论文总字数：18655字

摘 要

丙酮丁醇发酵有着悠久的历史，在上世纪五十年代以前，曾是仅次于酒精发酵的世界第二大发酵过程。但是，随着底物价格的提高和石油化学的优越性，丙酮丁醇发酵逐渐衰退下来。近年来，经济快速发展，伴随如此高速的经济发展便是石化资源的大量开采。而石化资源的开采及使用，也带来了能源的耗竭与环境的污染，全球变暖等问题。开发有利于可持续发展的储备技术广受关注，因而清洁、可再生的生物质能源——生物丁醇成为研究热点。而丙酮丁醇发酵作为生产生物丁醇的重要手段也重新引起了人们的广泛关注。但是，丙酮丁醇发酵目前还存在产物浓度低、产物回收成本高等问题制约了该产业的经济效益。

研究表明，丙丁梭菌细胞能够吸附在特定载体上，大量聚集后分泌胞外聚合介质EPS，进而促发菌体细胞以生物膜的形式生存，可明显增加高活性菌体密度，提高细胞耐受性和反应活性，对提高生物丁醇生产过程的时空效率具有重要意义。为了进一步研究丙酮丁醇梭菌发酵过程中的成膜现象，就需要一种可靠的活细胞标记物。绿色荧光蛋白因其对细胞无伤害且检测简单而十分适合作为标记物。本课题首次将绿色荧光蛋白转入丙酮丁醇梭菌中，确认方法的可行性，并为之后的研究奠定基础。

关键词：丙酮丁醇梭菌；绿色荧光蛋白；生物丁醇；生物膜

ABSTRACT

Acetone butanol fermentation has a long history. Before the 50s of last century, it was the second largest fermentation process in the world after alcohol fermentation. However, the fermentation of acetone and butanol gradually declined with the increase of substrate price and the superiority of petroleum chemistry. In recent years, the rapid economic development, accompanied by such rapid economic development, is a large number of petrochemical resources mining. The exploitation and utilization of petrochemical resources also bring energy depletion, environmental pollution, global warming and other issues. The development of reserve technology for sustainable development has attracted wide attention. Therefore, clean and renewable biomass energy, bio butanol, has become a hot research topic. And acetone butanol fermentation as an important means of production of biological butanol has also aroused widespread concern. However, there are still problems such as low concentration of products and high recovery costs of acetone butanol fermentation, which restricts the economic benefits of the industry.

Research shows that by Clostridium acetobutylicum cells can be adsorbed on a specific carrier, after the large accumulation of extracellular polymeric media EPS, so as to promote cell survival in the form of biofilm, can significantly increase the activity of high cell density, increase cell tolerance and reaction activity, to improve the efficiency of bio butanol production process time is important meaning. In order to further investigate the membrane formation during the fermentation of Clostridium butyricum, a reliable live cell marker is needed. Green fluorescent protein is well suited as a marker because it is harmless to cells and simple to detect. This topic for the first time transferred green fluorescent protein into Clostridium butyricum, confirmed the feasibility of the method, and laid the foundation for further research.

目 录

摘 要 I

ABSTRACT II

第一章 文献综述 1

1.1 引言 1

1.2 丙酮丁醇梭菌及生物膜简介 1

1.2.1 丙酮丁醇梭菌 1

1.2.2 生物膜 2

1.2.3 生物膜的形成过程 2

1.3 绿色荧光蛋白 3

1.3.1 简介 3

1.3.2 不同种类的荧光蛋白 4

1.3.3 对氧的需求 5

1.3.4 GFP的应用 5

第二章 材料与方法 7

2.1 材料 7

2.1.1 实验试剂和药品 7

2.1.2 实验仪器和设备 7

2.1.3 培养基 8

2.2 实验方法 8

2.2.1 目的基因查找 8

2.2.2 重组质粒构建 9

2.2.3 重组质粒的甲基化 14

2.2.4 重组质粒的电转化 14

2.2.5 荧光显微镜 15

第三章 结果与讨论 17

3.1 PCR电泳验证结果 17

3.1.1 重组质粒甲基化电泳结果 17

3.1.2 GFP片段PCR电泳结果 17

3.2 紫外灯下GFP的观察结果 18

3.3 荧光显微镜GFP成像的观察 18

3.4 讨论 19

第四章 结论与展望 20

4.1 结论 20

4.2 展望 20

参考文献 21

致谢 23

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