Stable Isotope Probing of Microbial Phospholipid Fatty Acids in Environmental Samples

  • Andrea WatzingerEmail author
  • Rebecca Hood-Nowotny
Part of the Methods in Molecular Biology book series (MIMB, volume 2046)


Phospholipid fatty acid (PLFA) extracted from environmental samples describe the microbial community pattern and are sensitive to monitor and quantify shifts in the microbial community. Linkage with the stable isotope technique adds a functional perspective and is frequently used to quantify carbon turnover in microbial communities and detect physiological changes. Here we present a PLFA extraction method by using an organic solvent water mixture, followed by lipid separation based on solid-phase extraction and an alkaline methylation. Finally, we provide a protocol for the carbon stable isotope measurements of the extracted fatty acid methyl esters (FAMEs) by gas chromatograph–isotope ratio mass spectrometer (GC-IRMS) and calculation of concentration and δ13CVPDB values.

Key words

Phospholipid fatty acid Stable isotope probing Lipid extraction Separation Solid-phase extraction Alkaline methylation Fatty acid methyl ester Microorganisms Environmental samples GC-IRMS 



The authors would like to thank all the coworkers and students for critical questions and comments when conducting PLFA extraction and GC-IRMS measurements, which over the years improved the protocol. The County of Lower Austria, NÖ Wirtschafts- und Tourismusfonds (WST3-F-5031245/003-2018) is acknowledged for funding.


  1. 1.
    Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917CrossRefGoogle Scholar
  2. 2.
    White DC, Davis WM, Nickels JD et al (1979) Determination of the sedimentary microbial biomass by extractable lipid phosphate. Oecologia 40:51–62CrossRefGoogle Scholar
  3. 3.
    Frostegård Å, Tunlid A, Bååth E (1991) Microbial biomass measured as total lipid phosphate in soils of different organic content. J Microbiol Methods 14:151–163CrossRefGoogle Scholar
  4. 4.
    Frostegård A, Tunlid A, Bååth E (1993) Phospholipid fatty acid composition, biomass, and activity of microbial communities from two soil types experimentally exposed to different heavy metals. Appl Environ Microbiol 59:3605–3617PubMedPubMedCentralGoogle Scholar
  5. 5.
    Frostegård Å, Tunlid A, Bååth E (2011) Use and misuse of PLFA measurements in soils. Soil Biol Biochem 43:1621–1625CrossRefGoogle Scholar
  6. 6.
    Zelles L (1999) Fatty acid patterns of phospholipids and lipopolysaccharides in the characterisation of microbial communities in soil: a review. Biol Fertil Soils 29:111–129CrossRefGoogle Scholar
  7. 7.
    Fernandes MF, Saxena J, Dick RP (2013) Comparison of whole-cell fatty acid (MIDI) or phospholipid fatty acid (PLFA) extractants as biomarkers to profile soil microbial communities. Microb Ecol 66:145–157CrossRefGoogle Scholar
  8. 8.
    Watzinger A (2015) Microbial phospholipid biomarkers and stable isotope methods help reveal soil functions. Soil Biol Biochem 86:98–107CrossRefGoogle Scholar
  9. 9.
    Boschker H, Nold S, Wellsbury P et al (1998) Direct linking of microbial populations to specific biogeochemical processes by 13C-labelling of biomarkers. Nature 392:801–805CrossRefGoogle Scholar
  10. 10.
    Brenna JT, Corso TN, Tobias HJ et al (1998) High-precision continuous-flow isotope ratio mass spectrometry. Mass Spectrom Rev 16:227–258CrossRefGoogle Scholar
  11. 11.
    Meier-Augenstein W (2002) Stable isotope analysis of fatty acids by gas chromatography–isotope ratio mass spectrometry. Anal Chim Acta 465:63–79CrossRefGoogle Scholar
  12. 12.
    Evershed RP, Crossman ZM, Bull ID et al (2006) 13C-labelling of lipids to investigate microbial communities in the environment. Curr Opin Biotechnol 17:72–82CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Institute of Soil ResearchUniversity of Natural Resources and Life Sciences ViennaTullnAustria

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