Study On Breeding Technology Of Laser-induced Candida Tropicalis And Its Phenol-degrading Characters

At present, the biological treatment technology is in the transitional period from mixed culture to pure culture. In this paper, two strains to degrade phenol with high activity were isolated and identified from acclimated activated sludge by using the traditional microorganism breeding technology. In between, the yeast Candida tropicalis was completely investigated for its characters of phenolic compounds biodegradation.In order to enhance the capacities of C. tropicalis to degrade phenolic compounds, He-Ne laser was utilized to irradiate wild-type C. tropicalis. The optimal irradiation parameters were obtained through a lot of parallel experiments as following: 10mW10-15min, 12.5mW10-15min and15mW10min. Correspondingly, high positive mutation frequency was obtained in this range. In phenol biodegradation tests, 70 positive mutants with high phenol biodegradation potential were screened. Phenol biodegradation stabilities were investigated and CTM2 was attained and used in the following studies for its foremost biodegradation potential and stability.Because of the definitive effects of phenol hydroxylase and catechol dioxygenase on phenol metabolism, the activities of these two enzymes were investigated. It showed the activities of these two enzymes were higher in mutant strain CTM 2 than in the wild strain, which was presumably due to the laser irradiation. Thus, the full-length sequence of phenol hydroxylase gene was successfully cloned by PCR method in wild and mutant C. tropicalis, respectively. The comparison of amino acid of phenol hydroxylase between wild-type strain and mutant strain CTM2 illustrated four amino acids were mutated after the strain was irradiated by He-Ne laser. In addition, heterogenous expression of phenol hydroxylase validated the truth that the enzyme activity varied after cell irradiated by laser.The characters of phenol, m-cresol and 4-chlorophenol biodegradation were contrastively studied between mutant strain CTM 2 and its parent strain. Their maximum phenol biodegradations were 2000 and 2600mg/L, and the maximum specific growth rates were 0.48 and 0.54 1/h, respectively. Besides, the biodegradation of the wild and mutant strains were also studied in dual-substratesystem, respectively. The experiments showed that the inhibitions to cell from m-cresol and 4-chlorophenol remarkably exceeded that from phenol. On the contrary, biodegradation for m-cresol or 4-chlorophenol was accelerated at the presence of phenol. Consequently, the studies of dual-substrate kinetics proved that the kinetic model proposed in this paper described compatibly the cell growth and substrate biodegradation of C. tropicalis and its mutant strain CTM 2.