Pyrolysis-assisted transesterification for accurate quantification of phospholipid fatty acids: Application to microbial community analysis in 1000-years paddy soil chronosequence

Abstract Phospholipid fatty acids (PLFAs) are widely used to assess soil microbial community and metabolic activity in various environmental samples. However, the transesterification of phospholipids into PLFAs still utilize homogeneous acid/alkaline catalyst, which may increase environment risk. This study developed and validated an accurate method for the determination of PLFAs in soil utilizing pyrolysis-assisted transesterification with diatomaceous earth. The thermolysis behavior of phospholipids, optimal pyrolysis conditions, tolerance against impurities, and recyclability of porous material were evaluated. Under optimal conditions, the transesterification can complete in only 8 min, with high impurity tolerance. The repeatability (relative standard deviation (RSD) between 2.59% and 12.12%), reproducibility (RSD between 5.22% and 16.22%), and linearity (r between 0.9259 and 0.9999) were excellent. This method was applied to analyze the PLFAs to evaluate microbial community of 1000-years paddy soil chronosequences and the relationships among soil chemical properties, enzyme activities and microbial community composition. Soil microbial biomass PLFAs increased significantly over time followed by slight fluctuation at 60–1000 years, but the ratios of soil fungal to bacterial and gram-positive to gram-negative PLFAs remained constant. In addition to soil total N and organic matter, soil enzyme activities related to N cycling metabolism also positively significantly influenced microbial communities. Overall, the developed PLFAs method using pyrolysis-assisted transesterification was environmentally friendly, efficient, accurate, stable, and suitable alternative to acid/alkaline transesterification of phospholipids.