【作者】 王亮；

【导师】 白凤武； 张华；

【作者基本信息】 大连理工大学 ， 生物化工， 2014， 博士

【摘要】 超高浓度(Very high gravity, VHG)发酵可以显著提高发酵终点乙醇浓度,节省精馏能耗,同时减少废糟液量。虽然VHG发酵技术已经在批式乙醇发酵中得到应用,但是批式发酵辅助操作时间长和人工成本高的特点,使其难以满足燃料乙醇大规模生产的需求,因此研究开发VHG乙醇连续发酵技术具有十分重要的意义。然而在酿酒酵母(Saccharomyces cerevisiae) VHG乙醇连续发酵过程中,包括残糖、乙醇和生物量浓度等发酵参数呈现长周期大振幅的振荡行为,不仅严重影响系统的乙醇发酵性能,而且不利于下游精馏装置的稳定运行。本研究通过过程工程策略、代谢轮廓及转录组分析对诱发S. cerevisiae VHG连续乙醇发酵过程振荡行为的机理进行了探讨。VHG乙醇连续发酵过程中酵母细胞受乙醇抑制和渗透压胁迫,且这两个胁迫因素是偶联的。通过向低糖(Low gravity, LG)培养基中添加乙醇,使乙醇抑制与渗透压胁迫解偶联,考察了乙醇抑制对连续发酵系统振荡行为的影响。实验结果表明LG培养基中添加30和50g/L乙醇后,能够使稳态运行的连续发酵系统产生振荡行为,而且高浓度乙醇引发的振荡行为更显著,当LG培养基中停止添加乙醇时,连续发酵系统逐渐恢复稳态。在VHG连续发酵系统偶联氮气气提,降低发酵液中自产乙醇浓度,可以有效弱化振荡行为。当添加乙醇浓度达到70g/L时,酵母细胞生长和乙醇发酵几乎被完全抑制。通过向LG培养基中添加S. cerevisiae不能代谢的木糖,模拟VHG连续发酵体系的渗透压环境,研究酵母细胞渗透压胁迫对系统振荡行为的影响,结果表明虽然渗透压胁迫对酵母细胞生长有一定的抑制作用,但不能诱发乙醇连续发酵体系的振荡行为。这些实验结果表明乙醇对酵母细胞的抑制作用是诱发VHG连续发酵系统振荡行为的主要原因。考察了菌种乙醇耐受性差异和培养基组成对VHG乙醇连续发酵系统振荡行为的影响,发现高乙醇耐性菌株S. cerevisiae BHL01和S. cerevisiae4126的VHG连续发酵体系均呈现振荡行为,而低乙醇耐性菌株S. cerevisiae288c发酵体系却维持在高残糖低乙醇浓度的拟稳态。在S. cerevisiae4126VHG乙醇连续发酵体系中,培养基中添加营养丰富的酵母粉和蛋白胨时,能够观察到周期性振荡行为,而使用合成培养基或停止通气时只能观察到类似阻尼振荡的行为,系统最终维持高残糖低乙醇浓度的拟稳态状态,进一步表明了VHG连续发酵体系中乙醇对酵母细胞的抑制作用是诱发周期性振荡行为的主要因素,而且这种振荡行为是营养和能量依赖的过程,并与菌株的乙醇耐受性有关。利用高乙醇耐性菌株S. cerevisiae BHLO1进行VHG乙醇连续发酵并偶联尾气循环气提,可以使发酵液中残糖降至0.10g/L以下,乙醇浓度和生产强度分别达到110.87g/L和2.99g/L/h,为VHG乙醇连续发酵技术开发提供了新思路。建立了以冷甲醇法和冷氯仿-甲醇-缓冲液法进行酵母细胞代谢淬灭和胞内代谢物提取及以乙腈和20mM乙酸铵溶液为流动相的选择反应扫描监测模式LC-MS/MS分析方法。对完整振荡周期关键节点取样,分析了糖酵解和下游乙醇生成途径关键代谢物及关键酶基因的转录。VHG乙醇连续发酵体系中,糖酵解耗能阶段代谢物GLC、G6P及F6P浓度变化与葡萄糖转运蛋白及己糖激酶编码基因HXTl和HXT3及11XK2和GLK1表达水平呈现V型,当发酵体系中乙醇变化速率最大时达到最低值,表明酵母细胞对葡萄糖的利用受到发酵体系中乙醇变化速率的调控。然而产能阶段代谢物GAP、G3P及PYR与磷酸果糖激酶催化反应产物FBP的浓度变化呈现N型,与耗能阶段代谢物浓度变化存在1/4周期差,表明酵母细胞胞内存在糖酵解振荡,且受到磷酸果糖激酶的调控。丙酮酸脱羧酶和乙醇脱氢酶是酵母乙醇发酵关键酶,其编码基因PDC1和ADHl的表达水平也呈现V型,表明乙醇生成受发酵体系中乙醇变化速率的调控。TCA循环顺时针途径代谢物AKG与SUC的浓度变化呈现倒S型,而逆时针途径代谢物ACCOA、OAA、FUM和MAL浓度变化则呈现N型,且与糖酵解途径代谢物FBP、GAP、G3P和PYR变化同步,表明VHG乙醇连续发酵体系中酵母细胞TCA循环不是闭合的,而是以逆时针途径为主。胞内CIT浓度达到10-20μmol/g(DCW),明显高于TCA循环其他代谢物浓度,且过氧化物酶体中柠檬酸合酶编码基因CIT2表达量明显高于线粒体中柠檬酸合酶编码基因CIT1,表明VHG乙醇连续发酵体系中酵母细胞内存在乙醛酸循环通量。VHG乙醇连续发酵体系中,胞外甘油平均浓度高达11.5g/L,甘油-3-磷酸脱氢酶编码基因GPD1表达量明显高于氧化还原调控基因GPD2,且与发酵体系中残糖浓度变化趋势一致,表明酵母细胞甘油合成主要用于调节细胞渗透压平衡。胞内TRE浓度达到40-80μmol/g(DCW),且与发酵体系中乙醇浓度的变化相关,海藻糖合成基因表达量明显高于糖原合成基因,表明海藻糖是VHG振荡过程中酵母细胞合成的主要抗逆物质。转录组数据显示,酵母细胞核糖体蛋白、核苷酸、氨基酸、细胞膜、细胞壁以及辅酶因子等合成途径相关基因的表达均呈现周期性变化,而且这些合成代谢过程存在明确的先后顺序,与胞内代谢物浓度的变化存在明显相位差,这表明VHG振荡不仅是酵母细胞发酵参数的波动,还是基因周期性表达行为。更多还原

【Abstract】 Very-high-gravity (VHG) fermentation can significantly improve ethanol titer to save energy consumption for ethanol distillation, and in the meantime reduce stillage discharge to save energy consumption for stillage treatment, which has been practiced in batch operation that is labor-intensive and time-consuming, and not suitable for fuel ethanol production at large scale, making it urgent for developing continuous VHG ethanol fermentation systems. However, the sustained oscillation of process parameters including residual glucose, ethanol and biomass concentrations was observed during continuous VHG ethanol fermentation by Saccharomyces cerevisiae. which not only compromised ethanol production, but also affected the distillation process. In this study, process engineering strategies, metabolic profiling and transcriptomic analysis were applied to explore mechanisms underlying this phenomenon.During