【作者】 任尚美；

【导师】 马霞；

【作者基本信息】 山东轻工业学院 ， 发酵工程， 2009， 硕士

【摘要】 γ-聚谷氨酸（γ-PGA）是一种水溶性的、可生物降解、可食用且对人和环境无毒的生物高分子物质,可由微生物聚合得到。这些特性使得γ-聚谷氨酸及其衍生物在食品、化妆品、医药、农业和水处理等领域有广阔的应用前景。为提高微生物发酵生产γ-聚谷氨酸的产量,本研究采用Bacillus subtilis NOBEL-007发酵制备γ-聚谷氨酸,并通过单因子试验及正交试验分析,得到枯草芽孢杆菌发酵生产γ-聚谷氨酸的最佳营养条件和培养条件。结果表明最佳营养条件为:葡萄糖40g/L,酵母膏5g/L,谷氨酸钠30g/L,NH₄Cl 3g/L,K₂HPO₄ 2g/L,MgSO₄ 0.25g/L;最佳培养条件为:培养温度37℃,摇床转速200r/min,装液量40mL（250mL三角瓶）,接种量为2%,培养48h,pH值7.0,此时γ-聚谷氨酸的产量最高,达到20.15g/L。为了确定Bacillus subtilis NOBEL-007发酵产品的结构组成,采用纸层析、FT-IR和NMR等多种测试手段对Bacillus subtilis NOBEL-007代谢产物γ-PGA结构进行了初步表征分析。纯化样品经水解后,纸层析谱图出现一个峰,与谷氨酸相同,说明其仅由谷氨酸聚合而成,红外光谱图的特征吸收峰与标准品一致,表明两者具有相同的化学结构,初步确定试验精制产品为γ-PGA。γ-PGA的核磁共振谱图进一步说明γ-PGA由谷氨酸组成。为深入了解分批发酵过程中Bacillus subtilis NOBEL-007的生长特性和提高γ-PGA的生产水平,在5L发酵罐上研究了pH、温度、搅拌转速、通气量等对菌体生长和γ-PGA产量的影响,研究表明最适条件为:pH7.0,温度37℃,搅拌转速和通气量分别是200r/min和l.0vvm。在优化后的培养条件下,γ-PGA的产量达到22.16g/L。根据分批发酵的实验数据,对Bacillus subtilis NOBEL-007合成γ-聚谷氨酸的发酵动力学特性进行了研究,通过Logistic方程,提出了发酵过程中菌体生长、γ-聚谷氨酸合成、基质消耗的动力学模型。应用MATLAB数值应用软件对实验数据进行处理,得到了Bacillus subtilis NOBEL-007分批发酵合成γ-聚谷氨酸的动力学模型参数。模型可表述为:菌体生长动力学模型:γ-PGA生成动力学模型:葡萄糖消耗动力学模型:对实验数据与模型进行比较,结果表明模型与实验数据能较好地拟合,相对误差较小,能很好地反映Bacillus subtilis NOBEL-007生产γ-PGA的分批发酵过程的动力学特征,为实验数据的模拟放大,以及从分批发酵过度到补料发酵乃至连续发酵提供了理论基础。研究了Bacillus subtilis NOBEL-007制备的生物絮凝剂γ-聚谷氨酸（γ-PGA）的絮凝活性。γ-PGA对高岭土、活性炭等悬浮液均有较高的絮凝活性,絮凝活性稳定,热稳定性好,当温度高于70℃时絮凝活性开始下降。采用10mg/L的γ-PGA溶液对活性炭的絮凝活性可达到90%以上,Mg²⁺、Ca²⁺、Na⁺、Fe³⁺等金属离子能不同程度增强γ-PGA的絮凝活性,其中Ca²⁺的助凝效果最佳,而Al³⁺、Fe²⁺则起削减作用。使用Ca²⁺作助凝离子可降低γ-PGA的用量,但Ca²⁺浓度过高会明显降低γ-PGA的絮凝活性。Ca²⁺浓度为10mM及介质溶液维持pH中性都有利于提高γ-PGA的絮凝活性。另外,还研究了γ-PGA的絮凝活性分布,实验证明絮凝活性主要分布于发酵原液及上清液中,而菌体细胞的絮凝活性一直很低。通过发酵过程中γ-PGA含量与培养液絮凝活性的关系中可得出,γ-PGA是产生絮凝现象的关键所在。更多还原

