(220g) Effect of Temperature, Analyte Concentration and Cell Growth Phase on the Luminescence of Pseudomonas Putida Tva8 Induced by Trichloroethylene

Han, J., Northeastern University
Bergman, K., Northeastern University
Applegate, B., Purdue University
Sacco, Jr., A., Northeastern University

The global market for biosensors and related materials is projected to grow from $6.1 billion in 2004 to $8.2 billion in 2009, at an average annual growth rate of 6.3%. Biosensors using whole microbial cells as the recognition element have been shown to exhibit potential to both detect the presence of certain chemical substances with high selectivity and determine their concentrations with high sensitivity (qualitative and quantitative sensing properties). The motivation for using living components stems from the bacterial self-assembly, self-repairing, and self-calibration capabilities, which are strongly affected by temperature, analyte concentration and cell growth phase. In the present work, the effect of these factors on the luminescent response of Pseudomonas putida TVA8 induced by trichloroethylene (TCE) has been investigated. The results obtained will aid in further development and applications of whole-cell based biosensors.

Bacterium P. putida TVA8 was genetically engineered to emit light at 490 nm in response to the presence of TCE. No cell growth inhibition was detected upon addition of up to 1000 ppbv TCE (testing concentration range). Initial cell number effect: Bioluminescence increased linearly with increasing initial cell concentrations. Therefore, specific bioluminescence, defined as bioluminescent response to viable cell concentration ratio was used to normalize light emission. Temperature effect: Cell population increased exponentially with temperature in the range of 21-34oC, and then decreased at 37oC. The optimal temperature for bioluminescence was determined to be 23oC. At this temperature, the bacteria could detect less than 25 ppbv TCE, and the specific bioluminescence measured 2 hours after the addition of 500 ppbv TCE was ~6,000- times higher than that of the uninduced control. Analyte concentration effect: Two linear regions of bacterial response to TCE concentration were detected. The linear regions were between 50-150 ppbv TCE and 200-1000 ppbv TCE at 21-25oC, and 100-200 ppbv TCE and 350-1000 ppbv TCE at 28oC. In both regions, specific bioluminescence increased linearly with TCE concentration. However, in the first linear region the light intensity remained low (~13-fold higher than the uninduced control measured after 2 hours at 23oC and 150 ppbv TCE); whereas in the second linear region the bioluminescence measured after 2 hours at 23oC and 1000 ppbv TCE was ~10,000-times higher than the control without TCE . Cell growth phase effect: Bioluminescence was also affected by the bacterial physiological states. No light response was detected in stationary phase. However, in logarithmic growth phase, cells responded to TCE induction stronger than in lag phase. In addition, the linear correlation between specific bioluminescence and TCE concentration was only observed in logarithmic phase. Response time: The luminescent response time was dependant on TCE concentration and cell incubation temperature. At 23oC, the delay period was ~1.5 hours, 50 minutes and 40 minutes in 100 ppbv, 500-2,000 ppbv, and 3,500-10,000 ppbv TCE, respectively. The instant inhibition of cell growth that occurred in TCE concentrations higher than 3,500 ppbv resulted in a slower bioluminescence increase compared to that detected for TCE lower than 2,000 ppbv in concentration due to possible toxicity.

The kinetics measured for both cell growth and bioluminescence was well described using Arrhenius expression and Michaelis-Menten expression. The experimental results together with the kinetic investigation provide guidelines for future biosensor design using whole cells. The study points to two major requirements which need to be incorporated into the biosensor: 1) temperature control unit and 2) cells maintained in the logarithmic growth phase which could be achieved using micro-chemostat.