| 题名 | 高温混菌发酵木质纤维素制备丁酸的研究 |
| 作者 | 张春辉 |
| 学位类别 | 博士 |
| 答辩日期 | 2010-05 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 杨芳晓 ; 马玉久 |
| 关键词 | 热纤梭菌 嗜热丁酸梭菌 丁酸 木质纤维素 固定化细胞 |
| 学位专业 | 环境科学 |
| 中文摘要 | 本 文 主 要 内 容 是 Clostridium thermocellum （ 热 纤 梭 菌 ） 和 Clostridiumthermobutyricum（嗜热丁酸梭菌）高温混菌发酵木质纤维素制备丁酸的研究。首先介绍了包括丁酸的用途，丁酸生产菌株，丁酸代谢途径以及代谢调控等在内的丁酸发酵的研究进展。也讨论了分批发酵、补料-分批发酵以及连续性发酵在丁酸发酵中的应用，以及相关基因工程技术在菌株改造方面的运用。通过统计学实验设计和分析，对 C. thermobutyricum 摇瓶发酵丁酸的培养基成分进行了优化。首先，采用 Plackett-Berman 设计，进行单因素实验，并根据实验结果进行分析，挑选出具有高度统计学显著效应的因子，进行响应面实验优化。通过一个四因素三水平响应面实验（Box-Behnken 设计），获得了最优的摇瓶发酵丁酸的培养基配方。预测值和实际值之间的高度关联性表明了实验模型的准确性和可信性。在 7.2 g/l K2HPO4，34.9 g/l 葡萄糖，20 g/l 酵母提取物和 15 g/l乙酸的条件下，获得了最大浓度的丁酸 12.05g/l，达到了预测值 12.13g/l 的 99%。利用5L全自动发酵罐对C. thermobutyricum丁酸发酵的起始葡萄糖浓度和pH条件进行了优化。60g/l葡萄糖在pH6.2时经过36小时发酵，获得最高浓度丁酸20.06g/l。在pH值从6.8降低至5.3的过程中，丁酸代谢流发生了流向变化，从主产物 丁 酸 转 换 到 了 乙 酸 为 主 产 物 。 另 外 ， 获 得 一 株 能 够 耐 受 50g/l 丁 酸 的 C.thermobutyricum菌株，该菌株在72小时内可以将60g/l葡萄糖转化为23.58g/l丁酸。热纤梭菌和嗜热丁酸梭菌混菌发酵木质纤维素制备丁酸的研究，主要是利用前者所分泌的纤维素酶，将纤维素水解为纤维二糖和葡萄糖，而嗜热丁酸梭菌则可以重新利用这两种糖，并将其转化为丁酸。通过单因素实验，对混菌发酵的温度，接种方式，起始纤维素浓度以及 pH值进行了优化。利用混合接种的方法，将发酵温度控制在55℃，并保持6.2的pH值，热纤梭菌和嗜热丁酸梭菌将60g/l的纤维素转化获得了最高16.02g/l浓度的丁酸。对预处理玉米秸秆和棉花秸秆进行了混菌发酵的实验。用不同浓度的稀NaOH、HCl和H2SO4溶液对原料进行了预处理。玉米秸秆经过0.1M的H2SO4溶液在121℃处理60分钟后，发酵可得到最高丁酸产量11.63g/l；棉花秸秆则是经0.55M的H2SO4溶液121℃处理40分钟后有最高产值——12.58g/l丁酸。由 C. thermocellum 和 C. thermobutyricum 组成的混菌发酵体系存在严重的产物抑制效应。 对丁酸在体系中的抑制位点作了研究。通过在实验中添加不同量的丁酸的方法，对丁酸在共发酵体系中的作用，对热纤梭菌生长和代谢的抑制效应，以及对热纤梭菌纤维素酶分泌量和酶活性等方面进行了实验，实验表明丁酸在混合发酵体系中的主要作用点是对纤维素酶活性的抑制，尤其是在丁酸超过16g/l时。利用粉碎并处理后的玉米秸秆作为固定化材料，制作了一个固定化反应器对C. thermobutyricum 发酵丁酸进行了研究。在 25 天的连续性反应中对 pH 和乙酸在丁酸发酵中的作用进行了考察。pH 会改变菌株代谢主流的流动方向。丁酸和乙酸的最大比值、丁酸和乙酸的最大总量、以及丁酸和乙酸最小比值分别出现在pH7.0、6.0 和 5.0。培养基中添加乙酸可以提高丁酸的终浓度，在添加 10g/l 乙酸并且 pH 值为 6.0 时，获得丁酸的最高值 15.82 g/l。这一固定化细胞反应器也被用来考察 Clostridium beijerinckii ATCC 55025进行丙酮、乙醇和丁醇发酵的实验。此反应连续进行了 20 天用以考察稀释率（D）在溶剂生产中的作用。 D=0.2 h-1 时，当溶剂浓度最高为 8.99g/l。D 值在 0.2 和1.0h-1 之间增高时，溶剂时空产率也随之提高，并且在 D=1.0h-1 时，得到最高时空产率 5.06 g l-1h-1。最高 的溶剂/葡萄糖产率出现在 0.25h-1 时，数值为 0.32g/g。 并通过扫描电子显微镜对生长时期的 C. thermobutyricum 和 C. beijerinckii 细胞吸附和形态变化进行观察。 |
| 英文摘要 | Several issues of butyric acid production with mixed Clostridium thermocellum and Clostridium thermobutyricum culture through co-fermentation are presented in this paper.The current progress of butyric acid fermentation including the utilization of butyric acid, the production strains, the metabolic pathway, and regulation are presented in the introduction. Process operation modes such as batch, fed-batch, and continuous fermentation are being discussed. Genetic engineering technologies for microbial strain improvement are also being discussed and fermentation systems have been recommended.The optimal medium components for butyric acid production by Clostridium thermobutyricum in a shake flask culture were studied using statistical experimental design and analysis. Plackett-Berman Design (PBD) is an efficient and effective approach to the systematic investigation and eveluation of the effects of medium components. Each component was tested at two levels. The most effective factors with high significance levels were selected by PBD for further Box-Behnken design (BBD) optimization. The optimal composition of the fermentation medium for maximum butyric acid yield, as determined on the basis of a three-level four-factor BBD, was obtained by response surface methodology (RSM). The high correlation between the predicted and observed values indicated the validity of the model. A maximum butyric acid yield of 12.05 g/l was obtained at K2HPO4 7.2 g/l, 34.9 g/l glucose, 20 g/l yeast extract, and 15 g/l acetate, which compared well to the predicated production of 12.13 g/l.The optimal fermentation condition was studied by optimization glucose concertration and pH in a 5L fermentor. The maximum butyric acid concertration of 20.06g/l in 36 hours was