【Abstract】 γ-Poly glutamic acid （γ-PGA） which can be produced by microbial, is a kind of biodegradable high molecular polymer.γ-Polyglutamic acid（γ-PGA） is a biopolymeric product exhibiting water solubility, biodegradation, edibility and non-toxicity to human and environment, making its wide application in many fields as food, cosmetics, medicine, agriculture and water treatment in the past decades.In this work, in order to improve production ofγ-polyglutamic acid, it was produced by Bacillus subtilis NOBEL-007, and the optimization of conditions of fermentation process were investigated. The production ofγ-PGA and biomass were greatly influenced by the composition of medium and culture conditions according to the results attained in flask liquid fermentation experiments, which were conducted to investigate the optimal process parameter forγ-PGA production. The highestγ-PGA yield was achieved in the conditions that describe as following: glucose 40g/L, yeast extract paste 5g/L, L-sodium glutamate 30g/L, NH₄Cl 3g/L, K₂HPO₄ 2g/L, MgSO₄ 0.25 g/L; inoculum level 2%, volume level 40mL （250mL flask）, temperature 37℃, rotate speed 200r/min, nitial pH 7.0 and culture time 48 h, then the yield ofγ-PGA reached 20.15 g/L.The characterization ofγ-PGA from B.subtilis NOBEL-007 was investigated by the paper chromatography, FT-IR and NMR. The paper chromatograph obtained from hydrolysate of tested polymer was only one peak and was coincident with the chromatograph of glutamic acid. It indicated the polymer was composed of glutamic acid. The FT-IR chromatograph of the polymer showed no difference from that ofγ-PGA standard sample. It indicated the polymer was likely to beγ-PGA. NMR chromatograph further proved that the polymer consisted of glutamic acid.In order to further kown the character of Bacillus subtilis NOBEL-007 and improveγ-PGA production in batch fermentation, the effects of different fermenatation conditions, such as pH, temperature, rotate speed and aeration rate was investigated with regard to cell growth andγ-PGA production in 5L jar fermentor scale. The final optimal conditions were determined as followed: pH 7.0, cultivation at 37℃, strring rate 200r/min and aeration rate 1.0vvm. The highest productivity ofγ-PGA （22.16g/L） could be abtained under the above optimal conditions.On the basis of Logistic and Luedelting-Piret equation and the experimental data of batch fermentation in the 5L fermentation reactor, the mathematical models were proposed to describe the fermentation process with Bacillus subtilis NOBEL-007. The parameters of the models were established by using MATLAB software with experimental data and the models.The modes consist of: Cell growth model: Production formation model: Substrate depletion model:The above models could fit the profiles of cell growth, production formation and substrate depletion well. It might be helpful to understand and guide further optimization of PGA fermentation in the future.we synthesized the flocculation activity of ploy（γ-glutamic acid）（γ-PGA） produced by Bacillus subtilis NOBEL-007.γ-PGA showed high flocculation activities in kaolin, activated carbon suspensions, and the flocculation activity ofγ-PGA is steay to hot, but began to decrease upon heating at 70℃. In activated carbon suspension, theγ-PGA flocculating activity attained upon 90% at concentration of 10mg/L, and the flocculating activity synergistically increased by the addition of metal cations such as Mg²⁺, Ca²⁺, Na⁺ and Fe³⁺, Ca²⁺ was the optimum cation that remarkably promoted the flocculating activity; while adding Al³⁺, Fe²⁺ was disadvantageous to them. Using Ca²⁺ cloud lower the cost by dropping the dosage ofγ-PGA, but the excessive Ca²⁺ could obviously restrain the flocculating activity ofγ-PGA. The flocculating activity ofγ-PGA could be improved by adding 10mM Ca²⁺ to the suspensions and keeping the solution netural pH value. Futhermore, the distribution of bioflocculant ofγ-PGA was also researched, the flocculating activity was tested main in culture broth and supernatant, while the flocculating activity of cells was always low. Through synthesizing the correlation ofγ-PGA concentration and the flocculation activity of culture, we could conclude thatγ-PGA was the factor which caused the flocculation in the cluture.更多还原