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题名	微生物法生产丁二酸过程工程研究
作者	王彩霞
学位类别	博士
答辩日期	2014-06
授予单位	中国科学院研究生院
导师	邢建民
关键词	丁二酸   生物质转化   超滤分离   发酵膜分离耦合   生物基PBS
其他题名	process Engineering of Efficient Succinic Acid Production via Microbial Fermentation
学位专业	生物化工
中文摘要	丁二酸是一种重要的平台化合物。利用微生物法生产丁二酸可以代替传统的石化途径。目前丁二酸的生物合成取得了很大的研究进展，但依然面临着许多问题。本研究从微生物菌种构建出发，探索利用生物质原料进行丁二酸生产，建立了丁二酸发酵膜分离纯化工艺。 构建了高产丁二酸大肠杆菌底盘微生物，考察了乳酸脱氢酶、乙酸激酶以及丙酮酸甲酸裂解酶等基因的删除对于大肠杆菌碳流变化的影响，结果表明乳酸脱氢酶（ldhA）的删除使乳酸浓度由13.36 g/L 降低到0.59 g/L，同时丙酮酸由无积累增加到1.7 g/L，乙酸浓度则由3.47 g/L 升高到4.97 g/L, 甲酸由1.73 g/L 升高到4.30 g/L。乳酸脱氢酶的敲除使得丙酮酸生成乳酸减弱，而其生成甲酸和乙酸增强，同时部分丙酮酸得以积累。乳酸脱氢酶和乙酸激酶双基因敲除的菌株生长变缓，在发酵方面，乙酸激酶的删除并没有显著降低乙酸浓度，丁二酸浓度有所提高，提高幅度仅2.3%，甲酸和丙酮酸含量有所降低。枯草芽孢杆菌丙酮酸羧化酶pyc和产琥珀酸放线杆菌130Z的pck分别导入乳酸脱氢酶敲除大肠杆菌MG1655菌株中，丁二酸产量分别提高7倍和6.17倍，同时甲酸和丙酮酸几乎无积累。 探索了利用热解生物油水相部分作为菌株生长和发酵底物，结果显示水相生物油可以作为菌体生长和丁二酸发酵的培养基，但是添加浓度不应高于25%。提出了生物质热裂解产物和酶解液用于产丁二酸发酵的思路，生物质经过酶解获得含糖酶解液和水相生物油部分共同作为底物发酵产丁二酸，在12.5%生物油浓度下，丁二酸发酵浓度达到11.5 g/L。探索离子液体AmimCl处理后的生物质作为丁二酸的发酵底物，结果表明离子液体AmimCl对松木有着很好处理效果，能够有效的破坏纤维素结晶度，处理后松木中纤维素含量增加，酶解率为61.7%，用离子液体处理后松木酶解液发酵产丁二酸，丁二酸浓度达20.7 g/L, 产率为0.9 g/L/h，糖酸转化率为0.99 mol/mol。离子液体对于玉米秸秆的处理效果一般，因此考虑组合预处理方式，结果表明离子液体AmimCl对于汽爆秸秆和高温水热秸秆处理效果比较好，用离子液体处理汽爆秸秆的酶解液发酵产丁二酸，丁二酸发酵浓度为17.0 g/L，产率为0.89 g/L/h，糖酸转化率为1.05 mol/mol。 将超滤技术用于丁二酸的分离纯化，综合比较了PES 100kDa、PES 10kDa、PES 30kDa以及RC 10kDa四种超滤膜在处理丁二酸发酵液时通量、蛋白截留率以及处理前后丁二酸浓度的变化，结果显示RC 10kDa和PES 10kDa的效果较好。与传统的离心相比，超滤在菌体和蛋白的截留率上都更具优势。利用线性阻力模型分析了造成四种超滤膜通量下降的主要阻力形式。利用Hermia’s model分析了四种超滤膜处理丁二酸发酵液过程中污染机制。结果表明，RC 10kDa and PES 30kDa为Complete blocking污染形式，PES 100kDa是 intermediate blocking的污染形式，PES 10kDa是caker layer 模型。膜污染前后接触角和粗糙度分析表明疏水性物质吸附于膜表面，是造成膜污染的原因。 将丁二酸超滤分离技术和发酵进行耦合，丁二酸发酵浓度提高了37%。考察了HPD-950树脂和活性炭不同添加量下对于发酵液脱色效果的影响，结果表明在1%的添加量以上，HPD-950对于发酵液的脱色效果优于活性炭。提出了丁二酸发酵-膜分离纯化研究工艺，即发酵-膜分离-脱色-浓缩-结晶，整个工艺对于丁二酸的收率在80-90%，纯度98%以上。结晶实验表明糖的存在容易使得丁二酸纯度降低。利用分离得到的丁二酸进行PBS实验，其氢谱核磁结果显示其成分与标品非常一致，Mw达到了150,000 g/mol。生物基丁二酸合成丁二酸二乙酯实验表明，其转化率为90%，选择性达到49%。 关键词：丁二酸，生物质转化，超滤膜分离，发酵-膜分离耦合，生物基PBS
英文摘要	Succinic acid (1, 4-butanedioic acid) is important for the synthesis of some valuable chemical derivatives, which can be used in food and pharmaceutical products, solvents, biodegradable polymers, surfactants and detergents. Due to its independence from petroleum, environmental benefit and CO2 sequestration, biological production of succinic acid has been investigated intensively and gained big progress during recent years. In this study, efficient succinic acid producing strains was contructed, biomass was used to produce succcinic acid, and more importantly membrane based succinic acid separation process was developed. Influence of ldhA and ackA deletion on bacterial growth and fermentation were studied. Lactic acid concentration decreased from 13.36 g/L to 0.59 g/L, meanwhile concentrations of pyruvic acid, acetic acid and formic acid increased with varying degrees due to the deletion of ldhA. Stains with ldhA and ackA deletion grows slowly than the control strain. There is no notable acetic acid concentration decrease after ackA blocked. And there is only 2.3% increase after the ackA deletion for succinic