The influence of afforestation on
the lignin content in the soil in the
Araguás catchment area
BACHELOR THESIS Afforestation is an important strategy that can enhance carbon sequestration not only in biomass but also in soils which will diminish global carbon dioxide levels in the air. This research is part of a larger research examining the effect of afforestation on the soils in the Araguás catchment area. The lignin content of the soil will be examined for the land use changes from abandoned agricultural land to afforested land with both the Scotts Pine and the Black Pine and from abandoned agricultural land to natural succession (re-vegetation) to mainly scrublands and grassland. The results will be compared to the lignin content of a native forest in the area. The method used is Curie-point pyrolysis wit tetramethylammonium hydroxide (TMAH). It was expected that the native forest will have the largest amount of plant litter resulting in higher degradation rates with an high decline in lignin content the deeper you get in the soil. The results showed that the P. nigra was the best afforestation practice for increasing the lignin content in the soil. The P. sylvestris was considered but proved to be even less successful than natural secondary succession. The native forest did not show accurate results probably caused by a sampling error. The meadows showed an increase in lignin content for the soil depths of >20 cm that was unusual and could not be explained by the S/G and
P/G ratios and the Ad/Al ratio. Probable is that the lignin accumulated on a mineral layer or on a layer with higher iron levels. Furthermore, the S/G, P/G did not show a significant difference. From this you can conclude that origin of the lignin has had no other source than the current vegetation. The Ad/Al ratios indicate levels of degradation that are not in line with the lignin content and could be explained by the total ketones count (TKC) in further research.
ROMY STIJSIGER (10113258) SUPERVISORS: DR. ESTELA NADAL ROMERO & DR. JULIAN CAMPO JUNE 25TH, 2015
26 1. Content
1. CONTENT... 2
2. INTRODUCTION... 3
3. SCIENTIFIC BACKGROUND AND RESEARCH...4
3.1 Research Area...5
3.2 The Research...5
4. MATERIALS AND METHODS...8
4.1 Curie-point Pyrolysis with Tetramethylammonium hydroxide (TMAH)...8
4.2 Lignin...9
4.3 Analysis Lignin Content...11
5. RESULTS... 13
5.1 Lignin Content...13
5.2 Origin of the Lignin...15
5.3 Degradation rates...16
5.4 Results ANOVA...17
6. DISCUSSION...18
6.1 Lignin content in afforested areas...18
6.2 Lignin content...18
6.3 Further Research...20
7. CONCLUSION...20
8. REFERENCES... 22
9. APPENDICES... 24
9.1 Appendix I : The lignin components...24
26 2. Introduction
Afforestation is a widely used technique to plant forests in areas where none had been before. Already in Europe, North America and Russia large areas are being remodeled into fresh new forests that can act as a carbon sink (Pérez-Crusado et al., 2014). Hence afforestation is an important strategy that can enhance carbon sequestration not only in biomass but also in soils which will diminish global carbon dioxide levels in the air. For this reason afforestation is one of the most credited strategies in the treaty most famously known for the breakthroughs in the fight against global warming, the Kyoto Protocol (Cerli et al., 2008).
Since the 1940s Spain has had an active afforestation program that aimed at the establishment of a solid forest-reserve able to supply a steady volume of industrial timber, to control erosion, to control flood and to create a "peasant" forest economy closely integrated with farming, whose immediate objective is to be the provision of domestic timber requirements (FAO, 2015a). The need for such a program was because the Mediterranean landscape includes few dense forests due to the semi-arid and subhumid climate, as well as the occurrence of widespread land abandonment in the region because of emigration flows from the rural areas towards the urban areas. The land abandonment caused large changes in land coverage and resulted in severe erosion and land degradation. The afforestation program active in the research area of the Araguás catchment area in Spain planted especially forests consisting entirely of Scots Pine (Pinus sylvestris L.) or Black Pine (Pinus nigra) on former agricultural land to counteract erosion and land degradation.
As afforestation influences the vegetation cover of the soil, it influences the input and therefore the levels of organic matter in the soil. The primary source of organic matter is the vegetal input that is then decomposed by living micro-organisms. Organic matter is important because it influences both the chemical and the physical properties of the soil. The properties that are primarily influenced by organic matter are: soil structure, moisture holding capacity, nutrient availability and diversity and activity of soil organisms (FAO, 2015b). The main aim of the
26
afforestation project was to inhibit erosion processes by increasing the organic matter and restoring the soil properties. Differences in organic matter composition can lead to various effects on decomposition rates and nutrient cycling in the soil. One of the components of organic matter is lignin. While the overall rate of organic matter gives an indication of the quantity of the organic matter, lignin gives a qualitative value on the status of organic matter and the soil. The degradation rate of lignin and the origin of the lignin can give an indication of the soil organic matter dynamics and its interaction with minerals.
