论文总字数：18536字

摘 要

作为一种重要的有机化工原料及中间体,丁二酸主要用于医药工业、食品工业、化学工业,以及合成一些重要的化工产品，在2004年被美国能源部认为是未来12种最有价值的生物炼制产品之一。

谷氨酸棒杆菌是目前发酵工业中一种非常重要的生产菌种，已经广泛应用于多种氨基酸及其相关衍生物的生产。谷氨酸棒杆菌发酵生产氨基酸是一个好氧过程，同时可产生大量的菌体细胞。由于谷氨酸棒杆菌在厌氧条件下培养具有良好的有机酸生产潜力，因此以其在厌氧条件下生产有机酸受到广泛关注。基于上述谷氨酸棒杆菌在好氧和厌氧条件下的不同特性，在好氧条件下发酵生产氨基酸，生产附加值高的产品，在好氧过程结束后收集菌体，经简单处理后，将菌体转入厌氧条件进行有机酸的生产，可有效地利用了生物质资源，这对于节约资源，提高谷氨酸棒杆菌的生物利用率，和降低丁二酸的生产成本都具有重要的意义。

本文研究表明：有氧种子培养时，通过优化尿素添加量及初始pH，有利于提高有氧发酵阶段的谷氨酸棒杆菌的生物量，进而有利于厌氧阶段对丁二酸的转化，其丁二酸生产性能不受有氧阶段产鸟氨酸性能的显著影响。

有氧发酵阶段形成的生物量直接关系厌氧发酵制备丁二酸的性能，当有氧发酵培养基中的生物素添加量为50~100ug/L时，其菌体浓度显著提高，可获得21~22g/L的丁二酸，对糖转化率约50%。厌氧条件下的初始糖浓度20~60g/L对丁二酸对糖转化率影响不显著，其转化率维持在49~51%，但当初糖浓度为60g/L时，加入碱式碳酸镁浓度较高，导致葡萄糖未完全消耗，且发酵结束时的生物量与产酸活力明显降低，72h仅产丁二酸25g/L。

针对以上问题，本文采用分批加入碱式碳酸镁的方式避免发酵初期pH过高对菌体活力带来损害，60 g/L的葡萄糖可完全消耗，最终48h产丁二酸29 g/L。本文研究结果表明：产鸟氨酸的谷氨酸棒杆菌Corynebacteria glutamicum 3663在完成有氧发酵制备鸟氨酸后可在厌氧条件下将葡萄糖转化为丁二酸，其转化率约50%，在此基础上开展深入研究将有利于降低生物基丁二酸的制备成本。

关键词：谷氨酸棒杆菌，丁二酸，厌氧发酵

Abstract

As an important organic chemical raw materials and intermediates, succinic acid is mainly used in the pharmaceutical industry, food industry, chemical industry, as well as the synthesis of a number of important chemical products, in 2004 by the US Department of Energy considered the future of the 12 most valuable biorefinery one product.

Glutamicum fermentation industry is a very important production strains, has been widely used in the production of a variety of amino acids and related derivatives. Glutamicum fermentative production of amino acids is an aerobic process, the cells produce large amounts of biomass. In recent years, scientists in Japan found that glutamicum under anaerobic conditions to cultivate good potential for organic production, the accumulation of large amounts of lactic acid, succinic acid. Therefore its production of organic acids under anaerobic conditions attracted widespread attention. Based on the above glutamicum different characteristics under aerobic and anaerobic conditions in the fermentation production of amino acids under aerobic conditions, the production of high value-added products, at the end of the process of collecting the aerobic bacteria, after simple treatment, the cells were transferred to the anaerobic conditions of the production of organic acids, can effectively utilize biomass resources, which for the conservation of resources, improve bioavailability glutamicum, and reduce the production cost succinic acid has important meaning.

This study showed that: aerobic seed culture, by optimizing the amount of urea and initial pH, help improve glutamicum biomass aerobic fermentation stage, and thus in favor of the anaerobic stage conversion of succinic acid, which succinic acid production performance is not significantly affected the production of ornithine aerobic stage performance.

When biomass aerobic fermentation stage performance is directly related to the formation of succinic acid anaerobic fermentation of preparation, when aerobic fermentation medium biotin dosage 50 ~ 100ug / L, which significantly increased cell concentration obtained 21 ~ 22g / L succinic acid, sugar conversion rate of about 50%. The initial sugar concentration under anaerobic conditions 20 ~ 60g / L of glucose to succinic no significant effect on the conversion rate, the conversion rate was maintained at 49 to 51 percent, but now the sugar concentration of 60g / L, adding magnesium carbonate higher concentrations, leading to the initial pH is too high (pH8.5), glucose is not completely consumed, and fermentation of biomass at the end of the

activity was significantly reduced acid production, 72h only produced succinate 25g / L.

To solve these problems, we use basic magnesium carbonate was added in portions of way to avoid the initial fermentation pH is too high cell viability cause damage, 60 g / L glucose can be completely consumed, ultimately producing succinic acid 48h 29 g / L. Results of this study showed that: the production of ornithine glutamicum Corynebacteria glutamicum 3663 after the completion of the preparation of ornithine may aerobic fermentation under anaerobic conditions for converting glucose to succinic acid, the conversion rate of about 50% in Based on this in-depth study carried out will help reduce the production cost of bio-succinic acid.

Keyword：Glutamicum，Succinate，Anaerobic fermentation.

目 录

摘 要 I

Abstract II

第一章 前言 1

1.1国内外谷氨酸棒杆菌的研究概况 1

1.1.1谷氨酸棒杆菌的特征 1

1.1.2谷氨酸棒杆菌的代谢工程的研究 1

1.1.3谷氨酸棒杆菌产丁二酸的研究 2

1.1.4谷氨酸棒杆菌厌氧条件下产丁二酸（发酵法） 3

1.2 本课题研究目的及内容 4

第二章 材料与方法 5

2.1材料 5

2.1.1主要试剂 5

2.1.2仪器设备 5

2.1.3菌种 6

2.1.4培养基与溶液配制 6

2.2培养条件 6

2.3 检测方法 7

2.3.1 生物量的测量 7

2.3.2葡萄糖含量的测定 7

2.3.3 发酵液中有机酸含量测量的测定 7

第三章 结果与讨论 8

3.1.种子培养阶段尿素浓度对厌氧发酵产丁二酸性能的影响 8

3.2 有氧种子培养初始pH对厌氧发酵产丁二酸性能的影响 8

3.3有氧发酵阶段生物素添加量对厌氧发酵产丁二酸性能的影响 9

3.4有氧阶段初糖浓度对厌氧发酵产丁二酸性能的影响 9

3.5 厌氧发酵阶段葡萄糖初始浓度对厌氧发酵产丁二酸性能的影响 10

3.6 厌氧发酵阶段分批加入碱式碳酸镁对丁二酸生产性能的影响 10

第四章 结论与展望 11

4.1结论 11

4.2展望 11

参考文献 12

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