【作者】 申渝；

【导师】 白凤武；

【作者基本信息】 大连理工大学 ， 生物化工， 2009， 博士

【摘要】 生产成本过高是制约燃料乙醇大规模推广应用的主要因素。目前以淀粉质为原料生产燃料乙醇的成本构成中,能耗成本约占30%,仅决于原料成本（约占60%）,因此降低能耗是降低燃料乙醇生产成本的主要方向之一。超高浓度乙醇发酵技术可以减少单位产量乙醇的物料总量,进而减少精馏过程及废糟液处理的能耗,是燃料乙醇生产领域最具发展前景的技术之一。超高浓度乙醇连续发酵过程中极易出现大幅度的参数振荡现象,影响发酵过程的稳定性,而对于诱发这类振荡现象的机理至今没有深入的研究报道。酵母细胞在超高浓度乙醇连续发酵过程中呈现的振荡现象,具有周期长、振幅大的特点,与文献中报道的酵母细胞连续培养时,在特定条件下由于不对称芽殖导致子母细胞分裂周期同步激发的周期短、振幅小的振荡行为不同。代谢通量分析表明在一个完整的振荡周期内,酵母细胞代谢状态没有根本性改变,乙醇代谢途径碳通量占总碳通量的比例始终保持在80%以上,占绝对优势;而振荡过程中酵母细胞代谢网络上关键代谢节点处碳通量分布呈周期性变化,反映了细胞在振荡周期内不同相位点上对能量和细胞组分需求的差异和调节过程.实验中证明了振荡过程中胞外乙醇是酵母细胞主要的抑制因素,而酵母细胞的代谢活性对乙醇抑制表现出100 h左右的滞后,受胞外乙醇刺激后胞内海藻糖积累等乙醇耐受机制的逐步启动,抵消了乙醇对酵母细胞的抑制作用,是这种振荡现象发生的本质原因。利用流式细胞分析技术对振荡过程中细胞群体周期分布分析,表明这种振荡行为发生时,不存在细胞周期同步化现象。建立了一套由搅拌式发酵罐和管式反应器串联组成的反应系统,开展酵母细胞超高浓度乙醇连续发酵的研究工作,尝试了利用填料吸附对细胞进行固定化处理来调控振荡过程。装填木块和聚氨酯填料的管式反应器对来自前面搅拌罐的振荡行为能够产生不同的影响,不同填料对酵母细胞生理参数影响的差异是造成这种效果的原因。经过木块填料吸附以后,酵母细胞的比糖消耗速率及乙醇耐受能力得到改善,能够对来自搅拌罐的强制振荡产生弱化作用,而聚氨酯填料则没有类似的效果。表面微观结构的差异是造成两种填料对酵母细胞生理参数影响效果不同的可能原因之一,木块填料表面的精细结构能够为酵母细胞提供了良好的附着表面,而聚氨酯填料孔隙较大,内壁光滑,对酵母细胞起到的只是截流效果。在0.027 h^-1和0.040 h^-1两个稀释速率下,通过不同的操作方式可以分别实现稳定和振荡两个发酵状态,这表明稀释速率和系统的初始状态都是超高浓度乙醇连续发酵过程振荡行为产生的必要条件。以不同的操作方式达到同一稀释速率,会造成系统的初始条件不同,进而产生了不同状态的发酵过程。在稀释速率0.04 h^-1时,与稳态发酵过程相比,振荡过程表现出更高的发酵效率和设备生产强度,单级搅拌罐中设备生产强度提高了12.3%。动力学分析发现在稀释速率0.04 h^-1时,与稳态发酵过程相比,振荡过程的动力学行为不仅存在滞后,而且在相同残糖和乙醇浓度条件下,酵母细胞平均比生长速率比稳态过程提高了53.8%,表明振荡过程能够在更高乙醇浓度条件下保证酵母细胞的平均比生长速率与稀释速率平衡,使连续发酵系统得以运行。组合式反应系统中引入振荡现象可以大幅度提高设备生产强度,缩短发酵时间,同时可以利用装填填料的管式反应器对振荡过程进行弱化,保证稳定的终点乙醇浓度和较低浓度的残糖,满足工业过程的要求论文工作进一步试图通过周期性切换稀释速率的方式对发酵过程施加强制振荡,并研究其对发酵性能的影响。在稀释速率为0.02 h^-1和0.04 h^-1两个稳态过程之间反复切换,保证一个周期内的平均稀释速率为0.03 h^-1,分别尝试了4 d、2 d和1 d三个周期。实验结果表明当强制振荡周期为4 d时,残糖、乙醇和生物量等发酵参数表现出规律性的振荡,且发酵性能优于稀释速率0.03 h^-1时的稳态过程,设备生产强度提高3.4%。随着振荡周期缩短,残糖等发酵参数变化的周期性也变得模糊,且系统的发酵性能不如稀释速率0.03 h^-1时的稳态过程,表明强制振荡周期必须满足酵母细胞的生理特点,周期太短既不能得到周期显著的振荡过程,也不能提高发酵效率。自发振荡周期是能够满足酵母细胞生理特点的最佳周期,强制振荡的周期长度只有在接近自发振荡周期时,才能对系统施加周期显著的振荡过程并提高发酵效率。更多还原

