Tolerance to sudden organic solvent shocks by soil bacteria anal characterization of Pseudomonas putida strains isolated from toluene polluted sites

Upon a sudden addition of toluene to soil (10% vol/wt) a significant propor tion (about1%) of the soil indigenous bacteria survived, the survival ones recolonized the soil to reach a high cell density. Two bacterial strains ca lled MTB5 and MTB6, which use toluene as the sole carbon and energy source, were isolated from the soil polluted with this large amount of toluene aft er 15 days incubation. The strains were taxonomically identified as Pseudom onas putida sensu stricto based on 16S rRNA sequences. Another strain (P. p utida R1) isolated from a biofilm washed with toluene-polluted waters was a lso studied regarding toluene degradation and toluene tolerance. All three strains used the toluene dioxygenase pathway for toluene metabolism. Strain P. putida MTB6 was able to grow on liquid medium with 10% (vol/vol) toluen e, whereas the other two strains did not grow at concentrations of toluene higher than 0.1% (vol/vol). All three strains grew in the presence of 1% (v ol/vol) ethylbenzene. After exposure to aromatic hydrocarbons, all three st rains isomerized the cis-fatty acid lipids C16:1 omega 9 and C18:1 omega 11 to their trans isomers. This change led to the rigidification of the ceil membrane. All three strains exhibited efflux pumps for the removal of tolue ne from the cell membrane, but the efflux systems appeared to be more effic ient in the MTB6 strain based on its higher tolerance to toluene and their increased capacity to remove toluene from the cell membranes. The solvent-t olerant MTB6 strain established best in soils polluted with toluene and min eralized this aromatic hydrocarbon in situ.