Figure 2

The R. opacus PD630 44B2 transposon mutant accumulates less triacylglycerides (TAGs) than the wildtype. A. Sudan Black staining of wildtype (WT) and 44B2 colonies grown on minimal medium supplemented with 4% (w/v) glucose and 0.15% (w/v) ammonium sulfate. The lipophylic dye Sudan Black was used to identify transposon mutants that accumulated less TAGs than the wildtype strain. Strains accumulating less TAGs absorb and retain less of the dye than the wildtype strain as can be seen in the 44B2 mutant colony. B. Thin-layer chromatography of the wildtype and 44B2 mutant grown for varying lengths of time. Lipid extracts from the two strains from progressive time points were resolved using a two solvent system to separate TAGs from other lipid species followed by charring. TAG standards were used to identify the Rf value for TAGs under the chromatographic conditions used. The 44B2 mutant accumulates less TAGs than the wildtype at all time points while the relevant abundance of other lipids remains largely unchanged. C. Biomass from kinetic assays was subjected to transesterification and the resulting FAMEs were separated via gas chromatography and detected using FID. The resulting spectrum was quantified against a standard curve of methyl esters. The wildtype strain produced more lipids than the 44B2 mutant at all time points reaching a maximum of 37.74% of total cell dry weight at 120 hours post inoculation as compared to the 44B2 mutant which peaked at 18.66% lipids.