In this research the influence of afforestation and land use changes on the lignin content in the soils is examined for the Araguás catchment area in the central Spanish Pyrenees. This research is part of a larger research examining the effect of afforestation on the soils in the Araguás catchment area over time (Med Afforest Project, PIEF-GA-2013-624974). The lignin content of the samples were examined for land use changes from abandoned agricultural land to afforested land with both the Scots Pine and the Black Pine and from abandoned agricultural land to natural re-vegetation (secondary succession). The results will be compared to the lignin content of a native forest in the area and agricultural land that is still used for growing grass for feeding livestock. The objective of the research is to examine if there are differences in the lignin content between land uses and in depth and how this relates to the best afforestation practice. Hypothesized was that the lignin content in depth would show the same trend as found by Thevenot et al. (2010), in which the first layers would show a significant peak of lignin content to which it would quickly decrease to a steady low-point in the deepest layers. The native forest was hypothesized to have the highest lignin content because this soil has had no disturbance in soil formation and vegetation cover. Secondly the forests of Scots Pine and Black Pine would have the highest lignin content because forests have a higher litter input than shrub land and meadows. The lignin content of the meadows is hypothesized to be higher than the lignin content of secondary succession because the soil has been left undisturbed longer. Lastly, the sites of the bare soil will have
26
the lowest amount of lignin content due to the absence of litter input for over 50 years.
3. Scientific background and research
Analyzing the effect of land use changes by examining the lignin content is part of an overarching research investigating the effect of afforestation on the soils in the Araguás catchment area. The Araguás catchment area is a small area in the central Spanish Pyrenees where widespread land abandonment and centuries of cultivation caused severe soil erosion. The national forest services were urged to afforest the land with Scots Pine and Black Pine. The original idea behind the choice for the Scots Pine and the Black Pine was that these were fast-growing conifers that subsequently would lead to rapid restoration of the abandoned land, therefore quickly restoring soil properties and hydrological processes. In this research that is conducted by Dr. Estela Nadal-Romero several components and processes are examined in order to answer the research questions of: (i) how do soil properties change after land abandonment in Mediterranean mountain areas?, and (ii) what is the impact of natural revegetation and afforestation on soil properties (physical and chemical) and aggregate stability?
3.1 Research Area
The Araguás catchment area in the Spanish Pyrenees has been cultivated for centuries until the 1950s with cereal crops. The different soils that are used to determine the effect of afforestation are soils of formerly abandoned agricultural lands that have undergone land use changes due to afforestation and natural revegetation. The abandoned agricultural lands are now covered with (i) Scots Pine forests, (ii) Black Pine forests and (iii) shrub land consisting of Genista scorpius, Juniperus communis, Rosa gr. canina and Buxus sempervirens, as well as some stands of small P. sylvestris. These areas are then compared to a site with (iv)bare soil that represents the former severely eroded agricultural
26
soil, to a site with a (v) native forest that represents undisturbed soil and a site of (vi) cultivated grassland representing the lignin content under prolonged cultivation. All sites have the exact same lithology to ensure that a comparison can be made. The soils have been severely cultivated in the past resulting into a thin and stony soil on an Eocene Flysch bedrock type. Characteristic for this region of the Central Pyrenees are the alternating sandstones with carbonate cementation and marl layers (Nadal-Romero et al., 2015). In figure 2 an overview is given of the different sites, their location and an illustration of their vegetation cover. The location of the native forest is not on the map, it is located 4 kilometers outside. However the lithology is identical and can therefore be used in the research.
3.2 The Research
In figure 1 an overview is given of the research into the effect of afforestation and land use changes on soil properties, hydrological processes, biodiversity and landscape. The physical soil properties which
are examined are aggregate stability, grain size distribution and bulk density.
