The applicability of accelerated solvent extraction (ASE) to extract lipid biomarkers from soils
Introduction
Organic matter in soils consists of a wide range of chemical components that originate predominantly from plant litter and the microbial biomass (Kögel-Knabner, 2002). The last decade has seen an increasing scientific interest in the organic chemical composition of soil organic matter (SOM) and changes therein upon soil biogeochemical processes (e.g., Gregorich et al., 1996, Guggenberger and Zech, 1999, Kögel-Knabner, 2002, Nierop et al., 2001, Wiesenberg et al., 2004b). Extractable lipids constitute a class of organic components in soils that has received particular attention. Reasons for this are amongst others the role of lipids in SOM accumulation and SOM stability (e.g., Naafs et al., 2004b, Poulenard et al., 2004, Rumpel et al., 2004), their significance for terrestrial food-web studies (e.g., Balser et al., 2005, Ruess et al., 2005), their influence on the fate of contaminants in soils (e.g., Chilom et al., 2005) and their use as vegetation tracers (e.g., Ficken et al., 1998, Van Bergen et al., 1997).
The use of lipids as vegetation tracers is based on the principle that plant-specific combinations of lipids are preserved in the soil and can serve as biomarkers to identify past vegetation compositions. Following our previous investigation of organic matter in a Dutch sandy soil under Corsican pine (Nierop and Verstraten, 2004), we considered applying this so-called biomarker technique to determine its past vegetation composition. However, we were faced with the challenge of isolating the selected lipid biomarkers from the soil matrix prior to analysis, as there is no unequivocal technique for this purpose. Instead, a wide range of extraction procedures are applied in contemporary practice, including Soxhlet extraction (e.g., Naafs et al., 2004a, Winkler et al., 2005), sonication (e.g., Dalton et al., 2005, Otto et al., 2005) and even simple shaking with solvent (Quenea et al., 2004). Soxhlet extraction has been used for the purpose of extracting lipids from soils for over 25 years (e.g., Jambu et al., 1978) and forms the basis of EPA method 3540C for the extraction of non-volatile organics from solids such as soils (EPA, 1996). As such, it is the most well-established of the methods mentioned.
While being a robust and well-established technique, Soxhlet extraction suffers from three main shortcomings: (i) the necessity of using relatively large extractant volumes of usually 250 mL or more; (ii) long analysis times of typically 16 h per analysis; and (iii) a difficulty to automate. A promising alternative is the relatively new technique of accelerated solvent extraction (ASE) (Richter et al., 1996). In short, ASE extracts samples under elevated temperature, while elevated pressure ensures that volatile extractants remain liquid. ASE can be completely automated, it employs very small extractant volumes (normally 5–30 mL) and has typical extraction times of less than an hour (Richter et al., 1996). As such the technique has the potential to overcome the main shortcoming of Soxhlet extractions. However, while the use of ASE to extract organic contaminants from soils is now reasonably well-established (Giergielewicz-Mozajska et al., 2001), its application to the extraction of soil lipids has received very little attention so far. To our knowledge only two studies have been published to date in which ASE was used to extract lipids from soil samples (Rumpel et al., 2004, Wiesenberg et al., 2004a), and no comparison with other techniques for this purpose has yet been made. Still, such a comparison of ASE with more established techniques is crucial if ASE is to be used in biomarker studies. The reason is that differences in extraction efficiencies for various types of lipids between ASE and other techniques would lead to a difference in the composition of the biomarker signal that is obtained.
Therefore, the purpose of the current study was to examine the efficiency of ASE to extract typical lipid biomarkers from a selection of soil horizons from a Dutch sandy soil under Corsican pine, using Soxhlet extractions as a reference. The biomarkers consisted of a selection from the following component classes: (i) straight-chain lipids; (ii) plant sterols; and (iii) terpenoids.
Section snippets
Description of site, soil profile and sampling procedures
The selected sampling site is a plot in ‘De Schoorlse Duinen’, a sand-dune area in The Netherlands near the village of Schoorl. The current vegetation on the study plot consists almost exclusively of Corsican Pine (Pinus nigra var. maritima) that was planted in 1929 (Nierop and Verstraten, 2004). The only undergrowth present in significant quantities is a moss layer situated between the litter (L) horizon and the F1 horizon. The soil was classified as a Haplic Arenosol (FAO-UNESCO, 1990) and a
Results and discussion
The concentrations of the selected biomarkers in the ASE and Soxhlet extracts represented as μg/g extracted soil material are presented in Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5. The values for the ASE extracts represent the average contents of the various biomarkers in the triplicate extracts with error bars indicating the standard error of the mean. Due to insufficient flushing during the filtering of one of the three low-pressure ASE extracts from the F1 horizon, probably a large fraction of
Conclusions
Altogether, we conclude that when investigating the combination of lipid biomarkers chosen in this study through extraction with the common extractant DCM/MeOH, overall ASE is a viable method to extract lipids from soils. The reasons are: (i) on average better extraction efficiencies, especially for the n-alkanes compared to the reference method of Soxhlet extractions; (ii) the reduced volumes of extractant (10–33 mL vs. 300 mL) as well as shorter extraction time (25 min vs. 16–24 h) compared to
Acknowledgements
We wish to thank Frans van der Wielen for his extensive experimental support. The Netherlands Foundation for the Advancement of Tropical Research (WOTRO) is gratefully acknowledged for their funding of this project under number WAN 75-406. We thank Fjällräven for their generous sponsoring in the form of clothing and gear.
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