) forests, whose role in the formation of carbon pools is usually not considered. It is noteworthy that, under the aerobic conditions of mountain landscape ecosystems with favorable hydrothermal conditions (spruce, walnut, steppe, and beechhornbeam), similar amounts of lignin phenols arrive onto the soil surface as falloff or mats (about 10 mg/g Corg) regardless of the content of lignin in the phytomass. Some authors [3, 14] believe that lignin can perform a regulatory function in different ecosystems by controlling the input of organic matter for humus formation. Lignin in forest litters and steppe detritus. The isolation of lignin oxidation products from the litters of different plant associations showed (Table 2) that their lignin parameters were less contrasting than those in plant tissues, and the content of lignin was significantly lower than in living plant tissues. The lowest content of lignin was typical for the F and H horizons of the forest litter in the spruce (3.06 mg/g Corg), cedar (3.08 mg/g Corg) [7], aspen (1.58 mg/g Corg), and beech (2.16 mg/g Corg) forests. The highest contents of lignin were found in the high-mountain ecosystems of thin juniper forests (62.56 mg/g Corg). The elfin birch forests (10.87 mg/g Corg), walnut forests (9.10mg/g Corg), and pine forests
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Table 1. Lignin parameters of plant tissues Plant tissue
Lignin N, % dry (VSC), weight mg/g Corg
V
S
C
mg/g Corg
S/V
C/V
K/F
V:S:C
Coniferous tissues Pine needles roots falloff Spruce falloff Juniper needles wood roots falloff Fir falloff Birch falloff Beech, hornbeam, chestnut falloff Walnut, apple tree falloff Aspen falloff Steppe aboveground phytomass steppe detritus Subalpine meadow aboveground phytomass waste roots 0–30 cm 30–60 cm Cultivated plants waste Steppe aboveground phytomass steppe detritus Subalpine meadow aboveground phytomass roots Alpine meadow aboveground phytomass waste roots 0–30 cm 30–60 cm
1.34 0.92 1.25
27.70 40.80 12.33
20.40 36.95 7.07
0 2.17 0
7.30 1.68 5.26
0 0.06 0
0.36 0.05 0.74
1.55 0 0
3:0:1 22 : 1 : 1 1:0:1
1.49
9.42
4.87
2.19
2.36
0.45
0.49
0.86
2:1:1
1.26 0.37 0.75 1.65
6.98 23.01 66.88 23.20
2.24 62.40 12.96 17.03
1.46 5.38 1.43 2.87
3.28 4.67 3.05 3.30
0.65 0.42 0 0.19
1.46 0.36 0 0.22
0.74 0.26 0.11 0.30
1:1:2 2:1:1 41 : 1 : 2 5:1:1
1.44
81.33
52.77 10.46 Deciduous tissues
18.10
0.13
0.34
1.28
5:1:2
0.32
24.54
8.84
12.58
3.12
1.42
0.35
0
3:4:1
1.23
10.41
5.41
4.16
0.84
0.77
0.16
0.95
6:5:1
0.98
10.18
6.50
2.99
0.69
0.46
0.11
0.44
9:4:1
0.23
9.34
3.60 5.20 Grass tissues
0.54
1.44
0.15
2.33
7:9:1
1.85 0.71
42.09 10.12
5.26 4.17
5.61 4.31
31.22 1.63
1.02 1.03
5.20 0.72
0.5 0.39
1:1:2 3:3:1
1.91 1.50 1.21 0.78
39.43 26.47 45.84 48.72
8.59 5.70 10.52 11.73
8.63 5.72 14.12 15.80
22.21 15.05 21.20 21.19
1.00 1.00 1.34 1.35
2.58 2.64 2.02 1.82
0.47 0.70 1.21 1.09
1:1:6 1:1:3 1:1:2 1:1:2
0.71
10.85
4.59 4.36 Herb tissues
1.90
0.95
0.20
0.96
2:2:1
1.62 0.71
25.00 13.06
9.91 5.71
11.08 5.72
4.01 1.64
1.12 1.00
0.40 0.29
0.09 0.69
2:3:1 4:4:1
2.05 0.94
19.44 19.17
6.45 4.14
8.03 6.89
4.96 8.14
1.25 1.66
0.77 1.97
0.87 0.28
1:2:1 1:1:2
2.07 0.44 1.11 1.48
35.90 81.68 72.02 48.48
5.79 21.24 10.05 8.48
10.46 38.39 25.21 17.29
19.65 22.05 36.76 22.71
1.81 2.73 2.51 2.04
3.39 0.77 3.66 2.68
0.91 1.08 0.55 0.55
1:2:3 1:2:1 1:2:4 1:2:3
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Table 2. Lignin in the F and H horizons of forest litters Ecosystem
Lignin (VSC), mg/g Corg
V
S
C
C S/V
C/V
K/F
N
V:S:C
mg/g Corg
