九州大学学術情報リポジトリ
Kyushu University Institutional Repository
火山灰土壌中における元素の長期的移動
ジッティア, ナウォディー, ウィジェシンハ
http://hdl.handle.net/2324/4475189
出版情報：Kyushu University, 2020, 博士（農学）, 課程博士 バージョン：
権利関係：
Long term movements of elements in Andosols
Wijesinghe Jithya Nawodi
2021
Long term movements of elements in Andosols
Wijesinghe Jithya Nawodi
Laboratory of Soil Science
Department of Agro environmental Sciences
Graduate School of Bioresources and Bioenvironmental Sciences Kyushu University
A dissertation submitted to the Graduate School of Bioresources and Bioenvironmental Sciences, Kyushu University, Japan, in partial fulfillment of the requirements for the degree
of Doctor of Philosophy
Declaration
I hereby declare that this submission is my own work in its entirety and that to the best of my knowledge, it contains no material previously published or written by another person nor material to a substantial extend has been accepted for the award of any other degree or diploma of the university of higher learning, except where due acknowledgement has been made in the text.
Wijesinghe Jithya Nawodi Date:
I certify that the declaration above by the candidate is true to the best of my knowledge and that this report is acceptable for evaluation for the degree of Doctor of Philosophy.
Prof. Syuntaro HIRADATE, PhD Date:
To the people of Sri Lanka and Japan
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List of Content
Acknowledgment………. ix
Abstract………. x
Chapter 1 ... 1
Background of the study ... 1
1.1. Elements in soil ... 2
1.2. Andosols ... 4
1.2.1. Andosols as a carbon store: Formation and translocation of SOC ... 6
1.2.2. Behavior of elements in Andosols ... 8
1.2.3. Suitability of Andosols in elemental studies ... 9
1.3. Fractionation of soil organic carbon ... 10
1.4. Radiocarbon (14C), 13C and 15N isotope ratio in soil carbon dynamics ... 12
1.5. Application of 13C Nuclear magnetic resonance (NMR) in soil organic carbon ... 14
1.6. Objectives of the study ... 16
1.7. Overview of this dissertation ... 16
Chapter 2 ... 18
Formation and mobility of soil organic carbon in a buried humic horizon of a volcanic ash soil ... 18
2.1. Introduction ... 19
2.2. Materials and methods ... 22
2.2.1. Study site and soil samples ... 22
2.2.2. Preparation of SOC fraction ... 24
2.2.3. Elemental analysis of C and N in SOC fraction ... 26
2.2.4. 14C dating ... 26
2.2.5. Solid-state 13C NMR measurement ... 28
2.2.6. Determination of rate of vertical translocation of SOC fraction ... 29
2.3. Results and discussion ... 30
2.3.1. C and N distributions in SOC fractions ... 30
2.3.2. Degree of biological transformation of SOC fractions ... 31
2.3.3. Vertical translocation of SOC fractions ... 39
ii
2.3.4. Chemical compositional feature of SOC fractions and their relationships with the
behavior in soils ... 45
2.3.5. Genesis of HA fractions in the present study ... 47
2.4. Conclusion ... 49
Chapter 3 ... 50
Depth profile of plant nutrients and acidity in a non-allophanic Andosol ... 50
3.1. Introduction ... 51
3.2. Materials and Methods ... 53
3.2.1. Study site and soil samples... 53
3.2.2. Chemical analysis ... 57
3.3. Results and Discussion ... 59
3.3.1. Depth profile of Alp, Fep, Alo, Feo, and Sio ... 59
3.3.2. Depth profile of soil acidity ... 64
3.3.3. Depth profile of soil exchangeable cations ... 67
3.3.4. Depth profile of soil phosphorous ... 75
3.3.5. Depth profiles of soil C and N ... 79
3.4. Classification of the soils in the present study ... 82
3.5. Conclusion ... 82
Chapter 4 ... 84
Summary and Conclusion ... 84
References ... 87
iii
List of Tables
Table 2.1. Averaged C and N recovery of humin, humic acid (HA), hydrophilic fulvic acid (FA1
and FA2), and hydrophobic fulvic acid (FA3 and FAIHSS) fractions prepared from eight sub- horizon samples from a buried humic horizon occurred between 147 and 187 cm depth of an Andosol near Towada volcano ... 31 Table 2.2. Selected chemical properties of eight sub-horizon samples from a buried humic horizon occurred between 147 and 187 cm depth of an Andosol near Towada volcano ... 32 Table 2.3. Averaged δ13C and δ15N values of humin, humic acid (HA), hydrophilic fulvic acid (FA1 and FA2), and hydrophobic fulvic acid (FA3 and FAIHSS) fractions prepared from eight sub-horizon samples from a buried humic horizon occurred between 147 and 187 cm depth of an Andosol near Towada volcano ... 33 Table 2.4. The C/N ratio of humin humic acid (HA), hydrophilic fulvic acid (FA1 and FA2), and hydrophobic fulvic acid (FA3 and FAIHSS) fractions prepared from eight sub-horizon samples from a buried humic horizon occurred between 147 and 187 cm depth of an Andosol near Towada volcano ... 36 Table 2.5. Distribution of carbon species and aromaticity of humin, humic acid (HA), hydrophilic fulvic acid (FA1 and FA2), and hydrophobic fulvic acid (FA3 and FAIHSS) fractions prepared from four sub-horizon samples (147-152, 162-167, 172-177, and 182-187 cm depths) from a buried humic horizon occurred between 147 and 187 cm depth of an Andosol near Towada volcano ... 37 Table 2.6. The rate of vertical translocation of humin, humic acid (HA), hydrophilic fulvic acid (FA1 and FA2), and hydrophobic fulvic acid (FA3 and FAIHSS) fractions prepared from eight sub-horizon samples from a buried humic horizon occurred between 147 and 187 cm depth of an Andosol near Towada volcano ... 41
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Table 2.7. Radiocarbon signature (as pMC: percent modern carbon) and 14C age of humin, humic acid (HA), hydrophilic fulvic acid (FA1 and FA2), and hydrophobic fulvic acid (FA3 and FAIHSS) fractions prepared from eight sub-horizon samples from a buried humic horizon occurred between 147 and 187 cm depth of an Andosol near Towada volcano ... 48 Table 3.1. Field description of fertilized grassland soil and secondary forest soil in Kuju highland, Oita Prefecture, Japan ... 55 Table 3.2. Application rates of chemical fertilizers (N, P2O5, and K2O), lime, and manure on reclaimed grassland soil in Kuju highland, Oita prefecture, Japan ... 56
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List of Figures
