Molecular and elemental composition of humic acidsisolated from selected soils of the Russian Arctic
Methods for assessing the degree of stabilization of polar soil organic matter. Analysis of the molecular and elemental composition of humic acids isolated from individual soils of the Russian Arctic. The essence of the concept of "aliphatic fragments".
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Molecular and elemental composition of humic acidsisolated from selected soils of the Russian Arctic
Authors:
Vyacheslav I. Polyakov, Nadezhda A.,
Chegodaeva, Evgeny V. Abakumov
Humic substances, isolated from selected soils of the Russian Arctic, were investigated in terms of molecular composition and stabilization rate. The degree of polar soil organic matter stabilization was assessed with the use of modern instrumental spectroscopy methods. The analysis of humic acid (HAs) preparations showed that aliphatic fragments prevail in the organic matter isolated in polar soils.
The predominance of aliphatic fragments was revealed in HAs from soils located in the coastal zone, which could be caused by regular refreshment of organic matter during sin-lithogenic process and processes of hydrogenation in HAs. Breaking of the C-C bonds and formation of chains with a high hydrogen content, which leads to the formation of aliphatic fragments in HAs, were noted. Data on the calculated atomic ratios of the elements in HAs are given and graphs show the main regularities in the formation of HAs and their properties. The integrated indicators of the molecular composition of humic acids of soils of the Russian Arctic are presented.
The paper contains 4 Tables, 4 Figures and 44 References.
Key words: CP/MAS 13C-NMR; humic acids; organic matter; permafrost-affected soils; Cryosol.
Funding: This work was supported by the Grant of Saint-Petersburg State University “Urbanized ecosystems of the Russian Arctic: Dynamics, state and sustainable development.”
The authors declare no conflict of interest.
Introduction
In the territories located at high latitudes of northern Europe, Greenland, Canada, Alaska and Russia, soils were formed during the Quaternary period, the diversity of which is mainly associated with the cryogenic processes within the pedosphere [1-2]. The area occupied by permafrost-affected soils is more than 8.6 million km2, which represents about 27% of the entire territory of the north [3]. According to the latest data, organic matter accumulated in the soil of the Arctic up to 1024 Pg (1 Pg = 1015g) stored in the upper 3 m of soil [1, 2, 4-8].
In the Arctic, the formation of soil and organic matter is influenced by cryogenic processes such as cryogenic mass exchange cracking, frost formation, soil swelling, cryogenic solifluction, thermokarst, etc. In permafrost-affected soils occupying about 65% of the territory of Russia, biogeochemical processes take place within the active layer of soil, the depth of the active layer depends on landscape position, the type of vegetation, and organogenic horizons [1, 6, 9]. Moreover, essential amounts of organic matter are stabilized within the frozen ground below the permafrost table. A common feature of the soil cover of the Arctic is its complexity associated with the permafrost processes in various bioclimatic and geological and geomorphological conditions. For arctic soils, as a rule, a high water content is typical, especially near the permafrost table, which appears to be a water barrier. As a result, gray color and redoxymorphic conditions appear associated with the gleying process, which is most pronounced on loamy and well-structured soils [10].
The soils in the cryolithozone are considered as carbon storages. The accumulation of organic carbon in the profile of arctic soils is associated with processes of permafrost retinization, processes of cryogenic mass exchange, with the formation of organic matter in situ from plant residues, as well as inheritance from a soil-forming rock [11-13]. For the soils of watershed positions of the Arctic and Subarctic regions, the formation of superficial humus enriched layer is typical with a weak intensity of decomposition of organic residues and humification processes.
Low average temperatures and a short vegetation season in permafrost areas cause organic matter to accumulate throughout the Quaternary period [2]. Biomass forms during a short growing season and initially accumulates in the upper layer of soil, thus creating an annual accumulation of organic matter, during which alluvial sedimentation of organic residues is also involved [2, 14-15].
Cryoturbation also leads to redistribution of organic matter into deeper soil horizons. Another process is the movement of organic matter in a dissolved state and its accumulation at the boundary with permafrost [2, 16-18]. During cryoturbation, fine fragments of organic material, separated from the lower parts of the surface horizons under the influence of ice, move inside the profile, mixing with the mineral matter of the underlying horizons; the result of this movement of organic masses along the profile is its compaction, homogenization and decomposition of plant residues [19]. As a result of slope processes, organogenic horizons are often buried under material that has fallen here as a result of solifluction. In contrast to cryoturbated material, buried organogenic horizons are characterized by high porosity, absence of excessive overcompaction, and plant residues are destroyed much less [19].
