Adaptive Laboratory Evolution of the Hydrocarbonoclastic Marine Strain Rhodococcus ruber MSA14 to High Pyrene Concentrations.

Rhodococcus ruber MSA14, isolated from oil-polluted coastal sediments in Baja California, Mexico, has previously been shown to degrade pyrene, highlighting its potential for polycyclic aromatic hydrocarbons (PAH) bioremediation. In the present study, MSA14 was subjected to adaptive laboratory evolution using progressively increasing concentrations of pyrene as the sole carbon and energy source, resulting in the isolation of a high-pyrene-adapted strain, R. ruber AD0911. Compared with the parental strain, AD0911 displayed improved growth and pyrene tolerance, modified colony morphology, enhanced biofilm biomass, altered biosurfactant production patterns, and increased oxidative stress tolerance. Whole-genome sequencing and breseq-based variant calling identified 75 mutations in AD0911 relative to MSA14, affecting membrane-associated proteins, transcriptional regulators, cell signaling, and stress-response components. These adaptive mechanisms identified in AD0911 provide new insight into the evolution of bacterial tolerance under high hydrocarbon stress and suggest that adaptation to extreme pyrene exposure was driven primarily by physiological optimization rather than changes in PAH catabolic enzymes.