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The importance of humin in soil characterisation: A study on Amazonian soils using different fluorescence techniques
Science of the Total Environment 537 (2015) 152–158
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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
The importance of humin in soil characterisation: A study on Amazonian soils using different fluorescence techniques Amanda Maria Tadini a,b,⁎, Gustavo Nicolodelli a, Stephane Mounier c, Célia Regina Montes d, Débora Marcondes Bastos Pereira Milori a a
Embrapa Agricultural Instrumentation, São Carlos, SP, Brazil Institute of Chemistry of São Carlos, University of São Paulo, São Carlos, SP, Brazil Laboratoire PROTEE, EA3819, Université de Toulon, CS 60584, 83041 Toulon CEDEX 9, France d Centro de Energia Nuclear na Agricultura and NUPEGEL, University of São Paulo, Piracicaba, SP, Brazil b c
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• Two fluorophores were observed in the structures of humin and the whole soil. • The fluorescence of the soil is strongly related to the fluorescence of the humin. • Humin fraction can represent 80-93% of fraction of the Amazonian SOM.
a r t i c l e
i n f o
Article history: Received 29 April 2015 Received in revised form 24 July 2015 Accepted 25 July 2015 Available online xxxx Editor: D. Barcelo Keywords: Humin Soil Humification Fluorescence CP/PARAFAC
a b s t r a c t Soil organic matter (SOM) is a complex mixture of molecules with different physicochemical properties, with humic substances (HS) being the main component as it represents around 20–50% of SOM structure. Soil of the Amazon region is considered one of the larger carbon pools of the world; thus, studies of the humic fractions are important for understanding the dynamics of organic matter (OM) in these soils. The aim of this study was to use laser-induced fluorescence spectroscopy (LIFS) and a combination of excitation–emission matrix (EEM) fluorescence with Parallel Factor Analysis (CP/PARAFAC) to assess the characteristics of humin (HU) extracted from Amazonian soils. The results obtained using LIFS showed that there was an increasing gradient of humification degree with depth, the deeper horizon presenting a higher amount of aromatic groups in the structure of HU. From the EEM, the contribution of two fluorophores with similar behaviour in the structures of HU and whole soil was assessed. Additionally, the results showed that the HU fraction might represent a larger fraction of SOM than previously thought: about 80–93% of some Amazon soils. Therefore, HU is an important humic fraction, thus indicating its role in environmental analysis, mainly in soil analysis. © 2015 Elsevier B.V. All rights reserved.
⁎ Corresponding author at: Embrapa Agricultural Instrumentation, São Carlos, SP, Brazil. E-mail address: [email protected] (A.M. Tadini).
http://dx.doi.org/10.1016/j.scitotenv.2015.07.125 0048-9697/© 2015 Elsevier B.V. All rights reserved.
A.M. Tadini et al. / Science of the Total Environment 537 (2015) 152–158
1. Introduction Soil organic matter (SOM) plays an important role in environmental sustainability, particularly in the carbon cycle in soil, which has been attracting considerable interest due to carbon accumulation/sequestration in soils. The main components of SOM are the humic substances (HS), which have defined physical and chemical characteristics, and are fractionated into humic acids (HA), fulvic acids (FA) and humin (HU) according to their solubility (Stevenson, 1994). Currently, there is scarce information available in the literature on the structure of HU compared to the other two fractions, even though the HU fraction makes up between 20% and 50% of the HS present in soil (Rice and MacCarthy, 1989; Stevenson, 1994; Rice, 2001; Yang et al., 2004; Nichols and Wright, 2006). HU is defined as the extracted portion that remains insoluble in aqueous solutions at all pH levels, and is operationally treated as the remaining SOM residue (Stevenson, 1994; Rice, 2001). This humic fraction is also known to strongly bind to hydrophobic organic compounds, and to present unchanged biopolymers, such as lignin and polysaccharides, in its structure (Nearpass, 1976; Chiou et al., 2002). Rice and MacCarthy (1989) demonstrated that HU had the characteristics of a lipid mixture of humic structures and mineral compounds (insoluble residue). Rice (2001) demonstrated the importance of a deeper knowledge of HU structures and their interactions with soil, allowing a better understanding of the role of SOM in carbon sequestration. Recent studies showed that the stability of HU in soil is not due to low humic reactivity, but to the agglomeration of humic materials responsible for particle aggregation mechanisms in the soil, which may represent the majority of the carbon present in the humified soil (Benites et al., 2003; Lombardi et al., 2006). According to Stevenson (1994), HU consists of a variety of materials, such as HA bound to mineral materials (clay) that were not separated in the extraction process. It is humic materials with high carbon content (more than 60%) that are insoluble in a basic medium, fungal melanins and/or paraffinic substances. According to this author, the soil HU can be divided into two fractions based on the separation of their microaggregates, which consists of a modified lignin polymer and other subcellular particles of plants retained as microaggregates. The determination of the optical properties of organic matter (OM) is an important method for the structural understanding of its fractions. Spectroscopic techniques, such as EPR, FTIR and 13C NMR spectroscopies, require the extraction and fractionation of the HS, making the soil analysis a limited, laborious process. However, the laser-induced fluorescence spectroscopy (LIFS) technique applied to soils is a new technique that has been shown to be effective in the analysis of SOM. It provides fast and sensitive results and it can be applied to samples without pre-treatment (Milori et al., 2006; Martins et al., 2011). Using LIFS, it is possible to determine the humification index of a sample, which is related to the concentration of rigid structures within, like quinone groups and aromatic rings. The three-dimensional fluorescence spectroscopy or emission– excitation matrix (EEM) is a selective and sensitive spectroscopic technique, whose spectra allow the composition and configuration of OM and HS of various origins to be determined (Coble, 1996; Filella et al., 2005; Ziegelgruber et al., 2013). The intensity, shift and position of the fluorescence peaks can be related to the presence of electron donor and/or acceptor groups, polycondensation, aromaticity, heterogeneity and chemical properties (Chen et al., 2002; Peuravuori et al., 2002; Sierra et al., 2005). Thus, the use of this technique on environmental samples, especially humic fractions, provides important data concerning the chemical structure of the present fractions. The EEM is a more complete technique of fluorescence analysis, and it allows a simple set or a mixture of fluorescent components present in humic fractions to be evaluated, thus providing a fingerprint of the sample (Chen et al., 2002; Sierra et al., 2005; Rodríguez et al., 2014; Zhu et al.,