Figure 1.1. Simplified schematic diagram of the terrestrial carbon cycle ... 4 Figure 1.2. Typical soil profile in Andosol with thick, dark, strongly humified A horizon (a) found in a secondary forest in Kuju highland, Oita prefecture, Japan and non-Andosol soil profile found in experiment forest area, Fukuoka prefecture, Japan (b) ... 5 Figure 1.3. Distribution of allophanic and non-allophanic Andosols in Japan ... 6 Figure 1.4. Thick humic horizons under thick tephra deposits found near Towada volcano, Aomori, Japan (a) and well-ordered horizons with distinct horizons in non-allophanic Andosol found in Tohaku, Japan (b) ... 10 Figure 1.5. Standard procedures of isolation and separation of humin, humic acid and fulvic acid fractions in soil accepted by the International Humic Substances Society. ... 11 Figure 1.6. A simplified representation of a typical NMR spectrum of SOC showing various chemical shift regions ... 15 Figure 2.1. Location of sampling site illustrated on isopach map of To-Cu and To-Nb tephras based on Machida and Arai (2003) (a) and sampling position in the soil profile (b). To-a:
Towada-a, To-b: Towada-b, To-Cu: Towada-Chuseri, To-Nb: Towada-Nambu ... 24 Figure 2.2. Fractionation and purification procedures of humin, humic acid (HA) and Fulvic acid (FA) fractions prepared from eight sub-horizon samples from a buried humic horizon occurred between 147 and 187 cm depth of an Andosol near Towada volcano ... 27
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Figure 2.3. Freeze dried fractions of humin, humic acid (HA) and Fulvic acid (FA) fractions prepared from eight sub-horizon samples from a buried humic horizon occurred between 147 and 187 cm depth of an Andosol near Towada volcano ... 28 Figure 2.4. Relationship between δ13C and δ15N values of humin, humic acid (HA), hydrophilic fulvic acid (FA1 and FA2), and hydrophobic fulvic acid (FA3 and FAIHSS) fractions prepared from eight sub-horizon samples from a buried humic horizon occurred between 147 and 187 cm depth of an Andosol near Towada volcano ... 35 Figure 2.5. Solid-state cross polarization magic angle spinning 13C nuclear magnetic resonance spectra of hydrophilic fulvic acid (FA1 and FA2), hydrophobic fulvic acid (FA3 and FAIHSS), and humic acid (HA) fractions prepared from a sub-horizon sample between 162 and 167 cm depth from a buried humic horizon occurred between 147 and 187 cm depth of an Andosol near Towada volcano ... 38 Figure 2.6. Comparison between deposition age of sub-horizon and 14C age of (a) humin, (b) humic acid (HA), (c and d) hydrophilic fulvic acid (c: FA1, d: FA2), and (e and f) hydrophobic fulvic acid (e: FA3, f: FAIHSS) fractions prepared from eight sub-horizon samples from a buried humic horizon occurred between 147 and 187 cm depth of an Andosol near Towada volcano ... 44 Figure 3.1. Location of Kuju highland, Oita Prefecture, Japan. Two soil samples were collected from a fertilized grassland soil (0-100 cm depth) and a secondary forest soil (0-110 cm depth) from the Kuju Highland, and the distance between the two sampling points was ca. 800 m .. 54 Figure 3.2. Sampling profile of fertilized grassland (a) and secondary forest (b) in Kuju highland, Oita Prefecture, Japan ... 56
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Figure 3.3. Comparison of depth profiles of sodium pyrophosphate extractable Al (a: Alp) and Fe (b: Fep), acid-oxalate extractable Al (c: Alo), Fe (d: Feo), and Si (e: Sio), ratio of Alp/Alo
value (f: Alp/Alo), and Alo + 1/2 Feo value (g: Alo+1/2Feo) between fertilized grassland soil collected from 0 - 100 cm depth and secondary forest soil collected from 0 - 110 cm depth in Kuju highland, Oita Prefecture, Japan ... 63 Figure 3.4. Comparison of depth profiles of (a) soil pH(H2O), (b) pH(KCl), and (c) exchangeable acidity between fertilized grassland soil collected from 0-100 cm depth and secondary forest soil collected from 0-110 cm depth in Kuju highland, Oita Prefecture, Japan ... 66 Figure 3.5. Comparison of depth profiles of (a) electrical conductivity, (b) base saturation, and (c) cation exchange capacity between fertilized grassland soil collected from 0-100 cm depth and secondary forest soil collected from 0-110 cm depth in Kuju highland, Oita Prefecture, Japan ... 69
Figure 3.6. Comparison of depth profiles of (a) exchangeable Ca2+, (b) exchangeable Mg2+, (c) exchangeable K+, and (d) exchangeable Na+ between fertilized grassland soil collected from 0- 100 cm depth and secondary forest soil collected from 0-110 cm depth in Kuju highland, Oita Prefecture, Japan. ... 72
Figure 3.7. Comparison of depth profiles of exchangeable cation ratios for (a) Mg2+/K+, (b) Ca2+/Mg2+, and (c) Ca2+/K+ between fertilized grassland soil collected from 0-100 cm depth and secondary forest soil collected from 0-110 cm depth in Kuju highland, Oita Prefecture, Japan ... 74
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Figure 3.8. Comparison of depth profiles of (a) available P (Bray II P), (b) total P, and (c) pH(NaF) values between fertilized grassland soil collected from 0-100 cm depth and secondary forest soil collected from 0-110 cm depth in Kuju highland, Oita Prefecture, Japan. The dotted lines indicate regression curves fit to power function model ... 78
Figure 3.9. Comparison of depth profiles of (a) total C, (b) total N, and (c) C/N ratio between fertilized grassland soil collected from 0-100 cm depth and secondary forest soil collected from 0-110 cm depth in Kuju highland, Oita Prefecture, Japan ... 81
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Acknowledgement
Firstly, I would like to express my sincere gratitude to my advisor Prof. Syuntaro Hiradate for the time, continuous outstanding support he devoted to my PhD study and related research and for his patience, motivation and immense knowledge. His guidance helped me in all the time of research and writing of manuscripts and this thesis.
Also, I would like to thank Associate Prof. Yuki Mori for his encouragements and guidance all the way through.
I am also grateful to all co-authors; Dr. Jun Koarashi, Dr. Mariko Atarashi-Andoh, Dr. Yoko Saito-Kokubu, Dr. Noriko Yamaguchi, Dr.Takashi Sase, Dr. Mamoru Hosono, Dr. Yudzuru Inoue and Mr Hiroyuki Asaoka for their valuable support and guidance throughout my study.
I would like to thank Prof. Yoshiyuki Shinogi and Associate Prof. Tsutomu Enoki for their valuable guidance to improve the thesis as reviewers.
I also express my deep gratitude to my former advisor Prof. Shin-Ichiro Wada for his guidance and motivation in all the time of my study in Kyushu University.
I thank my fellow lab mates for supporting me during my study and stay in Japan.
I would like to thank Asian Development Bank-Japanese scholarship programme (ADB-JSP) for providing me scholarship to study in Japan.
I would like to extend my gratitude to the people of Sri Lanka and Japan for funding my entire education.
Last but not the least, I would like to thank my family; my parents, my husband, my sisters, brothers and friends for supporting me spiritually throughout my PhD journey and my life in general.