26
The chemical soil properties studied will include the pH, total soil organic carbon, total nitrogen, organic matter and CaCO3. In the overarching
research the quantity of organic carbon was already measured however the exact composition is not known. In this paper that acts as an independent research in the overall research the exact composition of the lignin content in the soil is measured. Lignin is considered vital in the soil organic matter dynamics and is therefore important in examining the influence of afforestation and land use changes on the soil. Whilst the level of organic matter found in the soil influences the soil fertility, the lignin content can help to evaluate where this input originates as well as indicating the degradation rates of the different soils and their different depths. In doing so creating a better understanding of how the levels of organic carbon were influenced by afforestation and land use changes (Thevenot et al., 2010).
4. Materials and Methods
In order to analyze the effect of the land use changes and afforestation, the composition of the lignin content was measured for the different land cover types as described before. With first clearing the litter of the soil surface, soil has been collected from every site from a depth between 0-5, 5-10, 10-15, 15-20, 20-30, 30-40 and 40-50 cm to see how the lignin content changes in depth per land use.
4.1 Curie-point Pyrolysis with Tetramethylammonium hydroxide (TMAH)
The method used in this experiment is Curie-point pyrolysis with tetramethylammonium hydroxide (TMAH). TMAH is used for methylation of produced monomers simultaneously with pyrolysis turning phenolic acid and phenols into methyl esters and methyl ethers that can be
identified with gas chromatography (Kögel-Kabner, 2000, Mason et al.,
2009). Although the procedure is comparable with CuO oxidation in analyzing the lignin degradation,
TMAH-thermochemolysis is able to trace lignin when it has been degraded severely whereas in CuO oxidation it
remains undetected (Kögel-Kabner, 2000).
In preparation of the TMAH the samples were dried at low temperatures (drying at maximum temperature of 40 °C in an oven). The samples were grinded as fine as possible. The materials needed
were glass capillaries, pyrolysis wires 600°, small glass dish, spatula, metal tweezers and aluminium foil. The chemical used as an internal
standard was 5α-Androstane (C19H32 , CAS#
438-22-2) and the tetramethylammonium hydroxide (TMAH) was in a 25 % solution in water.
In preparation for the Pyrolysis 1-2 mg of the sample was mixed with 6 µl of androstane solution (100ug /l in cyclohexane) and 20 µl of TMAH solution (25% in water) to derivatize for 2 min. A
Figure 3 Soil samples being grinded
ferro magnetic wire was dipped into the paste of the sample or the sample slurry was spread on the wire with a spatula. The amount loaded to the wire depended on the amount of carbon in the sample, which determined the quality of the chromatogram. The wire was dried under a halogen lamp because water is very harmful for the GC column. The wire with the sample was placed in a glass capillary like shown in figure 4. The distance between the end of the wire with the sample and the end of the glass capillary should be 1 cm. The glass capillary is finally placed in the probe of the pyrolysis unit and inserted into the GC. The pyrolysis head will heat up for 5 seconds up to 250 °C. Then the sample is inserted into the GC column (Zebron, Zb-1MS) and gradually heated to 300 °C.
When the GC had analyzed the samples the mass spectrometer identified the peaks of the specific compounds and resulted into chromatogram and corresponding spectrum to one of the peaks. The peaks were examined and compared to an overview made by Poerschmann et al. (2008) and the retention times examined by Brock et al. (2015).
In table 1 an overview is given of the different lignin components that can be found after Curie-point pyrolysis with TMAH according to Poerschmann et al. (2008). In figure 6 the identified peaks with the lignin components is shown in the chromatogram of the 0-5 cm layer of the P. nigra.
4.2 Lignin
Plant litter is the source of soil organic matter whereas 20% of the input in soils is caused by lignin. Lignins are the most abundant aromatic plant components in terrestrial ecosystems (Thevenot et al., 2010). Lignins are found in the cell walls of vascular plants. The mechanical strength of a plant is the particular property of lignin. The mechanical strength causes plants to be less susceptible to herbivores, stem and leaf breaking, and pest injury (Gul et al., 2014). For this reason lignin is only degradable by a few organisms including a few bacteria such as Streptomyces sp. and Nocardia sp. and especially the brown-rot fungi and
white-rot fungi, of which only the white-rot fungi can mineralize the molecule (Thevenot et al., 2010).
In analyzing the lignin content the chemical structure and the compounds of lignin are used as biochemical indicators of origin and state of decomposition of lignin and SOM (Thevenot et al., 2010). Lignin consists of phenylpropanoid units and
monolignols with several linkages. The monolignols are the coniferyl alchol, the synapyl alcohol and the p-coumaryl alcohol. The lignols are incorporated into lignin resulting into units called guaiacyl (G units), syringyl (S units) and p-hydroxyphenyl (P units) (Gul et al.,
2014). The sum of these compounds is used for calculating the lignin content. The ratios between these components are used as indicators for the state of lignin degradation in the soil and the source of vegetation.
