The use of isotopic and lipid analysis techniques linking toluene degradation to specific microorganisms: applications and limitations

Phospholipid fatty acid (PLFA) analysis combined with 13C-labeled tracers has been used recently as an environmental forensics tool to demonstrate microbial degradation of pollutants. This study investigated the effectiveness and limitations of this approach, applied to the biodegradation of toluene by five reference strains that express different aerobic toluene degradation pathways: Pseudomonas putida mt-2, P. putida F1, Burkholderia cepacia G4, B. pickettii PKO1, and P. mendocina KR1. The five strains were grown on mineral salts base medium amended with either 10 mM natural or [13C-ring]-labeled toluene. PLFA analysis showed that all five strains incorporated the toluene carbon into membrane fatty acids, as demonstrated by increases in the mass of fatty acids and their mass-spectrometry fragments for cells grown on 13C-labeled toluene. Because of its ubiquitous presence and high abundance in bacteria, C16:0 fatty acid might be a useful biomarker for tracking contaminant degradation and 13C flow. On the other hand, the 13C-label (which was supplied at relatively high concentrations) generally exerted an inhibitory effect on fatty acid biosynthesis. Differences in fatty acid concentrations between cells grown on natural versus 13C-labeled toluene would affect the interpretation of lipid profiles for microbial community analysis as indicated by principal component analysis of fatty acids. Therefore, caution should be exercised in linking lipid data with microbial population shifts in biodegradation experiments with 13C-labeled tracers.