【作者】 薛闯；

【导师】 赵心清； 白凤武；

【作者基本信息】 大连理工大学 ， 生物化工， 2010， 博士

【摘要】 生产成本高是燃料乙醇生产面临的主要问题。由于能耗成本大约占燃料乙醇总生产成本的30%,仅次于原料成本,降低能耗成为燃料乙醇产业的重要需求。超高浓度发酵可以提高发酵终点的乙醇浓度,减少精馏操作的能耗及废槽液量,进而节省废糟液处理的能耗,是燃料乙醇创新技术发展的主要方向。由于高浓度乙醇对酵母细胞的强毒性作用,超高浓度乙醇会出现发酵速率慢,糖发酵不彻底的现象,不仅发酵罐设备生产强度显著降低,而且浪费原料。因此,开展酵母细胞乙醇耐性机理的研究,开发相应的调控策略,对燃料乙醇生产实现超高浓度发酵具有重要的理论和实际意义。本文以自絮凝酵母SPSC01为研究对象,使用合成培养基,研究了必需营养源(NH4)2SO4、K2HPO4和维生素及六种重要的二价金属离子(MgSO4·7H2O、CaCl2·2H2O、FeSO4·7H2O、CoCl2·6H2O、ZnSO4·7H2O和MnCl2·4H2O)对酵母细胞生长、乙醇耐性和发酵终点乙醇浓度的影响。实验结果表明：添加三种营养源对细胞生长和乙醇耐性提高有促进作用；Mg2+、Ca2+和Zn2+能提高酵母细胞的乙醇耐性和发酵终点乙醇浓度,但Mn2+、Fe2+和C02+对酵母细胞乙醇耐性没有明显影响,Co2+对提高发酵终点乙醇浓度有促进作用,Mn2+对乙醇浓度没有明显影响,Fe2+则降低发酵终点乙醇浓度。在此基础上,利用均匀设计法对与乙醇耐性和发酵终点乙醇浓度相关的七个因素进行优化,得到了用于预测酵母细胞乙醇耐性和发酵终点乙醇浓度的回归方程,对以乙醇冲击条件下酵母细胞存活率表示的最大乙醇耐性和发酵终点最高乙醇浓度进行了预测,结果分别为(91.1±1.74)%和47.90±0.95 g l-1,与实验值(90.2±0.89)%和47.15±0.85 g l-1吻合良好,比单因素优化获得的最大乙醇耐受性83.5%和发酵终点最高乙醇浓度43.70 g l-1相比有明显提高,表明这些因素之间存在协同效应,这些研究结果为提高发酵系统酵母细胞乙醇耐性和发酵终点乙醇浓度奠定了基础。在研究工作中首次发现了锌离子对提高酵母细胞乙醇耐性的促进作用,因此进一步考察了分别添加0.01、0.05和0.1g l-1硫酸锌对高浓度(培养基葡萄糖浓度245 g l-1)乙醇连续发酵的影响。实验结果表明：锌的添加能改变自絮凝酵母颗粒的平均粒度,提高其对乙醇和高温胁迫的耐性,使连续发酵终点乙醇浓度相应提高,因此调控培养基中的锌离子浓度,是提高絮凝酵母乙醇耐性的新途径；进一步的研究工作揭示,酵母胞内锌的积累能提高其麦角固醇和海藻糖的含量,这两种物质对提高酵母细胞乙醇耐性有重要的作用,和未添加锌离子的对照组相比,添加0.05 g l-1硫酸锌可以使发酵终点乙醇浓度提高8.4%,甘油浓度下降42.0%,抗乙醇和高温胁迫的耐性分别提高了20%左右。由于锌离子添加和自絮凝酵母粒度分布都影响其乙醇耐性,为了进一步揭示这一现象的机理,研究了培养基中添加0.05 g l-1锌离子和改变自絮凝酵母粒度分布对酵母细胞的保护作用及代谢通量的变化。为了区分这两个因素的影响,锌离子添加组颗粒大小调节为300μm,并与未添加锌离子的同一尺度自絮凝酵母乙醇发酵实验组进行比较。研究结果显示,与自絮凝酵母平均粒径为100和200μm的实验组相比,平均粒径为300μm的实验组乙醇浓度、酵母细胞存活率和糖吸收速率都达到最大,分别为110.0 g l-1、58.0%和286.69C-mmol l-1 h-1,而发酵副产物甘油、琥珀酸、乙酸和丙酮酸浓度分别降低了15.7%、24.5%、48.4%和50.0%。添加0.05g l-1硫酸锌使细胞内总麦角固醇和海藻糖含量分别增加了71.6%和96.5%,在发酵液中未检测到丙酮酸,甘油浓度降低了37.6%,乙醇浓度提高了9.1%,酵母细胞存活率提高了31.0%,葡萄糖代谢、麦角固醇、海藻糖和乙醇节点的碳流通量分别提高了5.3%、28.6%、43.3%和9.1%。此外,自絮凝酵母颗粒平均粒径的增加能增强反应器对细胞的截留作用,提高发酵罐内的生物量浓度,进而增加酵母颗粒群体对葡萄糖的表观吸收能力,使酵解途径碳流量增大,最终导致乙醇浓度的增加。锌离子添加和自絮凝酵母颗粒尺度增大导致乙醇浓度的增加,使胞内大分子生物合成如DNA、RNA、蛋白质和磷脂等节点的碳流量受到明显的抑制,用于维持代谢的ATP能耗增加,体现了高浓度乙醇产生的高负荷以及对细胞生长和发酵的强抑制作用。在超高浓度连续乙醇发酵过程中,酵母颗粒群体对葡萄糖的吸收速率仅为常规乙醇发酵条件下的1/2-1/3,因此提高超高浓度乙醇发酵中酵母细胞对碳底物的吸收能力是提高发酵终点乙醇浓度的关键。更多还原

