Comparison of two alkaline treatments in the

Geoderma 148 (2008) 167–172

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Geoderma j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / g e o d e r m a

Comparison of two alkaline treatments in the extraction of organic compounds associated with water repellency in soil under Pinus taeda Fabricio A. Hansel a,⁎, Cristiane T. Aoki a, Claudia M.B.F. Maia a, Anildo Cunha Jr. b, Renato A. Dedecek a a b

Embrapa Florestas, Estrada da Ribeira, km 111, cx 319, Colombo, PR, 88411 000, Brazil Embrapa Suínos e Aves, cx 21, Concórdia, SC, 89700 000, Brazil

a r t i c l e

i n f o

Article history: Received 14 March 2008 Received in revised form 28 August 2008 Accepted 2 October 2008 Available online 4 November 2008 Keywords: Lipids Biopolymer Cutin Suberin Hydrolysis Water repellent soil

a b s t r a c t Isopropanol:NH3 and Methanol:KOH (saponification) alkaline treatments are usually applied in the study of soil organic matter. The first is used in studies of soil water repellency, and the latter in the extraction of ester-bound lipids from soil. In this study, isopropanol:NH3 and methanol:KOH treatments were applied separately in a solvent extracted repellent soil, in order to compare their efficiency in the extraction of water repellent compounds. The soil sample was taken from a site under a 16 year old Pinus taeda stand. The amount and class of organic compounds released by each treatment were compared using gas chromatography-mass spectrometry (GC-MS). Both treatments resulted in wettable soil after alkaline extraction. In general, alkaline treatments yielded extracts with the same class of organic compounds. Alkanoic acids, α,ω-alkanedioic acids, hydroxyalkanoic acids, aromatic compounds, and alkanols were identified, indicating the preservation of suberin and cutin biopolyester in the soil. Large differences were observed in the amounts of ω-hydroxyalkanoic acids, as well as in the quantity and distribution of dihydroxyalkanoic and trihydroxyalkanoic acids. In contrast to methanol:KOH, isopropanol:NH3 was not efficient in the extraction of whole aliphatic biopolyesters, mainly pine cutin-related products. Methanol: KOH was more effective in hydrolysis. The presence of biopolyesters in water repellent soil under the P. taeda stand seems to play an important role in water repellency. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Water repellent soils, whose occurrence is widespread, are characterized by very slow water infiltration. Water repellency in soils leads to reduced infiltration capacity, enhanced surface flow and accelerated soil erosion, uneven soil wetting patterns, development of preferential flow, and accelerated agrochemicals discharge (Doerr et al., 2000). It is widely accepted that soil water repellency (hydrophobicity) is caused by hydrophobic and amphiphilic organic compounds deposited on the soil, and that they may originate from plant material (De Bano et al., 1970; Horne and McIntosh, 2000), soil organic matter residues (McGhie and Posner, 1981), and certain fungal or microbial species (Bond and Harris, 1964; Jex et al., 1985). A range of aliphatic chemical compounds has been associated with water repellency, including alkanes, alkanoic acids, and esters (Savage et al., 1972; Ma'shum et al., 1988; Franco et al., 2000; Morley et al., 2005). Several methods involving solvents have been used to extract organic compounds from soils associated with water repellency (Ma'shum et al., 1988; Hudson et al., 1994; Horne and McIntosh, 2000). Doerr et al. (2005) showed that an isopropanol:NH3 mixture ⁎ Corresponding author. Fax: +55 41 3675 5601. E-mail address: [email protected] (F.A. Hansel). 0016-7061/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.geoderma.2008.10.002