abserved when pH was set at 6.2 and the initial glucose concertration was 60g/l. A metabolic shift from butyric acid production to acetic acid production was induced by changing pH from pH6.8 to pH5.3 of the medium.Additionally, a butyrate-telorance C. thermobutyricum strain was obtained which could grow under 50g/l butyric acid condition. This strain converted 60g/l glucose to 23.68g/l butyric acid in 72 hours.Co-fermentation by mixed C. thermocellum and C. thermobutyricum culture from lignocellulose to butyric acid was studied. The former produced cellulase which hydrolyzed cellulose to glucose and cellubiose, and the hydrolyzed cellulose was re-utilized and converted to butyric acid by the latter strain.The tempreture, innoculum style, initial Avicel concertration, and pH value were optimized by single-factor experiments. The top butyric acid concertration was 16.02g/l when 60g/l Avicel was fermented at pH6.2 with the tempreture of 55℃ and mixed innoculum.The pretreated corn stalk and cotton stalk were fermented to butyric acid by mixed C. thermocellum and C. thermobutyricum culture. Diluted NaOH, HCl and H2SO4 solutions were used to pretreat the lignocellulose. The maximum butyric acid production is 11.63g/l when the corn stalk was hydrolyzed by 0.1M H2SO4 solution at 121 ℃for 60minutes. For cotton stalk, the top yield was 12.58g/l when the pretreatment condition is 0.55M H2SO4 solution maintained at 121℃ for 40 minutes.Theco-fermentationsystemcomposedbyC. thermocellum and C. thermobutyricum was seriously inhibited by product inhibition. The inhibition location of butyric acid in the co-fermentation system was studied. The effects on co-fermentation, C. thermocellum growth and metabism, C. thermocellum cellulose excretion and activity were tested through the addition of butyric acid with different concertration in the experiments. The results suggested that the primary effect of butyric acid in the system was the inhibition of cellulase activity, when its concertration was 16g/l and more.Corn stalk was used as a support to immobilize Clostridium thermobutyricum in the fermentation process of butyric acid production. The effects of pH and acetic acid on butyric acid production were examined in a steady-state 25-day continuous flow operation. A metabolic shift was induced by changing pH of the medium. The maximum ratio, the maximum yield, the minimum ratio of butyric acid and acetic acid were achieved at pH 7.0, 6.0 and 5.0, respectively. The addition of acetate in the medium resulted in an increased butyric acid final concentration, and the maximum total butyric acid concentration of 15.82 g/l was obtained at pH 6.0 with 10 g/l acetic acid in the medium.The immobilized bio-reactor filled with corn stalk was used by Clostridia beijerinckii ATCC 55025 in the fermentation process of acetone, butanol, and ethanol (ABE) production, too. A steady state 20 days continuous flow operation was operated to examine the effect of dilution rate on solvent production. The maximum total solvent concentration of 8.99 g l-1 has been obtained at dilution rate at 0.2 h-1. The increased dilution rate between 0.2 and 1.0 h-1 has resulted in an increased solvent productivity and the highest solvent productivity has been obtained as 5.06 g l-1h-1 with dilution rate of 1 h-1. The maximum solvent yield from glucose of 0.32 g.g-1 was observed at 0.25 h-1. Both Clostridium thermobutyricum and Clostridia beijerinckii cells adsorption and morphology change during the growth on corn stalk support were examined by the SEM. |
| 语种 | 中文 |
| 学科主题 | 公共技术 |
| 公开日期 | 2011-08-29 |
| 内容类型 | 学位论文 |
| 源URL | [http://ir.qibebt.ac.cn//handle/337004/327]  |
| 专题 | 青岛生物能源与过程研究所_技术转移部中试技术服务中心 |
| 推荐引用方式 GB/T 7714 | 张春辉. 高温混菌发酵木质纤维素制备丁酸的研究[D]. 北京. 中国科学院研究生院. 2010. |
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