【Abstract】 High cost is the biggest challenge in fuel ethanol production, in which energy consumption comprises about 30%, the second largest only after feedstock consumption, and thus saving energy consumption is one of the most effective strategies to make fuel ethanol more economically competitive. Very high gravity (VHG) fermentation can significantly increase ethanol titer in the fermentation broth, which not only saves energy consumption for the downstream distillation but also for the waste distillage treatment due to its significant reduction. However, high ethanol concentration severely inhibits yeast cells, and stuck fermentation frequently occurs, with more sugars unfermented and ethanol yield compromised correspondingly. Therefore, exploration of the mechanism underlying the ethanol tolerance of yeast cells is one of the prerequisites for developing effective VHG ethanol fermentation process.In the present work, the effect of nutrients including (NH4)2SO4, K2HPO4, vitamins and bivalent metal ions from MgSO4·7H2O, CaCl2·2H2O, FeSO4·7H2O, CoCl2·6H2O, ZnSO4·7H2O and MnCl2·4H2O on the growth of yeast cells, their ethanol tolerance and production was investigated using a defined medium and the self-flocculating yeast SPSC01. The experimental results indicated that the supplementation of (NH4)2SO4, K2HPO4 and vitamins stimulated the growth of yeast cells and improve their ethanol tolerance and production, correspondingly, so did the supplementation of Mg2+, Ca2+ and Zn2+. Although the supplementation of Mn2+, Fe2+ and Co2+ had no effect on the ethanol tolerance, Co2+ improved the ethanol production, but Mn2+ had no such an effect, and Fe2+ affected it negatively. The supplementation of the seven nutrients was further optimized by the uniform design, and regression equations for ethanol tolerance and production predictions were developed. The maximum ethanol tolerance characterized by the viability of yeast cells suffered from ethanol shock treatment and ethanol production were estimated to be (91.1±1.74)% and 47.90±0.95 g l-1, respectively, which were validated by the experimental results of (90.2±0.89)% and 47.15±0.85 g l-1, correspondingly. Meanwhile, compared with the maximum ethanol tolerance and production of 83.5% and 43.70 g l-1 achieved via the single factor optimization, improvement in the two parameters were observed, indicating the synergistic role of these factors on the yeast cells.Since the supplementation of Zn2+ significantly improved the ethanol tolerance and production of the yeast cells, zinc sulfate was thus supplemented at the concentrations of 0.01, 0.05 and 0.10 g l-1 to further investigate its impact on the ethanol fermentation. It was observed that Zn2+ affected the flocculation of the yeast, and improved its ethanol tolerance, thermal tolerance and ethanol production, indicating that a process engineering strategy for improving VHG ethanol fermentation could be developed based on the Zn2+ supplementation. Moreover, intracellular accumulation of Zn2+ stimulated the accumulation of ergosterol and trehalose, two components that are beneficial for yeast cells to protect from ethanol inhibition. In comparison with the control without Zn2+ supplementation, ethanol production was improved by 8.4%, thermal and ethanol tolerance by 20.0%, while glycerol production decreased by 42% under 0.05 g l-1 zinc sulfate supplementation conditions.Furthermore, the impact of the size of the yeast flocs on the ethanol fermentation was studied by controlling their size at 100,200 and 300μm, which was characterized by their average chord monitored by the focused beam reflectance measurement. For the yeast flocs with the size of 300μm,0.05 g l-1 zinc sulfate was supplemented, with no Zn2+ supplementation as the control. The experimental results illustrated that the yeast flocs with the size of 300μm exhibited maximum ethanol production, ethanol tolerance and glucose consumption, with 110.0 g l-1 ethanol produced,58.0% viability remained for the yeast cells after 20% (v/v) ethanol shock for 5 h and glucose uptake rate of 286.69 C-mmol l-1 h-1. Intracellular ergosterol and trehalose accumulations were improved by 71.6% and 96.5%, respectively, for the yeast flocs with the size of 300μm and supplemented with 0.05 g l-1 zinc sulfate, but glycerol decreased by 37.6%. No pyruvate accumulation was observed in the Zn2+ supplementation group, and ethanol production was increased by 9.1% with 31.0% improvement of the yeast cell viability. And in the meantime, carbon fluxes at the node of glucose uptake, ergosterol, trehalose and ethanol were enhanced by 5.3%,28.6%,43.3% and 9.1%, correnpondingly.Moreover, the retention of the self-flocculating yeast was improved for the yeast flocs with relatively large size and higher biomass concentration was achieved within the bioreactor, contributing to more glucose consumed and carbon flux to ethanol production. The decrease in the fluxes to the growth of the yeast cells such as DNA, RNA, protein and lipid biosynthesis as well as the increase of ATP consumption for the maintenance indicated that improved ethanol production resulted from Zn2+ supplementation and the increased yeast floc size raised more energy burden on the yeast cells, and glucose uptake of the yeast flcos was compromised under VHG conditions, making the improvement of glucose uptake rate the key to improve VHG ethanol fermentation.更多还原