Nowadays, the use of modern physicochemical research methods has made it possible to study the structure of HAs molecules in more detail without resorting to their destruction. The methods of infrared (IR), nuclear magnetic resonance (NMR) and electron paramagnetic resonance (ERS) spectroscopy can provide information not only about the qualitative set of the most important atomic groups and types of bonds, but also about the specific location of individual functional groups and molecular fragments. Among organic substances, humic acids are distinguished by the greatest biochemical resistance [20]. However, their composition and structure are variable and reflect the conditions of humus formation. The use of 13C-NMR spectroscopy allows the quantitative determination of structural and functional parameters of HAs, and, therefore, this method is used to evaluate their transformation under the influence of various factors, including man-made [19, 21-22].
13C-NMR spectroscopy also allows to obtain detailed and reliable information about the number of structural fragments and functional groups of HAs, which appear in the corresponding spectral ranges in the form of resonant signals of carbon atoms characterizing typical structural features: the presence of aliphatic, aromatic fragments and functional groups: -C=O) - aldehydes, ketones and quin- oid structures; (-COOH), (-NH2), (-OCH3), (-OH) - alcohols, carbohydrates and phenols [9, 17, 19, 23-24]. Studies of the principles of HAs structure, deciphering the intramolecular “state” allow going to the quantitative level of the description of the bound “structure - molecular weight”.
Thus, the main aim of this study was to determine the molecular composition of organic matter in selected soils of the Russian Arctic using CP/MAS (CrossPolarization/Magic Angle Spinning) 13C-NMR spectroscopy.
Materials and methods
The study area is located around the islands of the Russian Arctic, and include Vaigach and Kolguev islands in the Kara sea (70°00'04.3"N 59°40'22.4"E and 69°07'36.8"N 49°30'53.7"E), Andrey island in the Laptev sea and Kurungnakh island in the Lena River Delta (76°46'46.0"N 110°47'11.6"E and 72°21'46.7"N 126°08'23.3"E) and Sosnowiec island in the White sea (66°29'21.9"N 40°40'57.8"E) (Fig. 1).
The article presents single plot areas, confirming the statement about the diversity of soils in the Arctic, which is associated with the differentiation of climatic conditions in the latitudinal and meridional direction, and with a large variety of soil-forming rocks and topographic situations. Vaigach Island, which is located in the Western part of the Russian Arctic between the Barents and Kara Seas, has more humid climatic conditions than Kolguev Island, located in the Barents Sea. The annual precipitations of these islands are 250-350 mm and 210250 mm, respectively [25]. The thermal conditions are also different, for Vaigach Island (one latitude with the island of Sosnowiec), the average temperature of the warmest month is 5.8 °C, while for Andrey Island it is 2°C. Soil-forming rocks are represented either by sea loams of ancient sea terraces, or by skeletal deluvium of sandy and loamy composition. In the landscape, there is a pronounced microrelief associated with heaving mounds, the presence of structured soils, and also places of solifluction deformations.
Fig. 1. Location of the study area and the studied soils. 1 - Sosnowiec island; 2 - Kolguev island; 3 - Vaigach island; 4 - Andrey island; 5 - Kurungnakh island. Source: ESRI
The vegetation of the islands is represented mainly by arctic tundra communities with fragmented grass and shrub cover with the predominance of moss and lichen. Soils of the Lena River Delta form in strong Arctic weather conditions, mean annual air temperature is - (-13°C), mean air temperature of the warmest month (July) is - 6.5°C, and of the coldest month (January) is - (-32°C). The thickness of snow is 23 cm, the annual amount of precipitations is 323 mm (in summer it is 125 mm). Parent materials of the Lena Delta are presented by fresh alluvial material and loamy and sandy loamy sediments.