(7:3, v/v) was more effective in extracting organic repellent compounds than a range of organic solvents tested (i.e. hexane, dicholoromethane, toluene and isopropanol). In fact, the isopropanol:NH3 mixture is a method that has been commonly applied in the extraction of organic repellent compounds (e.g. Ma'shum et al., 1988; Franco et al., 2000; Mainwaring et al., 2004; Llewellyn et al., 2004). Franco et al. (2000) extracted organic compounds from soil using a sequential extraction procedure: first, chloroform was used and the fraction extracted was named “non-polar wax”; then, an isopropanol: NH3 mixture was employed and the fraction withdrawn was named “polar wax”. The polar wax fraction showed hydrophobicity and its chemical composition appeared to be similar to the plant material found in the soils from where the samples were taken (e.g. eucalyptus trees). Interestingly, non-polar wax can be directly associated with free (extractable) soil lipids, and the polar wax is easily linked with ester-bound lipids (Bull et al., 2000; Naafs and van Bergen, 2002; Nierop et al., 2003). Free soil lipids are extracted with organic solvents, or their mixtures (e.g. chloroform, dicholoromethane, chloroform: methanol and chloroform:acetone), and the ester-bound lipids (biopolyesters) are extracted with an alkaline treatment (saponification) (e.g. 1.0 mol L− 1 KOH in 96% methanol solution, 70 °C) (Holloway, 1982; Bull et al., 2000; Naafs and van Bergen, 2002; Nierop et al., 2003). The similarity between the polar wax fraction and ester-bound lipids is evident when both fractions are extracted with an alkaline

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treatment after organic solvent extraction, even with different chemical reagents and reaction conditions. Lipids are often used as biomarkers to trace sources of soil organic matter (van Bergen et al., 1997; Marseille et al., 1999; Puglisi et al., 2003; Otto and Simpson, 2005; Nierop et al., 2006). The main source of lipids in soil is vegetation, although certain classes of microorganisms and human interference can contribute, such as ergosterol, which may point to fungal activity (Puglisi et al., 2003). Ester-bound lipids are frequently found in soils – especially as a result of humification – but the majority of such moieties in soil have been related to cutin and suberin biopolyesters (Nierop et al., 2003). Cutin is an aliphatic biopolymer present in the above ground parts of a plant (leaves, needles, flowers, fruits), and suberin is a more complex biopolymer, composed of aliphatic and aromatic domains that form both protective and wound-healing layers in the bark, woody stem, and roots (Kolattukudy, 1980). The question raised is whether both alkaline treatments, methanol:KOH and isopropanol:NH3, are effective in the extraction of the same amount and class of organic compounds from a solvent extracted repellent soil. It is also of interest to verify whether the mediated methanol:KOH treatment turns the soil wettable. The present study's aim is to compare isopropanol:NH3 and methanol: KOH alkaline treatments in the extraction of water repellent compounds from a solvent extracted repellent soil samples taken from a Pinus taeda stand. The compounds yielded by each treatment, dissolved in chloroform, were studied using gas chromatographymass spectrometry (GC-MS). The results were also discussed in terms of the origin of compounds released by alkaline treatments. 2. Experimental 2.1. Soil sample and preparation Samples consisted of Lithosol (F.A.O.,1990) under a 16-year-old P. taeda stand located in Piraí do Sul, Paraná, Brazil (24°31′34″ S, 49°56′55″ W), with loamy texture (76% sandy, 8% silt, and 16% clay) and 36% organic carbon. Soil surface water repellency was classified on the field and in the laboratory as extremely repellent. Three samples of this hydrophobic soil (approximately 100 g) were taken from the top 5 cm between tree lines using Kopecky rings (5.8 cm diameter, 3 cm height). Samples were combined, air dried for three days and then sieved over a 500 μm sieve to remove coarse organic debris. 2.2. Soil extraction The soil sample (n = 2, 15 g) was soxhlet extracted for 12 h using chloroform:acetone (9:1, v/v) mixture to remove free soil lipids. Thereafter, soil insoluble residue was alkaline treated as follows: (i) isopropanol:ammonia: 5 g of dried residue (n = 3) was soxhlet extracted for 12 h using an isopropanol:ammonia solution (15,7 mol L− 1 NH3, 7:3, v/v). The samples were pre-wetted with the solvent mixture for 15 min prior to the soxhlet extraction. Extracts were collected and partially concentrated on a rotary evaporator and, after cooling and acidification to pH 1.0 with 12 mol L− 1 HCl, 5 mL of solvent extracted distilled water was added and the organic compounds were extracted with chloroform (3× 5 mL). The organic layers were combined and passed through an anhydrous NaSO4 column to remove residual water; solvent was removed under a gentle stream of nitrogen until completely dry. (ii) Methanol:KOH: 5 g of dried residue (n = 3) was alkaline treated for 30 min at 70 °C, using 10 mL of 1.0 mol L− 1 KOH in 96% methanol solution and mixing every 10 min. After cooling and decantation, the supernatant was removed and placed in a separate tube. The soil residue was further extracted with methanol:chloroform (1:1, v/v, 1 × 10 mL), and chloroform (2 × 10 mL). The combined extracts were acidified to pH 1.0 with 12 mol L− 1 HCl, and then 5 mL of solvent extracted distilled water was added. The released compounds were recovered in the chloroform

