Appearance of mutants of P. putida mt-2 during fed-batch cultivation. Colonies with a morphology similar to that of P. putida mt-2 on NB plates were replica plated on Tol plates and selective plates to determine their phenotype. Activity of XylE was determined by spraying Tol plates with replica-plated colonies with a catechol solution and checking for the formation of the yellow product HMS. Symbols: •, colonies able to grow on p-xylene and toluene and forming HMS after being sprayed with catechol (Tol+ p-Xyl+ XylE+ [wild-type]); □, colonies unable to grow on p-xylene and toluene (Tol− p-Xyl−); ▪, colonies able to grow on toluene, unable to grow on p-xylene, and not forming HMS when sprayed with catechol (Tol+ p-Xyl− XylE−); ○, colonies able to grow on toluene, unable to grow on p-xylene, and forming of HMS after being sprayed with catechol (Tol+ p-Xyl− XylE+); ↑, time at which TCE addition was started.