Soil samples are represented by upper horizons of Cryosol, mainly humus weak developed horizons (W) or gray humus horizons - AY (Umbric horizons). In most cases, these horizons are replaced by the horizon of CR, a horizon with pronounced signs of cryogenic mass exchange. Samples of soil were selected mostly in a polygonal tundra. Soil samples from surface horizons were collected during the summer of 2014-2015 during the Sea Expedition organized by the Arctic and Antarctic Research Institute. The soil description is presented in Table 1. The soils were identified according to WRB [26].
Soil samples were air-dried (24 hours, 20°C), grounded, and passed through a 2 mm sieve. Soil chemical routine analyses were performed using classical methods: C, H and N content was determined using an element analyzer (Euro EA3028-HT Analyser). Data were corrected for water and ash content. Oxygen content was calculated by difference of whole samples of mass and gravimetric concentration of C, N, H and ash. pH in water and in salt (soil-dissolvent ratios 1:2.5 in case of mineral horizons and 1:25 in case of organo-mineral horizons) suspensions was detected using a pH meter (pH-150 M).
Humic acids (HAs) were extracted from each sample according to a published IHSS protocol [27]. The soil or cryoconite samples were treated with 0.1 M NaOH (soil/solution mass ratio of 1:10) under nitrogen gas. After 24 hours of shaking, the alkaline supernatant was separated from the soil residue by centrifugation at 1.516 x g for 20 minutes and then acidified to pH 1 with 6 M HCl to precipitate the HAs. The supernatant, which contained fulvic acids, was separated from the precipitate by centrifugation at 1.516 x g for 15 minutes. The HAs were then dissolved in 0.1 M NaOH and shaken for four hours under nitrogen gas before the suspended solids were removed by centrifugation. The resulting supernatant was acidified again with 6 M HCl to pH 1 and the HAs were again isolated by centrifugation and demineralized by shaking overnight in 0.1 M HCl/0.3 M HF (soil/solution ratio of 1:1). Next, the samples were repeatedly washed with deionized water until pH 3 was reached and then finally freeze-dried. HAs extraction yields were calculated as the percentage of carbon recovered from the original soil sample [28]. Solid-state CP/MAS 13C-NMR spectra of HAs were measured with a Bruker Avance 500 NMR spectrometer in a 3.2-mm ZrO2 rotor. The magic angle spinning speed was 20 kHz in all cases and the nutation frequency for cross polarization was u1/2p 1/4 62.5 kHz. Repetition delay and the number of scans were 3 seconds. HAs extraction yields were calculated as the percentage of carbon recovered from the original soil sample.
Results and Discussion
Elemental composition of HAs
In general, the elemental composition of HAs is comparable with the previous data for soils of the polar zone [6, 18, 23, 29-31]. The elemental composition of HAs is the most important indicator determining the progress of humification, oxidation and degree of condensation of HAs [30-31].
The obtained data on HAs elemental composition, atomic ratios and degree of oxidation (W) are presented in Table 2.
Elemental composition of the studied humic acids from surface soil horizons.Gravimetric concentrations are given for C, H, O and N content. C/N, H/C, O/C,H/Cmod and W were calculated from mole fraction of C,H,O and N content.
H/C mod is the number of substituted hydrogen atoms in the humic acids; (H/C)mod = (H/C) + 2 (O/C) x 0.67; H/C and W indexes were calculated accordingto Orlov [43]. Sample numbers correspond to Table 1. SD±0.05 for N, H and C content
Table 2
The carbon content in the studied samples varies over a wide range from 26 to 52%. The studied HAs can be divided into two groups by carbon content, less than 37% and more than 37%. The first group includes samples from Kolguyev and Andrey islands, they are less charred, which is due to leaching of low carbon molecules from the organogeneous horizon. Samples from other islands have a higher carbon content and have a high degree of carbonization associated with humification processes.
The nitrogen content in all studied HAs is 4% lower. This is due to the low accumulation of nitrogen-containing compounds in plant residues and in preparations of humic substances.
The oxygen content in the studied soils also varied in a wide range from 33 to 64%. The highest oxygen values correspond to the preparations from Kolguev and Andrey islands in those samples where a low carbon content is observed. The high oxygen content is due to the better solubility of oxygen-enriched hydrophilic humic acid molecules and their migration to the underlying mineral horizons [32-36].