21 February 2021
Jithya Nawodi Wijesinghe Laboratory of Soil Science
Department of Agro environmental Science
Graduate School of Bioresources and Bioenvironmental Sciences
Kyushu University
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Abstract
Andosols are important agriculture soils in Japan and frequently characterized by thick humic horizons accumulating large amount of soil organic carbon (SOC). However, the genesis and accumulation processes of the SOC in Andosols are still unclear, and the information on the movement of the SOC would be a key to understand them. In Andosols, the movement of the other elements in the soil profiles should also be clarified, because the repeated application of agricultural materials including plant nutrients for agricultural production has caused environmental issues such as eutrophication due to discharge of these elements to surrounding areas. A soil profile with well-preserved horizons would provide a very good opportunity to study the long-term movement of such elements in soils, and a buried humic horizon under thick tephra deposits would be suitable for investigating movement of C with 14C dating technique with high accuracy since those covering tephra deposits have preserved past conditions from recent anthropogenic influences and external input of modern C. In the present study, the movement of C and other elements including Ca2+, Mg2+, Na+, and P were clarified by investigating 14C age of SOC fractions and depth profile of those elements in Andosols.
To clarify the mobility of SOC, a well-preserved buried humic horizon of an Andosol was collected from the depth between 147 and 187 cm at 5 cm-interval (total 8 sub-horizon samples), and SOC fractions were prepared from each sub-horizon sample by extraction and precipitation procedures, resulting in humin, humic acid, and four fulvic acid fractions. The average rate of vertical translocation of each SOC fraction determined by the 14C dating technique was very low ( 4 mm per century), implying that the vertical translocation of SOC would not be the main mechanisms for forming the thick humic horizons in Andosols. In addition, stable isotopic ratio of C and N revealed that most of the SOC fractions have not been well-metabolized, indicating that they have been fixed in situ right after photosynthesis by plants at the early stage of soil formation and chemically stabilized at the soil surface, by fire events to form charred
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materials, etc. Thus, in Andosols, high content of SOC found at deeper position than several cm depth from the soil surface would not be supplied through overlaying layers by infiltration but formed in situ on soil surface, and successive up-building accumulation of soil particles containing SOC would have contributed to the formation of the thick humic horizons.
To clarify the movement of the other elements, the depth profiles of available Ca2+, Mg2+, Na+, and P were investigated in Andosols, and they were compared between a secondary forest soil and an adjacent reclaimed grassland soil which has received fertilizers for a half century.
The comparison revealed that the Ca and Mg applied as fertilizer ingredients have reached at least 100 cm depth as exchangeable Ca2+ and Mg2+ in the fertilized grassland soil during a half century. Similar trend was observed for exchangeable Na+. Soil available P (Bray II P) was also observed to be increased at 80 to 100 cm depth in the fertilized grassland soil although the mobility was lower than those of the exchangeable Ca2+, Mg2+, and Na+.
Overall, the present study clarified the very low mobility of SOC in Andosols, together with higher mobility for available P and the highest for exchangeable Ca2+, Mg2+, and Na+. The findings in the present study would contribute to understand the mechanisms of SOC accumulation and adequate management of plant nutrients in Andosols for sustainable use.
1
Chapter 1
Background of the study
2
1.1. Elements in soil
Soils act as a main source of nutrients elements for plants. The main group elements involved in soil are carbon (C), nitrogen (N), potassium (K), phosphorus (P), calcium (Ca), magnesium (Mg), and sulfur (S). Soil elements composition strongly depends on the parent materials weathering processes, biocycling on additions via atmospheric wet and dry deposition due to natural sources such as volcanic eruptions (Martínez Cortizas et al., 2003). Recently, external factors such as anthropogenic activities i.e., agriculture, urbanization and industrialization etc., have highly influenced on the element composition of the soils (Memoli et al., 2018). In soil science, mobility or movement is defined as the capacity for elements, ions, and molecules to move from one compartment (soil atmosphere, soil solution, organic and mineral phases, and biota) of the soil to another (Juste, 1988).
With the increase of demand for food, most of the agriculture systems relying on chemical fertilizers and have increased agricultural productivity, but their application may change the composition of the elements in soil, their behavior and induce the leaching of elements as environmental pollutants. For example, phosphorous (P) leaching to the aquatic environment causes the eutrophication (Howell and Dove, 2017) However, studies on elements in soils are mainly focus on soil fertility or plant available nutrients (e.g., Littke et al., 2011). In addition, mechanism, distribution, and mobility of trace elements in soils and their effect on environment have been studied by many researchers (e.g., Carrillo-González et al., 2006). Studies dealing with long-term movement of elements in deep soil layers are poorly present in the scientific literature.
Soils are not only a nutrient source, but also are major absorbers, depositories, and releasers of organic carbon (OC) on the earth’s surface. The soil is the largest active terrestrial reservoir in the global carbon cycle (Figure 1.1). The estimated SOC in 1 m depth in the world’s soils is about 1550 Pg (Eswaran et al. 1993; Jobbagy and Jackson 2000) and about 2400 Pg in
3
2 m depth. The amount of OC in soils is more than four times that of carbon in terrestrial biota and three times that in the atmosphere. Therefore, soils have been received attention as a possible atmospheric CO2 sequester and convert it into soil organic carbon (SOC) which is long-lived i. e. soil carbon sequestration. The OC content in soils significantly varies, from less than 1% by mass in some arid-zone soils to 50% or more in waterlogged organic soils (Hillel and Rosenzweig, 2009) and differ in their potential of soil carbon sequestration. The 21st Conference of the Parties to the United Nations Framework Convention on Climate Change in Paris, 2015 implemented a concept called "4 per mille Soils for Food Security and Climate".
The 4 per mille or 4 per 1000 aspires to increase global soil organic matter stocks by 0.4 percent per year as a compensation for the global emissions of greenhouse gases by anthropogenic sources. Therefore, understanding the formation, accumulation, behaviour and composition of organic carbon in soils is important to implement those strategies.
However, most research on SOC has mainly focused on the topsoil layer, where SOC is more abundant and sensitive to changes (Batjes, 1996; Rumpel and Kögel-Knabner, 2011).
Several researchers have pointed out the importance of SOC in subsoil as it contributes more than half of the total SOC stock (Batjes, 1996; Eswaran et al., 1993; Hiederer, 2010). Therefore, in-depth understanding of the composition, behaviour and movement of the SOC in deep soil layers is vital importance in predicting future behaviour of terrestrial C sinks.
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Figure 1.1. Simplified schematic diagram of the terrestrial carbon cycle (source: U.S.
Department of Energy, Office of Science and IPCC, 2013; 2001).
1.2. Andosols
Andosols are soils that typically form in loose volcanic ejecta. They cover approximately 0.12 billion hectares of global ice-free land area (~1%) and generally support high population densities (Ping, 2000) due to high native fertility which supports for agriculture (Ugolini and Dahagren, 2002). They are characterized by andic properties and are classified as Andosols under one of the 32 soil reference groups recognized in the World Reference Base for Soil Resources (IUSS Working Group, 2006) or as Andisols; 11th soil order in USDA soil classification system (Soil Survey staff, 1990). Both soil names were derived from Japanese words meaning dark soil i.e., dark (An) and soils (do).
