RESUMO
A bacterial strain YHl that used p-tert-Butylcatechol (TBC) as the sole carbon and energy source was isolated from the activated sludge of the wastewater treatment plant of a chemical factory. The strain was identified as Pseudomonas corrugate by morphological characteristics, physiological and biochemical properties, BIOLOG and the 16S rDNA sequence analysis. The degration rate of TBC was above 82% with an initial concentration of 500 mg.L-1 at 24-36°C and an initial pH of 7. 0-10. 0. Through single-factor experiments of its degradation characteristics of strain YH1, the results showed that its optimal additional carbon and nitrogen sources were sucrose and tryptone, the optimal temperature was 30°C, the optimal initial pH was 7. 0, and the optimal inoculation volume was 2%. In order to improve the TBC degradation rate, the concentrations of sucrose and tryptone and initial pH were identified as the main factors by Placket-Burman Assay. Then these factors reached their optimal region by Steepest Ascent. Finally, the optimal levels of those main factors were further optimized using Box-Behnken design and response surface analysis. The optimal conditions were as follows: sucrose concentration 3% (ρ), tryptone concentration 1. 44% (ρ), TBC concentration 400 mg.L-1, initial pH value 8. 12, inoculation amount 2. 97% (φ), temperature 30°C, training time 96 h. Under the optimal conditions mentioned above, the TBC degradation rate reached 98. 21%. Enzymology analysis and localization experiments showed that the TBC-degrading enzymes were intracellular proteins and the synthesis of catechol 1,2-dioxygenase (C12O) could be induced by TBC. Through design of specific PCR primers for the degrading enzymes, the gene encoding the catechol 1,2-dioxygenase was amplified from YHL. It was found that genes encoding TBC-degrading enzyme were located on plasmids by the plasmid detection and elimination experiments. In addition, YH1 was tolerant to high concentration of NaCl and many kinds of heavy metal ions, and resistant to multiple antibiotics. This paper provided some information for the effective treatment of complex industrial wastewater.