The calculation of the degree of oxidation showed that most of the studied samples were in reduction conditions (from -0.1 to -0.4). The exceptions were the samples from Kolguev and Andrey islands, where the degree of oxidation reaches +2.3 and 0.7, respectively. This causes the migration of oxidized HAs down the profile. The weak reductions of preparations of HAs of organogenius horizons is determined by the produce of fresh organic residues and their weak humification in the specific bioclimatic conditions of the North.
One of the most widely used methods is a numerical description of the structure of humic acids in order to identify the patterns of their formation and transformation in the construction of Kleinhempel diagrams [37]. The method is based on a graphical representation of the data in the coordinates H/Cmod - O/C and serves a convenient technique for demonstrating the contribution of oxidation and condensation processes to changes in the elemental composition of HAs.
Based on Kleinhempel diagram (Fig. 2), it was found that in most of the studied soils the value of H/Cmod and О/С is relatively low, which indicates a low content of oxygen-containing groups in humic acids and their low migration ability. Samples from Kolguev and Andrey islands have relatively high values of H/C mod and O/C, which indicates that they have a high degree of hydromorphism due to low microbiological activity, which promotes a better preservation of carbohydrate and amino acid fragments in the structure of HAs. Relatively low values of H/Cmod indicate accumulation of aromatic fragments in the composition of humic acids.
Fig. 2. Elemental composition of the studied humic acids isolated from surface soil horizons. Gravimetric concentrations are given for carbon, hydrogen, oxygen and nitrogen content. sH/C mod - The number of substituted hydrogen atoms in the humic acids; (H/C) mod = (H/C) + 2 (O/C) x 0.67; Sample numbers correspond to Table 1.
The values of the H/Cmod and O/C ratios decrease, i.e. accompanied by an increase in the proportion of aromatic structures in humic acid molecules [33, 36, 38].
Characterisation of HAs by 13C-NMR spectroscopy
Spectra of the CP/MAS 13C-NMR are given in Fig 3. Various molecular fragments were identified by CP/MAS 13C-NMR spectroscopy (Table 3): carboxyl (-SOOR); carbonyl (-C = O); CH3-, CH2-, CH-aliphatic; -C-OR alcohols, esters and carbohydrates; phenolic (Ar-OH); quinone (Ar=O); aromatic (Ar-), which indicates a great complexity of the structure of HAs and the polyfunctional properties that cause their active participation in soil processes [23].
The chemical shifts are shown in Table 4. The aromatic group is calculated from the sum of the shifts of 110-185 ppm. Aliphatic fragments are calculated from the sum of the shifts of 0-110 ppm, 180-200 ppm, Al h, r + Ar h, r (total number of unoxidized carbon atoms) - The signals were summed over the regions 0-46 and 110-160 ppm, C, H-Al / O, N-Al. Signals from C, H-alkyls were summed in the range of 0-47 ppm. O, N-alkyl at regions 46-60 and 60-110 ppm [23]. The presence of all peaks of the carbon species which are required for identification of the studied substances as humic acids was revealed.
Aliphatic fragments of HAs were predominant up to 74% in the studied samples. In the studied soils, mineralization processes predominate. The low content of aromatic fragments due to low microbiological activity leads to accumulation of organic matter in the soil [18-19, 39]. The low content of carboxyl fragments (160-185 ppm) indicates that humic substances have low migration activity and are less enriched in oxygen-containing compounds, which is confirmed by elemental analysis data of HAs. To standardize the quantitative characteristics of HAs macromolecules, the following parameters were used: the ratio of carbon of aromatic structures to carbon of aliphatic chains - Ar/Al, the degree of decomposition of organic matter (C, H-alkyl/O, N-alkyl) and the integral indicator of hydrophobicity of HAs (Al h, r + Ar h, r). The integral indicator of hydrophobicity is shown in Figure 4.