The definition of Andosols in soil taxonomy is based on the existence of sub horizon meeting requirements of andic soil properties which have a cumulative thickness of 36 cm or more within 60 cm of either the mineral soil surface or the top of an organic layer (Soil Survey
1500- 2400 PgC Atmosphere
830 PgC
450-650 PgC 123
PgC/y
60 PgC/y
8 PgC/y
Net terrestrial
uptake 4 PgC/y
5
Staff, 1999). The relatively low bulk density values (≤0.90 g cm-3), high phosphate retention (>25-85%), presence of volcanic glass, and weathering products containing Al, Fe, and Si were identified as andic properties in Andosols (Soil Survey Staff, 2010). The distinctive morphological, physical, and chemical properties that found in Andosols are due to the formation of non-crystalline materials (e.g., allophane, imogolite, ferrihydrite, and Al/Fe complexes) and accumulation of organic carbon. A thick, humus rich, dark colored A horizon underlain by a brown Bw horizon is a typical horizon found in Andosols and it indicates the accumulation of large amount of organic C (Figure 1.2a).
Andosols are often divided into two groups based on their mineralogical composition i.
e. allophanic Andosol; a weakly acidic Andosol dominated by allophane and imogolite and non-allophanic Andosol; a strongly acidic Andosol dominated by Al-humus complex and 2:1layer silicates (Shoji et al., 1985).
(a) (b)
Figure 1.2. Typical soil profile in Andosol with thick, dark, strongly humified A horizon (a) found in a secondary forest in Kuju highland, Oita prefecture, Japan and non-Andosol soil profile found in experiment forest area, Fukuoka prefecture, Japan (b).
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Andosols in Japan are mainly distributed in Hokkaido, Tohoku, Kanto, and Kyushu prefectures (Figure 1.3). Cultivated andosols occupy approximately 1.35 million hectares, comprising 27% of total agricultural land use in Japan (Shoji and Takahashi, 2002). Among them about 50% are upland fields and about 10% are paddy fields (Japan Soil Analysis and Soil Preservation Council, 1991). Thus, Andosols are an important soil resource in Japan.
Figure 1.3. Distribution of allophanic and non-allophanic Andosols in Japan (Saigusa and Matsuyama, 1998).
1.2.1. Andosols as a carbon store: Formation and translocation of SOC
Andosols show the second highest SOC storage, with approximately 30 kg C m−2 on average aside from the Histosolsaccording to the result of the organic carbon mass estimation in soils around the world (Eswaran et al., 1993). Therefore, Andosols were reported as a carbon sink for atmospheric CO2, and the genesis and accumulation mechanisms of SOC in Andosols have been studied (e.g., Dahlgren et al., 2004; Ugolini and Dahlgren, 2002).
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Volcanic ash covers wide areas of Japan to significant depths and 31% of Japanese terrestrial area covered by Andosols (Obara et al., 2016). It has been reported that the abundance of volcanic ash in soil affects the organic carbon density of that soil, because volcanic ash absorbs organic matter and retains it in the soil in a stable form (Shoji et al., 1993). Morisada et al. (2004) estimated the mean organic carbon density of the forest soils associated with Andosols in Japan as 33.0 kg m-2 in 1m depth. The high organic carbon density of the forest soils in Japan is thought to be due to the influence of volcanic ash on soil (Forestry and Forest Products Research Institute, 1981; Kyuma, 1990). Moreover, Nakagami et al. (2009) also estimated average carbon stock in grasslands associated with Andosols in Japan and reported high carbon stock values in the upper 25 and 50 cm as 12.4 and 19.3 kg m-2, respectively.
Therefore, Andosols are important as a carbon sink in Japan.
Several hypotheses have been proposed for the mechanisms of the formation of humic substances in soil i. e., lignin, polyphenol and suger-amine condensation theory (Stevenson, 1994). In the case of Japanese Andosols, it has been assumed that grasslands plants as a major source of the SOC. It has been reported that burning and/ or fire as a major event with the production of charred fragments that eventually degraded into black humic acids and form typical black A horizon in Andosols (e.g., Shindo et al., 2004). However, recently, non- pyrogenic sources of humic acids also have been reported (DiDonato et al., 2016). The protection of SOC against the biodegradation by Al toxicity to microorganisms (Illmer et al., 2003), sorption of degraded enzymes and organic matter substrate, physical protection within microaggregates would help to accumulate large amount of SOC in Andosols. Stabilization of SOC in Andosols may occur through formation of Al-humus complexes and sorption to allophane, imogolite and ferrihydrite (Saggar et al., 1994).
Even though possible mechanisms of SOC accumulation and stabilization in Andosols have been discussed by several researchers (e.g., Ugolini and Dahlgren, 2002), in the case of
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formation of thick black horizons in Japanese Andosols has been still unclear whether the SOC has accumulated in situ with the deposition of volcanic ash or a part of SOC has been transferred from upper layers. As noted above, vertical translocation of SOC would be low due to stabilization mechanisms and it has been discussed in several studies (e.g., Ugolini and Dahlgren, 2002). However, the rates of vertical translation of SOC have not been estimated which would clarify the accumulation process of large amount of C in Andosols and formation of thick humic horizons.
1.2.2. Behavior of elements in Andosols
In general, Andosols are known to have excellent physical properties and relatively high native fertility which are favourable for agricultural production. Periodic addition of tephra can re- supply the potential nutrients and maintain the favourable fertility levels for crop production.
Lowe and Palmer (2005) reported the addition of large amount of sulfur and small quantities of Se, Mg and K to the soil due to volcanic eruption occurred in New Zealand.
However, the nutrient elements of these soils are not always high, because of a range of chemical limitations both acquired and inherited. For example, the presence of large quantities of amorphous clay minerals in Andosols leads to very high phosphorus (P) fixation capacities (Dahlgren et al., 2004). It means that even many agroecosystems in Andosols contain very high concentrations of P, only very little P is immediately available for plants (Borie and Rubio, 2003; Redel et al., 2007). Moreover, the high levels of exchangeable Al can be observed especially in non-allophanic Andosols which inhibit plant growth (Al toxicity) (Shoji and Takahashi, 2002; Dahlgren et al., 2004). Lowe and Palmer (2005) reported that lower exchangeable bases, especially K and other elemental deficiencies such as Mg, S, Co, Zn, and Cu in arable Andosols. Therefore, to fulfil the plant P requirements, addition of fertilizer including P fertilizer and to reduce the strong acidity, application of lime is often practiced for commercial plant productions in Andosols. The repeated addition of fertilizer causes
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environmental issues of surrounding area, such as eutrophication and subsequent decease in biodiversity (e.g., Scavia et al., 2014). Therefore, understanding the element mobility and their distribution in Andosols is important for healthy material cycling. The elemental composition in Andosols, the vertical distribution of major and trace elements in soils derived from volcanic deposits (Martı´nez Cortizas, 2003) and changes in element concentration during Andosols formation on tephra have been discussed (Nanzyo, 2007). However, studies on elemental movements in deep soil horizons in Andosols and effect of management practices like repeated application of fertilizer on behavior of elemental movement have not been discussed.
