石油烃污染土壤高效降解菌群构建与应用研究

Study on Construction and Application of High-Efficiency Degrading Microbial Communities for Petroleum Hydrocarbon-Contaminated Soil

  • 摘要: 石油烃泄漏易造成土壤功能退化、地下水污染等严重危害,自然降解周期长且治理难度大。物理与化学修复技术存在能耗高、易产生二次污染等局限,微生物修复因绿色环保成为研究热点,但传统筛选的菌株降解率低、修复周期长,亟待通过菌株改良与菌群构建技术,研发高效稳定的修复菌剂以破解实际治理难题。本文以保定周边及白洋淀流域石油烃污染土壤为样本,系统开展高效降解菌株筛选、改良、菌群构建及应用验证研究。通过富集培养、生理生化及16S rDNA鉴定,从34株菌株中筛选出6株优势降解菌(如肺炎克雷伯菌AY12、柯贝肠杆菌BY4等);经ARTP-紫外复合诱变,菌株降解率较原始菌株(产酸克雷伯氏菌BY6)最高提升37.67%,且连续转接10代后降解率波动≤3%,遗传稳定性良好。通过构建57种不同组合形式的复合菌群,筛选出针对柴油、煤油、汽油的高效降解菌群Y23、Y26、Y4,其降解率分别达70.94%、82.66%和76.04%;经正交试验优化接种量、温度、pH值和盐浓度等关键参数后,煤油降解率最高可达83.13%。室内修复实验中,采用多次投加菌剂的方式,30 d内对493 mg/kg汽油污染土壤、817 mg/kg柴油污染土壤的降解率分别达90.23%、81.43%。本研究构建的高效降解菌群及优化的应用技术方案,为石油烃污染土壤的高效治理提供了优质菌种资源与技术支撑。

     

    Abstract: The leakage of petroleum hydrocarbons easily causes severe hazards such as soil functional degradation and groundwater pollution. The natural degradation cycle is long, and the governance is difficult. Physical and chemical remediation technologies have limitations such as high energy consumption and the potential for secondary pollution. Microbial remediation has become a research hotspot due to its environmental friendliness. However, the strains traditionally selected have low degradation rates and long remediation cycles. There is an urgent need to develop efficient and stable remediation microbial agents by means of strain improvement and microbial community construction technologies to solve practical governance challenges. This study systematically carried out research on the screening, improvement, construction, and application verification of high-efficiency degrading strains, using petroleum hydrocarbon-contaminated soil samples from the Baoding area and the Baiyangdian Basin as the research objects. Through enrichment culture, physiological and biochemical identification, and 16S rDNA sequencing, 6 dominant degrading strains (such as Klebsiella pneumoniae AY12, Enterobacter cloacae BY4, etc.) were screened from 34 isolated strains. After ARTP-ultraviolet composite mutagenesis, the degradation rate of the strains was increased by up to 37.67% compared with the original strain (Klebsiella oxytoca BY6). Moreover, the degradation rate fluctuation was ≤3% after 10 consecutive subcultures, indicating good genetic stability. By constructing 57 different combinations of composite microbial communities, efficient degrading microbial communities Y23, Y26, and Y4 targeting diesel, kerosene, and gasoline were screened out, with degradation rates of 70.94%, 82.66%, and 76.04%, respectively. After optimizing key parameters such as inoculation amount, temperature, pH value, and salt concentration through orthogonal experiments, the highest kerosene degradation rate reached 83.13%. In the indoor remediation experiment, the multiple inoculation strategy of the microbial agent was adopted. Within 30 days, the degradation rates of gasoline-contaminated soil with an initial concentration of 493 mg/kg and diesel-contaminated soil with an initial concentration of 817 mg/kg reached 90.23% and 81.43%, respectively. The high-efficiency degrading microbial community constructed and the optimized application technical scheme developed in this study provide high-quality microbial resources and technical support for the efficient remediation of petroleum hydrocarbon-contaminated soil.

     

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