论文总字数：22279字

摘 要

细菌纤维素（bacterial cellulose, BC）是微生物合成的纤维素统称，有着许多植物纤维素无可比拟的优良性质，如高纯度、高结晶度、高聚合度、高吸水和保水能力、高拉伸强度和优良的生物相容性等。传统的生产BC方式为静态培养法，采用此方法发酵得到的BC呈现淡黄色或白色的膜状，具有表面积较大、聚合度高等优点。但是静态发酵也存在一些缺点，如发酵周期长、副产物较多、产量较低等。以提高BC产量为目的，本文以从红茶菌中筛选得到的、具有较高BC产量的A-4菌株为实验菌株，通过生理生化试验、分子生物学试验确定其系统进化学地位后，研究乙醇作为增效因子对BC产量的影响，并对乙醇作用机制进行了研究。实验结果表明，将A-4菌株的16S rRNA序列输入NCBI利用BLAST进行比对并构建系统进化树，确定A-4菌株属葡糖酸醋杆菌属（Gluconacetobacter. sp）；在一定范围内，乙醇能够显著提高BC产量，对A-4菌株而言，发酵培养基中的最适乙醇添加量为8 g/L，此时BC的干重、湿重产量分别为2.643 g/L、149.553 g/L，均比对照组提高了90 %以上，增产效果显著；此外乙醇作用机制的实验结果表明，乙醇促进醋酸菌发酵产BC的作用机制包括提高纤维素合成酶的酶活力、促进醋酸菌的菌体繁殖两方面。

关键词：细菌纤维素 葡糖酸醋杆菌 纤维素合成酶 静态发酵 分子生物学

Effect of ethanol for bacterial cellulose production

and its mechanism

ABSTRACT

Bacterial cellulose (BC) was a general name of the cellulose synthesized by different microbes contained genera Acetobacter, Gluconacetobacter, Agrobacterium, Sarcina. Compared to plant cellulose, BC has many unique physicochemical and mechanical properties, including higher purity and crystallinity, higher water holding and absorbing capacity, higher degree of polymerization and biological adaptability. The traditional production mode of BC was static fermentation, the BC formed by static fermentation displayed faint yellow or white membranes with high degree of polymerization and large surface area. There were some disadvantages of static fermentation such of long cycle, many by-products and low yield. In order to improving BC yield and overcome these fermentation disadvantages, A-4 strain isolated from traditional Kombucha was used for research after carrying out morphological and biochemical tests combined molecular biology identification. The effect of ethanol for BC production and the optimal adding ratio were researched. Based on these, the mechanism of ethanol for BC production was researched from several aspects contained promoting bacterial reproduction, improving enzyme activity and improving permeability of cell wall. The experimental results showed that A-4 strain was belonged to genus Gluconacetobacter. Ethanol could improve BC yield significantly and the optimal adding amount was 8g/L. In the case, the dry and wet weights were 2.643 g/L, 149.553 g/L, respectively. Compared to control group, the growth rates were more 90 %. And the mechanism of ethanol contained two aspects, that were promoting bacterial reproduction, improving the activity of cellulose synthase.

Key words: bacterial cellulose; Gluconacetobacter. sp; cellulose synthase; static fermentation; molecular biology

目 录

摘 要 I

ABSTRACT i

第一章 文献综述 1

1.1 细菌纤维素 1

1.2 BC的应用 1

1.2.1 BC在医学材料领域中的应用 1

1.2.2 BC在其它领域中的应用 2

1.3 BC的生产方式 3

1.3.1 静态发酵法 3

1.3.2 动态发酵法 3

1.4 本实验的研究背景与研究方法 4

第二章 实验材料与方法 5

2.1 实验材料 5

2.1.1 实验菌株 5

2.1.2 培养基 5

2.1.3 实验试剂 5

2.1.4 实验仪器 5

2.2 实验方法 6

2.2.1 菌株的活化 6

2.2.2 生理生化实验 6

2.2.3 分子生物学试验 6

2.2.4 种子液的制备与扩大培养 6

2.2.5 不同体积分数的乙醇溶液的配制 7

2.2.6 发酵培养基的制备与接种 7

2.2.7 BC膜的收获与处理 7

2.2.8 微生物生长曲线的测定 7

2.2.9 乙酸对纤维素合成和产酸代谢的影响 8

2.2.10 纤维素合成酶酶活力的测定 10

2.2.11 细胞壁通透性对BC产量的影响 10

第三章 实验结果与讨论 12

3.1 A-4菌株的生理生化实验 12

3.2 A-4菌株的分子生物学鉴定试验 12

3.3 发酵培养基中最适乙醇添加量的确定 12

3.4 乙醇对醋酸菌菌体繁殖的影响 14

3.5 细胞壁通透性对BC产量的影响 14

3.6 乙酸对BC产量的影响 15

3.7 乙酸对醋酸菌产酸代谢途径的影响 15

3.7.1 乙酸挥发性的确定 15

3.7.2 A-4培养液中乙酸含量的测定结果 16

3.8 乙醇和乙酸对CS活力的影响 16

第四章 结论与展望 18

4.1 结论 18

4.2 展望 18

参考文献 20

致 谢 24

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