%
Forest: spruce pine fir
3.06
1.24
1.14
0.68
0.92
0.55
0
2:2:1
3.45
0.17
13.70
6.77
3.73
3.19
0.55
1.13
0
2:1:1
2.85
0.17
5.77
3.45
1.57
0.75
0.46
0.22
0
5:2:1
31.24
0.19
walnut
9.10
4.33
3.74
1.03
0.86
0.24
0.72
4:4:1
6.33
0.20
beech–hornbeam– chestnut
2.16
0.71
0.73
0.17
0.58
0.17
0
4:4:1
3.23
0.21
aspen
1.58
0.74
0.37
0.46
0.50
0.62
2.08
2:1:1
2.37
0.22
birch
10.87
7.54
1.55
1.78
0.53
0.23
0.83
4:1:1
1.76
0.19
under the canopy
62.56
32.67
18.83
11.06
0.58
0.32
0.68
3:2:1
7.75
0.72
in a clearing
13.06
5.70
5.72
1.64
1.00
0.29
0.70
4:4:1
11.66
1.19
Grove:
Thin juniper forest:
(13.70 mg/g Corg) were characterized by a medium content of lignin in their litters. The composition of the coniferous litter was characterized by the accumulation of vanillic phenols (up to 33 mg/g Corg under the juniper canopy) and retained the tendency toward a decrease in the content of syringic and coumaric structures. The deciduous forest litter contained similar proportions of vanillic and syringic phenols and a lower content of ferulic acids (V : S : C = 4 : 4 : 1; 2 : 1 : 1). At the same time, the steppe detritus layers showed a clear accumulation of cinnamic phenols (Table 1) and especially ferulic acids (K/F < 1). Lignin in soils. Further transformations of lignin in soils are determined by the hydrothermal environmental conditions and the physicochemical properties of the soils controlling the microbial activity. No microorganisms capable of completely decomposing lignin are known. Lignin molecules are only partly decomposed by different soil organisms. Only the combined action of mixed microbial populations reaches the synergetic effect of the transformation of lignin structures into humus. It was experimentally proved that lignindecomposing microorganisms containing oxidative peroxidase-type enzymes only catalyze the breaking down of bonds in the lignin molecule. These ligninases excite the radical reactions that primarily occur in the lateral chains of α-β radicals and, to a lesser degree, methoxyl and cyclic structures [17]. The essential conditions for the oxidation of phenols by microorganisms are good aeration conditions and low concentrations of phenols: 0.025–0.050% for bacteria and up to 1% for mold fungi [9]. In forest soils, basidiomycetes such as white and brown rot fungi participate in the decomposition of lignin. White spongy rot fungi and actinoEURASIAN SOIL SCIENCE
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mycetes are less active in soils of pastures, but they are also important. Thermophilic streptomycetes significantly contribute to the decomposition of lignin under composting conditions. All the above microorganisms transform lignin into the water-soluble form by breaking the bonds of the methoxyl groups and introducing additional hydroxyl and other polar groups into the lignin molecule. Thus, the splitting and the destruction of the lateral chains primarily occur during the transformation of lignin polymers. The resulting intermediate unstable radicals add available oxygen or water, the primarily compact hydrophobic structures of lignin are strongly loosened, and the lignin solubility in water increases. The further addition of carboxyl and hydroxyl groups facilitates the participation of the polymer in chelation and humification. It is considered proved [17] that the multiple, gradual, and lasting transformation ensures the participation of lignin in the formation of soil humus, rather than separate phenolic monomers being repolymerized to