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2014). Moreover, the EEM spectrum can be used for the qualitative and quantitative characterisation of OM when combined with advanced multivariate statistical techniques, such as Parallel Factor Analysis (CP/PARAFAC), which can decompose the complex signal of the fluorescence spectra onto the combination of simple components. EEM-CP/PARAFAC is a potentially useful technique for the evaluation of complex samples, such as HS. This technique allows for the evaluation of the decomposition of the fluorescent-independent components of the complex EEM, which represent groups such as fluorophores (Luciani et al., 2008; Santín et al., 2009; Ziegelgruber et al., 2013; Zhu et al., 2014). Although this technique has not yet been applied to the characterisation of the HU fraction, it can provide further information on the optical characterisation of HU and its dynamics in the environment. To the best of our knowledge, there are no published studies using LIFS and EEM-CP/PARAFAC techniques with the objective to discover the structural analysis of HU fraction. The few studies found in the literature report on HU behaviour in adsorption and absorption processes with organic compounds and/or metals using chromatographic analysis, infrared spectroscopy and thermogravimetry (Ferri et al., 2005; Mecozzia et al., 2005; Jerzykiewicz, 2013; Zhang et al., 2015). LIFS and EEM-CP/PARAFAC techniques will make access to the structural analysis of HU fraction feasible, since it can be used on solid-state samples. Therefore, the main objectives of this study were: (1) to assess the characteristics of HU and the whole soil using LIFS; and (2) to evaluate the main components in this fluorescent HU fraction and compare them to the fluorescent components present in the soil using EEM associated with CP/PARAFAC. 2. Materials and methods 2.1. Study area The study area located north of Barcelos city, Amazon State, Brazil, at the coordinates 0°15′33.1″N and 62°46′27.6″W. The sedimentary cover of the rivers Branco and Negro with some younger depositional areas surrounding the Demeni river (Holocene alluvium of Demeni river) is the geological substratum (Pereira et al., 2015). The climate is typically equatorial and is characterised by an average temperature of 25 °C with high rainfall (around 3000 mm) throughout the year and a light dry season. Three soil types developed in the area according to IBGE (2008), oxisols, entisols and spodosol. Seven spodosol profiles with different soil drainage conditions were studied for characterisation of the area. For this study one soil profile of an overflood Humiluvic Spodosol was selected. The soil profile (0 to 350 cm of depth) presents sandy-loam texture and quartz is the prevalent mineral with kaolinite and gibbsite as accessory. The samples were collected by hand auger pits and due to the collapse of the sand of overlying spodic horizons, it was necessary reinforce the wall holes with PVC tubes. 2.2. Soil and humin sample preparation for analysis Sampling procedures, preservation and preparation of the samples followed the recommendations of official methods. The extraction and purification of HU followed the recommendations described by the International Humic Substances Society (IHSS), Rice and MacCarthy (1989) and Swift (1996). The pellets of whole soil and HU containing 30% boric acid were prepared for the analysis of LIFS and EEM, as observed in Fig. 1. 2.3. Elemental analysis For the chemical analysis of the elemental composition, the samples of the whole soil and HU were homogenised and grinded into particles
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Fig. 1. Scheme of the experimental procedure carried out in this study.
smaller than 106 mm. Then, 10 mg sample was weighed in tin capsules using the analytical balance; they were analysed by combustion at 1000 °C using an elemental analyser (Perkin Elmer model 2400). This procedure was performed in duplicate. 2.4. Inorganic residue 10 mg of HU extracted from soils was calcined at 700 °C in a muffle furnace for 4 h. The amount of OM by mass difference after calcination was then calculated, considering the final residue as inorganic matter (Griffith and Schnitzer, 1975).
2.6. 3D fluorescence spectroscopy Fluorescence measurements using a luminescence spectrometer (model LS50B Perkin Elmer) in soil and HU pellets were performed. The spectra were acquired in the scan range from 240 to 700 nm for emission and 220–510 nm for excitation. The spectra were obtained using a 290-nm cut-off excitation filter with an increment of 10 nm excitation and 30 scans. The spectra obtained using this technique were treated using the mathematical method known as parallel factor analysis (Luciani et al., 2008; Santín et al., 2009; Zhu et al., 2014).
2.5. Laser-induced fluorescence spectroscopy
3. Results and discussion
The LIFS spectra of the pellets were obtained by applying a diode laser centred at 405 nm with an average power of 20 mW; for the detection system, a spectrometer with a resolution of 4 nm (equipment developed by Embrapa Agricultural Instrumentation) was used. The laser beam was directed onto the pellet surface through six external optical fibres, and the fluorescence emission resulting from the decay of excited species was transferred to the spectrometer through a central optical fibre bundle. The measurement range was from 420 nm to 800 nm, and the selected integration time and average were 500 ms and 4, respectively, for all measurements. Four replicates were recorded for each sample and the averaged spectrum was used (Santos et al., 2015). The humification index (HLIFS) was determined by calculating the ratio between the area of the fluorescence emission spectrum (Ex: 465, Int from 450 to 700 nm) (ACF) and the amount of total organic carbon (in g kg−1) (TOC) present in the soil sample (Milori et al., 2006).