Figure 5 Structure of monolignols from Talbot et al. (2012)
Figuur 6 The lignin components identified in P.nigra, 0 cm
Table 1 The lignin components that can be found after Curie point pyrolysis. Poerschmann et al. (2008)
4.3 Analysis Lignin Content
After identification of the components the quantity is determined by calculating the area underneath the peak. This is done by the program called Xcalibur. A given amount is estimated for the area under the component and this is compared to the estimated area underneath the internal standard of 5α-Androstane. At most soil samples 0.6 µg of Androstane was added, however in some samples the amount of organic carbon in the soil was so low that fewer Androstane was added so the intensity would be congruent with the intensity of the components. There is no consensus among researches in the field of lignin on the best way to show the lignin content. Cerli et al. (2008) uses the units mg/g C and mg/ha, Mason et al. (2009) uses the units mg/100 mg TOC just like Ertel and Hedges (1984). Whereas Thevenot et al. (2010) uses g/kg OC a unit that is similar to the µg/mg that is used in this research. The reason for this choice is that Thevenot did a comparative analysis of a vast amount of researches all concluded into one graph with this unit. In this way the comparison with the other studies into the lignin content is more convenient. All the amounts per lignin component and the amounts of Androstane added can be found in Appendix I.
Furthermore the sum of the components in the table below is used in indicating the lignin content. These phenols indicate the lignin-derived material in the soil samples. The reason for this is that lignin is a large varied compound and there is consensus that the components in the
Table 2 The lignin components used in calculating the amount of lignin in the soil.
Lignin phenols Guaiacyl
G4 3,4-dimethoxybenzaldehyde
G6 3,4-dimethoxybenzoic acid methyl ester
G7 cis-1-(3,4-dimethoxyphenyl)-2-methoxyethylene G8 trans-1-(3,4-dimethoxyphenyl)-2-methoxyethylene G14 threo/erythro-1-(3,4-dimethoxyphenyl)-1,2,3-trimethoxypropane G15 threo/erythro-1-(3,4-dimethoxyphenyl)-1,2,3-trimethoxypropane Syringyl S4 3,4,5-trimethoxybenzaldehyde
S6 3,4,5-trimethoxybenzoic acid methyl ester
S7 cis-1-(3,4,5-trimethoxyphenyl)-2-methoxyethylene S8 trans-1-(3,4,5-trimethoxyphenyl)-2-methoxyethylene S14 threo/erythro-1-(3,4,5-trimethoxyphenyl)-1,2,3-trimethoxybenzene S15 threo/erythro-1-(3,4,5-trimethoxyphenyl)-1,2,3-trimethoxybenzene Coumaryl
P18 trans-3-(4-methoxyphenyl)-3-propenoic acid methyl
ester
G18 trans-3-(3,4-dimethoxyphenyl)-3-propenoic acid
table are definitely derived from lignin whereas other components that have been found can originate from other compounds in the soil or other sources aboveground.
All components were calculated to determine the overall lignin content of the different soils at their different depths as well as the ratio’s between components. The ratios between these components are used as indicators for the state of lignin degradation in the soil and the source of vegetation. The S/G and the P/G ratio’s which comprises the sum of the derived components of the lignin monomers syringyl, guaiacyl and p-hydroxyphenyl indicates the origin of lignin and therefore the vegetation. In essence, gymnosperms (e.g. conifers like the P.sylvestris and P. nigra) produce guaiacyl and no syringyl resulting in an S/G ratio close to 0. On the other hand angiosperms such as grasses, deciduous trees and
flowering plants produce both guaiacyl and syringyl resulting in an S/G ratio larger than 0. At last the P/G ratio of >0 indicates grasses, leaves and needles. They do not derive from woods resulting into P/G =0 (Ertel & Hedges, 1984).
Another ratio that is determined is the acid aldehyde ratio which will be indicated by the components of G6/G4 (3,4-dimethoxybenzoic acid methyl ester/3,4-dimethoxybenzaldehyde) and S6/S4
(3,4,5-trimethoxybenzoic acid methyl ester/3,4,5-trimethoxybenzaldehyde). The acid/aldehyde ratio of the guaiacyl lignin monomer and the syringyl
monomer describe the oxidative susceptibility of the lignin. The oxidative susceptibility correlates to the degradation rate in the soil. The higher the acid/aldehyde ratio, the higher the degradation rate (Poerschmann et al., 2008).