humic acids as was previously supposed (the repolimerization hypothesis), especially because no enzymes catalyzing the synthesis of humic acids in soils were found [13]. Typical structural fragments of lignin in soils are also recognizable for a long time, because fragments of partially decomposed lignin unavailable for microorganisms are stabilized on the external surfaces of metal oxides or clay minerals. We thoroughly studied the stabilization mechanisms of lignin structures with the formation of iron–manganic nodules as an example [4]. Different authors [17, 19] noted the good preservation of the composition ratios between the lignin phenols in soils. This fact can form the basis for diagnosing the origin of the lignin from the lignin parameters of the
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Table 3. Parameters of the lignin transformation in soils of the Northern Caucasus mountain ecosystems C Soil
Horizon, depth, cm
N %
Lignin (VSC), mg/g Corg
Vanillic acids/vanillin (Ac/Al)v
Syringic acids/syringic alS/V dehydes (Ac/Al)s
C/V
K/F V : S : C T, %
Steppe Mountain cher- Ad, 0–8 7.34 nozem A, 8–18 5.85 AB, 18–32 4.16
10.12 12.12 15.45
0.10 0.11 0.08
0.18 1.34 0.17
1.03 0.99 0.98
0.39 0.70 0.41
0.71 3 : 3 : 1 4.85 1.00 1 : 1 : 1 3.27 1.01 2 : 2 : 1 5.00
0.17 0.43
0.46 0.30
0.22 0.22
1.50 5 : 2 : 1 1.16 1.07 5 : 1 : 1 1.49
0.34 0.26
0.60 0.88
0.35 0.29
1.93 3 : 1 : 1 1.49 1.00 3 : 3 : 1 1.36
0.23 0.30 0.68
0.55 0.42 0.38
0.46 0.30 0.24
1.00 2 : 1 : 1 2.51 1.30 3 : 1 : 1 0.96 1.92 4 : 2 : 1 0.89
Fir forest Brown forest soil
A, 10–25 B, 25–35
2.38 1.75
0.18 0.15
1.86 1.09
0.13 0.13
Subalpine meadow Mountain-mead- A, 10–25 ow subalpine B, 30–40 soil
6.08 2.83
0.55 0.26
16.68 38.08
0.13 0.12 Alpine meadow
Mountain-mead- Ad, 0–10 ow Alpine soil A, 10–20 B, 20–30
6.45 6.75 2.53
0.57 0.60 0.23
4.37 4.84 2.32
0.11 0.13 0.14
Note: (VSC) total lignin oxidation products; (S/V) syringic phenols/vanillic phenols; (C/V) cinnamic phenols/vanillic phenols; (K/F) coumaric acids/ferulic acids; (T) percentage of changes in the lateral chains from the lignin-to-original plant tissue ratio.
plant tissues that remain in the soil. For example, the lowest syringic-to-vanillic phenol ratios (S/V < 0.5– 0.6) are typical for all the soils of coniferous ecosystems because of the absence of syringic phenols. The C/V ratio can be used for the separation of wood and nonwood materials in the soil organic matter, because cinnamic phenols are present only in the nonwood tissues of plants: it has the minimum value (0.22) in the brown soil of the fir forest and reaches 3.90 in the mountain-meadow Alpine soils. The p-coumaric-toferulic acid ratios are the maximum in the soils of the herbaceous ecosystems and tend to zero only in ecosystems with the predominance of grasses. For example, the buried humus horizons of mountain-meadow subalpine chernozem-like soils (5000–6000 years old [21]) are characterized by the accumulation of ferulic phenols at the complete absence of vanillin, syringic acids, and p-coumaric acids. These facts well agree with the hypothesis proved by us earlier [5, 21] about the steppe period of soil formation under warmer and drier climatic conditions compared to the present ones. We obtained similar results for the second humus horizons of the Bryansk opolie soils, in which the proportions of lignin phenols diagnose the steppe phase of pedogenesis at about 5000 years ago and the forest phase (with the predominance of broadleaved species) in the Late Holocene [4, 6].