Table 1 shows information on the chemical fractionating process and the values obtained by elemental analysis techniques for the different depths of the whole soil and for the extracted HU. For all the layers, the HU material represents more than 80% in mass, with the minimum value at the surface (80%) and the maximum at the bottom layer (93.46%). Based on these results, the organic material from the surface is assumed to be composed of carbon originating from HS for 41% and of non-humic carbon (probably labile to chemical and biological degradation) for 59%. This partition makes sense because, on the surface, there is a higher incorporation of fresh OM. In this layer, among stable carbon (HS) 63% belongs to the HU and 37% to HA. This shows that, on the surface, the ratio between carbon in HU and HA is almost 2:1 (around 1.7). In the 15 to 30 cm and 40 to 50 cm layers, carbon contents are much lower than on the surface, and they are basically stored as HA and HU. The contribution of HU for soil carbon in these layers was
Table 1 Values obtained by elemental analysis techniques and the values of the yields for the whole soil and the humin extracted from different depths. Samples/horizons (cm) Elemental analysis (C) %AHsoil %AFsoil % HUsoil % residual weight (estimated) %C in HA %C in FA %C in humin % C in soil %C soil from HA %C soil from FA %C soil humin %C soil residual (estimated) %C from HSs (estimated) %C from residual (estimated) Ash % Humin inorganic % Humin organic
0–15
15–30
40–50
260
350
9±1 0±1 80 ± 1 11 ± 1 38 ± 5 18 ± 1 7.2 ± 0.5 22.5 ± 0.9 3.4 ± 0.6 0.01 ± 0.01 5.8 ± 0.4 13 ± 3 41 ± 5 59 ± 7
0.9 ± 0.2 0.3 ± 0.2 83.9 ± 0.3 14.9 ± 0.4 49 ± 1 4.5 ± 0.2 0.35 ± 0.02 1.01 ± 0.09 0.4 ± 0.1 0.01 ± 0.01 0.29 ± 0.02 0.3 ± 0.2 72.7 ± 0.7 27.3 ± 0.3
1.4 ± 0.3 0.3 ± 0.3 88.8 ± 0.3 9.5 ± 0.5 52.8 ± 0.2 6.0 ± 0.1 0.31 ± 0.01 2.1 ± 0.2 0.74 ± 0.1 0.02 ± 0.01 0.275 ± 0.009 1±1 48 ± 1 52 ± 2
0.0 ± 0.5 0.7 ± 0.5 90.8 ± 0.7 9±1 43 ± 2 4.7 ± 0.2 0.07 ± 0.02 0.80 ± 0.03 0.01 ± 0.01 0.03 ± 0.02 0.06 ± 0.02 0.7 ± 0.2 9±5 90 ± 5
0.16 ± 0.02 0.43 ± 0.02 93.46 ± 0.03 5.95 ± 0.04 48 ± 5 6.2 ± 0.4 0.10 ± 0.02 1.2 ± 0.2 0.08 ± 0.01 0.030 ± 0.002 0.09 ± 0.02 1.0 ± 0.3 14.19 ± 0.04 85.8 ± 0.2
87.70 ± 0.01 12.30 ± 0.01
98.40 ± 0.01 1.60 ± 0.01
99.10 ± 0.01 0.90 ± 0.01
96.00 ± 0.01 4.00 ± 0.01
97.10 ± 0.01 2.90 ± 0.01
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between 27% and 42%, demonstrating again the relevance of this chemical fraction. When evaluating the ash content in HU of these layers, it was observed that this fraction was predominantly composed of minerals with little carbon content. In deeper layers, a very singular behaviour was observed. Around 85–90% of soil carbon in these layers does not come from HS. Some hypotheses can be formulated to justify this behaviour: 1. The chemical fractionation process can lead to a loss of carbon strongly complexed to metals, which must be present in abundance at depth; thus, the soil carbon measured is always greater than the sum of the carbon found in humic fractions, giving the impression that there is a great deal of labile carbon (~90%) in deeper layers. 2. This soil within the Amazonian ecosystem enables the percolation of labile carbon from the surface, which accumulates in deeper layers. 3. The water table, which passes just below the 350 cm layer, brings fresh OM to deeper layers. In the 350 cm layer, the contribution of HU to the humic carbon is around 50%. Thus, in this first analysis, the importance of HU in Amazonian soils is very clear. For this kind of soil, it is not possible to make a precise analysis of HS considering only the HA. These results emphasise the importance of HU; the development of techniques that allow the analysis of this material is, hitherto, scarcely examined. Fig. 2 shows the spectra obtained by LIFS of (a) the whole soil and HU extracted from soil at 0–15 cm (surface profile), and (b) HU extracted from soil at different depths (15 to 350 cm). In Fig. 2a, similar spectra were observed in the HU and whole soil samples, and a peak at 690 nm can be observed in the HU sample in the surface and 260 cm horizons. In the literature, the fluorescence peak at 690 nm refers to the chlorophyll present in the compounds of vegetable origins, such as plants (Lichtenthaler et al., 1999), and it can be associated in the fresher OM present in these horizons. In Fig. 2b, a band at 450–650 nm can be observed for deeper profiles of HU samples. Fig. 3 shows the humification index (points) and the LIFS area (square dashed) of the HU extracted from different depths of soil. An increase in the humification index with depth can be observed, which is related to the chemical characteristics of the samples, especially for HU fractions. When we apply the statistical analysis in these whole soil and HU fractions, we did not find a significant difference (p N 0.05). This behaviour for HU corroborates with Santos et al. (2015), who reported an increase of humification with depth in some Amazonian soils. An explanation for this behaviour could be that the presence of aromatic polycondensation in the OM is also present in the corresponding horizon (Tivet et al., 2013). Therefore, this increase in the humification index with depth can be attributed to, first, the
Fig. 3. Humification index (points) and the LIFS area (square dashed) obtained for the humin extracted from different depths of soil.
