论文总字数：32765字

摘 要

丁醇作为一种具有极大潜力的新型生物能源受到广泛关注，联合国国际能源署将丁醇列为第二代生物燃料。生物法制备丁醇可以降低对有限石油资源的消耗和依赖，发酵法生产丁醇也成了一大研究热点。而在发酵法生产丁醇的研究进展中发现，微生物发酵生产各种有用代谢产物时,其培养基组分往往种类繁多,且各组分间的相互作用也错综复杂。因此，优化微生物培养基的工作就显得尤为重要。现在数学统计中的多种优化方法己开始广泛地应用于微生物发酵培养基的优化工作中,其中以响应面方法的效果最为显著。本文也着重探讨响应面法优化发酵生产丁醇的工艺研究。

本文首先研究了生产丁醇的主要方法，生物合成法即发酵法。用于发酵法生产丙酮丁醇的菌种一般为Clostridium sp.(梭状芽孢杆菌)，其中最主要的当属拜氏梭菌Clostridium beijerinckii，与其他丁醇生产菌相比具有底物谱广、抗逆性强等特点。本文重点是将已选育到的阿魏酸抗逆菌株C. beijerinckii M11作为发酵菌株，利用响应面法优化甘蔗渣酸解糖液制备燃料丁醇的发酵工艺。

首先通过Plackett-Burman试验设计从5个因素中筛选出显著影响丁醇生产的3个因素，它们分别是CaCO₃、NH₄HCO₃、ZnSO₄·7H₂O，然后通过最陡爬坡试验和结果分析逼近极值区域，再根据中心点进行中心组合设计，确定影响因素CaCO₃、NH₄HCO₃、ZnSO₄·7H₂O的最佳浓度分别为3.14 g/L、2.16 g/L和0.16 g/L。最后经过模拟得出最优条件下丁醇发酵的预测浓度为6.75 g/L，验证实验结果为6.68 g/L，即实验结果与模拟预测值相吻合。

关键词：丁醇；拜氏梭菌；响应面

ABSTRACT

Butanol, as one of the important and potential biological energy has been classified as second generation of biofuel by the International Energy Agency. Biobutanol can reduce the consumption and dependency of the petroleum resources; hence butanol production through fermentation has become a hot pot of researchers. During the study of biobutanol, the products produced the variety of composition of medium affecting the microorganism mutually. It seems that optimizing the medium composition has become an important content. Now a variety of mathematical and statistical methods in optimization has been widely applied to optimize the work in microbial fermentation medium in which the response surface method is the most significant method. This paper focused on the optimization of butanol fermentation process using response surface methodology.

In this study we first studied the main method of producing biobutanol. Acetone-butanol fermentation of bacteria usually Clostridium sp. and compared with other butanol fermentation strains Clostridium beijerinckii has a characteristic of wide range of substrate utilization. The key point of this article is to optimize the fermentation process through response surface method of C. beijerinckii M11 which was a high ferulic acid tolerant strain.

First, using Plackett-Burman design to evaluate the influence of related 5 factors, CaCO₃、NH₄HCO₃、ZnSO₄·7H₂O were selected as the significant factors that affect the production of butanol. Second, the path of steepest ascent was undertaken to approach the optimum region of the three significant factors. Third, after Central Composite Design, the optimal concentration of nutrients was 3.14 g/L CaCO₃, 2.16 g/L NH₄HCO₃ and 0.16 g/L ZnSO₄·7H₂O. At last, a corresponding mathematical model was established to predict the fermentation experiment and maximum butanol yield of 6.75 g/L was acquired. Under the optimal conditions, the butanol concentration of verification test was 6.68 g/L, demonstrating that the mathematical model was reliable.

KEY WORDS: butanol；Clostridium beijerinckii；response surface method

目 录

摘 要 I

ABSTRACT II

第一章 文献综述 1

1.1 丁醇发酵概述 1

1.1.1 丁醇的性质 1

1.1.2 丁醇的主要用途 1

1.2 发酵法生产丁醇工艺的研究 2

1.2.1化学合成法 2

1.2.2 生物合成法 2

1.2.3 发酵方法 4

1.3 发酵生产丁醇的研究进展 6

1.3.1 原料来源 6

1.3.2 生物质发酵生产丁醇的现状研究 7

1.4 响应面法优化发酵产丁醇研究 8

1.4.1 响应面法介绍 8

1.4.2 响应面法优化发酵 8

1.5 研究内容与意义 9

1.5.1 研究内容 9

1.5.2 研究意义 9

第二章 材料与方法 11

2.1. 实验材料 11

2.2 实验试剂 11

2.3实验仪器与设备 12

2.4分析方法 13

2.4.1 pH的测定 13

2.4.2 葡萄糖、木糖及L-阿拉伯糖的测定 13

2.4.3 可溶性总酚(TPC, Total soluble Phenolic Compounds)的测定 13

2.4.4 菌体浓度的测定 14

2.4.5 产物检测 14

2.5 菌株培养基 14

2.6 培养方法 15

第三章 结果与讨论 16

3.1 单因素 16

3.2 Plackett-Burman实验 18

3.2.1 Plackett-Burman设计 18

3.2.2 Plackett-Burman试验设计及结果 19

3.3最陡爬坡实验及结果 20

3.3.1最陡爬坡实验设计 20

3.3.2 最陡爬坡实验结果 20

3.4 中心组合实验 21

3.5 模拟验证 26

3.6 本章小结 27

第四章 结论与展望 28

4.1 结论 28

4.2 展望 28

参考文献 29

致 谢 34

第一章 文献综述

1.1 丁醇发酵概述

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