Isolation, Identification And Characterization Of A Phenol-degrading Strain

The purpose of this dissertation is to investigate the characteristics of a newly isolatedBrucella sp. GXY-1 capable of degrading phenol. Physio-biochemical characterization and16S rDNA molecular identification of the strain were performed. The catechol1,2-dioxygenase activity of the crude enzyme was also investigated and the results indicatedthat the degradation of phenol was catalyzed by catechol 1,2-dioxygenase. Degradationperformances of GXY-1 immobilized on macro-pore carrier were studied.GXY-1 is an efficient phenol-degrading strain. It was identified as Brucella sp. accordingto morphological, physiological and biochemical characteristics and 16S rDNA sequenceanalysis. The 16S rDNA sequence of strain GXY-1 shared high similarity (99%) with that ofBrucella sp.CGL-1 and was submitted to GenBank (GenBank code EF514908).The growth and phenol degradation capability of strain GXY-1 were investigated. StrainGXY-1 is resistant to oxytetracycline and tetracycline, and sensitive to other antibiotics. Theoptimum conditions for the degradation of phenol were: 30℃, pH7.0, 150r/min and 10% ofinoculation amount, respectively. Under the optimal conditions, GXY-1 could decomposeover 99% of phenol (600 mg/L)within 60 h. GXY-1 could tolerate 1000 mg/L of phenolutilized as the sole carbon source. The effects of environmental factors on the growth andphenol degrading were investigated. The results indicated that externally added carbon sourceaccelerate the growth of GXY-1 and phenol degrading. However, externally added nitrogensource exhibited little effect. Ni⁺ and Co²⁺ inhibited the growth of GXY-1 due to their hightoxicity. GXY-1 lost their growth activity when the concentration of NaCl is 2%. GXY-1 isable to degrade other aromatic compounds, such as aniline, chlorobenzene, catechol, benzoicacid, naphthalene, phenanthrene,etc.The characteristics of catechol 1,2-dioxygenase in the degradation process was studied.It was demonstrated that the degradation of phenol was catalyzed by catechol1,2-dioxygenase. The optimal conditions for the enzymatic reaction were pH7.8 and 40℃.The enzyme activities were relatively stable and remained 50% under pH5.8～9.4 or 70℃.The effects of heavy metal ions on enzyme activities were not neglectable. Irons, such as Fe³⁺,Ba²⁺ and Mn²⁺ had a remarkalbe promotion effects on enzyme activities and Cu²⁺, Zn²⁺, Fe²⁺,Pb²⁺, Co²⁺ were just on the contrary. The kinetic constants of catechol 1,2-dioxygenase (K_m and V_max) were determined to be 33.72μmol/L and 13 mol/(L·min), respectively.The degradation of phenol by GXY-1 immobilized in macro-pore carrier was studied.The optimal degradation conditions for immobilized cells are obtained as follows: 25～35℃,pH7.0, shaking speed 150 r/min and the maximum concentration of NaCl 2%.Theinvestigation of degradation kinetics indicated that the degradation of phenol followed themodel of first order, reaction kinetics when the initial phenol concentration was lower than147.48 mg/L, while it followed the zero order kinetics when the initial concentration wasbetween 200.14 and 521.58 mg/L. Under the optimal conditions, the degradation kinetics ofimmobilized cells fits well with Andrews model.