phase, and two more extractions with chloroform (5 mL) were performed. Extracts were combined and dried through an anhydrous NaSO4 column; the solvent was removed with nitrogen. All alkaline extracts (n = 6) were dissolved in 1 mL toluene and then transesterified with 2 mL methanol:acetyl chloride mixture (10:1, v/v) for 12 h at 60 °C. After cooling, 1 mL KCl aqueous solution (10%, w/v) was added and the transesterified products extracted with chloroform (2 × 10 mL). Extracts were combined and passed through an anhydrous NaSO4 column and dried with nitrogen. The extracts were stored at −4 °C until needed for derivatization and analysis. Immediately before chromatographic analysis, the extracts were derivatized with 50 μL of BSTFA (N,O-bis(trimethylsilyl)trifluoracetamide, containing 1% of trimethylchlorrosilane) at 70 °C for 1 hour. The excess of BSTFA was evaporated with nitrogen, and the extracts were dissolved in 100 μL hexane and analysed using gas chromatograhymass spectrometry (GC-MS). 2.3. Gas chromatography-mass spectrometry (GC-MS) The derivative extracts were analysed using a Shimadzu GC-MS QP 2010, equipped with a capillary column Restek Rtx-5M (30 m × 0.25 mm, 0.25 μm film). The extracts were introduced via a split/splitless injector (1:100). A GC oven was programmed from 40 to 150 °C at 10 °C min− 1, then from 150 to 310 °C at 4 °C min− 1 and held at 310 °C for 20 min. Helium, at a constant pressure of 49.5 kPa, was the carrier gas. The interface and ion source temperatures were 300 °C and 200 °C, respectively. The quadrupole mass spectrometer was operated in the impact electronic mode at 70 eV, scanning the range m/z 50–600 in a 0.5 s cycle. Compounds were identified by computerised mass spectra library (NIST 05), interpretation of mass fragments and sequence of elution. Only compounds at concentration higher than 0.5% in all replicates were included in the quantification analyses. ω-Hydroxyhexadecanoic acid (ω-Hac16:0) was quantified using the total ion current (TIC) peak area and converted to compound mass using an external calibration curve. Concentrations of other compounds were calculated as a relative amount of ω-Hac16:0. Differences among alkaline treatments were evaluated by means of one-way ANOVA at probability levels of 5 and 10%. 2.4. Measurement of water repellency Water repellency levels of bulk and extracted soil were determined using the Water Droplet Penetration Time (WDPT) method. Methanol: KOH soil residue was washed with methanol:water (1:1, v/v) until pH 5.0. All soils were heated at 105 °C for 24 h and then equilibrated in a controlled atmosphere of 50% relative humidity for 24 h before the water repellency assessment. The WDPT consisted in the application of two drops (~ 65 μL) of distilled water on the sample surface and verification of the time taken for complete droplet penetration. Soils were classified according to the following ranges: wettable (0 to 5 s), slightly repellent (6 to 60 s), strongly repellent (61 to 600 s), severely repellent (601 to 3600 s), and extremely repellent (N3600 s). Water repellency class was based on the average of four measurements per sample, totalling 12 measurements per treatment. 3. Results and discussion After initial extraction with chloroform:acetone (9:1, v/v) to eliminate free soil lipids, the solvent extracted soil showed no difference in water repellency, continuing extremely repellent (WDPTN 3600 s), but nevertheless removed some organic material. Similar results were reported by Ma'shum et al. (1988), Franco et al. (2000), and Doerr et al. (2005) who concluded that non-hydroxylic solvents had little effect in reducing water repellency of soils, and that in some cases the extraction with solvents like hexane, and dichloromethane actually rendered the soil more water repellent.

