尾矿硫氧化微生物的分离鉴定与功能验证

doi:10.16258/j.cnki.1674-5906.2022.04.017

生态环境学报 ›› 2022, Vol. 31 ›› Issue (4): 785-792.DOI: 10.16258/j.cnki.1674-5906.2022.04.017

尾矿硫氧化微生物的分离鉴定与功能验证

李嘉义¹(), 孙蔚旻², 孙晓旭², 孔天乐¹, 李宝琴², 刘振鸿¹, 高品¹^,^*()

1.东华大学环境科学与工程学院,上海 201600
2.广东省科学院生态环境与土壤研究所,广东广州 510650

收稿日期:2022-01-26 出版日期:2022-04-18 发布日期:2022-06-22
通讯作者: *高品,男,教授,博士,研究方向为污染土壤化学与微生物修复技术。E-mail: pingao@dhu.edu.cn
作者简介:李嘉义（1998年生）,男,硕士,研究方向为尾矿中硫氧化过程的自养微生物驱动机制。E-mail: 1043547027@qq.com
基金资助:
国家自然科学基金项目(42107285);国家自然科学基金项目(U21A2035);国家自然科学基金项目(U20A20109);广西创新驱动发展专项资金项目(桂科AA17204076);广州市科技计划项目(202002020072)

Isolation, Identification and Functional Verification of Sulfur-oxidizing Microorganisms in Mine Tailing

LI Jiayi¹(), SUN Weimin², SUN Xiaoxu², KONG Tianle¹, LI Baoqin², LIU Zhenhong¹, GAO pin¹^,^*()

1. Donghua University, School of Environmental Science and Engineering, Shanghai 201600, P. R. China
2. Institute of Eco-environmental and Soil Sciences, Guangdong Academy of Sciences, Guangzhou 510650, P. R. China

Received:2022-01-26 Online:2022-04-18 Published:2022-06-22

摘要/Abstract

摘要：

尾矿污染是全球备受关注的环境问题之一,其产量大、污染风险高,对环境造成了严重的威胁。尾矿条件下的硫氧化过程会产生氢离子,导致尾矿酸化,进而使得金属污染物活化和迁移,对周边环境造成危害。硫氧化微生物作为硫元素生物地球化学循环的重要参与者,其硫氧化代谢途径是尾矿中硫氧化的主要组成部分。因此,加强对尾矿原位条件下微生物驱动的硫氧化过程的理解尤为重要。然而,目前对尾矿中硫氧化微生物的分离鉴定及其对原位环境的影响仍需进一步探讨和解析。针对这一问题,该文以“世界锑都”锡矿山的尾矿为研究对象,采用平板划线法,在硫氧化条件下对尾矿原位微生物种群进行分离纯化,并结合16S rRNA和硫氧化基因（soxB）序列分析对其进行物种鉴定。同时,构建硫氧化条件微宇宙实验体系,对两株具有硫氧化基因的纯菌进行功能验证。除此以外,通过构建尾矿原位条件模拟实验体系,探究尾矿酸化与硫氧化微生物之间的联系。结果显示,该研究成功得到了两株具有硫氧化基因的纯菌,分别为Methyloversatilis sp. LJY-1和Limnobacter sp. LJY-2。硫氧化实验进一步表明,LJY-1是首次被发现的具有硫氧化功能的Methyloversatilis属类,并且LJY-1的硫氧化能力明显强于LJY-2。基于尾矿原位条件模拟实验,证明在尾矿原位条件下,微生物驱动的硫氧化过程显著加剧了尾矿酸化（P<0.05）。针对尾矿不同阶段中含水率的差异,该研究进一步揭示了淹水条件下尾矿酸化过程显著快于湿润条件（P<0.05）。研究结果对尾矿场地的管理和修复具有重要意义。

关键词: 尾矿, 硫氧化微生物, 硫氧化基因, 土壤酸化

Abstract:

Mine tailing pollution is an environmental problem that has attracted worldwide attention. Its large output and high pollution risks were serious threats to the environment. Sulfur oxidation processes under the mine tailing conditions can produce hydrogen ions, leading to the acidification of mine tailing, which will activate and migrate metal pollutants and further cause harmful effects to the surrounding environment. As important participants in the biogeochemical cycle of sulfur, sulfur oxidizing microorganisms are the main components of sulfur oxidation under the mine tailing condition. Therefore, it is particularly important to strengthen the understanding of in situ microbe-driven sulfur oxidation processes in mine tailings. Till now, however, isolation and identification of sulfur oxidizing microorganisms in mine tailing and their impacts on in-situ environment still need to be further explored. Thus, this study collected the mine tailing samples from the "world antimony capital" tin mine as the research object. The plate scribing method was used to isolate and purify the in-situ microbial population of mine tailing under the conditions of sulfur oxidation, and identify its species by using the 16S rRNA and sulfur oxidation gene (soxB) sequence analysis. Then, the microcosm experimental system of sulfur oxidation condition was constructed to verify the functions of two pure bacteria with sulfur oxidation genes. In addition, the relationship between mine tailing acidification and sulfur oxidizing microorganisms was explored by constructing the simulation experimental system of mine tailing in-situ conditions. The results showed that two pure strains involving sulfur oxidation genes were successfully obtained, which were named as Methyloversatilis sp. LJY-1 and Limnobacter sp. LJY-2. We found that LJY-1 was the first discovered Methyloversatilis species with sulfur oxidation function, and its sulfur oxidation ability was significantly stronger than that of LJY-2. Based on the simulation experiments of mine tailing in situ conditions, it was proved that the microbe-driven sulfur oxidation process significantly aggravated the acidification of mine tailing (P<0.05). According to the differences in the water content of mine tailings, this study also revealed that the acidification process of mine tailing under flooding condition was significantly faster than that under the wet condition (P<0.05). The study is of great significance to the management and restoration of mine tailing sites.

Key words: mine tailing, sulfur oxidation microorganisms, sulfur oxidation gene, tailing acidification

中图分类号:

X172

李嘉义, 孙蔚旻, 孙晓旭, 孔天乐, 李宝琴, 刘振鸿, 高品. 尾矿硫氧化微生物的分离鉴定与功能验证[J]. 生态环境学报, 2022, 31(4): 785-792.

LI Jiayi, SUN Weimin, SUN Xiaoxu, KONG Tianle, LI Baoqin, LIU Zhenhong, GAO pin. Isolation, Identification and Functional Verification of Sulfur-oxidizing Microorganisms in Mine Tailing[J]. Ecology and Environment, 2022, 31(4): 785-792.

图/表 7

表1 硫氧化菌液体培养基和固体培养基

Table1 Sulfur-oxidizing bacteria liquid medium and solid medium

成分 Composition	液体培养基 Liquid medium/ (g∙L^-1)	固体培养基 Solid medium/ (g∙L^-1)
硫代硫酸钠 Sodium thiosulfate	5.0	5.0
磷酸氢二钾 Dipotassium phosphate	2.0	2.0
硝酸钾 Potassium Nitrate	2.0	2.0
硫酸镁 Magnesium sulfate	0.6	0.6
氯化铵 Ammonium Chloride	0.5	0.5
硫酸亚铁 Ferrous sulfate	0.01	0.01
琼脂 Agar	—	15

图1 soxB基因PCR扩增后电泳跑胶结果

Figure 1 Result of gel electrophoresis after PCR amplification of soxB gene

图2 硫氧化性能对比结果

Figure 2 Results of Sulfur oxidation performance comparison

图3 菌株Methyloversatilis sp. LJY-1基于16S rRNA基因序列同源性构建的系统发育树

Figure 3 Phylogenetic tree of Methyloversatilis sp. LJY-1 based on the 16S rRNA gene homology

图4 菌株Methyloversatilis sp. LJY-1基于soxB基因序列同源性构建的系统发育树

Figure 4 Phylogenetic tree of Methyloversatilis sp. LJY-1 based on the soxB gene homology

图5 淹水状态下微生物对土壤酸化的影响

Figure 5 The influence of microorganisms on soil acidification under flooded conditions

图6 湿润状态下微生物对土壤酸化的影响

Figure 6 The influence of microorganisms on soil acidification under humid conditions

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尾矿硫氧化微生物的分离鉴定与功能验证

Isolation, Identification and Functional Verification of Sulfur-oxidizing Microorganisms in Mine Tailing

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