【作者】 刘莎；

【导师】 傅大放； 许妍；

【作者基本信息】 东南大学 ， 建筑与土木工程（专业学位）， 2017， 硕士

【摘要】 多氯联苯(Polychlorinated biphenyls,PCBs)是一种典型的有机氯污染物,具有疏水、难降解、远距离传输等特性,容易在土壤和底泥中富集。河湖底泥中的多氯联苯可通过水体中的生物富集作用危害人类健康。在厌氧条件下,一些微生物可以利用多氯联苯作为其终端电子受体获取能量,从而实现多氯联苯的脱氯降解。然而,多氯联苯的微生物降解具有地点特异性。因此,针对特定底泥沉积物研究自然环境下脱氯微生物的作用与机理,是实现该地区多氯联苯污染底泥原位修复的关键基础工作之一。本文研究了三种地球化学条件下太湖底泥微环境中多氯联苯的微生物脱氯降解行为。三个反应组分别为外加PCBs母体的微环境(记为T-1实验组,即产甲烷条件下太湖底泥反应微环境)、外加PCBs母体以及Na2S04的微环境(记做T-S实验组,即硫酸盐还原条件下太湖底泥反应微环境)、外加PCBs母体以及FeOOH的微环境(记做T-Fe实验组,即三价铁还原条件下太湖底泥反应微环境),对多氯联苯在各实验微环境下的天然微生物降解情况进行为期66周的监测,共采集了 13个时间点的111个样品。将泥浆样品中的多氯联苯提取纯化后,参照美国环保署方法(EPA Method 8082)进行气相色谱检测,对降解产物定量分析;同时提取各样品的总菌DNA,进行高通量测序及荧光实时定量PCR实验,考察细菌群落结构随脱氯进程的变化,并筛选多氯联苯降解指示微生物/基因。主要结论如下:(1)脱氯速率由快到慢排序为T-1组>T-Fe组>T-S组。竞争性电子受体会抑制多氯联苯脱氯,且硫酸盐的抑制作用强于三价铁。66周内,T-1组、T-Fe组、T-S组多氯联苯总浓度由49.56 土0.38 mg/kg分别降至37.21±1.43 mg/kg、39.80土0.45mg/kg、43.43±0.63mg/kg。类二噁噪英毒性当量分析可知:T-l、T-Fe、T-S组分别在15、36、66周内降低了约80%,与脱氯速率的结果吻合。(2)高通量测序结果表明,第0、6、24、66周与脱氯相关的绿弯菌门(Chlorofexi),尤其是其中的Dehalococcoides有增长趋势。但已知的多氯联苯脱氯菌只占微环境体系中的少数,Dehalococcoides菌最高占细菌总数的4.13%,各实验组中更多的是不具备多氯联苯脱氯功能的细菌,如厚壁菌门梭菌目(Clostridiales)、δ变形菌亚门(δ-Proteobacteria)等微生物。(3)脱氯相关菌 Chloroflex/、Dehalococcoides 16SrRNA 基因、还原脱卤酶基因ardA、rdh12均能指示T-1组和T-Fe组底泥微环境脱氯反应的发生,但无法指示T-S组反应的发生,Chloroflexi 16S rRNA基因、ardA、rdh12可以指示T-S组脱氯反应的加快,且ardA、rdh12基因的所有指示均更加灵敏。(4)多氯联苯单体测定结果表明底泥微环境中存在多氯联苯邻位脱氯现象,但qPCR实验却发现已知的邻位脱氯菌o-17/DF-1 16SrRNA基因浓度很低,推测太湖底泥中存在其它未知的具有邻位脱氯功能的微生物,有待进一步研究。系统深入地研究底泥中的脱氯菌,对揭示多氯联苯的降解过程、开发相关原位修复技术具有重要作用。更多还原