acid fermentation. Pyc from Bacillus subtilis 168 and pck from Actinobacillus succinogenes 130Z was introduced into Escherichia coli with ldhA deleted. Succinic acid concentration showed significant increase after the introduction of these two genes, for 7 fold and 6.17 fold. In pyc overexpressed strain, formic acid and pyruvic acid decreased almost to zero. The use of AP-bio-oil for the production of succinic acid was investigated. The transgenic E. coli strain could grow in modified M9 medium containing 20 v/v% AP-bio-oil with an increase in OD from 0.25 to 1.09. And 0.38 g/L succinic acid was produced. With the presence of 4 g/L glucose in the medium, succinic acid concentration increased from 1.4 to 2.4 g/L by addition of 20 v/v% AP-bio-oil. When enzymatic hydrolysate of corn stover was used as carbon source, succinic acid concentration increased to 11.5 g/L when 12.5 v/v% AP-bio-oil was added. However, it decreased to 8 g/L when 50 v/v% AP-bio-oil was added. GC-MS analysis revealed that some low molecular carbon compounds in the AP-bio-oil were utilized by E. coli. Succinic acid fermentation using biomass pretreated by ionic liquids AmimCl was studied. Results revealed that pine wood pretreated by AmimCl was the best source for succinic acid production. With this enzymatic pine wood as nutrient source, 20 g/L succinic acid was produced with yield of 0.99 mol/mol and productivity of 0.9g/L/h. However, AmimCl had a very moderate effect in treating corn stover. Thus combination of AmimCl with other methods such as steam explosion(SE) and hot compressed water(HCW) was studied. With AmimCl and SE pretreated corn stover as the feedstock, 17 g/L of succinic acid was obtained with yield of 1.05 mol/mol and productivity of 0.89 g/L/h. Ultrafiltration was first investigated to clarify succinic acid fermentation broth. Results indicate almost all the microorganism cells (99.6%) were removed from the fermentation broth. Proteins were also removed effectively by all the membranes studied. The removal rate was 79.86% for PES 100 kDa, 86.43% for PES 30 kDa, 86.83% for PES 10 kDa, and 80.06% for the RC 10 kDa. After ultrafiltration, a clearer permeate was obtained compared with that from centrifugation. Resistance-in-series model was applied to determine the main factor that caused the operation resistance. Results indicated that most membranes tended to be fouled by cake layer or concentration polarization. Hermia’s model was also used to analyze the predominant fouling mechanism. Results showed that the fouling of RC 10 kDa and PES 30 kDa was controlled by the complete blocking model, while PES 100 kDa was controlled by the intermediate blocking and PES 10 kDa was controlled by cake layer. This conclusion was also proved by SEM photos. Both contact angle and roughness of most membranes increased after ultrafiltration.
语种	中文
公开日期	2015-07-08
内容类型	学位论文
源URL	[http://ir.ipe.ac.cn/handle/122111/15552]
专题	过程工程研究所_研究所（批量导入）
推荐引用方式 GB/T 7714	王彩霞. 微生物法生产丁二酸过程工程研究[D]. 中国科学院研究生院. 2014.

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