产电微生物胞内电子传递途径选择及胞外电子释放能力评估的研究

发布时间：2018-03-24 15:20

本文选题：产电微生物　切入点：CymA依赖性　出处：《江苏大学》2017年硕士论文

【摘要】：伴随着近年来在环境治理、纳米材料合成和生物能源等领域的广泛应用,产电微生物越来越受到研究者的关注。Shewanella是一种典型的产电微生物。它拥有强大的厌氧异化呼吸能力,具有广泛的环境适应性。但是目前对于Shewanella跨膜电子传递路径选择的研究还不是很透彻,特别是关于内膜蛋白CymA在跨膜电子传递过程中的作用尚存在许多争议。另外,电子胞外释放能力对于产电微生物的环境应用具有重要影响。但是目前已建立的评估微生物胞外电子释放能力的方法存在诸多缺陷,亟需发展一种便捷、高效、便宜的新方法来评估微生物的胞外产电能力。因此,本论文以模式产电微生物Shewanella oneidensis MR-1为研究对象,探究胞外电子释放过程对胞内电子传递途径选择的影响,并在此基础上建立了一种基于染料胞外还原的高通量比色法用以微生物胞外产电效能的高效评估。本研究初步得到以下几个结论:(1)胞内电子传递途径的选择受到胞外电子释放过程的影响。胞外电子受体性质决定了c型细胞色素CymA在电子跨膜传递过程中的重要性。CymA对于易利用的柠檬酸铁还原具有重要作用,但是对于难利用的Fe_2O_3胞外还原作用不大。通过添加胞外电子递质、提高柠檬酸铁浓度以及增加电极电势,加大胞外电子的释放速度,都能提高CymA在跨膜电子转运过程中的重要性。因此在胞外高电子传递速率条件下,胞内电子传递过程对CymA依赖性比较大。而在低电子释放速率条件下,其他的电子途径可以弥补CymA缺失的影响。(2)微生物胞外电子释放能力可以通过基于染料胞外还原的比色法进行高通量评估。实验证明不能进入细胞的强极性染料甲基橙(MO)和萘酚绿B(NGB)可以作为评估微生物胞外产电能力的指示剂。在厌氧条件下监测染料胞外还原速率不仅可以快速评估不同种微生物的胞外产电能力,还能区分出同种微生物的不同突变体之间产电能力的差异。活性污泥中的混合菌群对MO脱色效能与MFC产电趋势相耦合,进一步证明该方法不但可以评估纯菌的胞外产电能力,还可以评估混合菌群的胞外产电能力。此外,该方法还能优化碳源、温度等实验条件以提高微生物的胞外产电效能。因此本论文中所建立的高通量比色评估方法是一种简单方便、准确可靠、具有多功能利用的新型方法。
[Abstract]:In recent years, with the extensive application in the fields of environmental control, nano-material synthesis and bioenergy, the electroproducing microorganism has been paid more and more attention by researchers. Shewanella is a typical electric-producing microorganism. It has strong anaerobic dissimilatory respiration ability. However, the study of Shewanella transmembrane electron transport path selection is not very thorough, especially about the role of membrane protein CymA in the transmembrane electron transport process. In addition, there are still many controversies on the role of membrane protein CymA in the transmembrane electron transport process. The ability of extracellular electron release plays an important role in the environmental application of electrically producing microbes. However, there are many defects in the existing methods for evaluating the ability of electron release of microbes outside the cell, so it is necessary to develop a convenient and efficient method. Therefore, Shewanella oneidensis MR-1, a model electroproducing microorganism, was used to study the effect of extracellular electron release on the selection of intracellular electron transport pathway. On this basis, a high throughput colorimetric method based on dye extracellular reduction was established for the efficient evaluation of the extracellular electricity production efficiency of microorganisms. In this study, the following conclusions were preliminarily obtained: 1) the selection of intracellular electron transport pathway was evaluated. Effect of extracellular electron release process. The properties of extracellular electron receptors determine the importance of type c cytochrome CymA in electron transmembrane transport. CymA plays an important role in the readily available reduction of ferric citrate. By adding extracellular electron transmitters, increasing the concentration of ferric citrate and increasing the potential of electrode, the release rate of extracellular electrons was increased. Both can increase the importance of CymA in the transmembrane electron transport process. Therefore, at high extracellular electron transfer rate, the intracellular electron transfer process is highly dependent on CymA, but at low electron release rate, Other electron pathways can compensate for the loss of CymA. 2) the extracellular electron release ability of microorganisms can be evaluated by colorimetric method based on dye extracellular reduction. Under anaerobic conditions, monitoring the rate of extracellular reduction of dyes can not only rapidly evaluate the extracellular electricity production ability of different microorganisms, but also can be used as indicator for the evaluation of extracellular electricity production ability of different microorganisms. The mixed bacteria in activated sludge are coupled with the trend of MFC to decolorize MO. It is further proved that this method can not only evaluate the extracellular electricity production ability of pure bacteria, but also distinguish the difference of electricity production ability between different mutants of the same microorganism. It can also be used to evaluate the extracellular electricity production ability of the mixed bacteria. In addition, the method can also optimize the carbon source. The experimental conditions such as temperature are used to improve the extracellular electricity production efficiency of microorganisms. Therefore, the high throughput colorimetric evaluation method established in this paper is a simple, convenient, accurate and reliable method with multifunctional utilization.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X703;X172

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