contribution of fresh OM to the surface soil causing a dilution effect of the organic material, or second, the natural percolation of soluble HS, causing the accumulation of humified material at depth. The 3D fluorescence spectra of the whole soil (Fig. 4a) and HU fractions (Fig. 4b) for different layers (0–350 cm) are presented in Fig. 4. For a better understanding of the fluorescence spectra, the results were treated using CP/PARAFAC (EEM-CP/PARAFAC). The results showed the contribution of two fluorophores, which had core tensor consistencies (concordia) of 96.6% and 98.3% for the HU fraction and the whole soil, respectively. Fig. 5 shows the two fluorophores, (a) component 1 and (b) component 2, which are present in the structure of the whole soil and the HU fraction. Component 1 (λex/λem: 375/400 nm) refers to simpler structures present in the HS of terrestrial environments and component type C in the literature (Coble, 1996). Component 2 (λex/λem: 450/510 nm) refers to terrestrial HA and lignin derivatives, which are associated with a more complex and recalcitrant structure with fused aromatic rings and/or a combination of simple aromatic rings (Matthews et al., 1996). According to Matthews et al. (1996), HA extracted from soils are predominantly derived from lignin precursors (denominated peak L), which have the component structures of terrestrial plants. Grasset and Amblès (1998) studied the structural characteristics of HU and HA extracted from soil (north Correze, Plateau de Millevaches, France)
Fig. 2. Spectrum obtained by LIFS for the whole soil and the humin extracted from different depths (0 to 350 cm).
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Fig. 4. Total fluorescence spectra in the EEM mode obtained for (a) the whole soil and (b) the humin extracted from soil of different depths: (1) 0–15 cm, (2) 260 cm, and (3) 350 cm.
employing the thermochemolysis technique. Their results indicated that the lipid biopolymer lignin contributes to the formation of complex OM in soil, and that the ester and ether groups are clearly involved in the structure of HU and HA in the soil, and these groups can be origins microbiologic or metabolism derived of plants. Xiang-yun et al. (2014) studied carbon fixation by HS in soil under fertilisation and they found that low and high fertiliser rates were significant, highlighting the HU fraction, which was the main fraction responsible for carbon sequestration under long-term fertilisation of soil. The contributions of the two fluorophores (components 1 and 2) according to the depth in a) HU and b) whole soil are presented in Fig. 6. The HU and whole soil samples showed similar behaviours. OM, for both series, did not present any structural changes in the profile; the ratio between their main fluorophores throughout the soil profile remained almost constant. There were only changes in the amount
of fluorophores in the profile. The highest concentration of these fluorophores occurred in the 40–50 cm layer, and the lowest amount occurred at the surface, probably due to the initial stage of fresh OM degradation (more aliphatic). LIFS data was highly correlated with EEM-CP/PARAFAC data, particularly for HU (correlation coefficient of 0.92). 4. Conclusion Carbon quantification and humic fractions showed the importance of HU when studying OM in different horizons in Amazonian Soil. HU carbon is always one of the main pools of soil organic carbon for this ecosystem, contributing more than 40% of the carbon in HS. Particularly, in the analysed soil, the HU was the largest fraction, contributing 80–93% of the soil mass.
Fig. 5. Fluorescence Excitation Emission Matrix of the CP/PARAFAC components, (a) component 1 and (b) component 2, which are present in the samples of the whole soil and humin of the different depths.
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Fig. 6. Contribution of EEM-CP/PARAFAC components 1 and 2 from the samples of the (a) humin and (b) whole soil of the different depths.
Based on the EEM-CP/PARAFAC analysis, HU did not present any structural changes in the soil profile; the ratio between its main fluorophores remained constant. There were only variations in the amount of fluorophores in the profile. The highest concentration of these fluorophores occurred in the 40–50 cm layer. In the surface layer, the amount of these fluorophores was the lowest due to the abundance of fresh OM (more aliphatic). For soil, the EEM-CP/ PARAFAC analysis showed that HS at the deepest layer (350 cm) was quite similar to that from the 15–30 cm layer. This behaviour seems to agree with the soil carbon analysis, which suggests an accumulation of the humified OM during depth. LIFS results showed that both the whole soil and HU have quite similar spectral profiles, showing that HU is one of the main components of soil emission. This contribution is original, since there are few studies on HU characterisation, and there are no studies on HU fluorescence and its contribution to whole soil fluorescence emission. Thus, the use of EEM fluorescence spectroscopy with CP-PARAFAC and LIFS in soil analyses can provide information on the processes of HS transformation in this system. Furthermore, another advantage of these two techniques is that it allows a fast and low-cost analysis of the humic fractions present in the soil following the concept of green chemistry and the possibility of integrating a system for environmental use.
Acknowledgments The authors acknowledge the financial support for this work from the São Paulo Research Foundation (FAPESP) by project (Process 2011/03250-2) and scholarship (Process 2013/13013-3). We thank the Embrapa Agricultural Instrumentation for supplying the structure leading to the development of this research.
