Short Communication
Solvent-based washing removes lipophilic contaminant interference with phospholipid fatty acid analysis of soil communities

https://doi.org/10.1016/j.soilbio.2011.06.010Get rights and content

Abstract

Phospholipid fatty acid (PLFA) analysis is an informative method for characterising and quantifying changes in the phenotypic profile of the soil microbial community when soils are exposed to chemical toxicants and other xenobiotics. However, where such materials are lipophilic, a range of non-polar compounds can be co-extracted with PLFAs and can consequently mask PLFA chromatograms. We found this to be the case with the lipophilic anti-microbial compound triclosan, which can enter the soil via the addition of sewage sludge. A simple method of washing soil in solvent prior to extraction was developed in order to remove triclosan without altering the relative abundance of PLFAs. Three contrasting soils were spiked with 500 mg kg−1 of triclosan before being washed with methanol (MeOH), dichloromethane (DCM), hexane or aqueous solutions of these solvents. PLFAs were then extracted and analysed. All treatments were found to remove triclosan effectively, allowing all peaks to be identifiable. Whilst the polar solvents MeOH and DCM significantly altered the relative abundance of extracted fatty acids in most of the soils tested, soil washing with a small quantity of hexane was able to remove triclosan whilst best preserving the fidelity of the PLFA profiles.

Highlights

► A method for removing pollutants from soil prior to PLFA analysis is described. ► Triclosan is used as an example pollutant, but the method is generally applicable. ► All treatments removed triclosan effectively but some also altered PLFA abundance. ► Washing with hexane removed triclosan and preserved the fidelity of PLFA profiles.

Section snippets

Acknowledgements

We are grateful to Dr Raffaella Villa from Cranfield University for her generous advice and to the Engineering and Physical Science Research Council (EPSRC) and Unilever for funding. We are also grateful to Petra Marschner and an anonymous reviewer for their constructive comments which helped to improve this manuscript.

References (26)

Cited by (4)

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    Bacterial PLFA were determined from the sum of the Gram positive and Gram negative PLFA. The PLFA 18:1ω9c and 18:1ω9t were evaluated separately and tested for association with 18:2ω6, as the 18:1ω9t is sometimes cited along with the cis isomer as a fungal PLFA (e.g. Butler et al., 2011) and can vary with 18:2ω6 in diverse soils (Frostegard et al., 2011). These PLFA, however, may also be found in bacteria (Frostegard et al., 1993; Olsson et al., 1995; Frostegard and Baath, 1996), even in monocultures of bacterial isolates (Ringelberg et al., 1989) or other samples where 18:2ω6 is absent (Schoug et al., 2008).

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    We did, however, use these data to test for association with 18:2ω6 to determine specificity for fungi in these soils in order to evaluate our conservative approach. The PLFA 18:1ω9t is sometimes cited, along with 18:1ω9c, as a fungal PLFA (e.g. Butler et al., 2011) given their variation with 18:2ω6 in many soils (Frostegard et al., 2011). However, these PLFA are not exclusive to fungi in all environments (Frostegard et al., 1993; Frostegard and Baath, 1996; Olsson, 1999); we therefore did not define them as fungal PLFA.

  • The effect of triclosan on microbial community structure in three soils

    2012, Chemosphere
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    Initial trials showed that residual triclosan can be extracted with PLFAs which then co-elutes with the fatty acids on the gas chromatogram at high concentrations, masking the PLFA peaks. A simple method of soil washing was, therefore, developed to remove the triclosan without affecting the phenotypic profile (Butler et al., 2011b). Briefly, soil was shaken with 10 mL of hexane and 10 mL deionised water for 2 h at 300 rpm followed by centrifugation at 2000 rpm for 5 min to remove the aqueous phase and dried overnight at 37 °C to evaporate the organic phase, frozen at −80 °C for a minimum of 24 h and freeze-dried.

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