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United States Department of Agriculture

Agricultural Research Service

Research Project: CONTROLS ON MICROBIAL COMMUNITY STRUCTURE AND FUNCTION IN SOIL AND RHIZOSPHERE

Location: Sustainable Agricultural Systems Laboratory

Title: High throughput phospholipid fatty acid analysis of soils

Authors
item Buyer, Jeffrey
item Sasser, Myron

Submitted to: Soil Biology and Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 16, 2012
Publication Date: July 20, 2012
Citation: Buyer, J.S., Sasser, M. 2012. High throughput phospholipid fatty acid analysis of soils. Soil Biology and Biochemistry. 61:127-130.

Interpretive Summary: Phospholipid fatty acid (PLFA) analysis is commonly used to analyze the microbial communities of soil and other environmental samples. Lipids are extracted from soil and one class of lipids, phospholipids, are purified from the extract. A chemical reaction is used to convert the phospholipids into fatty acid methyl esters, which are then analyzed by gas chromatography. PLFA analysis yields information about the size and makeup of the microbial community which is very useful to scientists studying the effects of agronomic management systems on soil quality as well as more fundamental studies on soil biology. The standard PLFA protocol is rather slow, requiring 2 days to prepare 20 samples and approximately 15 hours to analyze those 20 samples by gas chromatography. We have developed a new method to prepare 96 samples in 2 days and analyze those 96 samples in 27 hours. This new method also reduces solvent consumption, thus lowering the cost of the analysis.

Technical Abstract: Phospholipid fatty acid (PLFA) analysis is widely used to characterize microbial communities in soil and other types of environmental samples. The analysis involves many steps and, as typically performed, 1.5 to 2 days are required to prepare a batch of approximately 20 samples, depending on the exact equipment employed in the laboratory. Gas chromatography or gas chromatography-mass spectrometry is then used to analyze the samples, requiring further time. We have developed a method for preparing 96 soil samples and blanks in 2 days. All drying and centrifuging steps take place in a centrifugal evaporator. Soil samples in test tubes are dried overnight and then a Bligh-Dyer lipid extraction is performed. The extract is dried, dissolved in chloroform, and loaded onto a 96-well solid phase extraction plate. Phospholipids are eluted into glass vials in a 96-well format, dried, and transesterified. The resulting fatty acid methyl esters are analyzed by gas chromatography using a 12-minute run time to identify fatty acids from 10 to 24 carbons long. Results are quantitated relative to an internal standard. This new method produces similar but not identical results compared to a standard method. The high throughput protocol may be useful to laboratories performing large numbers of PLFA analyses.

Last Modified: 12/20/2014
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