Fig. 3. CP/MAS 13C-NMR spectra of HAs from soils of the Russian Arctic. Number corresponds to Table 1; on the X-axis - Chemical shift, ppm; on the Y-axis Vaxis - Relative intensity, dimensionless
Chemical shifts of atoms of the 13C molecular fragments of humic acids
Percentage of carbon in the main structural fragments of humic acidsfrom the studied surface soil horizons (according to CP/MAS 13C-NMR data).Sample numbers correspond to Table 1; Ar - aromatic fraction;
Al-aliphatic fraction; Alhr + Arhr % - hydrophobicity degree; C, H-Al / O,
N-Al - the degree of decomposition of organic matter. SD±0.05
Table 5
Fig. 4. The diagram of integrated indicators of the molecular composition of humic acids. Sample numbers correspond to Table 1; Al h,r + Ar h,r indicates the total number of unoxidized carbon atoms
In the studied soils, a significant amount of aliphatic fragments in the ranges of 0-46 and 110-144 ppm is accumulated, which is due to the increased humidity in the Arctic sector. A comparative analysis of humic substances from different sectors of the Russian Arctic shows that, depending on the region, soils can accumulate different amounts of aliphatic and aromatic fragments of humic substances [18]. The Ar/Al ratio ranges from 0.35 to 1.04, which is caused by different climatic parameters, as well as soil-forming processes and activity of cryogenic processes. Soils, forming on sandy loam and silt loam classes of parent materials, have a relatively high degree of humification due to natural drainage and low degree of cryogenic processes. In soils from the islands of Sosnowiec, Vaigach, Kolguev and Andrey, the molecular and elemental composition is similar and is confirmed by data from other researchers working in the Arctic [11, 17-18, 3941]. Such soils develop according to the cryogenic type of soil formation. Soils from Kurungnakh island, located in the Lena River Delta, accumulate a relatively high content of aromatic fragments up to 51%, which is typical of the podzolic type of soil formation. Accumulation of aromatic fragments of HAs is associated with high microbiological activity, on the island of Kurunnakh, on sandy and loamy sandy soils and leads to favorable conditions for the microbiota [31].
A number of factors affect the qualitative composition of humic substances in soil, the leading features are hydromorphism and the quality of plant residues. The soils of the Arctic are mainly represented by moss-lichen communities, which are enriched with aliphatic compounds [42-44]. Under cryogenic processes, anaerobic conditions are formed in the soil, due to the nearness of permafrost table and excessive humidity. Such conditions decrease the microbiological activity, which decreases the rate of transformation of soil organic matter and promotes accumulation of paraffin fragments in the structure of humic substances. The processes of humification of plant residues in the soils of Kurungnakh Island, developing in the podzolic type of soil formation, proceed more intensively, contributing to the formation of aggressive humic substances enriched with oxygen-containing functional groups up to 15% and with a high content of aromatic fragments. Humic substances from the islands of Sosnowiec, Kolguev, Vaigach and Andrey have a lower degree of aromaticity and contain mainly non-oxidized aliphatic fragments up to 74% in their structure, which together with anaerobic conditions leads to the gleying process.
Conclusions
Structural characteristics and elemental composition of humic acids in soils formed in different sectors of the Russian Arctic have been studied. The analysis of the elemental composition showed an increase in the degree of hydromorphism in the soils of the islands of Sosnowiec, Vaigach, Kolguev and Andrey and is accompanied by an increase in values (H/Cmod), which is determined by the lower microbiological activity of Cryosol, which leads to a conservation of carbohydrate and amino acid fragments in the structure of HAs. Soils from the island of Kurungnakh, the Lena River Delta, revealed relatively low ratios of the values (H/Cmod) in the soils, which indicates an increase in the proportion of aromatic fragments in the structure of HAs.
Analysis of the molecular composition of HAs showed that humic acid molecules from the island of Kurungnakh are enriched with aromatic fragments relative to the humidification of soils from the islands of Sosnowiec, Vaigach, Kolguev and Andrey where aliphatic chains and structures like carbohydrates and amino acids prevail in the carbon skeleton. The structurally functional composition of humic acids in tundra soils is less enriched with aromatic structures. Excessive humidity leads not only to accumulation of humic substances, but also affects their molecular composition, namely, causes accumulation of humic substances in the soils enriched with aliphatic structures.
Analysis of the integral indicator of the hydrophobicity of HAs (Al h, r+Ar h, r) showed that HAs in the Arctic zone are resistant to oxidation (including microbial). Relatively high rates of C, H-Al/O, N-Al are observed in soils from the island of Kurungnakh in the Lena River Delta, which indicates a more significant degree of humification compared to other studied soils of the Russian Arctic.
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