【作者】 赵博；

【导师】 曲媛媛； 王晓坤；

【作者基本信息】 大连理工大学 ， 环境工程， 2024， 硕士

【摘要】 由于海洋中石油泄漏事件频发,多环芳烃等有机污染物通过潮汐、风暴等现象扩散到沿海地区,并通过各种迁移过程进入潮间带中。受潮汐影响,沉积物中微生物丰度较低,导致多环芳烃的自然衰减速度较慢,受污染的环境难以在短期内恢复。生物强化法凭借绿色友好优势,在潮间带污染修复中受到广泛关注。本研究以菲和芘作为模式底物,模拟了多环芳烃污染的潮间带环境,对筛选出的多环芳烃降解菌株进行复配,构建多环芳烃降解菌群,对污染潮间带沉积物进行120天的生物强化修复,对潮间带沉积物污染修复效果及微生物群落的演替特征进行考察。主要取得的研究结果如下:(1)从石油污染土壤中筛选出两株多环芳烃降解菌Cellulosimicrobium sp.RS和Brucella sp.BZ,两株细菌均能以菲、芘为唯一碳源生长,同时具有良好的耐盐性。将两株菌复配成多环芳烃降解菌群PP-RSBZ,并通过正交试验确定其最佳复配比例,通过单因素法考察p H和外加碳源浓度对菌群降解多环芳烃的影响。结果表明,在菌株RS:BZ=3:1(V/V)、p H为7、酵母浸粉浓度为0.3 g/L时,120 h内复合菌群对150 mg/L混合多环芳烃的降解率为52.46%,是单一菌株降解率的1.90-2.18倍。此外对两株细菌进行了全基因组分析,推测复合菌群对菲和芘的降解途径。(2)构建了模拟多环芳烃污染的潮间带沉积物的实验装置,并利用复合菌群PPRSBZ进行生物强化修复。经过120天的修复,生物强化组的多环芳烃降解率达到55.37%,显著高于对照组的29.93%(P<0.05)。在修复过程中,沉积物的电导率不断升高,p H值在7.1-8.0之间波动,总氮和总磷含量均略有减少,土壤脲酶、土壤多酚氧化酶、土壤过氧化氢酶和土壤蔗糖酶这四种土壤酶活性均有升高。(3)通过高通量测序技术考察了潮间带沉积物中微生物群落的结构与功能特征。在生物强化组中,外源菌群PP-RSBZ中的Cellulosimicrobium属丰度增加,在沉积物中成功定殖,而Brucella属未实现定殖,但在修复过程中起到了协同降解的作用。群落多样性分析表明,外源菌群促进了沉积物中微生物群落的演替,Dethiosulfatibacter、Halanaerobium等本土菌属成为了新的优势菌属,与外源菌群协同降解多环芳烃。此外,PICRUSt2预测发现,与对照组相比,生物强化组中ald B和pca GH等功能基因丰度显著上调,并且与多环芳烃降解相关的KEGG二级通路中的功能丰度上升。综上,本研究为低分子量多环芳烃的生物降解提供了新的菌株资源,同时为潮间带污染的生物强化修复提供了理论指导。更多还原

【Abstract】 Due to frequent oil spills in the ocean,organic pollutants such as polycyclic aromatic hydrocarbons(PAHs)are spread to coastal areas through tidal action,storms,and other phenomena,and enter the intertidal zone through various migration processes.Influenced by tidal action,the abundance of microorganisms in the sediment is low,leading to a slow natural degradation rate of PAHs and making it difficult for the polluted environment to recover in the short term.Bioaugmentation,with its environmentally friendly advantages,has received widespread attention in the remediation of intertidal pollution.This study used phenanthrene and pyrene as model substrates to simulate the intertidal environment polluted by PAHs,and the selected PAH-degrading bacterial strains were combined to construct a PAH-degrading bacterial consortium for 120 days of bioaugmentation remediation of polluted intertidal sediments,examining the remediation effects and the succession characteristics of microbial communities.The main research results are as follows:(1)Two PAH-degrading bacteria,Cellulosimicrobium sp.RS and Brucella sp.BZ,were isolated from oil-contaminated soil,both of which can grow with phenanthrene and pyrene as the sole carbon source and have good salt tolerance.The two strains were combined into a PAHdegrading bacterial consortium PP-RSBZ,and the optimal combination ratio was determined through orthogonal experiments.The effects of p H and additional carbon source concentration on the degradation of PAHs by the consortium were investigated using the single-factor method.The results showed that at a strain ratio of RS:BZ = 3:1(V/V),p H=7,and yeast extract concentration of 0.3 g/L,the composite consortium achieved a degradation rate of 52.46% for150 mg/L mixed PAHs within 120 hours,which was 1.90-2.18 times that of a single strain.In addition,whole-genome analysis was conducted on the two bacteria to infer the degradation pathways.(2)An experimental device simulating intertidal sediment polluted by PAHs was constructed,and the composite consortium PP-RSBZ was used for bioaugmentation remediation.After 120 days of remediation,the degradation rate of PAHs in the bioaugmentation group reached 55.37%,significantly higher than the control group’s 29.93%(P < 0.05).During the remediation process,the electrical conductivity of the sediment continued to increase,the p H between 7.1 and 8.0,the content of total nitrogen and total phosphorus decreased slightly,and the activities of four soil enzymes,including soil urease,soil polyphenol oxidase,soil catalase,and soil sucrase,all increased.(3)High-throughput sequencing technology was used to investigate the structure and functional characteristics of microbial communities in intertidal sediments.In the bioaugmentation group,the abundance of the genus Cellulosimicrobium in the exogenous degrading consortium PP-RSBZ increased,successfully colonizing the sediment,while the genus Brucella did not colonize but played a role in synergistic degradation during the remediation process.Community diversity analysis showed that the bioaugmentation of PPRSBZ promoted the succession of microbial communities in the sediment,with indigenous genera such as Dethiobacter and Halanaerobium becoming new dominant genera,working together with the exogenous consortium to degrade PAHs.In addition,PICRUSt2 prediction found that compared with the control group,the abundance of functional genes such as ald B and pca GH in the bioaugmentation group was significantly increased,and the functional abundance in the KEGG second-level pathways related to PAH degradation was significantly enriched.In summary,this study provides new bacterial resources for the biodegradation of low molecular weight PAHs and provides theoretical guidance for the bioaugmentation remediation of intertidal pollution.更多还原