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After the isopropanol:NH3 extraction, the soil residue became wettable (WDPT b 5 s), which is not surprising since isopropanol:NH3 is considered the most effective solvent mixture to extract water repellent compounds (Doerr et al., 2005). Similarly, the soil was classified as wettable after extraction with methanol:KOH, indicating that this treatment is also effective in the extraction of organic compounds involved in soil water repellency. However, isopropanol: NH3 (3.87 μg g− 1) yielded a lower amount of compounds than obtained by methanol:KOH (7.16 μg g− 1) (Table 1). Isopropanol:NH3 and methanol:KOH extracts were dominated by alkanoic acids, α,ω-alkanedioic acids, hydroxyalkanoic acids, aromatic compounds, and alkanols (Fig. 1). Extract compositions from each treatment were similar, considering the total concentration of alkanoic acid, α,ω-alkanedioic acid, aromatic compounds, and alkanols. In contrast, the total concentration of ω- and others hydroxyalkanoic acids (9,16-dihydroxyhexadecanoic acids and 9,10,18-trihydroxyoctadecanoic acid) were significantly different (p b 0.05). The methanol:KOH extraction was more efficient in freeing these compounds from the soil. ω-Hydroxyalkanoic acids were the most abundant organic compounds in both alkaline extracts (Table 1). The alkanoic acid distribution was very similar in both treatments and exhibited a saturated homologous series ranging from C12 to C30, with a strong even-over-odd predominance (Fig. 1). C15 and C17 iso and anteiso branched alkanoic acids were identified in both treatments, reflecting the C12 to C18 distribution, a bacteria input (Goossens et al., 1986; Zelles, 1999). However, C12 to C18 – but not branched alkanoic acids – can also be associated with cutin incorporation in soil, since they occur as minor components in cutin tissues (Holloway, 1982). The long chain alkanoic acids (NC20) are associated with higher plant inputs (Amblès et al., 1994). Suberin may contain homologous series of long chain alkanoic acid, with C22 and C24 as major monomers (Kolattukudy and Espelie, 1985). Interestingly, although no difference was detected in the total amount of alkanoic acid, C22 and C24 alkanoic acid concentrations were remarkably different in both extracts (p b 0.05), with isopropanol:NH3 extraction yielding lower amounts of such compounds. This indicates that suberin was not completely extracted by the isopropanol:NH3 treatment. The alkanoic acid distribution in the isopropanol:NH3 and methanol:KOH extracts shows that both methods, though with slight differences, extract the preserved suberin and cutin alkanoic acids from soil. Alkanoic acid

Table 1 Total amount and distribution of organic compounds released by alkaline treatments Isopropanol:NH3 (μg g− 1 ± sd) Methanol:KOH (μg g− 1 ± sd) a,⁎⁎

Total compounds Alkanoic acidsb, ns Distribution Alkanolsb, ns Distribution α,ω-alkanedioic acidsb, ns Distribution Aromaticsb, ns Distribution

ω-hydroxyalkanoic acidsb,⁎ Distribution Other hydroxyalkanoic acidsb,⁎ Distribution

3.87 ± 0.42 0.70 ± 0.12 C12–C30 0.16 ± 0,02 C18–C30 0.20 ± 0.03

7.16 ± 2.2 0.82 ± 0.17 C12–C30 0.23 ± 0.06 C18–C30 0.18 ± 0.05

C16–C30 0.12 ± 0.02 p-coumaric acid, ferulic acid, and ferulic acid derived product 0.85 ± 0.17

