Phospholipid-Linked+Fatty+Acid+(PLFA)+Profiling

PLFA is a type of lipid profiling analysis used to identify different types of microorganisms in a sample. It is commonly used in environmental sampling, especially when it is desirable to analyze a microbial community in situ (1). Because phospholipids are essential components of every living cell, they are useful biomarkers. PLFAs are considered to be representative of only the living microbial community, because they are not found in storage products and degrade quickly after the death of the cell (2). Different microbial populations can be identified by their characteristic lipid profile. This refers to the relative proportions of different kinds of phospholipids. Phoshpolipids are differentiated based on the structures of their fatty acid chains. These can be short, long, branched, saturated, etc. Examining the lipids of a community yields quantitative data on the relative proportions of different kinds of microbes in the community as well as biomass concentration of the community (3). Table 1 lists PLFAs that are characteristic of certain microbial taxa. Table 1: ** Type of Organism  ** || ** Characteristic PLFAs  ** || Eucaryotes (i.e. Protozoans and Fungi)  || Polyunsaturated  || Gram-Positive Bacteria  || Branched  || Gram-Negative Bacteria  || Cyclopropyl and Monounsaturated  || Actinomycetes  || 10-Methyl FAs  || (Moore-Kucera and Dick) Ratios of particular PLFAs can also be measured as indicators of nutritional or environmental stress in a microbial community (4). PLFAs can be used to assess changes in an environment, such as whether a site is aerobic or anaerobic, based on the signature lipids of the organisms present in the sample. It is most commonly used in soil microbial analysis (5) Table 2 lists PLFAs that indicate the effect of particular environmental stresses. Table 2: ** Stress Indicator  ** || ** Stress Factor  ** || Ratio >1 for //trans:cis// isomers of monounsaturated PLFAs  || Starvation/Water Stress  || Increased ratios of saturated:unsaturated PLFAs  || Other Environmental Stresses  || Increased levels of cyclopropyl FAs:monoenoic cyclopropyl FA precursors  || Other Environmental Stresses || (Moore-Kucera and Dick, Guckert et al) Based on the differences in PLFAs between different microbial taxa and the tendency of microbial populations to produce different PLFAs in the presence of different environmental pressures, researchers in the field of environmental microbiology often use PLFA profiling to assess shifts in microbial community structure. This method has been used to detect changes in environmental stress, including the introduction of pollutants into an ecosystem (6). The first step in PLFA analysis is to extract the lipid biomarkers from the cells in the community being analyzed. This is done by extracting the sample with organic solvents (commonly chloroform and methanol) and allowing the phases to separate into an aqueous and organic layer, with the PLFAs remaining in the organic layer. The organic phase is then dried and fractionated into neutral, glycolipid, and phospholipid fractions. Once the phospholipid fraction has been isolated, it can be analyzed with gas chromatography/mass spectrometry to find the relative proportions of different PLFAs based on their differing levels of volatility. The differences in fatty acid volatility based on their structural differences can be measured for a mixed sample in this step. Retention time is the time required for a fatty acid fraction to pass through the gas chromatograph; the shorter the retention time, the more volatile the compound. The relative intensity of the resulting peak is a measure of the relative amount of each compound. By this means, the number of different fatty acids and their relative abundances in a sample can be observed. (An example of a graph of retention time vs. relative intensity). 1) Tornabene TG. 1985. Lipid analysis and the relationship to chemotaxonomy. Methods in Microbiology. 18:209-234.   2) Moore-Kucera J, Dick RP. 2008. PLFA profiling of microbial community structure and seasonal shifts in soils of a Douglas-fir chronosequence. Microbial Ecology. 55:500-511.    3) White DC. 1983. Analysis of microorganisms in terms of quantity and activity in natural environments. //Society for General Microbiology Symposium//. 34:37-66.    4) Kieft TL, Wilch E, Oconnor K, Ringelberg DB, White DC. 1997. Survival and phospholipid fatty acid profiles of surface and subsurface bacteria in natural sediment microcosms. Applied and Environmental Microbiology. 63:1531-1542.   5) Hui Y, Cong CZ, Hui ZW. 2008. PLFA analysis and its applications in the study of soil microbial diversity. Acta Pedologica Sinica.    6) Hendrick DB, Pledger RD, White DC, Baross JA. 1992. //In situ// microbial ecology of hydrothermal vent sediments. FEMS Microbial Ecology. 101:1-10.   7) Guckert JB, Hood MA, White DC. 1986. Phospholipid ester-linked fatty acid profile changes during nutrient deprivation of //Vibrio cholera//: Increase in the //trans/cis// ratio and proportions of cyclopropyl fatty acids. Applied and Environmental Microbiology. 52:794-801.
 * Phospholipid-Linked Fatty Acid (PLFA) Profiling **