Published online 2 December 2005
Published in Soil Sci Soc Am J 70:57-63 (2006)
DOI: 10.2136/sssaj2004.0270
© 2005 Soil Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
Soil Chemistry
Organic Matter Study of Whole Soil Samples Using Laser-Induced Fluorescence Spectroscopy
Débora Marcondes Bastos Pereira Miloria,*,
Helder Vinicius Avanço Galetia,b,
Ladislau Martin-Netoa,
Jeferson Dieckowa,
Martha González-Péreza,c,
Cimélio Bayerd and
Júlio Saltone
a Embrapa Agricultural Instrumentation, P.O. Box. 741, 13560-970, São Carlos-SP, Brazil
b Federal Univ. of São Carlos, P.O. Box. 676, 13565-905, São Carlos-SP, Brazil
c Univ. of São Paulo, Chemistry Institute of São Carlos, P.O. Box. 369, 13560-970, São Carlos-SP, Brazil
d Soil Science Dep., Federal Univ. of Rio Grande do Sul, P.O. Box 15100, 90001-970, Porto Alegre-RS, Brazil
e Embrapa West Agriculture, 79804-970, Dourados-MS, Brazil
* Corresponding author (debora{at}cnpdia.embrapa.br)
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ABSTRACT
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Fluorescence spectroscopy relies on the fluorescence emitted by rigid conjugated systems and thus can be used to assess the soil organic matter (SOM) humification. This technique is generally applied to solution samples of humic substances, and so far no information exists about its applicability to whole untreated soil samples. The laser-induced fluorescence (LIF) spectroscopy is proposed as a novel technique to assess the organic matter humification in whole soil samples. We sampled the 0- to 2.5-, 2.5- to 5-, 5- to 10-, 10- to 15-, and 15- to 20-cm layers of three Oxisols of long-term experiments located in two sites of the Brazilian Cerrado. The humification index based on LIF spectroscopy (HLIF) of whole soil samples showed a close correlation with the humification indexes A4/A1, I465/I399, and A465 obtained after fluorescence spectroscopy analysis of the dissolved humic acids. The HLIF in soils under native cerrado or subjected to no-tillage increased from the top to the deepest layer, which is consistent with the deposition of labile organic matter from plant residues on the soil surface. The soils subjected to conventional tillage, however, showed relatively constant HLIF along the profile, possibly because homogenization imparted by disturbance of the arable layer. Accordingly, for the two top layers, the soils under no-tillage showed a lower HLIF than for conventionally tilled soils. Laser-induced fluorescence spectroscopy is a promising technique to assess humification in whole soil samples, particularly in Oxisols, which due to high concentration of Fe3+ are not feasible to electron spin resonance (ESR) and Carbon-13 nuclear magnetic resonance (13C NMR) spectroscopy, unless previous treatment is employed.
Abbreviations: ESR, Electron Spin Resonance • HLIF, Humification index based on Laser-Induced Fluorescence spectroscopy • LIF, Laser-Induced Fluorescence • NMR, Nuclear Magnetic Resonance • SOM, soil organic matter
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INTRODUCTION
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THE CHEMICAL RECALCITRANCE, like physical protection and organomineral interaction, is one of the stabilizing mechanisms of SOM (Christensen, 1996; Sollins et al., 1996) and, on account of being related to the composition of the organic matter, may be influenced by soil management systems. Recalcitrance is based on molecular-level characteristics of SOM, like elemental composition, molecular conformation and presence of functional groups (Sollins et al., 1996). During humification process, the increase of aromatic and alkyl structures (Baldock et al., 1992; Kögel-Knabner et al., 1992) and the increase of conformational disorders (Almendros and Dorado, 1999) are referred as performing significant roles in the organic matter resistance against biodegradation.
