Standardisation and implementation of an optimised method for lipid profiling of microbial communities

Abstract

Although the importance of phospholipid fatty acid (PLFA) analysis to characterise microbial communities has been recognised over the years, the vast number of modifications to this traditional method as well as discrepancies regarding its data interpretation have given rise to scepticism about the accuracy of PLFA analysis in environmental studies. Therefore, comprehensive literature-based studies were necessary to investigate these uncertainties. Additionally, new and modernised high-throughput approaches, such as metabolomics, holds promise for investigating microbial communities and there was a need to evaluate such an approach against traditional methods for characterisation of fatty acid methyl esters (FAMEs). The literature reviews conducted for this study evaluated i) different methods and their modifications in terms of the main steps of PLFA analysis, namely extraction, fractionation, derivatisation and quantification; and ii) recent applications of PLFA analysis in environmental studies with specific reference to data interpretation. Both reviews made significant contributions to summarise and clarify a vast body of literature on the topics and were published in the Journal of Applied Microbiology. During the experimental work, targeted analysis of selected FAMEs to characterise both homogenised soil and pure bacterial cultures were evaluated by comparing three methods, namely fractionated PLFA, total lipid extract (TLE) and metabolomics analysis. As an additional outcome, untargeted analysis was also investigated. The results showed that the extraction method and different derivatisation techniques had an effect on FAME concentrations and on repeatability between sample replicates. Furthermore, extraction method had a greater influence when analysing pure culture samples, while derivatisation technique was more important when analysing soil samples. The most appropriate derivatisation technique for fractionated PLFA analysis proved to be mild alkaline methanolysis, while methanolysis with oximation and silylation provided the best results for TLE analysis. The metabolomics-based approach benefited from the inclusion of an oximation step for derivatisation of a complete metabolite profile. An assessment of the applicability of the different methods to distinguish between soil microbial communities exposed to various soil fumigant treatments in a greenhouse study, showed that even though TLE analysis gave higher FAME yields than PLFA analysis, its discrimination potential between treatments were much lower. Therefore, PLFA analysis was recommended for FAME characterisation in microbial communities. The untargeted metabolomics analysis has potential in differentiating between different treatments, despite representing the larger soil community and not microbial communities per se. Based on all the obtained results, a standard operating procedure for the targeted analysis of FAMEs and the untargeted analysis of all metabolites from soil is proposed