Phospholipid-derived Fatty Acids - Phospholipid Fatty Acid (PLFA) Analysis

Phospholipid Fatty Acid (PLFA) Analysis

Phospholipid fatty acids (PLFA) are an essential structural component of all microbial cellular membranes. PLFA analysis is a technique widely used for estimation of the total biomass and to observe broad changes in the community composition of the living microbiota of soil and aqueous environments. There has been a surge of interest in PLFAs in recent years, evident from the large increase in peer-reviewed journal references on the subject. However, there is increasing concern that some researchers are assigning PLFAs to specific microbial classes when in fact those PLFAs are present in a broad range of life forms.

Phospholipids can occur in many biological classes (such as in plant roots, fungi as well as in soil bacteria), so care has to be taken in over-assigning PLFA biomarkers to the wrong class. Even though phospholipids occur in many different life forms, the fatty acid side chains between differing life forms can be quite unique. Polyunsaturated fatty acids (e.g. 18:3 ω3c) are found in plants, algae and cyanobacteria, but are often not present in bacteria. Monounsaturated fatty acids (particularly at the omega-7 position), odd-chain saturated fatty acids (e.g. 15:0), branched-chain fatty acids (mainly iso/anetiso and 10-methyl) and cyclopropane fatty acids (e.g. 19:0 cyclo ω7c) are mostly synthesized by bacteria.

The basic premise is that as individual organisms (especially bacteria and fungi) die, phospholipids are rapidly degraded and the remaining phospholipid content of the sample is assumed to be from living organisms. As the phospholipids of different groups of bacteria and fungi contain a variety of somewhat unique fatty acids, they can serve as useful biomarkers for such groups. PLFA profiles and composition can be determined by purifying the phospholipids and then cleaving the fatty acids for further analysis. Knowledge of the composition and metabolic activity of the microbiota in soils, water and waste materials is useful in optimizing crop production, in bioremediation and in understanding microbial ecosystems.

Analysis by PLFA has been the most widely used such technique due to the sensitive and reproducible measurement of the dominant portions of the soil microbiota and low cost per sample of the analysis. Sampling of soil populations by culturing has proven not cost effective and results in biased results due to the differing ease of culturing of some organisms. A new 96-well plate PLFA extraction procedure has been developed which represents a 4-to-5 fold increase in throughput over traditional PLFA extraction methods. This new method, coupled to new software tools for analyzing the PLFA data, will be useful to laboratories performing large numbers of PLFA analyses, or for laboratories wanting to begin PLFA research.