--> Comparing Different Extraction Methods for Analyses of Ester-linked Diglyceride Fatty Acids in Marcellus Shale

AAPG Eastern Section Meeting

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Comparing Different Extraction Methods for Analyses of Ester-linked Diglyceride Fatty Acids in Marcellus Shale

Abstract

This work contributes to the recent advances being made towards the comprehensive assessment of microbial biomass and diversity in the deep subsurface environments. Microbes have been known to inhabit the deep subsurface ecosystem, surviving severe conditions of starvation and extreme temperatures. While microbes have been known to survive in such environments, their existence in the shale ecosystem could be significantly compromised by the complex nature of the shale organic matter matrix and small pore sizes. Due to low biomass in these shales, it becomes very vital to develop methods that have the ability to yield higher and persistent recovery of microbial lipid biomarkers. In this study, three extraction methods i.e. modified Bligh and Dyer (mBD), Folch (FOL), and Microwave-Assisted Extraction (MAE) were used to extract the ester-linked diglyceride fatty acids (DGFA) from homogenized shale samples. Phosphate [Phos] and Citrate [Cit]) buffers were used for the mBD extractions together with MgCl2, Escherichia coli [E.coli] biomass, and phospholipid [POPC] spikes. To determine the variation in the different lipid profiles obtained from different methods, the extracted lipids were separated by column chromatography and the DGFA were eluted from the chloroform fraction and trans-esterified into fatty acid methyl esters (FAMEs) by methanolic potassium hydroxide. The FAMEs were then analyzed by gas chromatography mass spectrometry. The treatments had relatively comparable biomass yields and total lipid abundances except for the mBD-Cit and mBD+Phos+Mg2+, which had less yield compared to the other treatments. Based on the NMDS and PCA analysis, the differences in diversity of FAMEs between the different treatments were attributed to the less common fatty acids. For example, polyunsaturated, cyclopropyl, terminally branched saturated, and epoxy FAMEs were detected consistently in mBD-Phos+Ecoli, mBD-Phos + POPC, and Folch treatments while the Keto FAMEs were only captured in the mB&D-Phos+Mg2+ treatment. However, the mBD-Phos+POPC had the best reproducibility and yield among all the other methods, suggesting that the addition of a lipid spike helps with recovery of lipid biomarkers that would otherwise not be released from the shale matrix.