The absence of all phenols except for vanillic phenols in the brown-colored horizon buried in loess about 14000 years ago [21] indicates the existence of coniferous forest ecosystems in the late postglacial time. The acid–aldehyde ratio in the units of vanillin or syringyl as the degree of oxidation of the molecule is used for characterizing the rate of lignin decomposition and transformation in soils. Ertel and Hedges [16] convincingly showed that the content of aromatic acids relative to aromatic aldehydes increases with the degree of decomposition of organic matter and developed a formula for calculating the degree of transformation of the lateral chains in the biopolymer (the parameter T, %): í = 74 – (100 – ä)(1 + (ÄÒ/Äl)v)–1, where (ÄÒ/Äl)v is the ratio of vanillic acids to vanillic aldehydes, and K is the percentage of ketones in the original plant tissues. The results of using the above values are given in Tables 3 and 4. It follows that the higher the lignin contents (VSC), the lower the degree of oxidation of the biopolymer ((ÄÒ/Äl)v) and the lower the degree of transformation (T) typical for the mountain-meadow soils of Alpine and subalpine meadows, which well agrees with the retarded mineralization of the plant residues. The humid ecosystems of the Northern Caucasus region, which have low vanillic acid/vanillin ratios (0.08–0.13) and í values no higher than 5%, show a EURASIAN SOIL SCIENCE
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Table 4. Parameters of the lignin transformation in soils of the Tien Shan mountain ecosystems C Soil
Horizon, depth, cm
N %
Mountain chernozem
A, 12–38 AB, 38–53 B, 53–83 C, 97–110 [B], 110–120
4.52 3.53 3.97 3.55 2.44
0.48 0.27 0.20 0.07 0.04
Mountain-meadow subalpine chernozem-like soil
A, 4–35 [A], 35–50 AB, 50–67 B, 67–87 Bt , 87–97
5.21 2.05 1.17 1.10 1.41
0.61 0.21 0.13 0.12 0.14
Mountain-meadow subalpine chernozem-like soil
A, 10–33 [A], 33–52
3.62 0.36 2.56 0.31
Cinnamonic mountain soil
A, 15–30 [A], 30–60
2.35 0.23 1.19 0.12
Black-brown moun- A, 10–20 tain soil
4.50 0.34
Mountain-meadow subalpine soil
A, 5–20 [A], 20–50
9.08 0.82 0.71 0.08
Mountain-meadow Alpine soil
A, 3–20 AB, 20–35 BC, 35–50
4.02 0.41 3.03 0.30 1.27 0.15
Syringic Lignin Vanillic acids/sy(VSC), acids/vanringic alde- S/V mg/g illin hydes Corg (Ac/Al)v (Ac/Al)s Steppe 1.37 0.59 1.13 0.20 2.34 0.22 0 0 0.94 0.91 Meadow steppe 0.19 0.30 0.18 1.18 0.28 3.5 0.27 5.0 0.30 3.5 Pine forest 13.70 0.46 0.96 0.34 Thin Juniper forest 3.91 0.46 8.23 0.39 Walnut forest 7.79 0.27 Subalpine meadow 3.19 0.29 1.12 0.46 Alpine meadow 4.85 0.14 1.57 0.06 1.97 0.10
C/V
K/F
V:S:C
T, %
0.75 0.89 0.42 0 2.01
0.16 0.21 0.63 0 0.50
0.52 0.14 0.20 0 0.10
1.90 1.92 1.85 0 0
2:1:1 5:1:1 3:3:1 0:0:0 8:4:1
20.54 3.17 4.33 0 29.50
0.68 4.72 0.25 0.19 1.05
0.70 0.27 0.57 0.45 0.48
0.58 0.79 0.69 0.21 0.33
1.00 0 2.88 0 1.70
2:1:1 3:1:3 2:1:1 5:2:1 3:1:1
2.57 35.01 7.59 7.10 8.62
0.55 0.28
0.55 0.47
0.47 0.34
1.14 0
2:1:1 3:1:1
15.78 10.57
0.44 0.52
0.51 0.69
0.26 0.53
0.60 4 : 2 : 1 0.23 20 : 12 : 1
15.78 12.85
0.01
0.96
0
0.44 18 : 17 : 1
7.07
0.50 0.46
0.35 0.85
0.59 0.56
0.77 0.61
2:2:1 2:2:1
1.69 0.35 0.24
0.55 0.65 0.99
3.88 0.66 0.34
2.01 0.64 0.72
2:1:7 2:1:1 3:3:1
8.11 15.78 0 0 0
Note: (S/V) syringic phenols/vanillic phenols; (C/V) cinnamic phenols/vanillic phenols; (K/F) coumaric acids/ferulic acids; (T) percentage of changes in the lateral chains from the lignin-to-original plant tissue ratio.