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Contents lists available at ScienceDirect
Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
The importance of humin in soil characterisation: A study on Amazonian soils using different fluorescence techniques Amanda Maria Tadini a,b,⁎, Gustavo Nicolodelli a, Stephane Mounier c, Célia Regina Montes d, Débora Marcondes Bastos Pereira Milori a a
Embrapa Agricultural Instrumentation, São Carlos, SP, Brazil Institute of Chemistry of São Carlos, University of São Paulo, São Carlos, SP, Brazil Laboratoire PROTEE, EA3819, Université de Toulon, CS 60584, 83041 Toulon CEDEX 9, France d Centro de Energia Nuclear na Agricultura and NUPEGEL, University of São Paulo, Piracicaba, SP, Brazil b c
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• Two fluorophores were observed in the structures of humin and the whole soil. • The fluorescence of the soil is strongly related to the fluorescence of the humin. • Humin fraction can represent 80-93% of fraction of the Amazonian SOM.
a r t i c l e
i n f o
Article history: Received 29 April 2015 Received in revised form 24 July 2015 Accepted 25 July 2015 Available online xxxx Editor: D. Barcelo Keywords: Humin Soil Humification Fluorescence CP/PARAFAC
a b s t r a c t Soil organic matter (SOM) is a complex mixture of molecules with different physicochemical properties, with humic substances (HS) being the main component as it represents around 20–50% of SOM structure. Soil of the Amazon region is considered one of the larger carbon pools of the world; thus, studies of the humic fractions are important for understanding the dynamics of organic matter (OM) in these soils. The aim of this study was to use laser-induced fluorescence spectroscopy (LIFS) and a combination of excitation–emission matrix (EEM) fluorescence with Parallel Factor Analysis (CP/PARAFAC) to assess the characteristics of humin (HU) extracted from Amazonian soils. The results obtained using LIFS showed that there was an increasing gradient of humification degree with depth, the deeper horizon presenting a higher amount of aromatic groups in the structure of HU. From the EEM, the contribution of two fluorophores with similar behaviour in the structures of HU and whole soil was assessed. Additionally, the results showed that the HU fraction might represent a larger fraction of SOM than previously thought: about 80–93% of some Amazon soils. Therefore, HU is an important humic fraction, thus indicating its role in environmental analysis, mainly in soil analysis. © 2015 Elsevier B.V. All rights reserved.
⁎ Corresponding author at: Embrapa Agricultural Instrumentation, São Carlos, SP, Brazil. E-mail address: [email protected] (A.M. Tadini).
http://dx.doi.org/10.1016/j.scitotenv.2015.07.125 0048-9697/© 2015 Elsevier B.V. All rights reserved.
A.M. Tadini et al. / Science of the Total Environment 537 (2015) 152–158
1. Introduction Soil organic matter (SOM) plays an important role in environmental sustainability, particularly in the carbon cycle in soil, which has been attracting considerable interest due to carbon accumulation/sequestration in soils. The main components of SOM are the humic substances (HS), which have defined physical and chemical characteristics, and are fractionated into humic acids (HA), fulvic acids (FA) and humin (HU) according to their solubility (Stevenson, 1994). Currently, there is scarce information available in the literature on the structure of HU compared to the other two fractions, even though the HU fraction makes up between 20% and 50% of the HS present in soil (Rice and MacCarthy, 1989; Stevenson, 1994; Rice, 2001; Yang et al., 2004; Nichols and Wright, 2006). HU is defined as the extracted portion that remains insoluble in aqueous solutions at all pH levels, and is operationally treated as the remaining SOM residue (Stevenson, 1994; Rice, 2001). This humic fraction is also known to strongly bind to hydrophobic organic compounds, and to present unchanged biopolymers, such as lignin and polysaccharides, in its structure (Nearpass, 1976; Chiou et al., 2002). Rice and MacCarthy (1989) demonstrated that HU had the characteristics of a lipid mixture of humic structures and mineral compounds (insoluble residue). Rice (2001) demonstrated the importance of a deeper knowledge of HU structures and their interactions with soil, allowing a better understanding of the role of SOM in carbon sequestration. Recent studies showed that the stability of HU in soil is not due to low humic reactivity, but to the agglomeration of humic materials responsible for particle aggregation mechanisms in the soil, which may represent the majority of the carbon present in the humified soil (Benites et al., 2003; Lombardi et al., 2006). According to Stevenson (1994), HU consists of a variety of materials, such as HA bound to mineral materials (clay) that were not separated in the extraction process. It is humic materials with high carbon content (more than 60%) that are insoluble in a basic medium, fungal melanins and/or paraffinic substances. According to this author, the soil HU can be divided into two fractions based on the separation of their microaggregates, which consists of a modified lignin polymer and other subcellular particles of plants retained as microaggregates. The determination of the optical properties of organic matter (OM) is an important method for the structural understanding of its fractions. Spectroscopic techniques, such as EPR, FTIR and 13C NMR spectroscopies, require the extraction and fractionation of the HS, making the soil analysis a limited, laborious process. However, the laser-induced fluorescence spectroscopy (LIFS) technique applied to soils is a new technique that has been shown to be effective in the analysis of SOM. It provides fast and sensitive results and it can be applied to samples without pre-treatment (Milori et al., 2006; Martins et al., 2011). Using LIFS, it is possible to determine the humification index of a sample, which is related to the concentration of rigid structures within, like quinone groups and aromatic rings. The three-dimensional fluorescence spectroscopy or emission– excitation matrix (EEM) is a selective and sensitive spectroscopic technique, whose spectra allow the composition and configuration of OM and HS of various origins to be determined (Coble, 1996; Filella et al., 2005; Ziegelgruber et al., 2013). The intensity, shift and position of the fluorescence peaks can be related to the presence of electron donor and/or acceptor groups, polycondensation, aromaticity, heterogeneity and chemical properties (Chen et al., 2002; Peuravuori et al., 2002; Sierra et al., 2005). Thus, the use of this technique on environmental samples, especially humic fractions, provides important data concerning the chemical structure of the present fractions. The EEM is a more complete technique of fluorescence analysis, and it allows a simple set or a mixture of fluorescent components present in humic fractions to be evaluated, thus providing a fingerprint of the sample (Chen et al., 2002; Sierra et al., 2005; Rodríguez et al., 2014; Zhu et al.,
