Gas-phase environment activates an alternative catabolic route in toluene-degrading Acinetobacter

Volatile aromatic compounds are important industrial feedstocks but also major environmental pollutants, highlighting the need for bioprocesses for their removal and valorization. Although gas-phase bioprocesses offer practical advantages for handling poorly water-soluble and highly volatile substrates, how gas-phase environments alter microbial metabolism remains poorly understood. Here, we investigated the effect of gas-phase conditions on toluene metabolism in the highly adhesive aromatic hydrocarbon-degrading bacterium Acinetobacter sp. Tol 5. A mutant lacking todC1, which encodes an essential component of the toluene dioxygenase, failed to grow on toluene in liquid culture but retained the ability to grow on solid media under a toluene atmosphere. Consistent with this phenotype, the mutant showed no detectable toluene degradation in the liquid phase, whereas it degraded toluene under gas-phase conditions after a prolonged lag phase. Gas chromatography-mass spectrometry (GC-MS) analysis revealed the accumulation of o-cresol and p-cresol specifically in the mutant under toluene vapor, indicating that toluene metabolism had shifted to an alternative route involving cresol intermediates. In addition, transcriptome analysis identified strong induction of the mph operon encoding phenol monooxygenase (PMO), suggesting that PMO is a likely candidate enzyme mediating TDO-independent toluene oxidation under gas-phase conditions. Together, these results demonstrate that the gas-phase environment can activate an alternative catabolic route in Tol 5 that is not active during conventional liquid cultivation. Our findings highlight the importance of direct metabolic analysis under gas-phase conditions for understanding and designing bioprocesses using highly volatile substrates.