Electron spin resonance (Martin-Neto et al., 1998; Bayer et al., 2002b), Carbon-13 NMR (Pillon, 2000; Bayer et al., 2000) and fluorescence spectroscopy (Bayer et al., 2002a; Milori et al., 2002) have been used to assess the organic matter humification of tropical and subtropical soils subjected to different management systems. Despite the potentiality of 13C NMR and ESR techniques, both have limitations in investigating the organic matter composition of bulk samples or humic acid extracts of soils, like the tropical and subtropical Oxisols, which contain appreciable concentrations of paramagnetic Fe3+. To overcome such limitations, fluorescence spectroscopy, which is not affected by the paramagnetic Fe3+, seems to be an alternative method to study SOM humification in Fe oxide-rich bulk soil samples from different management systems, yet this subject has never been fully explored.
Several authors (Zsolnay et al., 1999; Kalbitz et al., 1999; Milori et al., 2002) have shown the potentiality of fluorescence spectroscopy to evaluate the humification degree of organic materials in solution samples. Zsolnay et al. (1999), by exciting the sample with ultraviolet radiation of 240 nm, observed that the fluorescence signal on the emission spectra of dissolved organic matter shifted toward longer wavelengths, showing progress in the humification process. Thus, the authors proposed a humification index based on the ratio between the area of the last quarter (A4: 570–641 nm) and the area of the first quarter (A1: 356–432 nm) of the emission spectrum. This index was called A4/A1.
Kalbitz et al. (1999), working with humic acids in solution sample, proposed another humification index based on the ratio between the fluorescence intensity at 400 and 360 nm (I400/I360) or 470 and 360 nm (I470/I360), in synchronous-scan excitation mode spectra. The authors proposed that the shift of the maximum fluorescence intensity from shorter to longer wavelengths was attributed to the presence of condensed aromatic systems.
Milori et al. (2002), working with dissolved humic acids adjusted to a concentration of 20 mg L–1 and to pH 8.0, observed that wavelengths in the blue region were more efficient to excite structures whose concentration had increased during the humification process. The authors have proposed that the area of a fluorescence spectrum obtained by excitation at the blue wavelengths is proportional to the humification degree of the sample and could thus be used as a humification index, referred as A465.
Assuming that fluorescence signals are emitted by conjugated rigid systems in individual molecules or structures, we hypothesized that such conjugated rigid systems should emit fluorescence signals notwithstanding if the sample is in solid or solution state. According to Krasovitskii and Bolotin (1988), when a substance passes from solid to liquid or to vapor state, or is dissolved, the fluorescence signal of this substance will still persist. This supports the application of fluorescence spectroscopy to investigate organic matter in whole soil samples.
This study aimed at showing the applicability of LIF spectroscopy for characterizing the SOM and for assessing its humification in bulk samples of soils subjected to different management systems, assuming that concentration of rigid conjugated systems increases with humification (Zsolnay et al., 1999; Kalbitz et al., 1999; Milori et al., 2002). We proposed a humification index, based on LIF spectroscopy, and compared it with the humification indexes proposed by Zsolnay et al. (1999), Kalbitz et al. (1999), and Milori et al. (2002). This novel technique can be innovative in SOM studies because the sample can be analyzed as a bulk, avoiding destruction by physical and chemical fractionations or treatments.
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MATERIALS AND METHODS
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Soil Sampling in Experimental Sites and Carbon Determination
Soil samples of the 0- to 2.5-, 2.5- to 5.0-, 5- to 10-, 10- to 15-, and 15- to 20-cm layers were collected in three long-term management experiments (A, B, and C) distributed in two sites of the Brazilian Cerrado region. The Exp. A and B have been performed for 5 and 13 yr, respectively, in the experimental field of Embrapa Agropecuária Oeste Research Center, near the town of Dourados, state of Mato Grosso do Sul. In Exp. A, we sampled the plots under conventional tillage (two disk-harrowings), permanent pasture (Brachiaria decumbens), and crop-pasture rotation [2 yr with oat (Avena sativa L.)/soybean (Glycine Max. L.) succession and 2 yr with Brachiaria decumbens pasture, both under no-tillage]; while in Exp. B, the plots subjected to conventional tillage (two disk-harrowings) and no-tillage, both plots cultivated with oat/soybean succession. The native cerrado soil, near both experiments, was sampled as representing the original soil conditions of this site.
