【作者】 舒成亮；

【导师】 岑沛霖； 徐志南；

【作者基本信息】 浙江大学 ， 生物化工， 2011， 硕士

【摘要】 丁酸是一种重要的有机酸,在化工、食品、农业、医药等领域有着广泛的用途。丁酸可通过化学合成法或微生物发酵法生产。发酵法存在产物浓度低、后处理量大等问题,实现工业化生产的成本较高。然而,发酵法生产丁酸条件温和,能耗低,污染小,原料来源广泛,生物源制品也更容易满足食品和医药级产品的要求。因此,近年来发酵法受到广泛的关注,其中,建立从发酵液高效提取丁酸的工艺已成为实现其工业化生产的关键。本文采用萃取和生物催化酯化等技术对丁酸分离的条件和策略进行了研究。在萃取法分离丁酸的研究中,经过筛选,选择三辛胺(TOA)为萃取剂,正辛醇为稀释剂。考察了TOA浓度、丁酸溶液pH、相比、温度等参数对水溶液中丁酸萃取分配系数的影响。获得的最佳萃取条件为：TOA浓度40%(v/v)、丁酸pH 2-3、相比0.8、温度25℃。发酵液中丁酸萃取和反萃研究结果表明,发酵液pH值为2-3,萃取相比为0.3时,能获得较高的分配系数(13.91),且可大量减少萃取剂的使用；经过两次萃取,发酵液中丁酸萃取率可达96.4%；以0.8 M NaOH为反萃剂,相比为1时,丁酸收率达到86.4%；研究表明,该萃取剂具有很好的再生性能。为探索固定化脂肪酶在酯化法分离丁酸中的应用,建立了脂肪酶固定化的新方法。以纤维织物(棉布)为载体,对Candida rugosa脂肪酶进行固定化。建立了聚乙烯亚胺(PEI)活化棉布、酶与PEI-棉布吸附、戊二醛交联的三步脂肪酶固定化方法。考察了PEI加入量、PEI溶液pH、载体及酶的加入量、戊二醛及稳定剂PEG2000的浓度对固定化的影响,获得最佳条件为：对0.1 g脂肪酶进行固定化,PEI加入量为10-20 mg,PEI溶液pH为8-10,载体/酶加入质量比为12-15,戊二醛浓度为0.1～0.5%,PEG 2000浓度为15 mg/mL。对固定化Candida rugosa脂肪酶催化丁酸乙酯的合成进行了研究。考察了底物浓度为0.5 M和0.8 M时,酶催化酯化反应过程。考察了反应溶剂、反应体系水活度、温度、酶用量、底物浓度和醇酸摩尔比等参数对反应的影响,获得的最佳反应条件为：环己烷为反应溶剂,高反应体系水活度(0.8-1.0),反应温度为25℃,酶加入量为0.6 g,丁酸和乙醇浓度分别为0.6 M和0.25 M。动力学研究表明,该反应符合双底物抑制Ping Pong Bi-Bi模型,且乙醇是比丁酸更强的抑制剂。该固定化酶具有较好的操作稳定性和储存稳定性。建立了固定化酶循环批次反应器并将其用于丁酸乙酯的合成反应,终产物浓度、产率和酯化率分别为0.85 M、1.45 mmol-h-1·g-1和70.6%。更多还原

【Abstract】 Butyric acid is an important organic acid which has various applications It can be produced with chemical synthesis or microbial fermentation. Fermentation method has some disadvantages such as low product concentration, complexity of post-processing and high cost for potential industrial application. However, fermentation method offers a production process with mild conditions, low energy consumption, low pollution and wide source of raw materials. Besides, the commercial price of butyric acid from microbial fermentation is high due to a strong preference by consumers and manufacturers for using bio-based natural ingredients in foods and pharmaceuticals. Therefore, much attention has been paid on fermentation method in recent years, and as a key step of this method, separation of butyric acid from fermentation broth with high efficiency is strongly demanded. In this study, extraction and esterification was adopted to isolate butyric acid, and several conditions involved in the separation process were investigated.In order to separate butyric acid with extraction method efficiently, trioctylamine (TOA) and n-octanol were adopted as extractant and diluent respectively. Effects of TO A concentration, pH of butyric acid solution, phase ratio and temperature on distribution coefficient were studied. The optimal conditions were:TOA concentration 40% (v/v), butyric acid solution pH 2-3, phase ratio 0.8, temperature 25℃. According to researches on extraction and back-extraction of butyric acid from fermentation broth, high distribution coefficient (13.91) was obtained with much less use of extractant at fermentation broth pH 2-3 and phase ratio 0.3; Extraction yield could be improved to 96.39% by adopting a two-stage extraction process. Further study showed that 0.8 mol/L NaOH was suitable as the stripping solution with a high recovery ratio (86.4%). The selected extractant can be regenerated well under the optimal conditions.To explore the performance of immobilized lipase in esterification method for butyric acid separation, a novel method for lipase immobilization was developed. Candida rugosa lipase was immobilized on fibrous material (cotton cloth) following a three-step procedure involving polyethylenimine (PEI) pretreating cotton cloth, adsorption of enzyme to PEI coated cotton cloth and glutaraldehyde cross-linking. Effects of amounts of PEI, support and enzyme, pH of PEI aqueous solution, concentrations of glutaraldehyde and stabilizing agent PEG 2000 on immobilization were investigated. The obtained optimal conditions were:0.1 g enzyme,10-20 mg PEI, PEI aqueous solution pH 8-10, mass ratio of support to enzyme 12-15, glutaraldehyde concentration 0.1-0.5%, PEG 2000 concentration 15 mg/mL.Catalytic performance of immobilized lipase in ethyl butyrate synthesis from butyric acid was evaluated. The time-course curves of esterification reaction were determined at various substrate concentrations (0.5 M or 0.8 M). Effects of reaction medium, water activity of reaction system, temperature, amount of immobilized lipase, substrate concentration and molar ratio of alcohol to acid on initial reaction rate and conversion yield were studied intensively. The optimal reaction conditions were:reaction medium cyclohexane, high reaction water activity (0.8-1.0), temperature 25℃, lipase amount 0.6 g, butyric acid concentration 0.6 M and ethanol concentration 0.25 M. A kinetic model of Ping Pong Bi-Bi mode with inhibition of both substrates was proposed and validated by experimental data. Furthermore, ethanol was a stronger inhibitor than butyric acid according to the fitting data of the model. Good operational and storage stability of the immobilized lipase were observed. Finally, a recycle batch reactor using immobilized lipase was developed for ethyl butyrate production. The achieved ethyl butyrat production revealed the promising potential of this immobilized lipase in practical applications.更多还原