微生物种间相互作用对水环境中锰的氧化过程与机制
文献类型：学位论文
| 作者 | 梁金松
|
| 学位类别 | 博士 |
| 答辩日期 | 2016-06 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 曲久辉 ; 柏耀辉 |
| 关键词 | 合作锰氧化，节杆菌，鞘氨醇盒菌，种间相互作用，分子机制 cooperative Mn II oxidation, Arthrobacter, Sphingopyxis, interspecific interaction, molecular mechanism |
| 其他题名 | The process and mechanism of MnII oxidation drived by microbial interspecific interactions |
| 学位专业 | 环境工程 |
| 中文摘要 | 二价锰（MnII）在自然水体中广泛存在，在微生物的催化作用下 MnII能被氧化生成生物氧化锰（MnIII及MnIV）。由于生物锰氧化物本身对污染物具有较强的氧化吸附能力，因此在水体净化上具有重要研究价值。以往国内外对锰氧化微生物的研究主要基于单个模式菌株的催化机理及生成的生物氧化锰的结构及化学特性，而对微生物相互作用导致的 MnII氧化过程缺乏深入研究。 本论文以复合菌系（节杆菌和鞘氨醇盒菌）为对象，首先探讨了“双菌株仅在混合培养体系产生锰氧化活性”的分子机制。结合传统微生物培养试验和现代分子生物学手段，发现 MnII氧化活性的产生必要条件是双菌活细胞的直接接触。进而采用改进的蛋白提取和纯化策略成功获取了双菌活细胞培养液中产生的MnII氧化蛋白。质谱鉴定结果表明，该蛋白属于胆红素氧化酶，来自节杆菌。利用基因组步移法获得了该基因的全长序列（2040bp），该基因被命名为 box。通过发育树分析发现该基因编码的蛋白序列与已知的锰氧化蛋白序列系统发育关系较远。利用RNA-seq技术考察混合培养体系中双菌的基因转录情况。结果显示，节杆菌中MnII氧化基因box在混合培养开始时处于沉默状态，而后被鞘氨醇盒菌激活（很可能通过释放压力因子），从而产生MnII为了探讨种间相互作用导致的 MnII氧化活性。氧化在实际水体中的发生过程，将双菌分别接种到地下水、生活污水及焦化废水，结合微生物群落表征及水质分析确定双菌 MnII氧化活性与菌株比例、水质的相互关系。结果表明，双菌能在三种实际水中氧化MnII；而水中营养物质浓度是影响 MnII氧化速率的重要因子。另外发现，双菌相互作用产生的生物氧化锰能促进水中 DOC和 TN的去除，具有深度净水的作用。 进一步发现双菌均为砷还原菌，能将AsV还原AsIII。在此基础上提出假设“种间相互作用改变水体中 As的归趋”。在含 AsV和 MnII体系中，比较了单菌与双菌培养体系中 As的转化和迁移。结果显示双菌培养体系中As的变化分三个阶段：1）AsV被双菌还原为 AsIII；2）双菌相互作用产生的锰氧化物氧化AsIII附AsV，As/Mn的吸附比例约为0.022；3）由于锰氧化物的还原性溶解及产生的MnII与As竞争吸附位点，使得原本吸附在锰氧化物的As发生解吸附。造成As转化和迁移的根本原因是双菌的砷还原活性及相互作用产生的锰氧化活性在培养过程的强弱变化。 |
| 英文摘要 | Manganese (MnII ) is widely distributed in various aquatic environments. MnII can be oxidized to MnIII adsorption of MnIV oxides play an important role in water purification. Many previous studies have focused on the single MnII-oxidizing microbe, i.e., the mechanism of biological MnII oxidation and the physical and chemical properties of formed biogenic Mn oxides. However, little is kown about the contribution of interspecific interactions between certain microbial species to MnII oxidation. We first explored mechanism of cooperative MnII oxidation between Arthrobacter sp. and Sphingopyxis sp. Combining the culture-dependent and molecular approaches, we find that the direct contact of two living strains’ cells is essential for the cooperative MnII oxidation.The MnII-oxidizing protein was extracted and purified using a modified protocol. Results of LC-MS/MS indicated that the -oxidizing protein is a bilirubin oxidase that only exists in Arthrobacter. The full sequence length of the MnII-oxidizing gene was identified (2040 bp) using genomic walking.The protein sequence encoded by the MnII-oxidizing gene (named as “box”) exhibits distinct phylogenetic relationships with known Mn(II)-oxidizing enzymes.Results of RNA-Seq further demonstrated that the box was silent in Arthrobacter monoculture and then activated by Sphingopyxis to induce MnII oxidation in the co-culture. Transcriptomic analysis by RNA-Seq further suggests the induction of box gene expression in Arthrobacter was associated with stress imposed by Sphingopyxis. To explore whether the cooperative MnII oxidation occured in natural aquatic ecosystem, we inoculated the two strains into three types of water samples (i.e.,groundwater, domestic sewage and coking wastewater) to the occurrence of MnII oxidation, and also investigate the relationships between MnII oxidation and the ratio of two strains, as well as MnII oxidation and water quality. Results showed the cooperative MnII oxidation could occur in the three actual water samples. The nutrient concentration in the water was a key factor affecting MnII oxidation rate. Additionally,it proved that the formed biogenic MnII oxides could further decrease the dissolved organic carbon (DOC) and total nitrogen (TN), thus resulting in a deep purification of water. Interestingly, the two strains are both Arsenate (AsV)-reducing bacteria, and are capable of reducing AsV to AsIII. Thus, we prpoposed and verified a hypothesis that microbial interspecific interactions may alter fate of AsV. Transformation and migration of As was compared between monocultures and co-culture of the two strains in the presence of AsV and MnII. The valent variation of As was divided into three phases in the co-culture. For phase 1, AsV was reduced to AsIII by the two strains.For phase 2, a portion of AsIII was oxidized to AsV and then adsorbed on MnII oxides produced by the two trains. The maximal adsorption ratio of As to Mn was 0.022. For phase 3, As was desorbed due to the reductive dissolution of Mn oxides and the competition for adsorption sites on Mn oxides between As and the released MnII. The underlying cause of valence state transformation and migration of As was determined by relative strength between AsV-reducing activity and MnII-oxidizing activity of the two strains. |
| 源URL | [http://ir.rcees.ac.cn/handle/311016/36889]  |
| 专题 | 生态环境研究中心_环境水质学国家重点实验室 |
| 推荐引用方式 GB/T 7714 | 梁金松. 微生物种间相互作用对水环境中锰的氧化过程与机制[D]. 北京. 中国科学院研究生院. 2016. |
入库方式： OAI收割
来源：生态环境研究中心
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