continuous VHG ethanol fermentation, yeast cells are suffering from ethanol inhibition and/or osmotic stress, which are associated in nature. The impact of ethanol inhibition on the process oscillation was studied by supplementing exogenous ethanol into the low-gravity (LG) medium containing-100g/L glucose, and experimental results indicated that steady state observed with continuous ethanol fermentation under the LG medium condition was disrupted, and process oscillation was triggered when30g/L ethanol was supplemented, which was augmented as ethanol supplementation was increased to50g/L. The steady state was restored when ethanol supplementations were stopped. In addition, when N2gas stripping was incorporated into the continuous VHG ethanol fermentation system to strip off ethanol produced during the fermentation, process oscillation was attenuated. As ethanol supplementation was increased to70g/L, no process oscillation was observed due to severe inhibition of ethanol in S. cerevisiae. On the other hand, non-fermentable xylose for S. cerevisiae was supplemented into the LG medium to simulate the osmotic stress under the VHG fermentation condition, no process oscillation was observed, although the osmotic stress inhibited yeast growth.The impact of ethanol tolerance with yeast strains and nutritional component of medium on the process oscillation was also investigated. Sustained oscillation was observed in continuous VHG ethanol fermentation with ethanol-tolerant strains S. cerevisiae BHL01and S. cerevisiae4126, but quasi-steady state with more residual glucose and less ethanol produced was developed with less ethanol-tolerant strain S. cerevisiae288c. During continuous VHG ethanol fermentation with S. cerevisiae4126. sustained oscillation was observed with the YPD medium supplemented with yeast extract and peptone, while damped oscillation and consequently quasi-steady state with more residual glucose and less ethanol were developed with the synthetic medium or under anaerobic conditions. These experimental results further validated that ethanol inhibition in yeast cells was the main reason for the process oscillation with continuous VHG ethanol fermentation, which was nutrition-and energy-dependent, and associated with ethanol tolerance of yeast strains as well. Furthermore, continuous VHG ethanol fermentation with the ethanol-tolerant strain S. cerevisiae BHL01was coupled with the off-gas stripping to alleviate ethanol inhibition, residual glucose less than0.10g/L and total ethanol of110.87g/L were achieved for the fermentation system, making its ethanol productivity improved to2.99g/L/h, providing a strategy for process development.In order to explore molecular mechanism underlying the process oscillation, five samples were collected within an intact oscillation period, and27intracellular metabolites and transcriptome of yeast cells were analyzed. The treatment with cold methanol (CM) and cold chloroform-methanol-buffer (CMB) was applied to metabolism quenching and extraction of intracellular metabolites for LC-MS/MS analysis with the select reaction monitor (SRM) mode and acetonitrile and20mM ammonium acetate at pH6.60as eluent.The concentrations of GLC, G6P and