Impacts of pressure on the activity of hydrocarbon degrading three strains of bacteria

Bioorthoganol Non-Canonical Amino Acid Tagging (BONCAT) was used in combination with fluorescence activated cell-sorting (FACS) to assess the impacts of elevated hydrostatic pressure on the protein activity of hydrocarbon degrading bacteria. Three study bacteria strains were used - Halomonas titanicae strain (Bead 10BA); Alcanivorax xenomutans strain (Bead 18); and Shewanella indica strain (Bead 36). Each strain was grown up to mid-log phase in ONR7a minimal medium supplemented with 50 mM hexadecane for each growth condition (aerobic flask, 0.1 MPa, 10 MPa, 25 MPa. BONCAT labeling using HPG was performed using the protocol outlined in Hatzenpichler et al. 2016, PNAS 113(28). Samples were then flow sorted using a ZE5 Cell Analyzer (BioRad) equipped with small-particle detection module. The DNA stain used was Hoechst 33342, and the BONCAT stain was an Alexa Flour 488 azide dye. Negative controls included PBS storage buffer and cultures not labeled with HPG, to define the level of nonspecific BONCAT stain fluorescence. The fraction of BONCAT positive cells was determined over time for the 0.1 MPa, 10 MPa, and 25 MPa incubations.