单环刺螠肠道电活性Shewanella marisflavi的氯霉素降解潜力研究
李新
学位类型硕士
导师刘芳华
2020-05-26
培养单位中国科学院烟台海岸带研究所
学位授予单位中国科学院大学
学位授予地点中国科学院烟台海岸带研究所
学位名称工程硕士
学位专业生物工程
关键词肠道电活性微生物胞外电子传递单环刺螠希瓦氏菌氯霉素降解Intestinal electrochemically active bacteriaExtracellular electron transfer, Urechis unicinctusShewanellaChloramphenicol degradation
摘要电活性微生物能够利用其细胞膜上的细胞色素c蛋白和导电纤毛，将底物氧化产生的电子从胞内传递到胞外电子受体，该过程被称为微生物胞外电子传递（Extracellular electron transfer，EET）。EET是一种新型微生物厌氧能量代谢方式，在环境修复及清洁能源产生等领域具有广阔的应用前景。近年来，在人和哺乳动物肠道中陆续发现新的电活性微生物和微生物胞外电子传递方式，电活性微生物在维持宿主的肠道菌群稳态和宿主健康过程中发挥着重要作用。单环刺螠是国家地理标志性海洋生物，富含多种营养成分和极高的研究价值，而关于单环刺螠肠道中电活性微生物的存在情况及其肠道微生物应用方向的探究还未见报道。本研究以单环刺螠作为研究对象，分离、筛选及研究该物种肠道中的电活性微生物的基础生理学及电化学特征，并初步探索其在氯霉素降解中的应用潜势，以期为肠道电活性微生物与宿主的相互作用研究提供新的思路。（1）单环刺螠肠道微生物的分离及鉴定。厌氧条件下对取自新鲜单环刺螠肠道中的微生物进行富集培养，通过稀释涂布和平板划线法从该肠道富集物中分离到5个形态、大小、颜色、透明度等明显不同的单菌落。通过16S rRNA序列比对及构建系统发育树进行分类鉴定，结果表明5株肠道微生物分别来自4个不同的菌属，包括希瓦氏菌属（Shewanella）、葡萄球菌属（Staphylococcus共2株）、赖氨酸芽孢杆菌属（Lysinibacillus）和芽孢杆菌属（Bacillus）。随后对5株肠道微生物的环境分布、电化学活性及应用潜势进行了分析；其中，与单环刺螠肠道希瓦氏菌和赖氨酸芽孢杆菌亲缘关系最为接近的Shewanella marisflavi、Lysinibacillus macroides均被证明具有EET能力；希瓦氏菌属是当前研究电活性微生物的模式菌之一，因此本论文选择该希瓦氏菌株Shewanella marisflavi作为后续实验的研究对象，并将其命名为Shewanella marisflavi UU-3-2。（2）单环刺螠肠道Shewanella marisflavi的电活性及生理学特征。对分离到的肠道微生物UU-3-2的基础生理特征和电化学活性进行检测分析。首先，革兰氏染色法结果显示该菌株为革兰氏阴性菌，扫描电镜（Scanning electron microscope，SEM）进一步观察确定其形态为短杆状，长度约2 μm，宽度约为0.5 μm，伴有端生鞭毛；其次，高效液相色谱（High performance liquid chromatography，HPLC）检测其无氧呼吸底物消耗及产物生成情况，结果显示在厌氧条件下该菌株可以乳酸盐为电子供体富马酸为电子受体生成乙酸盐及琥珀酸；随后，菲啰嗪显色法及甲醛肟显色法分别证明菌株UU-3-2能够在厌氧条件下还原可溶性电子受体柠檬酸铁（FeC6H5O7）及不溶性电子受体Fe2O3中的Fe(III)和MnO2中的Mn(IV)，表明该肠道菌株具有异化金属还原能力；最后，通过构建单室微生物燃料电池（Single-chamber Microbial Fuel Cells，SCMFCs）对UU-3-2进行电化学特征分析，结果表明菌株UU-3-2具有产电能力，最大电流密度为146 mA/m2，且通过循环伏安扫描（Cyclic voltammetry，CV）曲线突出的氧化还原峰推断UU-3-2能够分泌氧化还原物质。由此可知，菌株UU-3-2是一株具有异化金属还原能力及产电能力的肠道电活性微生物。（3）单环刺螠肠道Shewanella marisflavi的氯霉素降解潜力。向UU-3-2培养体系中添加不同浓度的氯霉素溶液（10、20、30 mg/L），通过HPLC检测氯霉素浓度变化情况，结果显示三种浓度氯霉素均可被降解。其中，当添加10 mg/L氯霉素时，该菌株12 h内氯霉素的去除率为78.7%。此外，实验进一步探究了培养基pH值、盐浓度、电子供体和电子受体浓度对菌株生长情况及降解氯霉素作用的影响。实验结果表明，菌株对氯霉素的去除率受到环境条件的影响。具体表现为，1）pH值为7.2中性条件时菌株生长情况及氯霉素去除量相比较pH值为6.5及8.4更佳，但菌株在偏酸或偏碱性条件下仍具备氯霉素降解能力；2）盐浓度的改变对菌株降解氯霉素的影响表现为，盐度为3%-5%时具有最佳降解效果（分别为77.4%和71.6%），且低盐（1%）或高盐（8%）条件下仍具备降解能力，表明该菌株能够耐受较为严苛的盐度环境；3）菌株UU-3-2在不同浓度电子供体和受体中降解氯霉素曲线显示，随着电子供体或电子受体浓度升高，氯霉素去除率增加；4）药敏实验检测菌株的抗生素敏感性及生物安全性，结果显示多种革兰氏阴性菌抗菌剂对菌株具有较强的抑制效果，表明该菌株虽然具备降解氯霉素能力，但仍可以被多种抗生素抑制，具有较高的安全性。以上结果表明菌株UU-3-2是一株具有降解氯霉素能力的肠道电活性微生物，在抗生素污染治理方面表现出一定的应用潜力。综上所述，本研究成功从单环刺螠肠道中分离并鉴定了5株肠道微生物，其中1株（UU-3-2）具有电化学活性，是一株电活性微生物。在海洋肠道电活性微 生物应用方向分析中发现，UU-3-2具有降解氯霉素的潜力，且具有广泛的环境适用性。本研究不仅将肠道电活性微生物的分离扩展到了海洋生物，为海洋肠道电活性微生物的生理学及电活性研究提供了一定的数据支持，而且对海洋电活性微生物在抗生素污染治理中的应用进行了早期探索，为该方向研究工作提供了前期基础数据。