In analyzing the results first a normality test was done to see what statistical test could be used. The dependent variable is the lignin content and the independent variables are land use and depth. The variance in results was examined with an ANOVA analysis to indicate if there is a significant difference and Tukey’s post-hoc test was used to examine the difference between the different soils. The significance was indicated by a p-level of 0.05 or lower. The correlation between the lignin content in the
soil and the carbon content of the soil was examined with the Pearson correlation test.
5. Results
Following the procedure resulted into a classification of the different lignin components in the soils and the amount in which they can be found. The native forest is referred to as forest, the cultivated grassland is referred to as meadows, the naturally re-vegetated shrub land is referred to REV, the P. sylvestris is referred to as P.S., the P. nigra is referred to as P.N. and the bare soil which represents the state before afforestation or secondary succession is referred to as Bare. The data on the organic carbon levels was provided by Nadal-Romero et al. (2015).
5.1 Lignin Content
The bare soil is the representative of the historic abandoned land and there was no lignin found in the soil samples of this site. Considering the bare soil, the effect of afforestation on the former abandoned agricultural is directly represented by the lignin content of the P. sylvestris and P. nigra. According to the organic carbon level the lignin should rise with both manual and natural re-vegetation. Afforestation with P. sylvestris results in an increase of 1.6% in organic carbon in the top layer (0 cm) and 1.5% in the second layer (0-5 cm). Afforestation with P. nigra results in an increase of 7.1% in organic carbon in the top layer and 4.6% in the second layer (0-5 cm). Natural re-vegetation increased the organic carbon level of the top layer with 2.6% and the second layer with 1.3%. The lignin content significantly differs between P. nigra and the native forest while in the carbon content the meadow significantly differs from the other land uses.
Figure 7 Figure 8 0 1 2 3 4 5 6 7 8 9 0 5 10 15 20 25 30 35 40 45 50
Lignin content (ug/mg soil)
FOREST MEADOWS REV
P.S P.N. Bare D e p th ( cm ) 0 1 2 3 4 5 6 7 8 9 0 5 10 15 20 25 30 35 40 45 50 Organic Carbon (%)
FOREST MEADOWS REV
P.S P.N. Bare D e p th ( cm )
Pearson correlation (r2) Significance (p) Native Forest 0.669 0.100 Meadows 0.314 0.448 Natural re-vegetation 0.662 0.074 P. sylvestris 0.704 0.051 P. nigra 0.893 0.003 Bare Soil -
-The Pearson correlation shows correlation that there is a high positive correlation the lignin content of P. sylvestris and the P. nigra. and the carbon content of the soils underneath the P. sylvestris and P. nigra. The Pearson correlation also shows correlation at a significant level between the lignin content and the carbon content of the P. nigra.
0 2 4 6 8 10 12 14 16 18 0 10 20 30 40 50
Lignin Conten (ug/mg carbon)
FOREST MEADOWS REV
P.S Bare P.N. D e p th ( cm ) Figure 9
The differences in the samples within soil depth and between the vegetation cover was tested with a two way Analysis of Variance
Table 3 Pearson correlation between the lignin content (µg/mg) and the carbon content of the soil (µg/mg).
(ANOVA). A significant difference is found between vegetation covers in the soil depths 20-50 cm. As can also be clearly seen in the graph, the ANOVA test shows that the lignin content of the meadows significantly differs from all the other vegetation types. Between the land uses significant differences can be found within depth in natural re-vegetation, within depth in P. nigra and within depth in the native forest. The results of the ANOVA test are also shown in table 4 on page 17.
5.2 Origin of the Lignin
0 0.5 1 1.5 2 2.5 3 3.5 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
Origin of the Lignin
Forest Meadows REV P.S P.N
P/G
S
/G
Figure 10
The origin of the lignin is portrayed in figure 10 by the syringyl/guaiacyl (S/G) ratio and the p-hydroxyphenyl/guaiacyl (P/G) ratio. The different boxes identify the different categories that can be differentiated according to the method used by Hedges & Mann (1979) and Cerli et al. (2008). The different categories are: gymnosperm wood (G), non-woody gymnosperm tissue (g), angiosperm wood (A) and non-woody angiosperm tissue (a). The P/G ratio and the S/G ratio do not show significant differences within one land use and between land uses on depth 0-15 and 15-20 cm.