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2014). Moreover, the EEM spectrum can be used for the qualitative and quantitative characterisation of OM when combined with advanced multivariate statistical techniques, such as Parallel Factor Analysis (CP/PARAFAC), which can decompose the complex signal of the fluorescence spectra onto the combination of simple components. EEM-CP/PARAFAC is a potentially useful technique for the evaluation of complex samples, such as HS. This technique allows for the evaluation of the decomposition of the fluorescent-independent components of the complex EEM, which represent groups such as fluorophores (Luciani et al., 2008; Santín et al., 2009; Ziegelgruber et al., 2013; Zhu et al., 2014). Although this technique has not yet been applied to the characterisation of the HU fraction, it can provide further information on the optical characterisation of HU and its dynamics in the environment. To the best of our knowledge, there are no published studies using LIFS and EEM-CP/PARAFAC techniques with the objective to discover the structural analysis of HU fraction. The few studies found in the literature report on HU behaviour in adsorption and absorption processes with organic compounds and/or metals using chromatographic analysis, infrared spectroscopy and thermogravimetry (Ferri et al., 2005; Mecozzia et al., 2005; Jerzykiewicz, 2013; Zhang et al., 2015). LIFS and EEM-CP/PARAFAC techniques will make access to the structural analysis of HU fraction feasible, since it can be used on solid-state samples. Therefore, the main objectives of this study were: (1) to assess the characteristics of HU and the whole soil using LIFS; and (2) to evaluate the main components in this fluorescent HU fraction and compare them to the fluorescent components present in the soil using EEM associated with CP/PARAFAC. 2. Materials and methods 2.1. Study area The study area located north of Barcelos city, Amazon State, Brazil, at the coordinates 0°15′33.1″N and 62°46′27.6″W. The sedimentary cover of the rivers Branco and Negro with some younger depositional areas surrounding the Demeni river (Holocene alluvium of Demeni river) is the geological substratum (Pereira et al., 2015). The climate is typically equatorial and is characterised by an average temperature of 25 °C with high rainfall (around 3000 mm) throughout the year and a light dry season. Three soil types developed in the area according to IBGE (2008), oxisols, entisols and spodosol. Seven spodosol profiles with different soil drainage conditions were studied for characterisation of the area. For this study one soil profile of an overflood Humiluvic Spodosol was selected. The soil profile (0 to 350 cm of depth) presents sandy-loam texture and quartz is the prevalent mineral with kaolinite and gibbsite as accessory. The samples were collected by hand auger pits and due to the collapse of the sand of overlying spodic horizons, it was necessary reinforce the wall holes with PVC tubes. 2.2. Soil and humin sample preparation for analysis Sampling procedures, preservation and preparation of the samples followed the recommendations of official methods. The extraction and purification of HU followed the recommendations described by the International Humic Substances Society (IHSS), Rice and MacCarthy (1989) and Swift (1996). The pellets of whole soil and HU containing 30% boric acid were prepared for the analysis of LIFS and EEM, as observed in Fig. 1. 2.3. Elemental analysis For the chemical analysis of the elemental composition, the samples of the whole soil and HU were homogenised and grinded into particles
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Fig. 1. Scheme of the experimental procedure carried out in this study.
smaller than 106 mm. Then, 10 mg sample was weighed in tin capsules using the analytical balance; they were analysed by combustion at 1000 °C using an elemental analyser (Perkin Elmer model 2400). This procedure was performed in duplicate. 2.4. Inorganic residue 10 mg of HU extracted from soils was calcined at 700 °C in a muffle furnace for 4 h. The amount of OM by mass difference after calcination was then calculated, considering the final residue as inorganic matter (Griffith and Schnitzer, 1975).
2.6. 3D fluorescence spectroscopy Fluorescence measurements using a luminescence spectrometer (model LS50B Perkin Elmer) in soil and HU pellets were performed. The spectra were acquired in the scan range from 240 to 700 nm for emission and 220–510 nm for excitation. The spectra were obtained using a 290-nm cut-off excitation filter with an increment of 10 nm excitation and 30 scans. The spectra obtained using this technique were treated using the mathematical method known as parallel factor analysis (Luciani et al., 2008; Santín et al., 2009; Zhu et al., 2014).
2.5. Laser-induced fluorescence spectroscopy
3. Results and discussion
The LIFS spectra of the pellets were obtained by applying a diode laser centred at 405 nm with an average power of 20 mW; for the detection system, a spectrometer with a resolution of 4 nm (equipment developed by Embrapa Agricultural Instrumentation) was used. The laser beam was directed onto the pellet surface through six external optical fibres, and the fluorescence emission resulting from the decay of excited species was transferred to the spectrometer through a central optical fibre bundle. The measurement range was from 420 nm to 800 nm, and the selected integration time and average were 500 ms and 4, respectively, for all measurements. Four replicates were recorded for each sample and the averaged spectrum was used (Santos et al., 2015). The humification index (HLIFS) was determined by calculating the ratio between the area of the fluorescence emission spectrum (Ex: 465, Int from 450 to 700 nm) (ACF) and the amount of total organic carbon (in g kg−1) (TOC) present in the soil sample (Milori et al., 2006).
