1株分离自海水中芘降解菌的鉴定及其降解特性研究

Isolation and identification of a pyrene-degrading bacterial strain in seawater and its pyrene biodegradation features

以芘为唯一碳源,对采自于天津港石油污染区的海水和土壤样品进行富集培养,分离到1株芘降解菌,经显微形态观察、生理生化鉴定1、6S rRNA基因序列的比对及系统发育进化的分析,确定该菌株为施氏假单孢菌Pseudomonas stutzeri,并采用室内培养方法,对其进行了芘降解性能的测定及降解途径的分析。结果表明,该菌株在以芘为唯一碳源的无机盐培养基中培养36 h后,对芘的降解率达到96%以上。该菌株具有邻二酚2,3-双加氧酶活力,且酶活力随芘质量浓度的增高而提高,可以确定该菌株是以邻苯二酚为中间代谢物对芘进行降解的。

Polycyclic aromatic hydrocarbons（PAHs） are common organic pollutants widely distributed in the natural environment.Their easiness to accumulate and be delivered by food chain and their carcinogenic and mutagenic features make them extremely harmful to human health and ecological environment.A large amount of wastewater containing PAHs flows into the ocean at last,which makes the study of PAHs pollution control of the sea a critical issue.But,due to the particularity and complexity of the marine environment,many regular physical and chemical approaches do not work.In recent years,remediation of the polluted environment with microbial degradation is considered to be the most efficient way to control PAHs contamination.Thus the study on PAHs microbial degradation has become one of the most active areas in marine environmental pollution research,and acquisition of high efficient PAHs-degradating bacteria is the key for biodegradation and bioremediation of PAHs.A pyrene-degrading bacterial strain B5 was separated through selectively enriched culture from the oil-contaminated sample from the Tianjin port.Its characteristics of growth and pyrene degradation ability are studied.This strain B5 grew very fast in culture solution with pyrene as the sole carbon source.The growth entered into logarithm growth phase after 12 hours inoculation and steady phase after 24 hours.The strain had high degradation action to pyrene and the degradation rate positively correlated to the bacteria quantity.In shaking culture conditions,the removal rate of pyrene for the pyrene solution of 44.1 mg/L initial concentration at 28 ℃ was approximately 29.0% after 12 hours culture and then increased quickly to 63.3% after 24 hours.When culture time reached 36 hours,the concentration of pyrene decreased to 1.8 mg/L and the removal rate was 96.3%.In order to determine the classification position of the strain,micro morphological observation was made and carbon usage was analyzed by Biolog system.The 16S rRNA of the strain was amplified by PCR and compared with those available in the GenBank databases.The strain belongs to Gram-negative bacterium without capsule and spore,and is aerobic.Biolog identification results showed that strains B5 could strongly use 41 of 95 kinds of carbon sources,and the similarity probability with Pseudomonas stutzeri was 100%.The 16S rRNA gene sequence analysis suggested that the strain was clustered together with Pseudomonas stutzeri in phylogenetic tree.Based on all these results,the strain B5 should be identified as Pseudomonas stutzeri.In order to explore the pathway of pyrene degradation,the activity of catechol 2,3-dioxygenase under different pyrene concentration was analyzed.The results showed that the activity of catechol 2,3-dioxygenase was increased with the increase of pyrene concentration.So we could speculate that the strain B5 degraded pyrene via catechol pathway.