C9–C30 0.17 ± 0.03 p-coumaric acid, ferulic acid, and ferulic acid derived product 2.03 ± 0.67

C12–C30 0.06 ± 0.01

C12–C30 1.82 ± 0.67

9,16-dyhydroxyhexadecanoic 9,16acid dyhydroxyhexadecanoic acid, 9.18dihydroxyoctadecanoic acid, 9,10,18trihydroxyoctadecanoic acid

a GC-MS data; bonly pure compounds provided by GC separation were computed. ⁎, ⁎⁎ are significantly different at p b 0.05 and p b 0.1 respectively, ns is non significant at level p b 0.1.

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distribution from C16 to C24 range has been identified in water repellent soils (Ma'shum et al., 1988; Horne and McIntosh, 2000; Morley et al., 2005). The total amount and distribution of alkanols were similar in both methods (Table 1). Homologues in the range of C15 to C32 were observed with a prevalence of even chain components. In general, alkanols are known as biopolyester-derived compounds (Kolattukudy, 1980), and the presence of such high range of components (C15–C32) is also indicative of cutin and suberin preservation in the soil. Apparently, alkanols have been detected in previous studies as a minor component in water repellent soil extracts (Mainwaring et al., 2004). Aromatic acids, i.e. hydroxycinnamic acid derivatives, were detected in both treatments in similar concentration and distribution (Fig. 1 and Table 1). Suberin is characterized by the presence of hydroxycinnamic acid derivatives domains, in addition to ω-hydroxyalkanoic acids and α,ω-alkanedioic acid domains (Kolattukudy, 1980). Therefore, the occurence of ferulic acid and p-coumaric acid probably reflects the contribution of suberin-derived components in soil, although other sources, e.g. lignin derived-products, cannot be totally excluded. The α,ω-alkanedioic acids showed a similar total amount and a slight difference in distribution between the treatments, where the methanol:KOH extract resulted in wider distribution (Table 1). C12 to C24 have been identified as major building blocks of suberin (Kolattukudy, 1980; Del Rio and Hatcheri, 1998), but C16 and C18 homologous has been already detected in Arabidopsis sp. leaf cuticule (Franke et al., 2005). α,ω-Alkanedioic acids can also originate from microbial oxidation of ω-hydroxyalkanoic acids (van Bergen et al., 1998), but different relative distribution of α,ω-alkanedioic acids and ω-hydroxyalkanoic acids practically excluded this source (data not shown). However, for the C9 and C10 components observed in the methanol:KOH extract, microbial oxidation of the C = C double bond of unsaturated fatty acid is the most probable origin (Grasset and Amblès, 1998; Regert et al., 1998; Naafs et al., 2004). The alkaline treatments were able to extract another important class of aliphatic compounds: the hydroxyalkanoic acids. The most important hydroxyalkanoic acids identified in the extracts were a series of ω-hydroxyalkanoic acids, 9,16-dyhydroxyhexadecanoic acid and 9,10,18-trihydroxyoctadecanoic acid (Fig. 1). There was a significant difference in the total amount of hydroxyalkanoic acids yielded by each treatment: concentration of ω-hydroxyalkanoic acids was twice as high in methanol:KOH extracts and the relative amount of other hydroxyalkanoic acids was thirty times greater (Table 1). The ω-hydroxyalkanoic acids had a similar range in alkaline extracts, but with a different relative distribution (Fig. 2). The isopropanol:NH3 extract showed a bimodal series of even numbered ω-hydroxyalkanoic acids ranging from C12 to C30, clearly maximizing at C16 and C24 (p b 0.05). A bimodal distribution, accompanied by C18 ω-hydroxyalkenoic acids, was observed in the methanol:KOH extract, but the ω-hydroxyalkanoic acids were maximized at C16 and C26, and the latter compound did not show any statistical difference (p N 0.1) when compared to C22 and C24 homologues (Fig. 2). ω-Hydroxyalkanoic acids are very abundant in cutin and suberin: the first has C16 and C18 as its principal components, while the presence of longer chain C16–C24 acids is more abundant in the latter (Kolattukudy, 1980; Holloway, 1982). A similar feature has been shown for Pinus species by Matzke and Riederer (1991) and Nierop et al. (2006), who identified, in roots and needles, depolymerisate products in the range of C16–C24 and C12–C16 ω-hydroxyalkanoic acids, respectively. C18 ω-hydroxyalkenoic acids were not detected in the isopropanol:NH3 extract; the origin of these compounds are also associated with cutin and suberin, either as original building blocks or artefacts of 18-hydroxy1,10-epoxyoctadecanoic acid (Santos Bento et al., 2001). Alternative sources of ω-hydroxyalkanoic acids, such as microbial oxidation of alkanoic acids, may play a role in this process, although probably not significantly, since there was no evidence of a precise relationship with

