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Modified Lipid Extraction Methods for Deep Subsurface Shale

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      Abstract

      Growing interest in the utilization of black shales for hydrocarbon development and environmental applications has spurred investigations of microbial functional diversity in the deep subsurface shale ecosystem. Lipid biomarker analyses including phospholipid fatty acids (PLFAs) and diglyceride fatty acids (DGFAs) represent sensitive tools for estimating biomass and characterizing the diversity of microbial communities. However, complex shale matrix properties create immense challenges for microbial lipid extraction procedures. Here, we test three different lipid extraction methods: modified Bligh and Dyer (mBD), Folch (FOL), and microwave assisted extraction (MAE), to examine their ability in the recovery and reproducibility of lipid biomarkers in deeply buried shales. The lipid biomarkers were analyzed as fatty acid methyl esters (FAMEs) with the GC-MS, and the average PL-FAME yield ranged from 67 to 400 pmol/g, while the average DG-FAME yield ranged from 600 to 3,000 pmol/g. The biomarker yields in the intact phospholipid Bligh and Dyer treatment (mBD + Phos + POPC), the Folch, the Bligh and Dyer citrate buffer (mBD-Cit), and the MAE treatments were all relatively higher and statistically similar compared to the other extraction treatments for both PLFAs and DGFAs. The biomarker yields were however highly variable within replicates for most extraction treatments, although the mBD + Phos + POPC treatment had relatively better reproducibility in the consistent fatty acid profiles. This variability across treatments which is associated with the highly complex nature of deeply buried shale matrix, further necessitates customized methodological developments for the improvement of lipid biomarker recovery.

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      Most cited references 78

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      A simple method for the isolation and purification of total lipides from animal tissues.

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        A rapid method of total lipid extraction and purification.

         E G BLIGH,  W. Dyer (1959)
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          Prokaryotes: the unseen majority.

          The number of prokaryotes and the total amount of their cellular carbon on earth are estimated to be 4-6 x 10(30) cells and 350-550 Pg of C (1 Pg = 10(15) g), respectively. Thus, the total amount of prokaryotic carbon is 60-100% of the estimated total carbon in plants, and inclusion of prokaryotic carbon in global models will almost double estimates of the amount of carbon stored in living organisms. In addition, the earth's prokaryotes contain 85-130 Pg of N and 9-14 Pg of P, or about 10-fold more of these nutrients than do plants, and represent the largest pool of these nutrients in living organisms. Most of the earth's prokaryotes occur in the open ocean, in soil, and in oceanic and terrestrial subsurfaces, where the numbers of cells are 1.2 x 10(29), 2.6 x 10(29), 3.5 x 10(30), and 0. 25-2.5 x 10(30), respectively. The numbers of heterotrophic prokaryotes in the upper 200 m of the open ocean, the ocean below 200 m, and soil are consistent with average turnover times of 6-25 days, 0.8 yr, and 2.5 yr, respectively. Although subject to a great deal of uncertainty, the estimate for the average turnover time of prokaryotes in the subsurface is on the order of 1-2 x 10(3) yr. The cellular production rate for all prokaryotes on earth is estimated at 1.7 x 10(30) cells/yr and is highest in the open ocean. The large population size and rapid growth of prokaryotes provides an enormous capacity for genetic diversity.
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            Author and article information

            Affiliations
            1Department of Geology and Geography, West Virginia University Morgantown, WV, United States
            2Civil, Environmental and Geodetic Engineering, The Ohio State University Columbus, OH, United States
            3Center for Environmental Biotechnology, University of Tennessee Knoxville, TN, United States
            Author notes

            Edited by: Jennifer F. Biddle, University of Delaware, United States

            Reviewed by: Helen F. Fredricks, Woods Hole Oceanographic Institution, United States; Greg F. Slater, McMaster University, Canada; Sabine Kerstin Lengger, Plymouth University, United Kingdom

            *Correspondence: Rawlings N. Akondi raakondinkerh@ 123456mix.wvu.edu

            This article was submitted to Extreme Microbiology, a section of the journal Frontiers in Microbiology

            Contributors
            Journal
            Front Microbiol
            Front Microbiol
            Front. Microbiol.
            Frontiers in Microbiology
            Frontiers Media S.A.
            1664-302X
            25 July 2017
            2017
            : 8
            5524817 10.3389/fmicb.2017.01408
            Copyright © 2017 Akondi, Trexler, Pfiffner, Mouser and Sharma.

            This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

            Counts
            Figures: 5, Tables: 3, Equations: 0, References: 78, Pages: 15, Words: 11895
            Funding
            Funded by: National Science Foundation 10.13039/100000001
            Award ID: 1342732
            Award ID: 1342701
            Award ID: 1205596
            Categories
            Microbiology
            Methods

            Microbiology & Virology

            deep subsurface, shale ecosystem, microbial biomass, plfa, dgfa

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