Contribution of calorimetry to real-time monitoring of Pseudomonas putida KT2440 growth under oxygen-limited conditions and heavy metal stress
摘要
Heat-conduction calorimeter measurements and appropriate data processing have been proposed to realize real-time assessment of aerobic growth of Pseudomonas putida KT2440 suspended in the minimal medium M1, containing D-glucose as a carbon source and nutrient, under combined oxygen-limited conditions and heavy metal stress. A commercial twin calorimeter, without stirring and injection systems, was adapted to simulate either oxygen-poor or uncontrolled O2 conditions. Monitoring of bacterial growth at specified time intervals by UV–Vis spectroscopy was conducted concurrently to first determine the duration of the different growth phases. In the same manner, the glucose uptake was followed by high-performance liquid chromatography. The time lag between the glucose uptake and the heat release, or even the secondary heat release during the stationary phase, was analyzed in line with the restricted availability of dissolved oxygen inside the calorimetric ampoule. An approximation was then proposed to estimate the average rate of heat release during the exponential growth phase based solely on calorimetric measurements. The effect of adding ionic Cd(II), Pb(II), and As(III) species to the M1 medium was subsequently studied using the calorimeter operating in an oxygen-limited regime. The half-maximal inhibitory concentration, IC50, was estimated from the recorded curves. The IC50 values were as follows: 139 ± 22 µmol L−1 for Pb(II), 22.5 ± 3.3 µmol L−1 for Cd(II), 138 ± 22 µmol L−1 for As(III), thus indicating the most harmful effect of cadmium species on the exponential growth of P. putida. The overall toxicity of metal species was an interplay between the direct perturbation of cellular processes and the phosphate depletion through precipitation.