significantly lower degree of biotransformation of lignin structures compared to the droughty ecosystems of the Tien-Shan. In the latter case, the vanillic acid/vanillin ratio varies from 0.2 to 0.9, and the í value reaches 20%. These results do not contradict our data on the humate character of the humus in the Tien Shan soils [2, 5] compared to the predominantly fulvate humus character in the soils of the Caucasus region [8]. The results suggest different characters of the organic matter transformation in the soils of different ecosystems. The steppe biotransformation of lignin is characterized by the highest degrees of biopolymer modification (í) in the considered vertical soil series (from 5% in the Caucasus region to 20% in the Tien Shan) and the highest values of the oxidation parameter EURASIAN SOIL SCIENCE
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((Ac/Al)v = 0.10–0.60) in the chernozems, chernozemlike soils, and cinnamonic soils. In the forest decomposition of lignin, í is about 1.5% for brown Caucasian soils and 15% for those of the Tien Shan; the (Ac/Al)v ratio has medium values. The meadow transformation type of lignin structures is characterized by near-zero T values and minimum acid/aldehyde ratios. The highest degrees of oxidation of lignin molecules ((Ac/Al)v = 5–6) and the highest values of the transformation index (T = 35%) are typical for the buried humus horizons (Tables 4, 5). In the previously studied second humus horizons of the Bryansk opolie soils, the acid/aldehyde ratio also exceeded that for the surface humus horizons, and the degree of molecule transformation reached 53% [4, 6]. This fact can be
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Table 5. Lignin oxidation products in the humic acid preparations
Soil
Horizon, depth, cm
13C
Lignin (VSC), mg/g Corg
( Ac/Al ) *v
V
S
C
V:S:C
NMR peaks, % of the spectrum area 56 ppm
147 ppm
Mountain chernozem
A, 12–38 [B], 120–137
3.32 0.025
1.15 5.00
0.73 0.012
0.95 0.006
1.65 0.007
1:1:2 2:1:1
3.15 4.14
5.85 3.40
Mountain-meadow subalpine chernozem-like soil
A, 0–23 [A], 37–50
5.10 0.96
0.51 0.99
1.48 0.27
1.93 0.33
1.70 0.37
1:1:1 1:1:1
3.33 2.62
4.00 5.05
Mountain-meadow subalpine soil
A, 5–35 [A], 35–57
3.66 0.10
1.60 6.20
1.59 0.72
0.56 0.01
0.75 0.01
2:1:1 5:1:1
3.37 1.48
6.23 8.18
Cinnamonic moun- A, 4–34 tain soil [A], 34–60
1.83 0.82
0.37 0.26
0.85 0.34
0.90 0.43
0.24 0.12
3:4:1 3:4:1
5.07 2.35
5.76 5.03
Mountain-meadow alpine soil
2.06
0.73
0.55
0.77
0.74
1:1:1
3.72
3.49
A, 3–20
Note: * (Ac/Al)v is the vanillic acids-to-vanillin ratio.
related not only to the diagenetic transformations of humus but also to the different hydrothermal potential of the soil-forming environment.
is typical for the humic acids from the cinnamonic soils of thin juniper forests and reflects the properties of the juniper tissues.
Lignin in humic acids of surface and buried soils. The humic acids isolated from the soils under study also contain aromatic phenols of lignin origin, which is indicative of their involvement in the formation of soil humus. Their presence was confirmed not only during the isolation of the humic acids (Fig. 1) but also by their NMR spectra (Fig. 2). It is known [20, 24] that the peaks at 147 ppm (aromatic molecular fragments) and 56 ppm (aliphatic molecular fragments) in the NMR spectra of the humic acids are due to compounds retaining their lignin nature. The data in Tables 5 and Fig. 2 show that the peak areas of lignin structures in the aromatic and aliphatic fragments are similar for the mountain-meadow Alpine soils. In the humic acids of chernozems, the contribution of aromatic lignin fragments to the nuclear part of the molecule is double that in the peripheral part of the molecule. In the buried horizons, the peak areas of lignin compounds in the nuclear fragments of humic acid molecules are larger than their halos for humic acids of the surface horizons by 5 times.
It was shown that the strong differences typical for the humic acids isolated from buried horizons are related to the increased proportion of vanillic phenols (up to 2 : 1 : 1 and 5 : 1 : 1). It can be supposed that the molecular structure of the humic acids keeps the memory of other humification types (the forest type about 200 years ago [21]), although the character of the diagenetic transformations also requires special consideration.
