EGU2020-17791, updated on 13 Jan 2021
EGU General Assembly 2020
© Author(s) 2021. This work is distributed under
the Creative Commons Attribution 4.0 License.

A unified protocol for the high-throughput measurement of PLFA, NLFA, GLFA and sterols from soil

Stefan Gorka, Alberto Canarini, Bruna Imai, Georg Teischinger, Sean Darcy, and Christina Kaiser
Stefan Gorka et al.
  • Centre for Microbiology and Environmental Systems Science, University of Vienna, Vienna, Austria (

Phospholipid fatty acids (PLFA) are widely used as biomarkers for soil microbial biomass. In more recent years, neutral lipid fatty acids (NLFA) have additionally been used as storage biomarkers. Both lipid classes are usually separated via silica solid phase extraction (SPE) after extraction with a mixture of chloroform, methanol and citric acid buffer. However, in recent years several studies reported incomplete or inconsistent separation of lipid classes, depending on minor differences in the polarity of the eluents used during the SPE. Moreover, while PLFA profiles have been tested on microbial pure cultures, the taxonomic specificity of NLFA is only assumed to equal that of PLFA.

Complementary to fatty acid based biomarkers, many studies quantify ergosterol as a reliable indicator for fungal biomass because the fungal-specific PLFA 18:1ω9 and 18:2ω6,9 also occur in plants, which compromises their use for detecting fungal biomass in plant tissue (for example mycorrhizal fungi in plant roots). Measuring ergosterol requires an additional extraction method, but existing protocols include silylation for further gas chromatography analysis and are thus not compatible with determining 13C by IRMS.

Here, we aimed to quantify the recovery of polar and non-polar lipid classes as well as ergosterol following lipid extraction and silica SPE fractionation. We used pure standards of representative phospholipids, glycolipids and neutral lipids with unique fatty acid chain lengths for unambiguous identification of the lipid class after SPE. Lipid fractionation was tested on a 96-well SPE plate with different eluents. Subsequently, we applied the modified method to characterize lipid fractions in microbial pure cultures from bacteria (Proteobacteria, Firmicutes, Actinobacteria), and saprotrophic and ectomycorrhizal fungi (Ascomycota, Basidiomycota).

Separation of lipid classes was achieved by successively eluting NLFA and sterols with a mixture of chloroform and ethanol (v:v = 98:2), glycolipid fatty acids (GLFA) with acetone, and PLFA with a mixture of methanol, chloroform and water (v:v:v = 5:5:1). GLFA were partially recovered in the NLFA or PLFA fraction depending on the nature of the lipid, which should be considered when interpreting PLFA data. Ergosterol recovery was unaffected by subsequent mild alkaline methanolysis of the NLFA fraction in which it was collected, allowing further analysis of both lipid classes in the same mixture. The gas-chromatographic method may be extended to elute both NLFA and (non-silylated) sterols in one run, assuming that the concentration of ergosterol in soil samples is high enough. Therefore, the method can be optimized by using an internal standard added to the NLFA fraction and simultaneously quantify ergosterol. Finally, we show how different lipid classes and attached fatty acid chains distribute in pure cultures of soil micro-organisms.

How to cite: Gorka, S., Canarini, A., Imai, B., Teischinger, G., Darcy, S., and Kaiser, C.: A unified protocol for the high-throughput measurement of PLFA, NLFA, GLFA and sterols from soil, EGU General Assembly 2020, Online, 4–8 May 2020, EGU2020-17791,, 2020

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Presentation version 1 – uploaded on 05 May 2020
  • CC1: Comment on EGU2020-17791, Sabine Lengger, 05 May 2020

    This is nice work! It's hard to get 100% recovery. Have you seen Heinzelmann et al. (2014) ? They have worked on marine lipids and also found incomplete recovery (

    I don't fully understand your comment about Ergosterol - it is easy to change the final derivatisation method to e.g. an acetylation, or measuring the pure compounds, no matter what the previous extraction procedure is like? Would you be able to clarify? 

    • CC2: Reply to CC1, Stefan Gorka, 05 May 2020

      Thank you, yes I know the Heinzelmann paper (which is very nice, since they measure the complete lipids). This was in fact one of the papers that led us to test the PLFA extraction protocol we use at our lab.


      Regarding your question:

      The idea was to measure Ergosterol on the GC-MS, since we already inject the NLFA-fraction anyway, and it would be very efficient to measure them both with a single injection (which was possible, since sterols needed a much higher temperature to elute). In principle, you could then even use the FAME internal standard to quantify ergosterol. Also, sterols are usually silylated for GC-MS analysis, but we are also aiming for GC-IRMS at some point, where it’s not good to inject silylated compounds.

      • CC3: Reply to CC2, Sabine Lengger, 06 May 2020

        Thanks for the reply!

        Yes, some don't like to use TMS for GC-C-IRMS as it can interfere with the reactor, but it's widely used and has been found to be reproducible. It's used in Bristol for example. I think the major problem is with amino acids and TMS. 

        But it isn't actually necessary to TMS derivatise sterols for GC - the classic technique is acetylation (which technically gives you fractionation but that has been reported to be insignificant) and you can also analyse without derivatisation (I've done that e.g. here ). I don't actually know why people are not doing this for IRMS also, but it might be that in these fractions there are a lot of other compounds that would cause issues.