Table 1 shows information on the chemical fractionating process and the values obtained by elemental analysis techniques for the different depths of the whole soil and for the extracted HU. For all the layers, the HU material represents more than 80% in mass, with the minimum value at the surface (80%) and the maximum at the bottom layer (93.46%). Based on these results, the organic material from the surface is assumed to be composed of carbon originating from HS for 41% and of non-humic carbon (probably labile to chemical and biological degradation) for 59%. This partition makes sense because, on the surface, there is a higher incorporation of fresh OM. In this layer, among stable carbon (HS) 63% belongs to the HU and 37% to HA. This shows that, on the surface, the ratio between carbon in HU and HA is almost 2:1 (around 1.7). In the 15 to 30 cm and 40 to 50 cm layers, carbon contents are much lower than on the surface, and they are basically stored as HA and HU. The contribution of HU for soil carbon in these layers was
Table 1 Values obtained by elemental analysis techniques and the values of the yields for the whole soil and the humin extracted from different depths. Samples/horizons (cm) Elemental analysis (C) %AHsoil %AFsoil % HUsoil % residual weight (estimated) %C in HA %C in FA %C in humin % C in soil %C soil from HA %C soil from FA %C soil humin %C soil residual (estimated) %C from HSs (estimated) %C from residual (estimated) Ash % Humin inorganic % Humin organic
0–15
15–30
40–50
260
350
9±1 0±1 80 ± 1 11 ± 1 38 ± 5 18 ± 1 7.2 ± 0.5 22.5 ± 0.9 3.4 ± 0.6 0.01 ± 0.01 5.8 ± 0.4 13 ± 3 41 ± 5 59 ± 7
0.9 ± 0.2 0.3 ± 0.2 83.9 ± 0.3 14.9 ± 0.4 49 ± 1 4.5 ± 0.2 0.35 ± 0.02 1.01 ± 0.09 0.4 ± 0.1 0.01 ± 0.01 0.29 ± 0.02 0.3 ± 0.2 72.7 ± 0.7 27.3 ± 0.3
1.4 ± 0.3 0.3 ± 0.3 88.8 ± 0.3 9.5 ± 0.5 52.8 ± 0.2 6.0 ± 0.1 0.31 ± 0.01 2.1 ± 0.2 0.74 ± 0.1 0.02 ± 0.01 0.275 ± 0.009 1±1 48 ± 1 52 ± 2
0.0 ± 0.5 0.7 ± 0.5 90.8 ± 0.7 9±1 43 ± 2 4.7 ± 0.2 0.07 ± 0.02 0.80 ± 0.03 0.01 ± 0.01 0.03 ± 0.02 0.06 ± 0.02 0.7 ± 0.2 9±5 90 ± 5
0.16 ± 0.02 0.43 ± 0.02 93.46 ± 0.03 5.95 ± 0.04 48 ± 5 6.2 ± 0.4 0.10 ± 0.02 1.2 ± 0.2 0.08 ± 0.01 0.030 ± 0.002 0.09 ± 0.02 1.0 ± 0.3 14.19 ± 0.04 85.8 ± 0.2
87.70 ± 0.01 12.30 ± 0.01
98.40 ± 0.01 1.60 ± 0.01
99.10 ± 0.01 0.90 ± 0.01
96.00 ± 0.01 4.00 ± 0.01
97.10 ± 0.01 2.90 ± 0.01
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between 27% and 42%, demonstrating again the relevance of this chemical fraction. When evaluating the ash content in HU of these layers, it was observed that this fraction was predominantly composed of minerals with little carbon content. In deeper layers, a very singular behaviour was observed. Around 85–90% of soil carbon in these layers does not come from HS. Some hypotheses can be formulated to justify this behaviour: 1. The chemical fractionation process can lead to a loss of carbon strongly complexed to metals, which must be present in abundance at depth; thus, the soil carbon measured is always greater than the sum of the carbon found in humic fractions, giving the impression that there is a great deal of labile carbon (~90%) in deeper layers. 2. This soil within the Amazonian ecosystem enables the percolation of labile carbon from the surface, which accumulates in deeper layers. 3. The water table, which passes just below the 350 cm layer, brings fresh OM to deeper layers. In the 350 cm layer, the contribution of HU to the humic carbon is around 50%. Thus, in this first analysis, the importance of HU in Amazonian soils is very clear. For this kind of soil, it is not possible to make a precise analysis of HS considering only the HA. These results emphasise the importance of HU; the development of techniques that allow the analysis of this material is, hitherto, scarcely examined. Fig. 2 shows the spectra obtained by LIFS of (a) the whole soil and HU extracted from soil at 0–15 cm (surface profile), and (b) HU extracted from soil at different depths (15 to 350 cm). In Fig. 2a, similar spectra were observed in the HU and whole soil samples, and a peak at 690 nm can be observed in the HU sample in the surface and 260 cm horizons. In the literature, the fluorescence peak at 690 nm refers to the chlorophyll present in the compounds of vegetable origins, such as plants (Lichtenthaler et al., 1999), and it can be associated in the fresher OM present in these horizons. In Fig. 2b, a band at 450–650 nm can be observed for deeper profiles of HU samples. Fig. 3 shows the humification index (points) and the LIFS area (square dashed) of the HU extracted from different depths of soil. An increase in the humification index with depth can be observed, which is related to the chemical characteristics of the samples, especially for HU fractions. When we apply the statistical analysis in these whole soil and HU fractions, we did not find a significant difference (p N 0.05). This behaviour for HU corroborates with Santos et al. (2015), who reported an increase of humification with depth in some Amazonian soils. An explanation for this behaviour could be that the presence of aromatic polycondensation in the OM is also present in the corresponding horizon (Tivet et al., 2013). Therefore, this increase in the humification index with depth can be attributed to, first, the
Fig. 3. Humification index (points) and the LIFS area (square dashed) obtained for the humin extracted from different depths of soil.