On the whole, the humic acids are similar in the content of lignin oxidation products and the lignin parameters (Table 5) to those of the soil samples; they inherit the characteristic properties of the plant tissues but show a higher order of structural lignin fragments. Their proportions of vanillic, syringic, and cinnamic phenols are similar (1 : 1 : 1) for the different types of herbaceous ecosystems, from steppe to subalpine meadow, and characterize the herbaceous plant tissues, as was shown above. The composition ratio of 3 : 4 : 1
It also follows from the data in Table 5 and Fig. 3 that the humic acids have a more acid phenol mixture compared to the soils: the humic acid preparations have higher acid/aldehyde ratios than the soil extracts. Thus, our results confirm that the increasing carboxylation of the lignin residues is the major process of their transformation into humus. The destruction reactions can primarily originate in the lateral chains of lignin and result in the formation of phenylcarboxylic acids as the first specific compounds. The data in Fig. 3 show that the amount of aromatic acids increases with respect to that of aldehydes when the degree of organic matter transformation in the “plant tissues–litter–soil–humic acids– buried humic acids” series increases in all the objects of the study regardless of the total lignin content and reaches its maximum in the humic acid preparations from the buried soils. This fact confirms the formation of similar humic substances of specific nature in soils, which involves, along with other aromatic compounds, lignin from higher plants. EURASIAN SOIL SCIENCE
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(a)
ppm
ppm
200
150
200
100 (b)
150
50
100
50
0
0
Fig. 2. 13C NMR spectra of humic acids from (a) the surface and (b) buried horizons of mountain-meadow subalpine chernozemlike soil of the Tien Shan.
CONCLUSIONS Within the ranges of the regional climatic conditions, the biochemical composition of the plants in different ecosystems has a decisive effect on the character of the humification and determines the humus formation mechanism and the structure of the soil humic acids. According to the values of the lignin parameters—the VSC (the total content of lignin oxidation products) and the C/V (cinnamyls/vanillins), the S/V (syringils/vanillins), the K/F (coumaryls/feruls), and the acid/aldehyde ratios—different types of plant tisEURASIAN SOIL SCIENCE
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sues (coniferous and leafy, woody and nonwoody, and herbal and grass) and lignin transformation (steppe, forest, and meadow) can be specified in the soils. The proportions of lignin phenols remain unchanged in the litters, soils, buried horizons, and humic acids for a long time. Therefore, the proportions of lignin phenols in the paleosols and sediments can be used as diagnostic criteria of the terrestrial vegetation types in past epochs. The lignin of the highest plants participates in the formation of specific soil humus compounds, including
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Vanillic acids-to-vanillin ratio
7 6 5 4 3 2 1
0 Plant tissues
Soil
Buried humic acids Humic acids
Litter 1
2
3
4
5
Fig. 3. The vanillic acids-to-vanillin ratios at different stages of the lignin transformation in soils: (1) mountain chernozem (steppe); (2) mountain-meadow subalpine soil; (3) cinnamonic soil (juniper forest); (4) mountain-meadow alpine soil; (5) mountain chernozem-like soil (pine forest).
aliphatic and aromatic fragments. The proportion of aromatic carbon of lignin origin increases with time due to the decrease in its content in the peripheral fragments of molecules. The degree of oxidation of the biopolymer increases in the following series and with time: plant tissues–litter–soil–humic acids–buried humic acids. ACKNOWLEDGMENTS This work was supported in part by the Russian Foundation for Basic Research (project nos. 04-0449727 and 08-04-00809) and the German Academic Exchange Service (DAAD). REFERENCES 1. A. Blazhei and L. Shutyi, Phenolic Compounds of Plant Origin (Mir, Moscow, 1977) [in Russian]. 2. A. S. Vladychenskii, Soil Formation in the Mountains, G. V. Dobrovol’skii (Ed.), (Nauka, Moscow, 1998) [in Russian]. 3. T. I. Evdokimova and N. O. Kovaleva, “The Role of the Biological Cycling of Substances in the Evolution of Landscapes in the Northern Tien Shan,” Vestn. Mosk. Univ., Ser. 17: Pochvoved., No. 3, 24–29 (1999). 4. I. V. Kovalev and N. O. Kovaleva, “Biochemistry of Lignin in Soils of Periodic Excessive Moistening (from the Example of Agrogray Soils in Opolie Landscapes of the Russian Plain),” Pochvovedenie, No. 10, 1205–1216 (2008) [Eur. Soil Sci. 41 (10), 1066–1076 (2008)]. 5. N. O. Kovaleva and T. I. Evdokimova, “Characterization of Organic Matter in Mountainous Soils on the Northern Slope of Kirgiz Ridge, Tien Shan,” Pochvovedenie, No. 10, 1239–1247 (1995). 6. N. O. Kovaleva and I. V. Kovalev, “Aromatic Lignin Structures in the Organic Matter of Gray Forest Soils,” Vestn. Mosk. Univ., Ser. 17: Pochvoved., No. 2, 23–29 (2002).