其他摘要Electrochemically active bacteria (EAB) are capable of transferring electrons generated by intracellular organic matter oxidation, to extracellular electron acceptors by conductive pills (e-pili) and the cytochrome c on the outmost membranes, this process is defined as microbial extracellular electron transport (EET). Microbial EET is a new manner of anaerobic energy metabolism, which has broad applied prospects in environmental restorations and clean energy generation. In recent years, the EAB presenting extracellular electron transfer capability have been widely reported in the intestines of human beings and other mammals. EAB play an important role in maintaining the gut microbiota homeostasis and health of hosts. Urechis unicinctus is a marine organism of national geographical possessing dozens of nutrients and high research values. However, the existence and applied potentials of EAB in the gut of Urechis unicinctus has not been reported. Here, Urechis unicinctus was selected as experimental samples. Subsequently, the EAB in the intestine was screened and isolated. In addition, the basic physiological and electrochemical activities of the EAB was depicted. Furthermore, the degradation capacity of chloramphenicol by the intestine EAB was investigated. This study underlights the essential functions of EAB in the intestine gut of Urechis unicinctus and provides an insight into the interactions between EAB and hosts.(1) Isolation and identification of gut microbes from Urechis unicinctus. The intestinal enrichments were obtained by dissecting fresh Urechis unicinctus under anaerobic conditions. Five single colonies with distinct differences in morphology, size, color and transparency were then successfully isolated from the intestinal enrichment by dilution coating and plate streaking. 16S rRNA sequence alignment and phylogenetic tree analysis show that the 5 isolates were from 4 different genera, including Shewanella, Staphylococcus (2 strains), Lysinibacillus, and Bacillus. Moreover, the environmental distribution, electrochemical activity, and applied potential of the 5 species were analyzed. Shewanella marisflavi and Lysinibacillus macrolides showing the closest relationship with the 5 isolated intestinal bacteria, had been proved to possess EET ability. Shewanella is used as a model genus to study EAB, therefore, in this study Shewanella marisflavi UU-3-2 was chosen as a key species in the following experimental processes.(2) Electrochemical and physiological characters of Shewanella marisflavi UU-3-2 isolated from Urechis unicinctus. The basic physiological and electrochemical activity of UU-3-2 were tested. Firstly, the isolate is gram-negative identified by gram staining, and then the morphology was depicted by scanning electron microscope (SEM), The species is a short rod, about 2 and 0.5μm in length and width, respectively accompanied by flagella on one of its ends. Secondly, high performance liquid chromatography (HPLC) was used to analyze the metabolites, the results show that, sodium acetate and succinic acid were the terminal metabolic products as sodium lactate and fumaric acid acting as the electron donor and acceptor, respectively. Subsequent ferrozine assays demonstrate that the strain is capable of reducing the Fe(III) in