Experiment C has been conducted for 7 yr in the experimental field of Schneider Logemann Farm, near the town of Costa Rica, also in the state of Mato do Grosso do Sul. Here we sampled the conventional tillage (two heavy disk-harrowings) and no-tillage plots, cultivated with maize and soybean in the summer, while in the winter the area was left fallow. The native cerrado soil, near the experimental field, was also sampled.
In both sites the soil is classified as Oxisol, according to the Soil Taxonomy, or Rhodic Ferralsol, according to the FAO classification scheme, and contains >600 g kg–1 of clay in the 0- to 20-cm layer. For this same layer, the Fe oxide content in the Dourados site is 86 g Fe kg–1, while in the Costa Rica site it is 210 g Fe kg–1. The climate is similar in both sites, being classified as tropical, with precipitations concentrated in summer (Aw, according to Köppen classification).
Soil samples were air dried at room temperature, crushed with a wood roll to pass a 2-mm mesh and stored in plastic pots.
The C concentration was determined through the Walkley-Black dichromate oxidation method (Nelson and Sommers, 1996).
Laser-Induced Fluorescence Spectroscopy
Soil sample aliquots of approximately 0.5 g, after being composited from the three experimental replications and further grinded to pass a 250-µm mesh, were pressed into pellets of 1-cm diam. and 2-mm thickness, which were then inserted into a home-assembled apparatus to run LIF measurements (Fig. 1
). The samples were excited with 351-nm ultraviolet radiation emitted by an Ar laser equipment (Coherent Innova 90–6, Coherent Inc., Santa Clara, CA), with exit power of 400 mW. A prism was placed in front of the laser exit to remove background gas fluorescence. The back scattering fluorescence emitted by excited samples was collected through a convergent lens and focused on the slit of a monochromator (focal distance of 240 mm, 1200 g mm–1 and blaze in 500 nm– CVI). Signals were multiplied by a Hamamatsu photomultiplier (Hamamatsu, Hamamatsu City, Japan), adjusted to the maximum sensitivity in the visible region (530 nm), and filtered and amplified by a lock-in amplifier. The system functioning and the data acquisition were controlled through a home-developed software. The spectral resolution was adjusted to 4 nm.
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Fig. 1. Schematic representation of the home-assembled apparatus for laser-induced fluorescence (LIF) spectroscopy.
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To confirm if LIF signals originated from organic matter, we analyzed two soil samples before and after two organic matter removal treatments and compared the results. One sample was treated with hydrogen peroxide (2 g of soil in 10 mL of H2O2 solution) and the other one was thermally treated (700°C for 2 h).
Fluorescence Spectroscopy of Dissolved Humic Acids
To validate the applicability of LIF spectroscopy to assess organic matter humification in whole soil samples, the humification indexes obtained by this technique were compared with the humification indexes estimated after fluorescence spectroscopy analysis of corresponding dissolved humic acids. Humic acids were extracted from soil samples, composited from the three experimental replications, according to the methodology proposed by the International Humic Substances Society (Swift, 1996). The humic acids were brought to a concentration of 20 mg L–1 and to pH 8 by diluting them in a solution of 0.05 mol L–1 NaHCO3.
Fluorescence spectra in the emission and synchronous-scan excitation modes were acquired in a PerkinElmer LS-50B luminescence spectrophotometer (PerkinElmer, Wellesley, MA). The emission and excitation slits were adjusted to a band-width of 7 nm. A scan speed of 200 nm min–1 was selected for both monochromators. To obtain the A4/A1 index (Zsolnay et al., 1999), the area of the last quarter of the spectrum (570–641 nm) acquired in the emission mode with excitation at 240 nm was divided by the area of the first quarter (356–432 nm). The A465 index (Milori et al., 2002) corresponded to the area of the spectrum acquired in the emission mode with excitation at 465 nm. Spectra in the synchronous-scan excitation mode acquired with a 
of 55 nm were used for calculating the I454/I399 index (Kalbitz et al., 1999), obtained from the ratio between the signal intensity at 454 nm and the signal intensity at 399 nm.
