The aim of the present study was to explore the difference in toxicity mechanism of TiO2 nanoparticles (NPs) at low concentrations (≤1 μg mL−1), in a freshwater bacterial isolate, Bacillus licheniformis, under light (UV-illuminated) and dark (non-illuminated) conditions. Standard plate count and MTT assays showed the dose dependent decrease in the bacterial cell viability. The difference in reduction of cell viability under light (20.7%) and dark conditions (21.3%) was statistically non-significant at 1 μg mL−1 concentration and 2 h interaction period. The fluorescence microscopy of the NP interacted cells (1.0 μg mL−1, 2 h) under light and dark conditions showed the mixture of live and dead cells. A significant dose dependent increase in intracellular ROS generation compared to control was noted. The ROS level after 2 h of interaction was significantly higher under light conditions (7.4 ± 0.13%) as compared to dark conditions (4.35 ± 0.12%). The LDH analyses confirmed a statistically significant increase in membrane permeability under dark conditions compared to the light conditions. The NPs were stable against aggregation in sterilized lake water matrix for a period of 24 h, under both light and dark conditions. However, in the presence of bacterial cells an elevated rate of sedimentation was noted under dark conditions. The electron microscopic (SEM, TEM) observations suggested the concentration buildup of NPs near the plasma membrane leading to internalization. The zeta-potential analysis proved that NP attachment was not charge based. The FTIR studies demonstrated the possible involvement of surface functional groups in the attachment. The concentration of dissolved Ti4+ ions was found to be negligible during the test period. The dominant cytotoxicity mechanism under light conditions was found to be ROS generation, whereas, NP attachment to the cell membrane leading to membrane damage significantly contributed in dark conditions.