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Fig. 1. Partial total ion current (TIC) showing the major compounds identified in the isopropanol:NH3 (A) and methanol:KOH (B) extracts. Peak identities: h, hydroxycinnamic acid derivatives; ○, α,ω-alkanedioic acids; ■, alkanoic acids; ●, ω-hydroxyalkanoic acids; □, alkanols; ⁎, contaminants. CX above the peaks refers to the total number of carbon atoms, and numbers after the colon refers to the number of double bonds. Iso and anteiso branched alkanoic acids are denoted as “i” and “a” respectively.

the distribution of alkanoic acids (data not shown). Nierop et al. (2003) demonstrated that a large majority of ester-linked aliphatic moieties disappeared from the soil after the methanol:KOH alkaline treatment, pointing that this treatment is an efficient approach to release aliphatic ester-bound components from cutin and suberin present in soils. Based on this, the difference seen in the amount extracted and the ωhydroxyalkanoic acids distribution in both methods is due to the fact that the isopropanol:NH3 treatment depolymerized part of the aliphatic ester-bound components (e.g. cutin and suberin) preserved in the soil. Other hydroxyalkanoic acids identified here consisted mainly of 9,16dihydroxyhexadecanoic acid, 9,18-dihydroxyoctadecanoic acid, and 9,10,18-trihydroxyoctadecanoic acid (Fig. 1, Table 1). Comparisons in the distribution of these compounds showed considerable differences between the treatments (p b 0.05). 9,18-Dihydroxyoctadecanoic acid, and 9,10,18-trihydroxyoctadecanoic acid were not present in detection levels in the isopropanol:NH3 extract, and only a small amount of 9,16dyhydroxyhexadecanoic acid (0.06± 0.01 μg g− 1) was found (Fig. 1a). In contrast, 9,16-dyhydroxyhexadecanoic acid was the major compound identified in the methanol:KOH extract (1.30 ± 0.45 μg g− 1) (Fig. 1b). These compounds are common constituents of suberin and cutin tissues (Matzke and Riederer,1991), although dihydroxyhexadecanoic acids (i.e. x,16-C16 family) and 9,10,18-trihydroxyoctadecanoic acid have been

identified as major components of plant cutin (Kolattukudy, 1980; Holloway, 1983; Goñi and Hedges, 1990; Nierop et al., 2006). For example, whereas pine needles (cutin) have abundant 9,16-

Fig. 2. Distribution and abundance of major ω-hydroxyalkanoic acids yielded by alkaline treatments in the water repellent soil.

