【作者】 王进；

【导师】 刘慧霞；

【作者基本信息】 广西大学 ， 制糖工程， 2004， 硕士

【摘要】 本文首先综述了甘蔗糖蜜酒精废液治理技术和酒精发酵中各类优良酵母菌株的性能及其选育方法。在分析测定了糖蜜及其酒精废液中主要有机与无机成分后研究了单因素对废液回用的极限值，在此基础上提出了利用原生质融合技术构筑具备强自絮凝能力的酒精酵母，对融合株进行多次物理和化学诱变，选育出了适应酒精废液回用环境的能力较强的目的菌株。探讨了以O₃／H₂O₂法对酒精废液进行预处理，利用目的菌株和分批加糖相结合的方法来实现酒精废液回用发酵的新工艺。论文的主要研究内容和结果如下： （1）模拟废液中所含的主要金属元素和主要有机酸进行添加影响因素探讨，找出了主要的对酵母发酵有较大影响的单因素极限值，论述了各影响因素对发酵产酒过程的生物化学效应的反应机制。为进行菌种驯化、酒精废液有用成分回收利用、有害成分的控制和废水回用量提供了参考依据。实验结果为K⁺、Na⁺、Ca²⁺、Mg²⁺、Fe²⁺、Cu²⁺、Zn²⁺、Al³⁺、Pb²⁺、As³⁺对甘蔗糖蜜酒精发酵产酒的抑制点分别为：850、1300、800、500、600～700、11～13、1200、600、21、3mg／L。K⁺对废液回用的影响最大。其次是Ca²⁺、Mg²⁺、Cu²⁺对废液回用有一定的影响。草酸、乳酸、柠檬酸、醋酸、富马酸对糖蜜酒精发酵产酒的抑制点分别为0.9％、3.5％、0.9％、0.3％、1.4％。乳酸、醋酸为有机酸最主要影响因素，对发酵的抑制作用很明显，因此要实现酒精废液高回用率，还需要在废液净化时设法降低其中的有机酸含量，或通过驯化提高菌种适应高浓度有机酸的能力。 （2）分别建立了粟酒裂殖酵母属（Schizosaccharomyces pombe）变异株和酿酒酵母属（Saccharomyces cerevisiae）变异株的遗传标记。利用原生质体融合技术进行属间原生质融合，得到融合子数株后再将其通过连续传代15代，确认获得融合子遗传稳定且自絮凝能力强的融合株XDST。 （3）对融合株XDST进行多次物理和化学诱变，使融合株XDST耐盐和有机酸能力较酒精酵母Q1有较大幅度的提高。在此基础上使用常规的连续培养驯化法，提高了目的菌株整体适应酒精废液回用恶劣环境的能力。目的菌株的酒精废液回用率从30％提高到了50-60％（采用双浓度流程工艺）。本斯值测定法测得目的菌株XDST的本斯值为2.2，为强自絮凝性。为以后其应用于工业化生产的固定化酵母技术奠定了基础。 （4）利用选育出的目的菌株用分三批加糖的新方法代替传统的双浓度流程发酵工艺。该工艺属于高浓醪发酵，能实现酒精废液以80％代替稀释清水回流发酵。通过正交试广西大学硕士学位论文糖蜜酒精废液回用与发酵酵母菌种选育的研究验得到目的菌株的最适发酵条件和发酵工艺为:·pH 5.0，发酵温度34℃，酒精废液回用代替清水率80%，初糖添加量M，二20 mL的分批加糖方式。该方式也是最接近均匀进料的工业生产方式。 （5）利用高级氧化法，即氏/H 202对酒精废液进行脱盐、降色、除COD，为多次糖蜜酒精废液回用发酵奠定基础。利用正交试验进行03州202处理糖蜜酒精废液的技术条件研究，以臭氧化氧气流量、HZOZ添加量、温度、废水在反应器的停留时间为工艺控制参数，以出水的脱色率和coo去除率作为指标确定最佳工艺。确定不同因素对废液处理效果影响（COO去除率和脱色率）主次顺序为:温度一时间一臭氧化氧气流量^H202添加量。当臭氧化氧气流量为0.07ma/h、HZQZ添加量为0.ZmL、温度50oC、反应时间75min时Coo去除率和脱色率均为最高，分别达21.41%和87.48%。经该工艺参数处理后酒精废液并结合分批加糖工艺废液回用率可达80%。 （6）对糖蜜酒精废液回用极限与回用新模式进行了探讨。根据物料衡算，分析了废液循环。从理论和实践上都说明了糖蜜酒精废液回用前的初处理也是可行的。最后提出了可实现酒精废液以80%回用率回流发酵，使废液循环回用可达多次的酒精废液回用发酵新工艺流程: 分三批加糖一卜XDST菌株发酵一奋上清酪液与经离心分离后的下层醒液的清液混合蒸酒-卜部 今分热废液先0a/H 202处理后鼓coZ冷后通。2回流更多还原