1.2.3. Suitability of Andosols in elemental studies
The accumulation of large quantities of OC in subsoil horizon in Andosols, provide a unique opportunity to study SOC in subsoil horizons. The soil horizons in Andosols under thick tephara deposits is suitable for investigating the genesis and accumulation mechanisms of SOC in natural conditions (Figure 1.4a), because theses horizons have covered and preserved past conditions from external inputs of recent C and recent anthrophonic influences. Such kind of conditions in soils are important to use emerging technologies like 14C dating technique to investigate the SOC dynamics with high accuracy.
In addition, multiple sequences of horizons with typically distinct horizon boundaries in Andosols (Figure 1.4b) can be observed due to the intermittent deposition of tephra and top- down soil formation. Therefore, Andosols with well-ordered horizons from surface to the deep soil layers (250+ cm depth) provide a rare opportunity to study movements of elements in subsoil horizons.
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Figure 1.4. Thick humic horizons under thick tephra deposits found near Towada volcano, Aomori, Japan (a) and well-ordered horizons with distinct horizons in non-allophanic Andosol found in Tohaku, Japan (McDanial et al., 2012) (b).
1.3. Fractionation of soil organic carbon
To study the composition, structure, history etc., of SOC, first it should be separated and isolated from the inorganic components of the soil. The SOC can be mainly isolated by chemical and physical fractionation. Historically SOC has been chemically fractionated into three
0
100
200
300
humic soil tephras To-a
AD 915 To-b 2.2
To-Nb 8.3 kyr BP To-Cu 5.3 kyr BP
(a)
(b)
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fractions; humin, humic acid (HA), and fulvic acid (FA) fraction based on their solubility in acid alkaline solutions (Conte et al., 1997) (Figure 1.5). Physical fractionation is based on aggregate size, particle size, and density and are widely used in recent studies on SOC (e.g., Plaza et al., 2019).
Figure 1.5. Standard procedures of isolation and separation of humin, humic acid and fulvic acid fractions in soil accepted by the International Humic Substances Society.
Soil
Insoluble organic Soluble organic
Extract with alkali (NaOH)
Humin (Precipitated)
Treat with acid (HCl, pH 1.0)
Humic acid (Precipitated)
Crude Fulvic acid (Soluble)
hydrophobic resin (e.g. XAD-8)
elute with 0.1 M NaOH
Fulvic acid
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Even though humin and HA fractions have been isolated as precipitates, preparation of FA fractions has been difficult, because FA are soluble in both alkaline and acidic solution, resulting in difficulty of dehydration and demineralization. Isolation of fulvic acid by adsorption on hydrophobic resin has been employed and accepted by the International Humic Substances Society as a standard procedure. However, several researchers have pointed out that adsorbed fraction of the fulvic acid on resin should be as hydrophobic fulvic acid (e.g., Hiradate et al., 2006) because the fraction not adsorbed by the resin should be more hydrophilic than the adsorbed fraction and it is typically discarded. Therefore, new adsorption and precipitation procedure to isolate fulvic acid has been proposed by Hiradate et al. (2007) and fulvic acid fractions were further separated into hydrophilic and hydrophobic fulvic acid fractions.
1.4. Radiocarbon (
14C),
13C and
15N isotope ratio in soil carbon dynamics
Radiocarbon (14C) is a tool to study the dynamics of C in soils on decadal to millennial timescales. 14C nucleus is unstable and will spontaneously emit an electron (β particle), decaying to 14N with a half-life of 5730 years by the interaction of cosmic rays with the atmosphere and earth’s surface. Once C atoms removed from contact or substrate with the atmosphere and stored with no further exchange with other reservoirs. Then, the time period since removal of C atoms from substrate could be determined from the degree to which the
14C/12C ratio has been reduced (by radioactive decay) compared with the original atmospheric CO2 from which it was derived.
Although the first radiocarbon measurements in soil organic matter were made in the 1960s (e.g., Campbell et al., 1967), the recent development of accelerator mass spectrometry (AMS) has led to a rapid expansion in the use of 14C to study organic matter dynamics in terrestrial ecosystems, because of smaller sample size requirements and faster throughput capabilities. For C pools like soil organic matter, which constantly receive new C in-puts from plants and lose C through decomposition, the 14C/12C ratio in a given organic matter pool
13
reflects both the rate of decomposition and radioactive decay (Trumbore, 2000; Torn et al., 2009). Recently, 14C dating technique has been used in most research to study the residence times of soil organic matter (e.g., Dwivedi et al., 2017), to estimate the turnover of SOC (Ahrens et al., 2015), and to assess pedogenesis process in humic horizons (Inoue et al., 2011). However, few studies have been used 14C age to estimate vertical translocation of SOC (Marin-Spiotta et al., 2011).
With respect to natural abundance of C isotopes in the environment, 98.9% of C exists as12C, 1.1% as13C. During kinetic and thermodynamic processes, such as biochemical reactions, phase changes, or diffusion, heavier isotopes are normally discriminated against the lighter counterparts, because of the higher kinetic energy of the latter. As a consequence, the lighter isotopes accumulate relative to the heavier ones in the reaction products.
The isotope composition is expressed as value, which was introduced by Craig (1953) and which can be calculated according to equation (1)
𝛿13C‰ = [ 𝑅𝑆𝑎𝑚𝑝𝑙𝑒
𝑅𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑−1] × 103
where R Sample and R Standard correspond to the molar ratio of the heavier to the lighter isotope (equation. 2) with R Standard being equal to 0.0112372.
𝑅 = 13𝐶
12𝐶
The photosynthetic fixation of CO2 leads to a discrimination against the heavier isotope in the assimilated products. As a consequence, the range of δ13C value in plant material varies between –10‰ and –34‰. Therefore, δ13C mean values have been assigned as –27‰ for C3
plants and –13‰ for C4 plants.
(1)
(2)
14
The stable-isotope analysis has been mainly used to quantify sequestration and turnover of specific organic sub-stances, to trace the origin of organic substances, and to reconstruct landscape history and climate. For example, isotopic values of δ13C‰ and δ15N‰ have been widely used to determine the source plants of soil organic carbon (Katsumi et al., 2015;
Hiradate et al., 2004) and assessing the degree of decomposition and humification of organic matter within different soil profiles (Panichini et al., 2012; Marin- Spiotta et al.,2009; Kramer et al., 2003; Krull et al., 2002). In addition, these isotope ratios serve as indicators of relative
degree of microbial processing, which SOC has undergone before reaching its present state.
The δ15N‰ value is known to increase with biological transformation (Minagawa and Wada, 1984; Wada et al., 2013). Decomposition and recycling of organic matter, by organisms meditated by enzymatic reactions result in enrichment of heavier carbon and nitrogen isotopes due to preferential stabilization (Wada et al., 2013). This means that both δ13C‰ and δ15N‰
values should be increase when organic matter has been recycled by enzymatic reactions even in soil. Therefore, the increase of δ13C‰ and δ15N‰ values in SOC fractions would provide strong evidence of biological transformation of SOC.
