We hypothesize that O 2 uncoupling and the ensuing formation of ROS limits the fitness of Acidovorax sp. strain JS42 in a substrate-dependent manner which would also correlate with the adaptation of mutant strains to alternative substrates. To examine O 2 uncoupling as a measure for the characterization of microbial adaptation processes, we compared the oxygenation efficiencies obtained in vitro (in enzyme assays) with in vivo parameters for microbial activity (as NO - 2 formation rates) and growth (from inverse doubling times). The in vivo parameters were taken from previously experiments with evolved Acidovorax strains and are referred to here collectively as NT + strains. ) became especially evident in the doubly-substituted oxygenase variant L238V-M248I but not so much in M248I. This trend implies that the increase in dioxygenation activity and thus NO - 2 formation rate in vivo could have been the consequence of the decrease of O 2 uncoupling that we quantified in vitro. Deviations from the linear correlation illustrate that additional factors than the oxygenation efficiency of the variants are relevant for microbial activity; a finding that points to the inherent limitations of assessing substrate specificity exclusively from the activity of the dioxygenase.