BACKGROUND: In cometabolic transformation of 4-chlorophenol (4-cp) in the presence of phenol and sodium glutamate (SG), a new biphasic growth pattern has been reported. This study investigates the physiological changes of Pseudomonas putida P8 during the biphasic growth by means of 2-dimensional gel electrophoresis (2-DE) and MALDI-TOF analyses. RESULTS: A total of 49 protein spots were selected and identified in the 2-DE gels from P. putida P8 grown on a mixed substrate containing 200 mg L-1 of phenol, 200 mg L-1 of 4-cp and 1000 mg L-1 SG. Among them, 16 protein spots were found differentially expressed in the two exponential growth phases during the biphasic growth, including six catabolic enzymes (DmpC, DmpD, DmpE, DmpF, DmpG and AspA) for substrate utilization. The expression of other proteins involved in detoxification and stress responses, carbohydrate and energy metabolism, and environmental information processing as well as a multifunctional xenobiotic reductase (XenA) was quantitatively analyzed and discussed. CONCLUSION: The expression levels of the identified catabolic enzymes during growth in the two growth phases correlated well with the substrate utilization patterns observed in previous kinetics studies. Furthermore, the results show that cells growing on a mixture of aromatic substrates undergo significant physiological changes. (C) 2009 Society of Chemical Industry