1.5. Application of
13C Nuclear magnetic resonance (NMR) in soil organic carbon
SOC can be studied using a variety of spectroscopic (fluorescence spectroscopy, Infrared spectroscopy, X-ray spectroscopy, nuclear magnetic resonance spectroscopy, mass spectroscopy) and wet-chemical techniques (e.g., acid hydrolysis). The progressive advancement and enhancement of these techniques, particularly spectroscopic techniques have developed to study not only molecular characteristic of SOC but also to explore structural properties, in various medium; solids, liquid, and gaseous states (Preston, 1996; Bencze et al., 2007). Amongst these spectroscopic techniques, nuclear magnetic resonance (NMR) spectroscopy is strongly emerging as the premier tool for investigating the chemical
15
composition, molecular properties, and functionalities of a range of substances including SOC.
NMR spectroscopy can be performed in both liquid and solid state depend on the nature of the material being analysed and the subject of the enquiry. An advantage of solid-state 13C NMR techniques in SOC analysis is the ability to analyse insoluble samples. The cross-polarisation magic-angle spinning (CPMAS) technique in NMR spectroscopy allows for a considerable signal enhancement during investigation of solid samples at natural 13C abundance (Preston, 2001) and provides semi-quantitative evaluation of carbon distribution of heterogeneous solid samples. The solid-state CPMAS 13C NMR has been used for identifying the structural differences and carbon distribution of SOC fractions (Iimura et al., 2012; Hiradate et al., 2007).
An example solid-state CPMAS 13C NMR spectrum of SOC is shown in Figure 1.6.
Figure 1.6. A simplified representation of a typical NMR spectrum of SOC showing various chemical shift regions.
On the spectrum, distinct bands represent different chemical shifts which indicate specific organic compounds that are present in the sample. The resonance signal intensity of each chemical shift
-50 0
50 100
150 200
250
carbonyl aromatic O-alkyl alkyl
chemical shift (ppm)
250 200 150 100 50 0 -50
16
region generally indicates the proportion of a particular compound or groups in the sample although overlaps between regions can occur. The alkyl region on the spectrum (0-45 ppm) consist of compounds such as fatty acids, waxes, and resins which are derived from plant remains and microbial products. The O-alkyl C (45-110 ppm) region represents structures which contain materials such as sugar moieties and
peptide like structures that are easily decomposed by soil microbial community hence has a shorter turnover/residence period in the soil. The region between 110-165 ppm represents C structures of aromatic C region. The aromatic C region represents recalcitrant component of soil organic matter relative to other compounds and there has a longer residence period in the soil. It is mainly enriched from pyrogenic materials like charcoal.
1.6. Objectives of the study
The objectives of the present in the study are (1) to clarify the mobility of SOC fractions in a buried humic horizon and (2) to clarify the long-term movements of plant nutrients in Andosols.
To the clarify mobility of SOC fractions in humic horizons, soil samples were collected from buried humic horizon derived from a volcanic ash of Towada volcano, Amori prefecture, Japan and 14C age of SOC fractions were investigated.
To clarify the long-term movements of plant nutrients in Andosols, soil samples were collected from fertilized grassland and adjacent secondary forest in Kuju highland, Oita Prefecture, Japan and depth profiles of plant nutrients were investigated.
1.7. Overview of this dissertation
This dissertation describes the long-term movement of elements in Andosols.
Chapter 1 outlines a brief introduction on elements in soil, capacity of soil to store carbon, unique characteristics of Andosols, distribution of Andosols in the world and Japan, and role of Andosols as a potential carbon store. This chapter describes the possible mechanisms of formation and translocation of SOC in Andosols and related studies. Chapter 1 further explains the suitability of andosols in elemental studies. This chapter briefly explains the historical and
17
current available methods to isolate SOC, principles of radiocarbon (14C), 13C and 15N isotope ratio, 13C nuclear magnetic resonance (13C NMR) and their applications in SOC studies. Finally, chapter 1 describes the objectives of the present study.
Chapter 2 outlines the characterization of SOC by 13C and 15N isotope values, 14C age, and solid -state CPMAS 13C NMR spectroscopy. Procedure for the isolation and preparation of SOC fractions are discussed in detail. The order of the degree of biological transformation of SOC fractions is clarified. Rates of vertical translocation of SOC fractions are calculated, compared with each fraction, and most mobile SOC fractions are identified. A possible formation mechanism of thick humic horizon in Andosols is proposed. The chemical compositional feature of SOC fractions is analysed with solid-state CPMAS 13C NMR spectroscopy and their relationships with the behavior in Andosols is discussed. Finally, this chapter discusses the genesis of humic acid fractions.
Chapter 3 outlines the depth profiles of soil acidity, exchangeable cations (Ca2+, Mg2+, K+ and Na+), available phosphorous, total C, N, pyrophosphate-extractable Al (Alp) and Fe (Fep), acid- oxalate-extractable Al (Alo), Fe (Feo), and Si (Sio) of fertilized grassland and secondary forest.
Mobility of each elements in grassland and secondary forest is compared. Finally, effect of long-term effect of management agriculture practices on classification of the Andosols is discussed.
Chapter 4 explains the general summery of the thesis and conclusion.
18
Chapter 2
Formation and mobility of soil organic carbon in
a buried humic horizon of a volcanic ash soil
19
2.1. Introduction
Volcanic ash soils or Andosols comprise are relatively small area (0.12 billion hectares) among 14 billion hectares of the global terrestrial area (Dahlgren et al., 2004), but they contain several times higher concentration of soil organic carbon (SOC) than adjacent non-Andosols (Eswaran et al., 1993). It has been estimated that they stored approximately 75 Pg of SOC among 1500 Pg of global SOC in 0 to 100 cm depth of their soil profiles (Dahlgren et al., 2004). Therefore, Andosols have been receiving intensive attention as a carbon sink for atmospheric CO2, and the genesis and accumulation mechanisms of SOC in Andosols have been studied (e.g., Dahlgren et al., 2004; Ugolini and Dahlgren, 2002).
A soil horizon under thick tephra deposits is suitable for investigating the genesis and accumulation mechanisms of SOC in natural conditions, because the age of C can be determined with 14C dating technique with higher accuracy than recent C and the covering tephra deposits have preserved past conditions from recent anthropogenic influences and external input of recent carbon. Inoue et al. (2011) investigated a 40 cm-thick buried humic horizon of an Andosol near Towada volcano, Aomori, Japan, which is interlayered between Towada-Chuseri pumice (5.39 ± 0.14 kyr BP; Hayakawa, 1983) and Towada-Nambu pumice (8.37 ± 0.17 kyr BP; Hayakawa, 1985). They collected eight sub-horizon samples from the buried humic horizon at 5 cm-interval and investigated 14C age of particulate SOC contained in each of the sub-horizon sample and clarified up-building pedogenesis of the soil horizon with a rate of about 30 mm per century. In the present study, we further investigated the same sub- horizon samples to clarify the genesis and mobility of SOC fractions in the buried humic horizon by isolating SOC fractions and investigating 14C age and stable isotopic ratio of 13C (δ13C) and 15N (δ15N).
