Organic Matter Study of Whole Soil Samples Using Laser-Induced Fluorescence Spectroscopy De ´bora Marcondes Bastos Pereira Milori,* Helder Vinicius Avanço Galeti, Ladislau Martin-Neto, Jeferson Dieckow, Martha Gonza ´ lez-Pe ´ rez, Cime ´lio Bayer, and Ju ´ lio Salton ABSTRACT 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 tech- nique 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 (H LIF ) of whole soil samples showed a close correlation with the humification indexes A 4 /A 1 ,I 465 /I 399 , and A 465 obtained after fluorescence spectroscopy analysis of the dissolved humic acids. The H LIF in soils under native cerrado or subjected to no-tillage increased from the top to the deepest layer, which is consistent with the deposi- tion of labile organic matter from plant residues on the soil surface. The soils subjected to conventional tillage, however, showed relatively constant H LIF along the profile, possibly because homogenization im- parted by disturbance of the arable layer. Accordingly, for the two top layers, the soils under no-tillage showed a lower H LIF 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 Fe 31 are not feasible to electron spin resonance (ESR) and Carbon-13 nuclear magnetic resonance ( 13 C NMR) spectroscopy, unless previous treat- ment is employed. T HE CHEMICAL RECALCITRANCE, like physical protec- tion and organomineral interaction, is one of the stabilizing mechanisms of SOM (Christensen, 1996; Sol- lins 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 humifica- tion process, the increase of aromatic and alkyl struc- tures (Baldock et al., 1992; Ko ¨ gel-Knabner et al., 1992) and the increase of conformational disorders (Almen- dros and Dorado, 1999) are referred as performing sig- nificant roles in the organic matter resistance against bio- degradation. 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. De- spite the potentiality of 13 C 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 Fe 31 . To overcome such limitations, fluorescence spectros- copy, which is not affected by the paramagnetic Fe 31 , seems to be an alternative method to study SOM humifi- cation 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 sig- nal 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 (A 4 : 570–641 nm) and the area of the first quarter (A 1 : 356–432 nm) of the emission spectrum. This index was called A 4 /A 1 . 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 (I 400 /I 360 ) or 470 and 360 nm (I 470 /I 360 ), in synchronous-scan excitation mode spectra. The authors proposed that the shift of the maximum fluorescence intensity from shorter to longer wavelengths was attrib- uted to the presence of condensed aromatic systems. Milori et al. (2002), working with dissolved humic acids adjusted to a concentration of 20 mg L 21 and to pH 8.0, observed that wavelengths in the blue region were more efficient to excite structures whose concen- tration had increased during the humification process. The authors have proposed that the area of a fluores- cence spectrum obtained by excitation at the blue wave- lengths is proportional to the humification degree of D.M.B.P. Millori, H.V.A. Galeti, L. Martin-Neto, J. Dieckow, and M. Gonza ´ lez-Pe ´rez, Embrapa Agricultural Instrumentation, P.O. Box. 741, 13560-970, Sa ˜o Carlos-SP, Brazil; H.V.A. Galeti, Federal Univ. of Sa ˜o Carlos, P.O. Box. 676, 13565-905, Sa ˜o Carlos-SP, Brazil; M. Gonza ´ lez-Pe ´rez, Univ. of Sa ˜o Paulo, Chemistry Institute of Sa ˜o Car- los, P.O. Box. 369, 13560-970, Sa ˜o Carlos-SP, Brazil; C. Bayer, Soil Science Dep., Federal Univ. of Rio Grande do Sul, P.O. Box 15100, 90001-970, Porto Alegre-RS, Brazil; J. Salton, Embrapa West Agricul- ture, 79804-970, Dourados-MS, Brazil. Received 12 Aug. 2004. *Cor- responding author (debora@cnpdia.embrapa.br). Published in Soil Sci. Soc. Am. J. 70:57–63 (2006). Soil Chemistry doi:10.2136/sssaj2004.0270 ª Soil Science Society of America 677 S. Segoe Rd., Madison, WI 53711 USA Abbreviations: ESR, Electron Spin Resonance; H LIF , Humification index based on Laser-Induced Fluorescence spectroscopy; LIF, Laser- Induced Fluorescence; NMR, Nuclear Magnetic Resonance; SOM, soil organic matter. Reproduced from Soil Science Society of America Journal. Published by Soil Science Society of America. All copyrights reserved. 57 Published online December 2, 2005