PHYSIOLOGICAL STRESS IN BATCH CULTURES OF PSEUDOMONAS-PUTIDA 54G DURING TOLUENE DEGRADATION

Physiological stress associated with toluene exposure in batch culture s of Pseudomonas putida 54G was investigated. P. putida 54G cells were grown using a continuous vapor phase feed stream containing 150 ppmv or 750 ppmv toluene as the sole carbon and energy source. Cells were e numerated on non-selective (R2A agar plates) and a selective minimal m edium incubated in the presence of vapor phase toluene (HCMM2). Differ ential recovery on the two media was used to evaluate bacterial stress , culturability and loss of toluene-degrading capability, A majority o f the bacteria were reversibly stressed and could resume active colony formation on selective medium after passage on non-selective medium. A small fraction of the bacterial cells suffered an irreversible loss of toluene degradation capability and were designated as Tol(-) varian ts, Numbers of stressed organisms increased with duration of toluene e xposure and toluene concentration and coincided with accumulation of m etabolic intermediates from incomplete toluene degradation. Respiring cell numbers in the batch cultures decreased as injury increased, indi cating a possible relationship between respiring and injured cells. Ra te expressions for injury, for formation of Tol(-) variants and for gr owth of Tol(-) variants were determined by calibrating a theoretical m odel to the results obtained. These rate expressions can be used to ca librate bioreactor models, and provide a basis for better design and c ontrol of bioremediation systems.