【Abstract】 Polychlorinated biphenyls(PCBs)are typical organochlorine contaminants with hydrophobic,non-degradable and long-distance transport properties,which are easily adsorbed in soil and sediment.PCBs in the sediments have adverse effects on human health through bioaccumulation in aquatic systems.Previous studies revealed that certain microorganisms are able to ultilize PCBs as their terminal electron acceptor to obtain energy.This results in PCB dechlorination.However,microbial dechlorination of PCBs is relatively site specific.Therefore,the study of the function and mechanism of dechlorination microorganisms in natural environment is one of the key steps for in-situ bioremediation of PCB contaminated sediments.In the present study,we investigated microbial dechlorination of PCBs under three different geochemical conditions in Lake Taihu sediment microcosoms.The experiments were set as group 1 with spiked PCBs(recorded as T-1,also as methanogensis condition of the Lake Taihu sediment microcosms),group 2 with spiked PCBs and Na2SO4(recorded as T-S,also as sulfate reduction condition of the Lake Taihu sediment microcosms),and group 3 with spiked PCBs and FeOOH(recorded as T-Fe,also as ferric iron reduction condition of the Lake Taihu sediment microcosms).Microcosms were set up in triplicates to explore the dechlorination of PCBs.Over a time course of 66-weeks,111 samples were collected at 13 time points.Gas chromatography analysis was performed according to the EPA method 8082 after the extraction and purification of PCBs in sediment slurries and the daughter PCBs were quantified indivually.DNA of samples was extracted for high-throughput sequencing and qPCR analysis.The shifts of microbial communities along with dechlorination were carefully examinated.Moreover,target microorganisms/genes were screened to identifiy the suitable biomarkers for PCBs dechlornation.The findings of this study are as follows:(1)The dechlorination rates were T-1>T-Fe>T-S.Competing electron acceptor partially inhibited PCB dechlorination and the inhibition by sulfate was more than that of ferric iron.After 66 weeks,the total concentrations of PCBs in T-1,T-Fe and T-S decreased from 49.56 ± 0.38 mg/kg to 37.21 ± 1.43 mg/kg,39.80 ± 0.45 mg/kg,43.43 ± 0.63 mg/kg,respectively.The dioxin-like toxic equivalency of PCBs decreased about 80%in the T-1 group after 15 weeks of incubation,in the T-Fe group at the end of 36 weeks,in the T-S group after 66 weeks of incubation,which was consistent with the order of PCBs dechlorination rates.(2)The results of high-throughput sequencing of samples of week 0,week 6,week 24 and week 66 revealed that the percentage of Chloroflexi over total bacteria,especially the percentage of Dehalococcoides increased with the dechlorination process.The known microorganisms,which can dechlorinate PCBs,only accounted for a small proportion of the microbial community.Among all the analyzed samples,Dehalococcoides accounted for 4.13%of the total bacteria at most.Bacteria such as Clostridiales,δ-Proteobacteria,that do not have the dechlorination function were dominant in the microbial communities.(3)The 16S rRNA gene of Chloroflexi(CHL),Dehalococcoides(DHC),reductive dehalogenase genes ardA,rdh12 were suceesuflly used to indicate the dechlorination in T-1 and T-Fe group but not in T-S group.The 16S rRNA gene of Chloroflexi(CHL),and reductive dehalogenase genes ardA,rdh12 were able to be linked to the rapid stage of dechlorination in the T-S group.Compared with Chloroflexi and Dehalococcoides,these two reductive-dehalogenase genes were more sensitive as dechlorination biomarkers.(4)The ortho dechlorination of PCBs was confirmed by gas chromatography analysis,but the concentrations of known ortho dechlorinating bacteria o-17/DF-116S rRNA gene were very low.This suggested that there might be other unknown microorganisms with ortho dechlorination function in Lake Taihu sediments,which need to be studied further.The study of dechlorination microorganisms is crucial for comprehensive understandings of the dechlorination processes of PCBs,as well as the development of in-situ bioremediation technology.更多还原