(511l) Physiological Understanding Of P. Putida P8 In Biphasic Growth On Mixture Of Phenol, 4-Chlorophenol And Glutamate

Bin, C., National University of Singapore
Loh, K. C., National University of Singapore

Biphasic growth of P. putida P8 in a ternary co-metabolic system containing 4-chlorophenol (4-cp), phenol and sodium glutamate was reported previously#. While concurrent utilization of phenol and glutamate was observed in P. putida grown on mixtures of phenol and glutamate, in the presence of 4-cp, a new growth pattern emerged. This growth pattern, characterized by two exponential growth phases separated by an intermediate lag phase, manifested under certain unique concentration combinations of the three substrates. It was found that cells preferentially utilized phenol in the first exponential growth phase while glutamate was mainly utilized in the second growth phase. Through experimental observations and kinetics modeling, it was inferred that the biphasic growth was due to the disparity of the toxicity of 4-cp to phenol-oxidizing and SG-oxidizing enzyme activities. While this was sufficiently accepted, it was rather general in its meaning. In addition, although lots of work has been done in microbial degradation of mixture of aromatic compounds, little is known about the physiological changes that occur during the growth on mixed substrates. In this research, P. putida P8 cells were cultured in minimal media supplemented with a mixture of 1000 mg/L of glutamate, 200 mg/L of phenol and 200 mg/L of 4-cp. Cells were harvested at both of the first (GP-I) and the second (GP-II) mid-exponential phases at specific growth rate of 0.10 h-1 and 0.22 h-1 with OD600 of around 0.10 and 0.53, respectively. Two-dimensional gel electrophoresis (2-DE) was used to resolve the proteins in cell lysates. Forty-nine protein spots whose averaged normalized quantities in GP-I were at least 1.5-fold or higher and 0.67-fold or lower versus GP-II were excised from the gels for identification using MALDI-TOF MS. Among them, 16 spots were found differentially expressed with p < 0.05 during the Student's t-test. Based on identities and functions of the proteins which have shown differential expression, the global physiological changes within the cells during the biphasic growth were elucidated to better understand the toxicity imposed by 4-cp and also to ascertain the long term stability of the treatment process that employs these microorganisms. Furthermore, the information obtained from this research represents the beginning contributions to scientific knowledge for a mixed pollutant co-metabolic system on a proteome level.

#Wang, S. J. and K. C. Loh (2000). New cell growth pattern on mixed substrates and substrate utilization in cometabolic transformation of 4-chlorophenol. Water Res., 34(15), 3786-3794. Wang, S. J., K. C. Loh, et al. (2003). Prediction of critical cell growth behavior of Pseudomonas putida to maximize the cometabolism of 4-chlorophenol with phenol and sodium glutamate as carbon sources. Enzyme Microb. Technol., 32(3-4), 422-430.