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RESULTS AND DISCUSSION
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Source of the LIF Signals
A very broad LIF signal in the visible region was observed in the emission mode spectra of the untreated whole soil sample (Fig. 2
). On the other hand, a very low intensity signal was observed for the sample in which the organic matter was removed by hydrogen peroxide treatment (Fig. 2a), while any signal could be recorded on the spectrum of the thermally treated sample (Fig. 2b). These results show that LIF signals emitted by whole soil samples after excitation with ultraviolet radiation (351 nm) are unambiguously due to the organic matter fraction.
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Fig. 2. Laser-induced fluorescence (LIF) spectra of the untreated whole soil sample compared with the LIF spectrum (a) of the hydrogen peroxide-treated sample and (b) of the thermally treated sample. The fluorescence emission intensity is given in arbitrary units (a.u.).
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Correlation between LIF Signal and Carbon Concentration
There was a linear correlation between the C concentration and the area of the LIF spectra for whole soil samples (Fig. 3
), however, with a large dispersion of points. It is important to emphasize that the area of the LIF spectra measure C in fluorescent organic structures and not the total organic C. Only C present in more complex or rigid structures, such as structures with aromatic rings and quinone groups, can be observed through this technique. Thus, the dispersion indicates that there is more information in the LIF signal area besides C content.
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Fig. 3. Relationship between the C concentration and the corresponding area of the LIF spectra (ALIF) of whole soil samples. ALIF is given in arbitrary units (a.u.).
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According to Milori et al. (2002), fluorescence emission of humic acid in solution when excited at near ultraviolet or blue wavelengths brings information about more humified structures. This postulate can be applied to the fluorescence of the whole soil samples. In the case of organic fluorophores, the structure of an individual molecule is the responsible for fluorescence. Therefore, when a substance passes from the solid crystalline state into a melt or vapor, or is dissolved, its fluorescence persists (Krasovitskii and Bolotin, 1988). In this way, LIF spectroscopy using whole soil can also be used to evaluate humification degree of SOM. However, to compare fluorescence spectra of whole soil for different samples, it is necessary to know the total soil C contents to normalize the fluorescence signal. This normalization would be similar to work with solutions at the same concentration, as was used in A465 index (Milori et al., 2002). Therefore, that variation in soil normalized emission intensity can be associated to structural changes.
Organic Matter Humification and Soil Management
To evaluate the humification of SOM, we divided the area of the LIF spectrum of each soil sample by the corresponding C concentration (Table 1) and obtained a normalized fluorescence signal, which was then considered as being the HLIF of SOM. This index also refers to the recalcitrance due to rigid conjugated systems present, especially in aromatic structures. The recalcitrance imparted by aliphatic structures, possibly derived from lipid compounds (Baldock et al., 1992; Kögel-Knabner et al., 1992), is not taken into account by the herein proposed index.
There was a close correlation between the HLIF of the whole soil samples and the humification indexes A4/A1 (Zsolnay et al., 1999), I465/I399 (Kalbitz et al., 1999), and A465 (Milori et al., 2002) of the corresponding humic acid samples (Fig. 4 , 5,
and 6)
. The only exception was for the correlation between HLIF and A4/A1 in Costa Rica site (Fig. 4a), which was not as close as in the other cases and no clear explanation could be provided. In spite of that, results proved the possibility of using LIF spectroscopy as a tool for investigating the humification of organic matter in whole soil samples, without the need of chemical extractions. The possibility of using whole soil samples is particularly relevant in studies of tropical Oxisols, whereby the 13C NMR and the ESR spectroscopy generally fails if organic matter is not separated from the Fe oxide-rich mineral phase.
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Fig. 4. Correlation between the humification index determined by LIF spectroscopy (HLIF) in whole soil samples and the humification index A4/A1 (Zsolnay et al., 1999) determined by conventional fluorescence in the corresponding humic acid samples at (a) Costa Rica and (b) Dourados sites. The indexes are expressed as arbitrary units (a.u.).