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dyhydroxyhexadecanoic acid isomers, pine roots (suberin) do not contain or have small amounts of 9,16-dyhydroxyhexadecanoic and 9,10,18-trihydroxyoctadecanoic acids. However, this is not the case for some angiosperm species. Naafs et al. (2005) used ratios to evaluate changes in molecular composition of ester-bound compounds in soil, and considered that suberin was a predominant source of longer chain ω-hydroxyalkanoic acids (NC20), while the presence of 9,16-dihydroxyhexadecanoic acid was associated with cutin input in the soil (e.g. ω-hydroxydocosanoic acid:9,16-dyhydroxyhexadecanoic acid). Thus, a relative dominance of cutin over suberin was clearly reflected in the methanol:KOH extracts (Table 1), while the opposite was seen with isopropanol:NH3 due to the low amount of 9,16-dyhydroxyhexadecanoic acid in the extract, suggesting that it was not able to efficiently extract cutin derived products from the soil. The difference in dihydroxyalkanoic acids and trihydroalkanoic acid distribution in both treatments can be explained by the fact that these cutin derived compounds are part of the biopolyester structure core (Nierop et al., 2003) and that they are more protected from hydrolysis. This reinforces that the isopropanol:NH3 treatment is less effective in hydrolysis. Moreover, the significant difference in the amount of ω-hydroxydocosanoic acid (Fig. 2), C22 and C24 alkanoic acids (p b 0.05) extracted suggests that suberin derived compounds are only partially extracted with isopropanol:NH3. Oxygen-based compounds of α,ω-Alkanedioic acids and hydroxyalkanoic acids were not previously reported as organic constituents in water repellent soils. However, although Morley et al. (2005) detected polar compounds containing oxygen-based functional groups, no association has been made between these polar compounds and α,ω-alkanedioic acids and hydroxyalkanoic acids. Based on biomarker compounds identified in soils and their relative distributions, incorporation of P. taeda cutin and suberin into the soil can be well characterized by the presence of chemical constituents usually detected in gymnosperms (Goñi and Hedges, 1990; Matzke and Riederer, 1991; Nierop et al., 2006). However, the ranges of biomarkers identified are not exclusively present in Pinus species, and incorporation from different sources (i.e. fungi and mosses) may also be expected. Pinus is a plant associated with water repellency because of its considerable high amount of resins, waxes and aromatic oils (Doerr et al., 2000); nevertheless, it seems that besides these components, incorporation and preservation of cutin and suberin in soil may also have an important role in water repellency under pinus plantations. Interestingly, depolymerization provided by the isopropanol:NH3 treatment was enough to break the hydrophobicity, suggesting that the loss of part of the building block composition of cutin and mainly suberin may be able to render the soil wettable. The relationship between P. taeda needle (cutin) and roots (suberin) with soil water repellency is currently being investigated. 4. Conclusion The main conclusions drawn from the molecular characterization of soil extracts obtained from different alkaline treatments are summarized as follows: • Both alkaline treatments rendered the soil wettable. • Both alkaline treatments extracted the same classes of organic compounds from the solvent extracted soil, excluding C18 di and trihydroxyalkanoic acids. • Alkaline treatment demonstrated that cutin and suberin were preserved in the soil. • Based on isopropanol:NH3 released products, partial biopolyester depolymerization was enough to render the soil wettable. • Alkaline treatment with isopropanol:NH3 was not efficient in the extraction of whole aliphatic biopolyesters from soil, indicating that methanol:KOH was a more effective hydrolysis treatment.

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• Apart from these plant biopolyesters, the presence of iso and anteiso C15 and C17 alkanoic acids suggested that microbial activity also occurs in the repellent soil. • The influence of biopolyesters in the soil water repellency needs to be better evaluated in future studies, mainly in soil under pinus culture. For example, high amounts of preserved cutin and suberin in soil can be the main constituent of the macromolecular hydrophobic organic layer that impedes water penetration in the soil. Acknowledgements This study was supported by the Agrogases project funded by Embrapa and the Ministry of Agriculture of Brazil. Iguaçu Celulose e Papel Ltda is thanked for soil samples. Cristiane T. Aoki is very grateful to the CNPq for her scholarship. References Amblès, A., Jambu, P., Parlanti, E., Joffre, J., Riffe, C.,1994. Incorporation of natural monoacids from plant residues into an hydromorphic forest podzol. Eur. J. Soil Sci. 45, 175–182. Bond, R.D., Harris, J.R., 1964. 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