5.3 Degradation rates 0 5 10 15 20 25 30 0 5 10 15 20 25 30 35 40 45 50
Natural re-vegetation
Ad/Al G Ad/Als 0 5 10 15 20 25 30 0 5 10 15 20 25 30 35 40 45 50P. sylvestris
Ad/Al G Ad/Als 0 5 10 15 20 25 0 5 10 15 20 25 30 35 40 45 50P. nigra
Ad/Al G Ad/Als Figure 11The G6/G4 ratio (3,4-dimethoxybenzoic acid methyl ester/3,4-dimethoxybenzaldehyde) and the S6/S4 ratio (3,4,5-trimethoxybenzoic acid methyl ester/3,4,5-trimethoxybenzaldehyde) represent the
acid/aldehyde ratio of the guaiacyl lignin monomer (indicated with Ad/AlG)
and the syringyl monomer (indicated with Ad/AlS). The acid/aldehyde ratio
0 5 10 15 20 25 30 0 5 10 15 20 25 30 35 40 45 50
Native Forest
Ad/Al G Ad/Als 0 5 10 15 20 25 30 0 5 10 15 20 25 30 35 40 45 50Meadows
Ad/Al G Ad/Alsdescribes the oxidative susceptibility of the lignin and therefore the degradation rate. The results of the ANOVA show that through the entire depth there are not significant differences within land uses. Between the land uses on the depth of 0-15 cm no significant difference was found, however on the depth of >20 cm a significant difference was found between the P. sylvestris and the P. nigra.
All the data of the ratios was collected in a table in Appendix II. 5.4 Results ANOVA
Table 4 shows the results of the ANOVA test on the land uses. The asterisk (*) shows if there is a significant difference in one variable in the depth within one land use.
Table 4
Lignin S/G P/G Ad/AlG Ad/AlS
Native Forest 0.013* 0.133 0.286 0.279 0.747 Meadows 0.090 0.772 0.286 0.335 0.181 Natural re-vegetation 0.046* 0.685 0.239 0.178 0.225 P. sylvestris 0.112 0.259 0.115 0.237 0.147 P. nigra 0.045* 0.685 0.239 0.178 0.225 Bare Soil - - - -
-Table 5 shows the results of the ANOVA test showing no significant differences between land uses in the variables on soil depths of 0-15 cm.
Table 5
0-15 cm Lignin S/G P/G Ad/AlG Ad/AlS
Total Land
uses 0.286 0.190 0.081 0.173 0.762
Table 6 shows the results of the ANOVA test showing significant differences between land uses in the variables of lignin content and Ad/AlG ratio on soil depths of 15-50 cm..
Table 6
15-50 cm Lignin S/G P/G Ad/AlG Ad/AlS
Total Land
6. Discussion
Lignin is considered vital in the soil organic matter dynamics and is therefore important in examining the influence of afforestation and land use changes on the soil. The lignin provides information about the origin of the plant-derived organic matter and the state of degradation.
6.1 Lignin content in afforested areas
The effect of afforestation on the lignin compounds have been tested in the literature. Although there is consensus among researchers on the effect of different vegetation types and land uses on the lignin content and the occurrence of the different lignin monomers there is still a high variation within these studies. Lignin content can also highly vary between different climates and different soil characteristics such as soil depth, clay content and particle size (Thevenot et al., 2010). However within this study the characteristics are all the same except for vegetation type and land use. According to Thevenot et al. (2010) the overall lignin content can be related to the organic carbon content and the nitrogen content in the soil, suggesting a higher content in forests than in arable soils. However the evaluation of 29 different studies concluded that the lignin content was the highest in arable land, then grassland and forests. It was suggested that this was related to the difference in decomposition conditions and organic matter sources.
6.2 Lignin content
The carbon content in the soil show that the P. nigra would have the best potential in increasing the organic carbon levels in the soil. In addition, the P. nigra has the highest lignin content in the soil between the depths of 0-15 cm, which is in compliance with the Pearson correlation of the P. nigra. However below 15cm the lignin content decreases significantly. The diminishing state can be explained by the acid/aldehyde ratio. The ratios are in the first four layers rather low indicating low oxidative susceptibility, while on the other hand when the depth of 15 cm is surpassed the acid/aldehyde ratio is 7 times higher. This indicates that the lignin input of P. nigra is high but the degradation rate is low in the
top layers and the degradation rates in the lower layers is higher resulting in lower lignin levels. The high degradation rates found in the depths of >20 cm are significantly different from the data of the topsoil and could be an indication of an old soil that developed previous to the afforestation and cultivation.