Historically, SOC has been studied by separating into three fractions: humin, humic acid (HA), and fulvic acid (FA) fractions depending on their solubility against acid and alkaline
20
solutions (Conte et al., 1997), although this technique has been critically disputed by many researchers (e.g., Kleber and Lehmann, 2019; Olk et al., 2019). Humin fraction is insoluble in both acid and alkaline solutions, HA fraction is alkali-soluble and acid-insoluble, and FA fraction is soluble in both acid and alkaline solutions. Because FA fraction has high solubility against water, it would play an important role in forming soluble complexes with metals and hydrophobic organic chemicals (Iimura et al., 2012; Maie et al., 2004; Stevenson, 1994). The standard method for preparing the FA fraction has been established by International Humic Substance Society (IHSS, http://www.humic-substances.org/) (hereafter referred as FAIHSS), but this procedure isolates a part of hydrophobic FA fractions and most of hydrophilic FA fractions are typically discarded (Hiradate et al., 2006). To recover other FA fractions than FAIHSS, Hiradate et al. (2006) proposed applying the crude FA on a column filled with hydrophobic resin, eluting the column with water with controlling the pH of the eluent, and precipitating the eluted FA fraction by partial neutralization for preparing hydrophilic (FA1 and FA2) and hydrophobic (FA3 and FAIHSS) FA fractions, and the present study followed this procedure.
The δ13C and δ15N values have been used for estimating the source plants (Hiradate et al., 2004; Katsumi et al., 2015; Yoneyama et al., 2001) and the degree of decomposition and humification of SOC (Kramer et al., 2003; Krull et al., 2002; Marin-Spiotta et al., 2009;
Panichini et al., 2012). These isotopic ratios serve as indicators of relative degree of microbial processing, because recycling of organic matter by organisms results in enrichment of heavier
13C and 15N stable isotopes due to their preferential stabilizations (Wada et al., 2013). Therefore, the simultaneous increase of δ13C and δ15N values in SOC fractions corresponds to the successive transformation of SOC by biological processes among the fractions. Soil humic substances have been assumed to be transformed from plant residues through decomposition and polymerization reactions in soils, which would be closely related with biological and
21
enzymatic reactions. Considering from the differences in molecular weight of SOC fractions, Miltner et al. (2012) assumed that FAs would be formed first, followed by HA and finally humin, although this has not yet been confirmed with sufficient evidence. In the present study, we determined the δ13C and δ15N values of SOC fractions prepared from the buried humic horizon samples to assess the degree of biological transformation that occur during decomposition of the SOC fractions.
In addition to the high C content of Andosols, the thick humic horizon is also the distinct feature. Although Inoue et al. (2011) have confirmed the up-building formation of the thick buried humic horizon of the Andosol from Aomori, Japan, the accumulation process of the soil organic matter (SOM) in the horizon has been still unclear whether the SOM had accumulated in situ with the deposition of volcanic ash or a part of SOM had been transferred from upper layer. The mobility of the SOC in Andosols has not been reported. To answer this question, it would be necessary to clarify the rate of vertical translocation of SOC in soil profile. Therefore, in the present study, we tried to measure the mobility of the SOC fractions isolated from the buried humic horizon from the Andosol profile, i.e., the isolated SOC fractions from eight sub horizon samples were analyzed with 14C dating technique and mean vertical translocation rate was estimated for each SOC fraction.
The aims of the present study were to clarify the degree of biological transformation and mobility of SOC fractions in a thick buried humic horizon of a soil derived from volcanic ash in Aomori, Japan, by investigating δ13C and δ15N values and 14C dating. Solid-state 13C nuclear magnetic resonance (NMR) spectra were also measured for selected SOC fractions to interpret the formation processes and mobility of them.
22
2.2. Materials and methods
2.2.1. Study site and soil samples
The soil samples used in the present study were the same as used in Inoue et al. (2011), which had been collected from a buried humic horizon derived from a volcanic ash of Towada volcano, Aomori prefecture, Japan.
The buried humic horizon had occurred between 147 and 187 cm depth, which had been packed by overlaid Towada-Chuseri pumice (5.39 ± 0.14 kyr BP; Hayakawa, 1983) and underlaid Towada-Nambu pumice (8.37 ± 0.17 kyr BP; Hayakawa, 1985) (Figure 2.1). Eight subhorizon samples had been collected at 5 cm-interval within the 40 cm thick of the buried humic horizon. The sub-horizon samples had been air-dried, sieved through a 2-mm mesh, and subjected to further analysis. The present vegetation of the soil sampling site is secondary forest dominated by Quercus serrata (Sase and Hosono, 1996), but the past vegetation during the formation of the buried humic horizon (5.4 to 6.8 kyr BP) has been reported to be grassland dominated by non-Bambusoideae grass, mainly Panicoideae, as evidenced by δ13C values and phytolith analysis (Inoue et al., 2011).
The grassland vegetation would be maintained during the formation of the buried humic horizon without significant changes, because the δ13C values of the bulk soil samples of the humic sub-horizons are uniform and stable (−21.7 to −22.4‰, Inoue et al., 2011). The grassland would have been maintained by prehistoric human activities, and the humic horizon would be preserved by the thick pumice layer after the burial (Inoue et al., 2011; Sase and Hosono, 1996).
In the buried humic horizon, contamination of young C from roots would be negligible, because the horizon has been preserved in the deep horizon (between 147 and 187 cm depth) and no root was observed there.
The buried humic horizon has reported to show pH (NaF) value of 10.0 to 10.8 (Inoue et al., 2011), indicating the formation of noncrystalline materials from volcanic ash. The mean
23
annual temperature is 10.2 ± 0.7 °C and the mean annual rainfall is 1353 ± 204 mm, based on the data from nearby Kawamorita weather station, at Sannohe, Aomori prefecture (monthly average for the 63-year period between 1956 and 2019; Japan Meteorology Agency, 2020). It is classified into a temperate-humid climate with a udic moisture regime and mesic soil temperature regime (Soil Survey Staff, 2010).
(a)
24 (b)
Figure 2.1. Location of sampling site illustrated on isopach map of To-Cu and To-Nb tephras based on Machida and Arai (2003) (a) and sampling position in the soil profile (b). To-a:
Towada-a, To-b: Towada-b, To-Cu: Towada-Chuseri, To-Nb: Towada-Nambu. The deposition ages of To-a* and To-b** are calculated 14C ages and cited from Machida et al. (1981) and Oike and Shoji (1974), respectively. The deposition ages of To-Cu*** and To-Nb*** are non- calculated 14C ages and cited from Hayakawa (1983;1985).
2.2.2. Preparation of SOC fraction
A 20 g portion of the powdered sub-horizon sample was extracted with 160 mL of 0.1 M NaOH in the presence of 3% NaCl overnight. The supernatant was obtained by centrifugation (2380×g,
0
100
200
300
Depth (cm)
Depth (cm) Sample code
humic horizon tephras (pumice) To-a
AD 915* To-b 2.2**
To-Nb
8.37 kyr BP***
To-Cu
5.39 kyr BP***
-147
-152 -157
-162 -167 -172
[
-177
-182
-187
8-1 8-2 8-3 8-4 8-5 8-6 8-7 8-8
25
15 min). This extraction procedure was repeated three times, and the supernatant obtained from each extraction was mixed all together. The residue was suspended in a small amount of distilled water, neutralized with 6 M HCl, washed with distilled water at least three times, and freeze-dried (humin fraction).
