Tracing 2,4-D metabolism in Cupriavidus necator JMP134 with 13C-labelling technique and fatty acid profiling

The use of stable isotope probing of fatty acid methyl esters (FAME-SIP) is a powerful tool to study the microorganisms involved in xenobiotic biodegradation in soil. Nevertheless, it is important to determine how representative these molecules are of microorganisms both qualitatively and quantitatively. Using Cupriavidus necator JMP134 as a simple experimental model, we showed that the (13)C-labelling technique can be used both at a global (here defined as cellular, medium and CO(2)) and molecular level to study the metabolism of 2,4-Dichlorophenoxyacetic acid (2,4-D). Although isotopic fractionation among substrate, biomass and FAME were observed, this technique could be used when using a highly (13)C-labelled substrate. Global (13)C analyses gave similar results to those obtained with traditional (14)C-labelling methods. After 10 days of incubation 59% of ring-C was mineralized and about 30% remained in the liquid medium. A maximum of 11% was incorporated into the biomass after 3 days. The assimilation yield of chain-C into the biomass was about half that of ring-C, suggesting a preferential use of chain-C for energy acquisition. Molecular analysis of the lipid fraction evidenced that the incorporation of the labelled 2,4-D did not correspond to a bioaccumulation of pesticide residues but to the metabolism of the 2,4-D carbons for FAME synthesis. Provided the labelling is located on the benzenic ring, the assessment of (13)C-FAME is a robust method to quantify the incorporation of (13)C into the whole microbial biomass. However, the variability of the (13)C incorporation among FAME due to physiological processes has to be considered in complex biological systems. The coupling of bulk and molecular studies with a simple model as C. necator JMP134 is a good approach for testing FAME-SIP.