多环芳烃降解菌的分离鉴定及其生理特性研究

Study on isolation, identification and physiological characteristics of PAHs-degrading bacteria

从兰州石化污水处理厂的污泥中分离出一株能分别以萘、蒽为唯一碳源生长的细菌(BDP01).通过菌株形态观察及16S rDNA序列比对,确定该菌株属于假单胞菌属的绿脓杆菌(Pseudomonas aeruginosa).菌株在LB培养基中48h内呈对数生长,48h到72h进入生长稳定期,72h后开始进入衰亡期;其最佳培养温度为30℃,最适生长pH为中性或弱碱性,该菌能在萘和蒽的质量浓度分别为100mg·L-1和50mg·L-1的无机盐培养基中良好生长,与已报道的其他微生物比较,BDP01在较短时间内具有较强的降解多环芳烃能力,1%的接种量15d内可降解89.64%的萘和77.21%的蒽.采用PCR和琼脂糖凝胶电泳技术检测到菌株基因组中有邻苯二酚2,3-双加氧酶(C23O)基因.测序结果与NCBI数据库发布的C23O基因序列相似度为92%.该酶在20～65℃以邻苯二酚为底物时,细胞裂解液中酶活力变化范围为13.10～540.86U.

A strain, designated as BDP01, was isolated from the sludge of Lanzhou Sewage Disposal Plant. It utilized naphthalene and anthracene as the sole carbon sources for its growth. Based on the morphological characterizations and alignment analysis of the 16S rDNA sequence, BDP01 was identified as Pseudomonas aeruginosa. The growth curve of the strain was in logarithmic phase within 48 h, in stationary phase after 48 h to 72 h, and in decline phase after 72 h, in LB liquid medium. The strain grew well in inorganic salt liquid medium containing 100 mg · L-1 naphthalene or 50 mg · L-1 anthracene. The optimal growth temperature was 30 ℃ and the optimal growth pH value was 7~8, a neutral or alkalescent condition. The strain had a strong ability to biodegrade polycyclic aromatic hydrocarbon. In the present experiment, when the inoculum concentration of the strain was 1%, the degradation rates of naphthalene and anthracene were 89.64% and 77.21%, respectively, after cultured for 15 days. Furthermore, catechol 2, 3-dioxygenase gene sequence of the strain was detected using the PCR method and agarose gel electrophoresis. The gene sequencing and alignment analysis showed that catechol 2, 3-dioxygenase gene sequence of the strain had 92% similarity with the closest C23O gene sequence in the NCBI. With catechol as the substrate, the activity of catechol 2, 3-dioxygenase in the strain cellysis solution was 13.10~540.86 U at 20~65 ℃ reactive temperature.