Stabilization of Recombinant Bioluminescent Bacteria for Biosensor Applications

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Abstract
The effects of growth media, drying protocols, and storage conditions on the long-term survival and activity of Escherichia coli, which harbors genetic fusions of inducer-responsive promoters to bacterial luminescence (lux) genes, were evaluated. Bacteria were grown in a regular or high-salinity (0.5 M NaCl) complex medium (Luria–Bertani, LB) or minimal medium (M-9), freeze-dried or dried by vacuum under ambient temperatures, and stored at –20°C or 37°C. The survival rates of vacuum-dried and freeze-dried bacteria grown in LB ranged from 1% to 2% and 19% to 20%, respectively, and were not as affected by medium salinity. Survival rates of vacuum-dried and freeze-dried bacteria grown in M-9 ranged from 22% to 75% and 45% to 63%, respectively. Here, however, high salinity promoted survival of vacuum-dried bacteria but negatively affected the survival of freeze-dried bacteria. Survival rates of vacuum-dried bacteria stored at 37°C were substantially higher than those of freeze-dried bacteria and were maximal with high-salinity M-9-grown bacteria achieving 37% and 4.7% after 10 and 35 days, respectively. Bioreporter activity was typically correlated to survival but was affected by growth media composition and drying protocols. Interestingly, growth in high-salinity M-9 medium, which typically promoted high survival rates and improved storage stability, caused a substantial decrease in activity of tested bioreporter strains. Our results demonstrate the need to further optimize growth conditions and drying protocols for improved short- and long-term stability of our dried bioreporter bacteria, especially upon storage under ambient conditions.