Tolerance to Sudden Organic Solvent Shocks by Soil Bacteria and Characterization of <i>Pseudomonas putida</i> Strains Isolated from Toluene Polluted Sites

Abstract

Upon a sudden addition of toluene to soil (10% vol/wt) a significant proportion (about 1%) of the soil indigenous bacteria survived, the survival ones recolonized the soil to reach a high cell density. Two bacterial strains called 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 after 15 days incubation. The strains were taxonomically identified as Pseudomonas putida sensu stricto based on 16S rRNA sequences. Another strain (P. putida R1) isolated from a biofilm washed with toluene-polluted waters was also 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) toluene, 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% (vol/vol) ethylbenzene. After exposure to aromatic hydrocarbons, all three strains isomerized the cis-fatty acid lipids C16:1ω9 and C18:1ω11 to their trans isomers. This change led to the rigidification of the cell membrane. All three strains exhibited efflux pumps for the removal of toluene from the cell membrane, but the efflux systems appeared to be more efficient in the MTB6 strain based on its higher tolerance to toluene and their increased capacity to remove toluene from the cell membranes. The solvent-tolerant MTB6 strain established best in soils polluted with toluene and mineralized this aromatic hydrocarbon in situ.