【作者】 王文婷；

【导师】 景传勇；

【作者基本信息】 山东大学 ， 环境工程（专业学位）， 2023， 硕士

【摘要】 西藏是我国典型的高砷地热生态系统。近年来,该地区砷污染严重,危害了当地居民的健康,解析砷污染机制迫在眉睫。研究报道,砷还原微生物驱动了砷的释放和迁移,其可将稳定、低毒的五价砷还原为流动性强且毒性高的三价砷。然而,西藏热泉系统中微生物驱动的砷还原机制研究还十分有限。系统研究西藏热泉生态系统中微生物及其砷还原机制可深入解析该地区砷释放的机制,为砷污染防治及控制提供新思路。本研究以西藏日多温泉处的厌氧沉积物为研究对象,构建以乙酸作为电子供体和碳源,As(Ⅴ)为电子受体的厌氧微宇宙培养体系,模拟As(Ⅴ)在天然厌氧环境中的还原过程,用以揭示厌氧生境下介导砷还原的功能微生物特征及其代谢活性。具体而言,通过稳定性同位素示踪技术(DNA-SIP)耦合16S rRNA高通量测序,靶标厌氧热泉生境中的As(Ⅴ)还原功能的核心微生物物种;进一步结合宏基因组测序技术明确此类功能微生物介导的砷还原微生物学过程及其代谢机制。本研究发现:在单一砷污染寡碳营养的西藏日多温泉处,微生物可以利用乙酸作为碳源和电子供体,驱动As(V)的还原过程;DNA-SIP对As(Ⅴ)还原功能菌株进行标记,并通过16S rRNA扩增子测序发现该污染土壤中驱动厌氧砷还原过程的优势功能微生物分别是Pseudomonas和Thermincola两类细菌菌属,占比分别为66.2%和22.3%。通过宏基因组测序分析同样证实了上述关键微生物在砷还原中的重要作用,Pseudomonas和Thermincola菌属同时包含砷呼吸还原酶的相关基因和砷的外排途径的酶的相关基因,这一结果表明微生物通过砷的呼吸还原途径实现砷的还原,同时呼吸过程为微生物生长提供能量,通过多种外排机制缓解高砷的压力。综上所述,多样化的砷代谢途径使它们成为西藏热泉沉积物中砷还原代谢的主导微生物。此外,在这两个菌属中鉴定到的碳固定相关基因、氮转化、硫转化相关基因和其他金属抗性基因表明协同碳、氮、硫等元素转化代谢,是寡营养环境中的优势物种的生存策略。后续还可以对Pseudomonas和Thermincola等菌群进行进一步的纯菌分离培养,并应用于污染场地,应用于实际的土壤微生物修复工程中。本文通过DNA-SIP与高通量测序(16S rRNA和宏基因组)相结合的微生物学前沿方法,示踪介导砷还原过程的核心微生物,探明西藏热泉沉积物土著微生物参与的砷迁移转化过程,并深入挖掘厌氧条件下土壤中砷还原功能微生物的代谢机制。研究结果有望为热泉环境中砷的微生物修复提供理论支撑,有助于缓解砷的污染问题,具有重要的环境修复意义。更多还原

【Abstract】 Tibet is a typical high-arsenic geothermal ecosystem in China.In recent years,the serious arsenic pollution problem in the region has endangered the health of local residents.Therefore,resolving the mechanism of arsenic contamination is urgently needed.Studies have reported that arsenic-reducing microorganisms drive the release and transport of arsenic,which can reduce stable,low-toxicity pentavalent arsenic to mobile and highly toxic trivalent arsenic.However,research on microbial-driven arsenic reduction mechanisms in Tibetan hot spring systems is limited.A systematic study of microorganisms and their arsenic reduction mechanisms in Tibetan hot spring ecosystems will reveal the mechanisms of arsenic release in the region and provide new insights for arsenic pollution prevention and control.This study constructed an anaerobic microcosm culture system to simulate the anaerobic environment at the sediment near Riduo hot spring in Tibet.Acetic acid was used as an electron donor and carbon source and As(Ⅴ)as an electron acceptor to verify whether microorganisms are involved in the process of arsenic reduction.The stable isotope tracing technique(DNASIP)was used to identify functional strains of As(Ⅴ)-reducing microorganisms by labeling them with 13C-acetate.The functional microorganisms mediating anaerobic arsenic reduction processes and their metabolic mechanisms in arsenic-contaminated environments under thermal spring systems were explored in combination with metagenomic high-throughput sequencing.In this study,we found that microcosm culture experiments at a single arseniccontaminated oligo carbon nutrient-rich hot spring in Riduo,Tibet,showed that microorganisms in the system could use acetic acid as a single carbon source and electron donor to drive the As(Ⅴ)reduction process.DNA-SIP experiments showed that microorganisms could successfully label As(Ⅴ)-reducing functional strains by assimilating 13C-acetic acid and incorporating isotopically labeled carbon into their genomic DNA.16S rRNA amplicons sequencing showed that the dominant functional microorganisms driving the anaerobic arsenic reduction process in this contaminated sediment were two genera of bacteria,Pseudomonas and Thermincola,respectively.The metagenome sequencing results revealed that Pseudomonas and Thermincola genera contain both genes related to the arsenic respiration reduction pathway and the arsenic efflux pathway.Their diverse arsenic metabolism pathways make them the main microorganisms responsible for arsenic reduction in Tibetan hot spring sediments.The identification of carbon fixation-related genes,nitrogen transformation,sulfur transformationrelated genes,and other metal resistance genes in these two genera suggests that they may respond to this oligotrophic hot spring environment through other alternative non-arsenic transformation pathways,contributing to their environmental dominance in the genus.Further pure bacterial isolation and culture of Pseudomonas and Thermincola can be followed and applied to arsenic-contaminated sites for implementation into practical soil microbial remediation projects.Therefore,this project traces the core microorganisms involved in the arsenic reduction process by a cutting-edge method combining high-throughput sequencing technology and DNA-SIP.The association between indigenous microorganisms and arsenic transport and transformation in Tibetan hot spring sediments was investigated.The metabolic information of arsenic-reducing microorganisms in sediments under anaerobic conditions was also explored.The results of this study are expected to provide theoretical support for microbial remediation of arsenic in hot spring environments,which is of great environmental remediation significance for mitigating arsenic pollution problems.更多还原