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One limitation of employing lux bioreporters to monitor in situ microbial gene expression in dynamic, laboratory-scale systems is the confounding variability in the luminescent responses. For example, despite careful control of oxygen tension, growth stage, and cell number, luminescence from Pseudomonas putida RB1353, a naphthalene-degrading lux bioreporter, varied by more than sevenfold during saturated flow column experiments in our laboratory. Therefore, this study was conducted to determine what additional factors influence the luminescent response. Specifically, this study investigated the impact of temperature, pH, and initial cell number (variations within an order of magnitude) on the peak luminescence of P. putida RB1353 and the maximum degradation rate (V(max)) during salicylate and naphthalene catabolism. Statistical analyses based on general linear models indicated that under constant oxygen tension, temperature and pH accounted for 98.1% of the variability in luminescence during salicylate catabolism and 94.2 and 49.5% of the variability in V(max) during salicylate and naphthalene catabolism, respectively. Temperature, pH, and initial substrate concentration accounted for 99.9% of the variability in luminescence during naphthalene catabolism. Initial cell number, within an order of magnitude, did not have a significant influence on either peak luminescence or V(max) during salicylate and naphthalene catabolism. Over the ranges of temperature and pH evaluated, peak luminescence varied by more than 4 orders of magnitude. The minimum parameter deviation required to alter lux gene expression during salicylate and naphthalene catabolism was a change in temperature of 1 degrees C, a change in pH of 0.2, or a change in initial cell number of 1 order of magnitude. Results from this study indicate that there is a need for careful characterization of the impact of environmental conditions on both the expression of the reporter and catabolic genes and the activities of the gene products. For example, even though lux gene expression was occurring at approximately 35 degrees C, the luciferase enzyme was inactive. Furthermore, this study demonstrates that with careful characterization and standardization of measurement conditions, the attainment of a reproducible luminescent response and an understanding of the response are feasible.

applied and environmental microbiology

Effect of Temperature, pH, and Initial Cell Number on luxCDABE and nah Gene Expression during Naphthalene and Salicylate Catabolism in the Bioreporter Organism Pseudomonas putida RB1353