【Abstract】 In this paper, molasses alcohol waste water controlling approaches and the characters of various good alcohol yeast strain including their breeding selectively methods were surveyed at first. Then on the basis of finding out the limit value of the single factor to the alcohol fermentation after analysis and determination of main inorganic and organic matter in molasses and its alcohol ic waste water , construct i on of yeast cells to self-flocculate by protop last fusion was put forward in this paper. Then the yeast strain suitable for molasses alcoholic waste water recycling was breed after physical and chemical mutagenization adopted to several times. And design a new operation process of the thin stillage recycling fermentation pretreated with O3/H2O2 combined with fusants and adding sugar method. The main research contents and results of the thesis are reported as following:(1) According to different quantities were added to molasses al coholic waste water through imitating the metallic ions and organic acids content in alcoholic waste water, find out the limit value of the single factor and point out each influence factor to the alcohol fermentation and its reaction mechanism of biochemistry effect. That provides reference for taming yeast strain , the control of the harmful ingredients and thin still age recycling ratio. Under the laboratory term, the limit value of K+, Na+, Ca2+ , Mg2+ , Fe2+ , Cu2+ , Zn2+, Al3+ Pb2+ As3+ to the alcohol fermentation respectively is 850, 1300, 800, 500, 600~700, 11~13, 1200, 600, 21, 3mg/L K+ is the most great factor to the alcohol fermentation, Ca2+,Mg2+ ,Cu2+ are in the next place of influence factors. The Iimit value of oxalic acid, lactic acid, citric acid, acetic acid, fumaric acid to the alcohol fermentation respectively is 0. 9%, 3. 5%,0. 9%,0. 3%,1. 4%. Lactic acid and acetic acid are considered as the key influential factor in organic acids. Therefore it is necessary to reduce organic acids contain or to improvethe ability of yeast strain suitable for high density organic acids through taming yeast strain in order to realize high thin stillage recycling ratio.(2) Genetic markers of Schizosaccharomyces pombe and Saccharomyces cerevisiae have been establ ished. Fusant has been obtained by protoplast fusionand by going down to 15 generations. At last , the fusant XDST has good genetic stable fermentation capacity and can self-flocculate strongly.(3) The fusant XDST achieves higher density salt and organic acids tolerance compared with original alcohol yeast yeast 01 after physical and chemical mutagenization adopted to several times. Then the objective yeast strain more suitable for molasses alcoholic waste water recycling was breed on the basis of fusant XDST through continuous taming technique. The ratio of thin stillage recycling fermentation of the objective yeast strain is improved from 30% to 50-60%(adopting the double concentration flow). The berns value is 2.2. Theobjective yeast strain can flocculate well by itself at the end of fermentation. That will be helpful for the strain to be used as a cell immobilization method in industrial production.(4) A new operation of three times adding sugar method was put forward instead of traditional double concentration flow technique making use of the trait of the objective yeast strain more suitable for molasses alcoholic waste water recycling. From the whole fermentation flow (ferment 72 hours), the process is thick mash fermentation and realized the target of the ratio 80% of thin stillage recycling fermentation. The optimum fermentation conditions and technique were gained through the orthogonal experiments: initial pH 5.0, 37 C fermentation , the ratio of thin stillage recycling fermentation 80%, adding sugar in batch with the initial sugar concentration M=20 mL That is the most similar to industrial production.(5) Making use of O3/H2O2process of advanced oxidation processes desalt, decolour and reduce COD of molasses alcohol ic waste water , which makes prepared for thin stillage recycling severa更多还原