The lignin content of the natural re-vegetated sites (REV) shows the same trends as the P. nigra with a high lignin content in the top soil and a low lignin content in the deepest layers. This is also a trend according to the research of Thevenot et al. (2010) in which the results of 29 researches were reviewed. The lignin levels on the top of the soils are also high but these are being degraded by UV-light causing the highest peak in lignin to be slightly underneath the top. Expected was that all the different land uses show the same trend. However, the meadows show a rise in lignin content in the soil layer >20 cm. Although the meadows have the highest carbon content in this part of the soil, it does not significantly differ from the other land uses, whereas the difference in the lignin content between the meadows and the other sites is significant. The result could be explained by that this is an older part of the soil where not a lot of degradation took place causing the lignin to accumulate. If so the S/G ratio and the P/G ratio would have to show a significant difference between the soil of the meadows 0-20 cm depth and 20-50 cm depth however it does not show any significant differences in the different land uses. Concluding that the origin of the lignin is the same throughout the entire depth for every type of vegetation. This means that there is no prove for older layers in the soil that may cause higher or lower levels of lignin content. Another cause for the high lignin contents in the sub soil in the meadows can be that the degradation rates are lower. However the acid/aldehyde ratios of the meadows didn’t show a significant difference between the top soil and lower soil. Another explanation can be that the grasses in the meadow had a top layer with
high lignin content and high Ad/AlG and these lignin compounds are then
drained through the soil by rain. This causes the decrease in lignin content whereas it then in the deepest layers accumulates. The
accumulation can be caused by a mineral layer, higher water content, higher carbohydrate levels or higher levels of iron. However these correlations will be examined in the further ongoing research of Estela Nadal-Romero.
The Ad/Al ratio did not always correlate with the amount of lignin in the soil. The reason for this is that for calculating the lignin content in the soil only certain compounds of lignin were added because there is consensus in the literature about the certainty that these compounds actually derive from lignin. The other compounds which were not used in calculating the lignin content are called the total ketones count (TKC). The TKC is not taken into account because their specific origin does not always necessarily link to the lignin content. However in further research the TKC will be taken into account to see if there is correlation between degradation rates and the new lignin content. The aim is to see if higher lignin content leads to higher or lower degradation rates that could not be explained with the original lignin content levels used in this research.
The Native Forest in the region has unexpectedly low organic carbon levels. The forest soil has remained undisturbed for years and yet the organic carbon levels together with the lignin levels remain lower than all the other sites except the bare soil. Within the soil a significant difference has been found between the top layer of 0-15 and 15-50 cm depth resulting from a minor difference between the two means. The low levels in the native forest can be explained by a sampling error of the soils. The density of the native forest was very high causing sites that were sampled to be easier accessible and to be at an erosion point where carbon could have been eroded away.
Previous research on the topic of the effect of afforestation on the lignin content consists of other soil types and climate. According to Mason et al. (2009) ‘afforestation has had a significant effect on phenolic degradation dynamics in peaty gley soils’. Although Masons research has a different environment and situation it showed that input of litter increased and the degradation of guaiacyl and syringyl increased. Cerli et al. (2006) researched the changes in organic matter in an age sequence
on former agricultural soils in Norway. She cites that gymnosperm derived lignin was more apparent when aged increased. Our research was new in examining the effect of afforestation on the lignin content in Mediterranean mountain areas. Results show that the P. nigra has the highest lignin content following the common trend of higher lignin content in the top soil and lower lignin content in deeper soils. Secondly the secondary succession shows the second-best results in increasing the lignin content which are better than the afforestation practice of P. sylvestris. In conclusion the best afforestation practice in the Mediterranean mountain areas is P. nigra, then natural revegetation and then the P. sylvestris.
6.3 Further Research
This research will be published and further research will include the trend of the lignin content in the soil and the degradation rates being
compared to other variables such as pH, CaCO3, EC, C/N, Total Nitrogen,
organic carbon, inorganic carbon, P, N, bulk density, field capacity and the clay/silt/sand levels of the different land uses native forest, the meadows, natural re-vegetation, P. sylvestris and P. nigra.
