论文总字数：25649字

摘 要

当前社会的快速发展导致了能源耗竭和环境污染等问题，开发新型清洁可再生能源的研究备受关注，因此生物丁醇走进研究人员的视野。丙酮丁醇梭菌作为ABE（丙酮-丁醇-乙醇）发酵的重要工业菌株在发酵过程中会分泌胞外聚合物，产生生物被膜。本研究以丙酮丁醇梭菌为研究对象，探讨其组氨酸蛋白激酶CA_C2730对生物被膜的形成等生理性状影响。

（1）首先，对丙酮丁醇梭菌的CA_C2730基因进行敲除，构建突变菌株Δ2730。将表达质粒pSY8-2730电转化如突变菌株中，得到回补菌株c-Δ2730。将表达质粒pSY8-2730电转化入野生菌株B3中，构建过表达菌株B3-pSY8-2730。

（2）然后，对其形成的生物被膜进行检测，发现突变菌株Δ2730产膜量下降约98%，回补菌株c-Δ2730产膜量只有野生菌株的30%。利用测定发酵产物和残余糖浓度对其发酵性能进行测定，发现突变菌株Δ2730丁醇产量提高30%，回补菌株c-Δ2730出现“酸崩溃”现象。用透射电镜观测其细胞形态，发现突变菌株Δ2730缺失鞭毛结构。所以对其半固体平板上的运动能力进行测试，发现突变菌株Δ2730无明显运动，过表达菌株B3-pSY8-2730菌落面积扩大约5倍，运动能力明显上升。

（3）最后，根据实验结果与数据，组氨酸蛋白激酶CA_C2730通过调节菌株鞭毛结构的形成与运动能力从而控制其生物被膜的形成。

本研究探讨了组氨酸蛋白激酶CA_C2730对丙酮丁醇梭菌的生物被膜形成机制，给生物被膜的工业化应用提供了理论依据。

关键词：丙酮丁醇梭菌 生物被膜 组氨酸蛋白激酶

Effect of histidine protein kinase CA_C2730 on the biofilm formation of Clostridium acetobutylicum

Abstract

There are many questions such as energy depletion and environmental pollution,because of the rapid development of society. Researching for new clean renewable energy has been followed. Therefore, researchers begin to take attention to biobutanol. Clostridium acetobutylicum, as an important industrial strain of ABE (acetone-butanol-ethanol) fermentation, secretes extracellular polymeric substances during the fermentation process, producing a biofilm. In this study, we investigated the glutamine kinase CA_C2730 in acetone butanol affects physiological properties such as formation of biofilm.

(1) First, knockout of CA_C2730 gene of acetone butanol sovereign is knocked out, and Δ2730 is constructed. The plasmid pSY8-2730 is electrically converted as a mutant strain to obtain a c-Δ2730. The recombinant pSY8-2730 was transfered electricity to the wild strain B3.

(2) it was found that the amount of the membrane produced by the Δ2730 decreased by about 98%, and that the amount of the membrane produced from the c-Δ2730 was only 30% of the wild strain. The fermentation performance was determined by measuring the fermentation product and residual sugar concentration, and it was found that the mutant strain Δ2730 butanol production increased by 30%, and the supplementary strain c-Δ2730 had an “acid collapse” phenomenon. With the cell morphology, tem observation found that the mutant strain Δ 2730 lack flagella structure. Therefore, when the motor ability on the semi solid plate was tested, it was found that the mutant strain. Bacterial strain b3-psy8-2730 increased the bacterial area by about 5 times and increased the motor capacity.

(3) Finally, based on the experimental results and data, the histidine protein kinase CA_C2730 controls the formation of the biofilm by regulating the formation and movement ability of the flagella structure of the strain.

This study explored the biofilm formation mechanism of histidine protein kinase CA_C2730 on Clostridium acetobutylicum, and provided a theoretical basis for the industrial application of biofilm.

Key Words: Clostridium acetobutylicum Biofilm Histidine protein kinase

目 录

摘 要 I

Abstract II

第一章 文献综述 1

1.1生物被膜简介 1

1.2生物被膜组分 1

1.2.1胞外聚合物（EPS） 1

1.2.2 EPS的组成 2

1.3生物被膜的形成过程 4

1.4丙酮丁醇梭菌简介 5

1.4.1丙酮丁醇梭菌B3简介 5

1.4.2丙酮丁醇梭菌的CA_C2730基因 5

1.4.3双组分信号传导系统 6

1.5实验思路与目的 6

1.5.1实验思路 6

1.5.2实验目的 6

第二章 实验材料与方法 7

2.1实验材料 7

2.1.1菌株、质粒和引物 7

2.1.2实验仪器 8

2.1.3实验试剂 9

2.1.4培养基 11

2.2实验方法 13

2.2.1培养方法 13

2.2.1.1大肠杆菌培养方法 13

2.2.1.2 丙酮丁醇梭菌厌氧培养发酵方法 13

2.2.2工具酶与试剂盒 14

2.2.3 CA_C2730基因突变菌株Δ2730的构建方法 14

2.2.5 CA_C2730过表达菌株B3-pSY8-2730的构建 18

2.2.6生物被膜的定量检测 19

2.2.7测定其发酵性能 19

2.2.7.1发酵产物的测定 19

2.2.7.2残余糖浓度的测定 20

2.2.8使用透射电镜（TEM）观察菌株形态 20

2.2.9半固体平板测定其运动能力 21

第三章 实验结果与讨论 22

3.1菌株构建结果 22

3.1.1构建突变菌株Δ2730的结果 22

3.1.2回补菌株c-Δ2730构建结果 22

3.1.3过表达菌株B3-pSY8-2730构建结果 23

3.2 CA_C2730基因的失活使菌株生物被膜形成能力下降 23

3.3 CA_C2730基因的失活提高了菌株的发酵效率 24

3.3.1Δ2730菌株在发酵过程中菌体密度大幅增加 25

3.3.2Δ2730菌株发酵产物浓度高于野生菌株B3 25

3.4 CA_C2730基因的失活使丙酮丁醇梭菌无法产生鞭毛结构 27

3.5 CA_C2730基因的失活使菌株运动能力下降 28

第四章 结论 30

参考文献 32

致谢 35

第一章 文献综述

1.1生物被膜简介

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