【Abstract】 The widely using of fuel ethanol was limited by its high production cost.Energy cost accounted for 30%of total production,only after that of raw material consumption,was the second largest in ethanol production cost.Very high gravity（VHG） ethanol fermentation was the most promising technology for energy saving in distillation of fermented broth and waste water.Markedly oscillatory behaviors of parameters,which destroyed steady run of fermentation process,occurred usually in continuous ethanol fermentation under VHG condition,and no rational explanation was given yet.Parameter oscillations characterized by long oscillation periods and large oscillation amplitudes were observed in the continuous ethanol fermentation with Saccharomyces cerevisiae under VHG conditions,and the mechanism was speculated to be different from those oscillations observed in the continuous culture of the yeast,which were triggered by the synchronization of the cell cycles and characterized by short oscillation periods and small oscillation amplitudes.Metabolic flux analysis（MFA） was applied to the oscillatory VHG ethanol fermentation system,and the results indicated that the carbon fluxes at the keynotes within the metabolic network oscillated correspondingly,with over 80%of the total carbon flux to the ethanol production to generate energy to support the metabolism of the yeast cells. Ethanol was validated to be the main inhibitor of the yeast cells under oscillatory conditions, and the overall metabolic activity of the yeast cells was found not exactly out of phase but lag behind the ethanol concentration accumulated within the fermentation system and its inhibition on the yeast cells as well,which experimentally supported the mechanistic speculation for the process oscillation:ethanol inhibition in yeast cells and the lag response of the yeast cells to the ethanol inhibition.The synchronization of the intracellular trehalose,an effective protectant of yeast cells to environmental stresses,with ethanol production further supported this mechanistic speculation since time was needed for the stress protectant to provide protection again the ethanol inhibition.And in the meantime,analysis of the yeast cell cycle using the flow cytometry approach showed that no cell cycle-dependent synchronization of the daughter and mother cells occurred within the duration of the oscillation.A bioreactor system composed of a stirred tank and tubular bioreactors in series was established,and the impact of yeast cell immobilization with supporting materials on the oscillation of the fermentation generated within the tank ahead was investigated through packing the tubular bioreactors with wood chips and polyurethane particles,two packings with significant difference in their surface properties and microstructures.It was found that the immobilized yeast cells with the wood chips effectively attenuated the oscillation,and sugar consumption and ethanol tolerance were improved compared with the tubular bioreactor without packing,which could be the root reason for the oscillation attenuation.Either oscillatory or steady state could be generated at the dilution rates of 0.027 and 0.04 h^-1,which depended on the initial conditions applied to the fermentation system. However,the productivity increased by 12.3%compared to the steady state,when the diluation rate of 0.04 h^-1 was applied to the fermentation system under oscillation conditions. Further investigation revealed that besides the lag response of the yeast cells to ethanol inhibition under the oscillation conditions,the oscillatory dynamic kinetics of the yeast cells possessed advantages over that under steady state conditions,with 53.8%increase in the specific growth rate,indicating the fermentation system could be operated at higher dilution rates without washing out of the yeast cells.Introduction of such oscillations in a CSTR multistage tubular reactors system for ethanol continuous fermentation using VHG medium was experimentally proven to be practical.Fermentation time was shorten remarkably in oscillatory mode compared to steady states by greatly increased the fermentation efficiency. Oscillations introduced were attenuated successfully by wood chips packed tubues,and steady final ethanol content and low level of final residue sugar which were satisfied to the industrial standards were realized.Forced oscillations were created by applying the dilution rates of 0.02 and 0.04 h^-1 to the fermentation system periodically.Compared with the fermentation system operated under steady state at the dilution rate of 0.03 h^-1,improved fermentation performance was achieved when the period of 4 d was applied to the periodic change of the two dilution rates,with a increases of 3.4%for ethanol productivity of the fermentation system,while no improvement was observed for another two fermentations under the oscillation periods of 2 and 1 d, respectively,indicating the effectiveness of the forced oscillation in improving the fermentation performance could be achieved when the forced oscillation period is close to the oscillation period naturally developed by the yeast cells within the fermentation system.更多还原