Application of the Solvent Effect on Bioluminescent Reporter Bacteria as a Real-Time Membrane Toxicity Assay

Bioluminescent bioreporters are widely used across various scientific disciplines due to the well-characterized bacterial bioluminescence mechanism. However, solvent-induced membrane perturbations may confound the use of bioreporters in assessing cellular toxicity from environmental contaminants. This study investigated the solvent effect, wherein membrane damage increases intracellular availability of bioluminescent reaction precursors, increasing the light produced. A new online in-situ monitoring system was also tested with multiple bioluminescent reporters, including a newly constructed Pseudomonas fluorescens M3A strain, exposed to toluene, trichloroethylene, acetone, phenol, and creosote derived from beechwood tar. Additional tests included the introduction of carbon nanotubes, fullerene, and fullerenol. A solvent effect was confirmed by the detection of increased bioluminescent signal and the occurrence of fatty acid release (P < 0.05). Phenol (25 ppm), a benchmark for bactericidal activity, demonstrated luminescence enhancement via the solvent effect. Membrane toxicity assays showed that P. fluorescens M3A responded sensitively to sublethal and lethal membrane disruptions, whereas V. fischeri MJ1 did not exhibit a solvent effect, and its luminescence changes were not correlated with viability (P > 0.05). These results indicate that P. fluorescens M3A is a sensitive biosensor for detecting environmental contaminants and identifying both lethal and sublethal membrane perturbations. The findings underscore essential considerations when utilizing bacterial bioluminescence as a proxy for gene expression or cellular physiology.