Extraction and methylation parameters of phospholipid fatty acid analysis and their effect on total yield and community structure

Phospholipid fatty acids are membrane compounds of microbial cell walls and the structure of individual compounds is indicative for specific microbial groups. The extraction and analysis of phospholipid fatty acids in soils improved our understanding of factors driving microbial abundance and community composition. Despite the wide application of this method, important pitfalls persist which impede comparability of PLFA results between studies.

Here, we show that there was an effect of freeze-drying on the community composition. However, compared to the effect of using of old extraction solution (4 weeks) and two different methylation procedures, this effect seems negligible. Using old extraction solution, the overall yield of PLFA was 12% lower and and we observed significant differences in the relative abundances of functional microbial groups. But most importantly, base catalyzed methylation yielded 35% less PLFA compared to acid catalyzed methylation and the relative abundances of all microbial groups were completely different. Our results show that it is crucial to keep the analytical parameters constant to capture subtle treatment effects and that especially the use of different methylation methods prevents comparability between studies.