contribution of fresh OM to the surface soil causing a dilution effect of the organic material, or second, the natural percolation of soluble HS, causing the accumulation of humified material at depth. The 3D fluorescence spectra of the whole soil (Fig. 4a) and HU fractions (Fig. 4b) for different layers (0–350 cm) are presented in Fig. 4. For a better understanding of the fluorescence spectra, the results were treated using CP/PARAFAC (EEM-CP/PARAFAC). The results showed the contribution of two fluorophores, which had core tensor consistencies (concordia) of 96.6% and 98.3% for the HU fraction and the whole soil, respectively. Fig. 5 shows the two fluorophores, (a) component 1 and (b) component 2, which are present in the structure of the whole soil and the HU fraction. Component 1 (λex/λem: 375/400 nm) refers to simpler structures present in the HS of terrestrial environments and component type C in the literature (Coble, 1996). Component 2 (λex/λem: 450/510 nm) refers to terrestrial HA and lignin derivatives, which are associated with a more complex and recalcitrant structure with fused aromatic rings and/or a combination of simple aromatic rings (Matthews et al., 1996). According to Matthews et al. (1996), HA extracted from soils are predominantly derived from lignin precursors (denominated peak L), which have the component structures of terrestrial plants. Grasset and Amblès (1998) studied the structural characteristics of HU and HA extracted from soil (north Correze, Plateau de Millevaches, France)
Fig. 2. Spectrum obtained by LIFS for the whole soil and the humin extracted from different depths (0 to 350 cm).
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Fig. 4. Total fluorescence spectra in the EEM mode obtained for (a) the whole soil and (b) the humin extracted from soil of different depths: (1) 0–15 cm, (2) 260 cm, and (3) 350 cm.
employing the thermochemolysis technique. Their results indicated that the lipid biopolymer lignin contributes to the formation of complex OM in soil, and that the ester and ether groups are clearly involved in the structure of HU and HA in the soil, and these groups can be origins microbiologic or metabolism derived of plants. Xiang-yun et al. (2014) studied carbon fixation by HS in soil under fertilisation and they found that low and high fertiliser rates were significant, highlighting the HU fraction, which was the main fraction responsible for carbon sequestration under long-term fertilisation of soil. The contributions of the two fluorophores (components 1 and 2) according to the depth in a) HU and b) whole soil are presented in Fig. 6. The HU and whole soil samples showed similar behaviours. OM, for both series, did not present any structural changes in the profile; the ratio between their main fluorophores throughout the soil profile remained almost constant. There were only changes in the amount
of fluorophores in the profile. The highest concentration of these fluorophores occurred in the 40–50 cm layer, and the lowest amount occurred at the surface, probably due to the initial stage of fresh OM degradation (more aliphatic). LIFS data was highly correlated with EEM-CP/PARAFAC data, particularly for HU (correlation coefficient of 0.92). 4. Conclusion Carbon quantification and humic fractions showed the importance of HU when studying OM in different horizons in Amazonian Soil. HU carbon is always one of the main pools of soil organic carbon for this ecosystem, contributing more than 40% of the carbon in HS. Particularly, in the analysed soil, the HU was the largest fraction, contributing 80–93% of the soil mass.
Fig. 5. Fluorescence Excitation Emission Matrix of the CP/PARAFAC components, (a) component 1 and (b) component 2, which are present in the samples of the whole soil and humin of the different depths.
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Fig. 6. Contribution of EEM-CP/PARAFAC components 1 and 2 from the samples of the (a) humin and (b) whole soil of the different depths.
Based on the EEM-CP/PARAFAC analysis, HU did not present any structural changes in the soil profile; the ratio between its main fluorophores remained constant. There were only variations in the amount of fluorophores in the profile. The highest concentration of these fluorophores occurred in the 40–50 cm layer. In the surface layer, the amount of these fluorophores was the lowest due to the abundance of fresh OM (more aliphatic). For soil, the EEM-CP/ PARAFAC analysis showed that HS at the deepest layer (350 cm) was quite similar to that from the 15–30 cm layer. This behaviour seems to agree with the soil carbon analysis, which suggests an accumulation of the humified OM during depth. LIFS results showed that both the whole soil and HU have quite similar spectral profiles, showing that HU is one of the main components of soil emission. This contribution is original, since there are few studies on HU characterisation, and there are no studies on HU fluorescence and its contribution to whole soil fluorescence emission. Thus, the use of EEM fluorescence spectroscopy with CP-PARAFAC and LIFS in soil analyses can provide information on the processes of HS transformation in this system. Furthermore, another advantage of these two techniques is that it allows a fast and low-cost analysis of the humic fractions present in the soil following the concept of green chemistry and the possibility of integrating a system for environmental use.
Acknowledgments The authors acknowledge the financial support for this work from the São Paulo Research Foundation (FAPESP) by project (Process 2011/03250-2) and scholarship (Process 2013/13013-3). We thank the Embrapa Agricultural Instrumentation for supplying the structure leading to the development of this research.
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