论文总字数：19159字

摘 要

产琥珀酸放线杆菌是一种野生的细菌菌株，具有高效生产丁二酸，发酵产生的杂质少，可以利用多种碳源以及廉价的可再生资源作为原料等特点，被誉为琥珀酸最有效的天然生产者之一。但是，在产琥珀酸放线杆菌培养时，由于菌株的生长过程中会产生丁二酸或其他副产物，这些酸性物质逐渐积累导致pH降低，菌株生长受到影响，导致丁二酸产率降低。本课题以储藏的产琥珀酸放线杆菌NJ113为探究对象，将菌株置于长期酸性环境下，分析和寻找适应酸性环境的突变菌株，以此提高菌株的耐酸能力。

本课题通过连续传代培养，逐步降低培养环境pH值，希望能收获生长良好并且高产丁二酸的菌株。经实验发现，连续传代培养后的菌株在pH 5.8发酵培养50 h后，葡萄糖消耗量13.18 g/L，丁二酸的产量达5.17 g/L，收率39.22 g/g，原始菌株在pH 5.8，培养50 h后葡萄糖消耗量12.98 g/L，丁二酸的产量为4.41 g/L，收率33.97 g/g，丁二酸产量增加了17.23%，收率增加了15.45％。

关键词：琥珀酸放线杆菌；丁二酸；酸胁迫；PH控制

Study on the training and acid resistance of Actinobacillus succinogenes NJ113

Abstract

Actinobacillus succinogenes is a wild bacterial strain with high-efficiency production of succinic acid, less impurities produced by fermentation, can use a variety of carbon sources and cheap renewable resources as raw materials, etc., is known as the most succinic acid One of the effective natural producers. However, during the cultivation of Actinobacillus succinogenes, succinic acid or other by-products are produced during the growth of the strain. These acidic substances gradually accumulate, resulting in a decrease in pH, which affects the growth of the strain and reduces the yield of succinic acid. This subject takes the stored Actinobacillus succinogenes NJ113 as the research object, puts the strain in a long-term acid environment, analyzes and looks for the mutant strain that adapts to the acid environment, so as to improve the acid tolerance of the strain.

In this project, through continuous subculture, the pH value of the culture environment is gradually reduced, hoping to harvest strains that grow well and produce succinic acid with high yield. It was found through experiments that the strains after continuous subculture were fermented and cultured at pH 5.8 for 50 h. The glucose consumption was 13.18 g / L, the yield of succinic acid reached 5.17 g / L, the yield was 39.22 g / g, and the original strain was at pH 5.8 After 50 hours of cultivation, glucose consumption was 12.98 g / L, succinic acid production was 4.41 g / L, yield was 33.97 g / g, succinic acid production increased by 17.23%, and yield increased by 15.45%.

Keywords: Actinobacillus succinate; succinic acid; acid stress; PH control

目录

摘要 I

Study on the training and acid resistance of Actinobacillus succinogenes NJ113 II

Abstract II

目录 I

第一章 前言 1

第二章 文献综述 2

2.1 丁二酸 2

2.1.1 丁二酸的理化性质 2

2.1.2 丁二酸的生产 2

2.1.3 丁二酸的用途 3

2.2 产琥珀酸放线杆菌简介 4

2.2.1 产琥珀酸放线杆菌特点 4

2.2.2 产琥珀酸放线杆菌发酵产丁二酸途径 4

2.3 微生物抵御酸胁迫机制的研究 5

2.3.1 酸胁迫产生的原因 6

2.3.2 酸胁迫的危害 6

2.4 微生物抵御酸胁迫的途径 7

2.4.1 细胞膜和细胞壁的变化 7

2.4.2 增强质子泵 7

2.4.3 改变基因表达及蛋白调控 8

2.5课题来源及研究内容 8

2.5.1课题来源 8

2.5.2本课题研究内容 8

第三章 材料与方法 9

3.1菌株 9

3.2主要实验试剂 9

3.3主要实验仪器 10

3.4培养基 10

3.5 培养方法 11

3.5.1种子培养 11

3.5.2连续发酵传代培养 11

3.6分析方法 11

2.4.1 pH的测定 11

2.4.2 OD的测定 11

2.4.3葡萄糖含量的测定 11

2.4.4有机酸含量的测定 11

第四章 结果与讨论 12

4.1 pH为6.8时的生长曲线，耗糖以及产物的分布 12

4.2 连续传代培养 13

4.2.1 pH为6.4时，培养菌株的OD以及葡萄糖变化情况 13

4.2.2 pH为6.1时，培养菌株的OD以及葡萄糖变化情况 14

4.2.3 pH为5.8时，培养菌株的OD以及葡萄糖变化情况 15

4.3连续传代培养后菌株与原始菌株在pH为5.8时发酵对比 16

4.4讨论与展望 17

参考文献 18

致谢 21

第一章 前言

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