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Fig. 5. Correlation between the humification index determined by LIF spectroscopy (HLIF) in whole soil samples and the humification index I454/I399 (Kalbitz et al., 1999) determined by conventional fluorescence in the corresponding humic acid samples at (a) Costa Rica and (b) Dourados sites. The indexes are expressed as arbitrary units (a.u.).
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Fig. 6. Correlation between the humification index determined by LIF spectroscopy (HLIF) in whole soil samples and the humification index A465 (Milori et al., 2002) determined by conventional fluorescence in the corresponding humic acid samples at (a) Costa Rica and (b) Dourados sites. The indexes are expressed as arbitrary units (a.u.).
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The HLIF for the native cerrado and no-tillage soils in Dourados (Fig. 7a
and 7b) and in Costa Rica (Fig. 7c) showed a tendency to increase from top to deeper layers. This is a consistent result, considering that the proportion of particulate organic matter fraction, and thus of labile compounds like carbohydrates and peptides, decreases in depth.
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Fig. 7. Soil organic matter humification index obtained through LIF spectroscopy (HLIF) as affected by land use and soil management systems in (a) experiment A and (b) experiment B of Dourados and (c) in the experiment of Costa Rica. The HLIF is expressed as arbitrary units (a.u.).
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In pasture and crop-pasture rotation treatments in Dourados (Fig. 7a), the HLIF was lower in the top layer, increased in the 2.5 - to 5-cm layer and remained relatively unchanged in deeper layers, except for an additional increase observed in the 5- to 10-cm layer of pasture soil. The lowest humification in the top layer is also attributed to a higher proportion of particulate organic matter in this layer.
In conventional tillage treatments (Fig. 7), the HLIF was rather uniform along the soil profile, except in the top layer of the Exp. A of Dourados (Fig. 7a), where a higher value for HLIF was observed. This uniformity of humification along the profile is consistent to the homogeneity imparted by tillage disturbances on the 0- to 20-cm layer of these soils.
In the two top layers, the soil in conventional tillage treatment presented higher HLIF than the soil under no-tillage (Fig. 7b and 7c). The same trend was observed between the soil under conventional tillage and those under pasture and crop-pasture rotation (no-tillage) in the Exp. A of Dourados (Fig. 7a). This can also be attributed to a higher proportion of particulate organic matter in the two layers of the soil under no-tillage than in soil subjected to conventional tillage. In deeper layers, however, there was trend for HLIF to be similar between these two tillage systems or lower in conventional tillage (Fig. 7b and 7c).
Higher humification degree, as measured by fluorescence spectroscopy of dissolved humic acids, was also reported for a subtropical Acrisol under conventional tillage than the counterpart subjected to no-tillage (Bayer et al., 2002a). Similar trend was found when the humification degree was evaluated through ESR spectroscopy (Bayer et al., 2002b). However, the higher humification, which is commonly found in conventionally tilled soils does not mean that conventional tillage increases SOM stability. On the contrary, it can clearly indicate that other protection mechanisms, like physical protection in aggregates, are failing to protect the most labile fractions of the organic matter.
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CONCLUSIONS
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The fluorescence signal emitted from whole soil samples excited with near ultraviolet-blue radiation is due to the organic matter.
The LIF spectroscopy is useful to assess the organic matter humification in whole soil samples. This is particularly relevant for soil samples containing appreciable amounts of paramagnetic metals, samples that can hardly be analyzed by ESR or 13C NMR spectroscopy in their original states.
The organic matter in soils subjected to conventional tillage is more recalcitrant than in soils managed under no-tillage. Disking operations possibly increase the decomposition of labile organic matter, so that recalcitrant remains tend to comprise a higher proportion of the SOM stock.
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ACKNOWLEDGMENTS
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This work was supported by FAPESP 1998/14270-8 and 03/06084-0; Embrapa MP1 01.02.1.03.02.05 and MP3 03.02.2.23.00.01.CNPq fellowship of L. Martin-Neto, C. Bayer, and J. Dieckow.
Received for publication August 12, 2004.
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ABSTRACT
INTRODUCTION