soluble ferric citrate(FeC6H5O7) and insoluble Fe2O3 under anaerobic conditions. Meanwhile, the formaldoxime assays show that Mn(IV) in insoluble MnO2 could be reduced by this species. These results indicate that UU-3-2 has the capacity in dissimilatory metal reduction. Finally, a maximum current density (146 mA/m2) and secreted redox substances were detected using single-chamber microbial fuel cells (SCMFCs) and cyclic voltammetry (CV) detection, respectively. Therefore, a line of the above evidence implies that UU-3-2 is an intestinal EAB presenting electricity production and dissimilatory metal reduction.(3) Study on chloramphenicol (CAP) degradation potential by Shewanella marisflavi UU-3-2. The variations of CAP concentrations were monitored by HPLC. The results manifest that chloramphenicol was degraded by the EAB S. marisflavi UU-3-2, and the removal rate of chloramphenicol (10 mg/L) peaked at 78.7% within 12 h. In addition, the effects of pH, the concentrations of salt, electron donor and electron acceptor on the chloramphenicol degradation and bacterial growth were exploited. The results show that, (1) The optimum pH value for bacterial growth and CAP degradation was at pH 7.2 while the species still could degrade chloramphenicol under acidic or alkaline conditions; (2) the salt concentration assay imply that the strain presented the best CAP degradation efficiency (77.4%) at salinity of 3%. Though CAP degradation was inhibited at low (1%) or high (8%) salinity, the strain still kept reduced CAP degradation and physiological activity, indicating that the strain can tolerate to some extent severe environmental conditions; (3) the CAP degradation curve show that the degradation rate was proportional to the increased concentrations of electron donors and acceptors; 4) the antibiotic sensitivity and biosafety were analyzed by the drug sensitivity test, the results show that UU-3-2 was sensitive to scores of G- bacteria antibiotics, testifying to that although this isolate had the ability to degrade CAP it may had a high biological safety. In conclusion, UU-3-2 is an EAB with the capacity of CAP degradation, indicating its applied potential in antibiotic pollution remediation.In this study, five intestinal microbes from Urechis unicinctus were successfully isolated and identified. Among them, UU-3-2 was confirmed to be an EAB with a maximum current density about 146 mA/m2. Furthermore, UU-3-2 show to some extent applied potential in antibiotics pollution remediation as the capability in CAP degradation. This work extends EAB from terrestrial niches to marine ecosystems, providing basic physiology and pathology data on the studying of EAB in the intestinal gut of marine organisms.
语种中文
文献类型学位论文
条目标识符http://ir.yic.ac.cnhttp://ir.yic.ac.cn/handle/133337/25257
专题中科院烟台海岸带研究所知识产出_学位论文

通讯作者李新推荐引用方式
GB/T 7714李新. 单环刺螠肠道电活性Shewanella marisflavi的氯霉素降解潜力研究[D]. 中国科学院烟台海岸带研究所. 中国科学院大学,2020.


PDF全文下载地址:

点我下载PDF