Methylation procedures affect PLFA results more than selected extraction parameters

Microorganisms are key players in organic matter and nutrient cycles of terrestrial ecosystems. The analysis of microbial membrane lipids, phospholipid fatty acids (PLFAs) has strongly improved our understanding of how microbial processes contribute to these cycles. The analysis has proven to yield robust results, but adaptations of analytical parameters to laboratory needs might lead to pitfalls and impede comparability of PLFA results between different studies. Here, we show how a set of four analytical parameters (freeze-drying vs. field moist, amount of sample extracted, age of solvent mixture, and methylation methods) influence the quantitative and qualitative results of PLFA analysis. Freeze-drying vs. field moist samples and the amount of sample extracted had only minor effects on PLFA concentrations and recovery of the microbial community structure. Nevertheless, these parameters are important to consider, especially if treatment effects in an experiment are expected to be low. The use of a four weeks old extraction solution resulted in 12% lower PLFA concentrations as well as significant differences in the relative abundance of functional microbial groups. This suggests that extraction solution should be prepared on the day of extraction or that the different components of the extraction solution should be added sequentially to the sample. Most importantly, the choice of the methylation method led to differences in both, PLFA concentrations (35%) and the relative abundance of functional microbial groups, making comparisons between studies difficult. Our study provides a valuable ranking of parameters that need to be considered during PLFA method implementation in a laboratory and also highlights the fact that comparability of studies using different methylation methods might be limited.