7. Conclusion
Afforestation is a common practice used for erosion control by increasing the organic matter in the soil. The lignin content of a soil influences the soil dynamics of organic matter and is vital for understanding the origin and degradation rate of the organic matter. In this research the influence of afforestation and land use changes on the lignin content was examined for Araguás catchment area in the central Spanish Pyrenees. The results showed that P. nigra was the best afforestation practice for increasing the lignin content in the soil. P. sylvestris was considered but proved to be even less successful than natural secondary succession. The numbers were compared to a native forest site and grassland site. The native forest did not show accurate results probably caused by a sampling error.
The meadows showed an increase in lignin content for the soil depths of >20 cm that was unusual will be examined in further research.
8. References
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Cerli, C., Celi, L., Kaiser, K., Guggenberger, G., Johansson, M. B., Cignetti, A., & Zanini, E. (2008). Changes in humic substances along an age sequence of Norway spruce stands planted on former agricultural land. Organic Geochemistry, 39(9), 1269-1280.
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http://www.fao.org/docrep/x5386e/x5386e02.htm#TopOfPage
FAO (2015b) The Importance of soil organic matter. Key to
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Mason, S. L., Filley, T. R., & Abbott, G. D. (2009). The effect of afforestation on the soil organic carbon (SOC) of a peaty gley soil using on-line thermally assisted hydrolysis and methylation (THM) in the presence of 13 C-labelled tetramethylammonium hydroxide (TMAH). Journal of Analytical and Applied Pyrolysis, 85(1), 417-425.
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Poerschmann, J., Rauschen, S., Langer, U., Augustin, J., & Górecki, T. (2008). Molecular level lignin patterns of genetically modified Bt-maize MON88017 and three conventional varieties using tetramethylammonium hydroxide (TMAH)-induced thermochemolysis. Journal of agricultural and food chemistry, 56(24), 11906-11913.
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9. Appendices
9.2 Appendix II : The ratios and degradation rates
Native Forest Meadows Natural
revegetation S/ G P/G Ad/Al G Ad/ Als S/ G P/G Ad/Al G Ad/ AlS S/G P/G Ad/Al G Ad/Al S 0 0.8 9 0.86 3.66 2.04 0.73 0.17 5.09 1.25 5 0.5 7 0.20 5.54 1.55 0.52 20.2 28.58 2.70 0.62 0.09 6.57 2.88 10 0.7 3 0.00 2.62 0.37 2.10 20.4 10.96 3.38 0.92 0.02 17.50 1.24 15 0.1 5 0.00 1.73 7.15 0.96 10.3 6.48 2.37 0.94 0.04 3.31 4.73 20 0.1 7 0.00 6.06 4.09 0.84 40.0 8.42 2.59 0.80 0.00 9.02 1.94 30 0.2 9 0.00 3.32 1.01 1.77 10.1 11.84 2.35 0.72 0.00 1.72 1.90 40 0.1 8 0.00 9.57 2.73 0.93 0.12 11.17 2.37 - - 0.00 0.00 50 0.2 3 0.00 3.71 1.64 0.63 0.40 4.36 2.35 - - 0.00 0.00 Mea n 0.3 3 0.03 4.65 2.6 5 1.0 8 0.3 1 10.68 2.5 2 0.05 0.7 9 5.40 1.74
P. sylvestris P. nigra Bare Soil
S/ G P/G Ad/AlG Ad/ Als S/G P/G Ad/AlG Ad/ AlS S/G P/G Ad/Al G Ad/Al S 0 0.6 2 0.23 4.23 4.2 9 0.1 6 0.1 2 0.00 0.0 0 0 0 0 0 5 0.1 5 0.22 1.46 0.0 0 0.1 5 0.3 3 4.93 0.9 6 0 0 0 0 10 0.9 0 0.11 5.30 1.9 2 0.3 2 0.3 2 3.69 1.0 1 0 0 0 0 15 0.5 2 0.23 4.81 4.4 3 0.4 8 0.0 0 3.63 1.3 2 0 0 0 0 20 0.1 5 0.00 0.00 0.0 0 0.4 9 0.1 9 22.81 2.3 5 0 0 0 0
30 0.1 2 0.10 0.68 0.0 0 1.2 0 0.1 1 9.58 9.8 7 0 0 0 0 40 - -5.16 0.8 8 0.1 4 0.0 0 9.92 1.8 2 0 0 0 0 50 - -0.00 0.0 0 0.0 0 0.0 0 3.83 2.7 8 0 0 0 0 Mea n 0.1 5 0.41 2.70 1.7 4 0.3 7 0.1 3 7.30 2.5 1 0 0 0 0