F6P associated with energy-consuming in the glycolytic pathway and the expression levels of HXT1, HXT3, HXK2and GLK1, encoding glucose transporter and hexokinase, respectively, were characterzed by the V profile, and their minimal levels were observed at maximal ethanol gradient, suggesting that glucose utilization in yeast cells was regulated by ethanol gradient in the continuous VHG fermentation system. However, the concentrations of GAP, G3P and PYR with energy-producing and FBP produced by the phosphorylation of fructose catalyzed by phosphofructokinase (PFK) showed the N profile, with1/4-period difference to the profile of metabolites with energy-consuming, indicating that metabolic oscillation existed in yeast cells and regulated by PFK. The expression levels of PDC1and ADH1, encoding pyruvate decarboxylase and ethanol dehydrogenase, respectively, were also the V profile, indicating that ethanol production was co-regulated by ethanol gradient in the continuous VHG ethanol fermentation.The concentrations of AKG and SUC in the clockwise-branch of the TCA cycle showed the invert S profile, but those of ACCOA, OAA, FUM and MAL in the counter clock branch exhibit the N profile, synchronizing with that for FBP, GAP and PYR in the glycolytic pathway, indicating that the TCA cycle in yeast cells was incomplete during the continuous VHG ethanol fermentation, and that the counter clockwise branch was the main flux direction The concentration of extracellular CIT fluctuated in the range of10-20μmol/g(DCW), which was much higher than those observed with other metabolites in the TCA cycle. Moreover, the expression level of CIT2, encoding peroxisomal citrate synthase, was much higher than that of CIT1, encoding mitochondrial citrate synthase, revealing that glyoxylate cycle worked in yeast cells during the continuous VHG ethanol fermentation. During continuous VHG ethanol fermentation, the average concentration of extracellular glycerol was as high as11.5g/L. The expression level of GPD1, encoding glycerol-3-phosphate dehydrogenase associated with osmoregulation, was much higher than that of GPD2encoding glycerol-3-phosphate dehydrogenase involved in the redox balance, which also synchronized with the profile of residual glucose, suggesting that glycerol in yeast cells was synthesized mainly for osmoregulation. The concentration of intracellular TRE reached40-80μmol/g(DCW), which synchronized with the profile of ethanol production. The expression levels of genes associated with trehalose synthesis were much higher than those in glycogen synthesis, indicating that trehalose synthesis was mainly for ethanol tolerance in the continuous VHG ethanol fermentation.Transcriptomic analysis showed that the expression levels of genes associated with the syntheses of ribosomal proteins, nucleotides, amino acids, coenzymes, cell membranes and walls oscillated sequentially during the process oscillation, and significant phase shifts with respect to the profiles of intracellular metabolites were observed. Therefore, the oscillatory behavior of yeast cells under continuous VHG ethanol fermentation conditions not only was reflected in the process parameters but also in the levels of gene expression.更多还原