Viability and Luciferase Activity of Freeze-Dried Recombinant Biosensor Cells for Detecting Aromatic Hydrocarbons

Aromatic hydrocarbons are of major concern among genotoxic chemicals due to their toxicity and persistence. Some microorganisms can utilize aromatic hydrocarbons as carbon and energy sources by inducing expression of catabolic operon(s). The XylR regulatory protein activates transcription of the catabolic enzymes to degrade BTEX (benzene, toluene, ethylbenzene, and xylene) from its cognate promoters, Pu and Ps upon exposure of the cells to the aromatic hydrocarbons. The activity of XylR on the promoters was previously monitored using luciferase luc reporter system. The xylR, its promoter Pr and the promoter Po for the phenolic compound catabolic operon were introduced upstream of firefly luciferase luc in the pGL3b vector to generate about 7.1 kb of pXRBTEX. Here E. coli harboring the plasmid was freeze-dried under various conditions to fin,d optimal conditions for storage and transport. The cell viability and luciferase activity were maintained better, when the cells were freeze-dried at -7$0^{\circ}C$ in the addition of the 10% skim milk or 12% sucrose. However, coaddition of protectants such as 10% skim milk plus 10% glucose or 12% sucrose plus 10% glucose, resulted in much better viability and bioluminescence activity compared with the effect of single addition of each protectant. In addition, it was shown that the freeze-dried cells maintained almost intact bioluminescent activities and cell viability for at least 1 week after freeze-drying. This work demonstrated that the properly freeze-dried recombinant bacterial cells could be utilized as a whole-cell biosensor for simple and rapid monitoring of BTEX in the environment.