7. N. O. Kovaleva and I. V. Kovalev, “Biotransformation of Lignin in Forest Soils,” Lesovedenie, No. 3, 57–63 (2006). 8. N. O. Kovaleva and Yu. M. Kosareva, “Paleosols of the Kyafar (Karachai) Medieval Settlement of Alan Tribes,” in The Role of Soils in the Biosphere, Proc. of the M.V. Lomonosov Institute of Ecological Soil Science, No. 8 (MAKS Press, Moscow, 2007), No. 8, pp. 102–113 [in Russian]. 9. M. M. Kononova, Soil Organic Matter: Nature, Properties, and Study Methods (Nauka, Moscow, 1963) [in Russian]. 10. S. M. Manskaya and L. A. Kodina, Geochemistry of Lignin (Nauka, Moscow, 1975) [in Russian]. 11. D. S. Orlov, Soil Humus Acids and the General Theory of Humification (Mosk. Gos. Univ., Moscow, 1990) [in Russian]. 12. D. S. Orlov and L. A. Grishina, Practical Manual on Humus Chemistry (Mosk. Gos. Univ., Moscow, 1981) [in Russian]. 13. N. L. Radyukina, A. V. Sof’in, N. N. Kudryavtseva, et al., “Modern Concepts of the Biochemical Processes in Soil,” Vestn. Mosk. Univ., Ser. 17: Pochvoved., No. 2, 13–19 (2001). 14. Regulatory Role of Soil in the Functioning of Taiga Ecosystems, G. V. Dobrovol’skii (Ed.), (Nauka, Moscow, 2002) [in Russian]. 15. W. Amelung, Zum Klimaeinflüb auf die Organische Substanz Nordamerikanischer Präieböden. Bayreuth, 1997. 16. J. R. Ertel and J. I. Hedges, “The Lignin Component of Humic Substances: Distribution among the Soil and Sedimentary Humic, Fulvic and Base-Insoluble Fractions,” Geochim. Cosmochim. Acta 48, 2065–2074 (1984). 17. K. Haider, Von der Toten Organischen Substanz zum Humus, Z. Pflanzenernähr. Bodenk, 162, 363–371 (1999). EURASIAN SOIL SCIENCE
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TRANSFORMATION OF LIGNIN IN SURFACE AND BURIED SOILS 18. J. I. Hedges and D. C. Mann, “The Characterization of Plant Tissues by Their Lignin Oxidation Products,” Geochim. Cosmochim. Acta 43, 1803–1807 (1979). 19. I. Kögel, “Estimation and Decomposition Pattern of the Lignin Component in Forest Soil,” Soil Biol. Biochem., No. 18, 589–594 (1986). 20. I. Kögel-Knabler, W. Zech, and H. G. Hatcher, “Chemical Composition of the Organic Matter in Forest Soils: The Humus Layer,” Pflanzenernähr. Bodenk, No. 151, 331–340 (1988). 21. N. Kovaleva, “Northern Tian-Shan Paleosoil Sequences as a Record of Major Climatic Events in the Last 30 000 Years,” Revista Mexicana de Ciencias Geologicas 21 (1), 71–78(2004).
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22. A. Rodionov, W. Amelung, I. Urusevskaja, and W. Zech, “Klimaeinflüb auf Lignin und Polysacharide in Partikelgröben-Fractionen Zonaler Steppenböden Rüblands,” Plant Nutr. Soil. Sci., No. 162, 563–569 (1999). 23. S. Waksman, Humus. Origin, Chemical Composition, and Importance in Nature, 2nd ed. (Bailliere Tindall, London, 1938). 24. W. Zech, M.-J. Johansson, L. Haumaier, and R. Malcolm, “CPMAS 13C NMR an IR Spectra of Spruce and Pine Litter and of the Klason Lignin Fraction at Different Stages of Decomposition,” Pflanzenernähr. Bodenk., No. 150, 262–265 (1987). 25. F. Ziegler, I. Kögel, W. Zech, “Alteration of Gymnosperm and Angiosperm Lignin during Decomposition in Forest Humus Layers,” Pflanzenernähr. Bodenk., No. 149, 323–331 (1986).