The mixed supernatant was acidified to pH 1.0 with 4 M HCl and allowed to stand overnight. The precipitated fraction was separated by centrifugation (2380×g, 15 min; crude HA). The supernatant fraction was filtered through a 0.2 µm-pore cellulose acetate membrane filter (47 mm φ, Toyo Roshi Kaisha Ltd, Japan) (crude FA). The crude HA was re-dissolved in a small amount of alkaline solution of pH 13.0 by adding 4 M NaOH and centrifuged to remove small soil minerals. Then the supernatant was acidified to pH 1.0 by adding 4 M HCl and allowed to stand overnight, and again it was centrifuged to obtain supernatant and precipitate.
The supernatant was collected and added to the crude FA. The dissolution-precipitation cycle was repeated five times until final supernatant becomes light yellow in color. The precipitate was re-dissolved in a small amount of NaOH solution and centrifuge (2380×g) for 2 h to remove small soil minerals. Finally, the supernatant was acidified to pH 1.0 by 4 M HCl, washed with distilled water, and freeze dried (HA fraction).
The collected crude FA was further separated by applying the adsorption/desorption and precipitation procedures proposed by Hiradate et al. (2007). The crude FA was loaded on a glass column (20 mm of internal diameter, 300 mm of length) filled with approximately 25 mL of Amberlite XAD-8 resin (Rohm and Haas, Philadelphia, PA, USA) which had been washed sequentially with 0.1 M NaOH, 0.1 M HCl, and distilled water. The eluted non-adsorbed fraction was collected and partially neutralized to pH 5.0 with 4 M NaOH. The precipitate was collected by centrifugation (2380×g, 15 min) (FA1 fraction). The column was eluted with 400%
of column volume (100 mL) of 0.1 M HCl, and the eluted fraction was precipitated by adjusting the pH of the solution to 5.0 with 4 M NaOH and collected by centrifugation (2380×g, 15 min)
26
(FA2 fraction). The column was eluted with 400% of column volume (100 mL) of distilled water, and the eluted fraction was precipitated by adjusting the pH of the solution to 5.0 with 4 M NaOH. The precipitate was collected by centrifugation (2380×g, 15 min) (FA3 fraction).
Finally, the column was eluted with 50 mL of 0.1 M NaOH, and the eluted fraction was passed through an Amberlite IR 120 resin (H+ form) (FAIHSS fraction) (Figure 2.2). All FA1, FA2, FA3, and FAIHSS fractions were freeze-dried (Figure 2.3).
2.2.3. Elemental analysis of C and N in SOC fraction
The total C and N contents of each SOC fraction were measured using an elemental analyzer (Vario PYRO cube, Elementar, Germany). The δ13C and δ15N values of each SOC fraction were analyzed using an isotope ratio mass spectrometer (IsoPrime100, Isoprime Ltd., UK) connected with an elemental analyzer (Vario PYRO cube), with an analytical uncertainty of<0.1‰ (1SD).
2.2.4. 14C dating
The 14C age of the SOC fraction was determined by an accelerator mass spectrometer at Tono Geoscience Center, Japan Atomic Energy Agency (Saito-Kokubu et al., 2015). The SOC sample was wrapped in a tin capsule and combusted at 920 °C in an elemental analyzer (vario MICRO cube, Elementar). The resulted gas was passed through a reduction column and a separation column to purify CO2 from the gas. The purified CO2 was introduced into an automated graphitization equipment (AGE3, Ion plus AG, Switzerland) in which CO2 was converted to graphite by reducing CO2 with hydrogen gas at 580 °C for 2 h in the presence of iron powder as catalyst (Wacker et al., 2010). Radiocarbon 14C was then measured on graphite targets. The 14C data was reported as conventional 14C age (yr BP) with an analytical uncertainty (2σ) of 14C age less than ± 280 yr BP.
27
Figure 2.2. Fractionation and purification procedures of humin, humic acid (HA) and Fulvic acid (FA) fractions prepared from eight sub-horizon samples from a buried humic horizon occurred between 147 and 187 cm depth of an Andosol near Towada volcano.
Extract HA and FA from soil with 0.1 M NaOH in the presence of 3% NaCl 20 g of soil sample
Acidify the extractant (HA + FA) to pH 1.0 with 4 M HCl
Apply the supernatant to XAD-8 resin
Elute the resin with 0.1 M HCl
Elute the resin with H2O
Elute the resin with 0.1 M NaOH
Extractant (HA + FA) precipitated fraction
supernatant (Crude FA) precipitated fraction (Crude HA)
adsorbed fraction non-adsorbed fraction
adsorbed fraction eluted fraction
adsorbed fraction
eluted fraction
adjust pH 5.0, centrifuge, and freeze-dry
(FA3)
adjust pH 5.0, centrifuge, and freeze-dry
(FA2)
eluted fraction
pass through an Amberlite IR 120 resin (H+- form) and freeze-dry (FAIHSS)
adjust pH 5.0, centrifuge, and freeze-dry
(FA1)
neutralize to pH 6.0, centrifuge, and freeze-dry
(humin)
centrifuge and freeze-dry (HA)
28
Figure 2.3. Freeze dried fractions of humin, humic acid (HA) and Fulvic acid (FA) fractions prepared from eight sub-horizon samples from a buried humic horizon occurred between 147 and 187 cm depth of an Andosol near Towada volcano.
(a) humin (b) HA
(c) FA1 (d) FA2
(e) FA3 (f) FAIHSS
29 2.2.5. Solid-state 13C NMR measurement
A powdered sample of SOC fraction (ca. 20 mg) was tightly packed into a high-speed spinning NMR tube (made of zirconia, JEOL, Tokyo, Japan) and introduced into a FT NMR system (ECAII-600, JEOL). Solid state cross polarization magic angle spinning (CPMAS) 13C NMR signals were recorded at 150.9 MHz, with a contact time of 1 ms, an observation band of 90.6 kHz, 1,024 observation points (resolution;88 Hz), an acquisition time of 11.3 ms, a pulse interval of 3 s, 3,000-77,000 scans (analytical time; 2.5-75 h), and 15 kHz of magic angle spinning, with ramp technique. A broadening factor of 200 Hz was used in the Fourier transformation procedure. Chemical shifts were quoted with respect to tetramethylsilane (0 ppm) but were determined by referring to an external standard of adamantane (29.50 ppm). The chemical shift regions 0-45, 45-110, 110-165, and 165-190 ppm were assigned to alkyl C, O- alkyl C, aromatic C, and carboxyl C, respectively (Hiradate et al., 2007).
2.2.6. Determination of rate of vertical translocation of SOC fraction
The rate of vertical translocation of each SOC fraction was calculated by the following equation:
Rate of vertical translocation
= [current depth of SOC fraction- depth of original deposition]
/ [ 14Cage of SOC fraction] (1)
The depth of original deposition has been reported to be closely related with the 14C age of deposition of the corresponding buried sub-horizon, as follows (Inoue et al., 2011):
[depth of original deposition] = 0.0263 × [14C age of deposition] + 5.646 (2)
