寒区降解多环芳烃耐冷菌株的分离鉴定及特性

Isolation,identification and characterization of cold resistance and PAHs-degrading bacteria

多环芳烃(Polycyclic Aromatic Hydrocarbons,PAHs)是土壤中一种典型的持久性有机污染物。典型寒区东北地区因农业投入品不合理使用、污灌等造成农田土壤中含有大量致癌、致畸与致突变的多环芳烃,针对寒区气候特征致使农田中微生物降解多环芳烃效果不佳,难以改善农田土壤环境并降低食品风险的关键问题,基于目前常用商品化的降解微生物多来自于温暖地区,且难以适应寒区气候的特性,该研究以PAHs典型污染物-菲(PHE)为研究底物,驯化温度15℃,筛选分离出适应寒区低温环境的7株菌。经鉴定及降解性能研究,筛选了3株在温度20℃、接种量5%、pH值为8、底物浓度500 mg/L,以及外源物质腐殖酸的促进的条件下PHE降解率达到80%的高效降解耐冷菌。以上3株菌两两之间与三者组合均无拮抗关系。对菌株进行碳源的广谱性分析,菌株对2-5环多环芳烃降解率可达15%~85%之间,在将菌株应用至20℃土壤环境时,60 d可降解土壤中75%的PHE。该耐冷菌群适应条件符合寒区农田土壤实际环境,研究结果对黑土地区土壤多环芳烃污染的微生物修复提供了一定的基础资料。

Polycyclic aromatic hydrocarbons(PAHs)are highly toxic organic contaminants.The ubiquitous class of aromatic compounds is usually composed of 2 to 7 aromatic rings in nature.The accumulation of PAHs has been widely spread in farmland soil in recent years.The reason can be that the various wastes and gas pollutants have been ever-increasing beyond the repairing and purification capacity of the environment in the world.Microbial remediation of PAHs can be expected to serve as high efficiency,low cost,and no secondary pollution.Among them,biodegradation has been the most potential soil remediation to eliminate the PAHs pollutants in the environment.Particularly,the PAHs have posed an enormous safety risk to the farmland soil ecosystems in Northeast China.The farmland soil contains a large number of carcinogenic,teratogenic,and mutation polycyclic aromatic hydrocarbons at present,due to the unreasonable use of agricultural inputs and sewage irrigation.However,the most commonly-used,commercial and microbial microorganism degradation of PAHs is confined to the climatic characteristics of the cold region in the farmland.It is a high demand for efficient PAH-degrading bacteria in the natural environment,particularly for the PAH-contaminated soil in the northern cold area.In this study,a typical pollutant of PAHs,phenanthrophene(PHE)was used as the substrate,in order to reduce the food risk of the farmland soil environment.The domestication temperature was 15℃for the seven cold-resistant bacteria.The PHEs as the sole carbon and energy sources were screened from the soil.The bacterial mixtures were obtained from an equal volume mixture of seven strains.The high-performance liquid chromatography was then utilized to optimize the use conditions for the most effective strains or mixtures of strains.Three highly effective PHE-degrading capabilities(named DX-1,DX-2,and DX-3)were identified and screened out by the morphological observation and 16S rRNA gene sequencing.16S rRNA gene sequencing analysis was performed on the DX-1,DX-2,and DX-3,which were identified as Achromobacter sp.,Stenotrophomonas sp.,and Pseudomonas sp.,respectively.After identification and degradation performance,three strains with no antagonistic relationship between two and three combinations were selected,where the degradation rate of PHE reached 80%at 20°C,5%inoculum,pH=8,500mg/L substrate concentration,and the humic acid as exogenous substance.The broad-spectrum analysis of the carbon source of the strain showed that the cold-resistant bacterium degraded the PAHs of 2-5 loops between 15%and 85%.75%PHE was degraded in the 60d,when the strain was applied to the soil at the temperature of 20℃.Consequently,the cold-resistant bacterium can be expected for the adaptation conditions of the actual soil environment.The finding can provide the immobilized strain and theoretical basis for the microbial remediation of PAH pollution in the contaminated black soil in cold areas.