非粮原料燃料乙醇发酵关键技术的研究

生物质燃料乙醇是一种高度清洁的交通液体燃料，是减少温室气体排放，缓解大气污染的最佳技术选择。以非粮原料生产燃料乙醇可以在进行能源生产的同时保证粮食安全，有利于产业的可持续发展。在众多的非粮原料中，甘薯是我国开发潜力最大的生物质能源作物之一。我国占世界甘薯种植总面积和产量的90％。同时，甘薯的单位面积燃料乙醇产量远大于玉米和小麦。其成本是目前酒精中最低廉的，因此利用甘薯生产乙醇是发展生物质燃料乙醇的首要选择。目前采用薯类全原料主要采用分批发酵生产乙醇，其技术水平低，发酵强度低，一般在0.7－2.5g/（L•h），乙醇浓度低，甘薯发酵乙醇为6－8％(v/v)，能耗高，环境负荷大，污染严重。针对上述问题，本文从菌株选育、原料预处理、中试放大、残糖成分分析等方面进行研究。 为了研究乙醇发酵生产规模扩大过程中，大型发酵罐底部高压条件下，CO2对酵母乙醇发酵的影响，我们通过CO2 加压的方法进行模拟试验，研究结果表明，发酵时间随压强的升高而逐渐延长，高压CO2 对乙醇发酵效率影响不大，在0.3 MPa 以下时，发酵效率均可达到90%以上。高压CO2 对发酵的抑制作用是高压和CO2 这两个因素联合作用的结果。高压CO2 条件下，酵母胞外酶和胞内重要酶类的酶活均表现出特征性。0.2 MPa 下，酶活性的变化趋势和0.1 MPa 条件下的较为一致。而0.3 MPa 下的酶活变化趋势与0.4 MPa 下的酶活更为接近。通过全基因表达分析发现在CO2 压力为0.3 MPa 下，乙醇发酵途径中多个基因表达量下调，同时海藻糖合成酶和热激蛋白基因表达量上调。 筛选耐高温的乙醇酵母菌株能够解决糖化温度和发酵温度不协调的矛盾，实现真正意义上的边糖化边发酵。高温发酵还能够降低发酵时的冷却成本，实现乙醇的周年生产。本研究筛选出一株高温发酵菌株Y-H1，进而我们对该菌株的胞外酶和胞内乙醇代谢重要酶类的酶活性进行了分析。结果表明Y-H1 能够在40 ℃条件下正常进行乙醇发酵，发酵33h，最终乙醇浓度达到10.7%（w/w），发酵效率达到90%以上。同时发酵液最终pH 在3.5 左右，显示菌株具有一定的耐酸性能力。同时观察到40 ℃下，菌株的胞外酶和胞内乙醇代谢重要酶类的酶活性发生了变化，乙醇发酵途径中关键酶基因表达下调，而海藻糖合成酶与热激蛋白基因表达量上调，这些结果为进一步研究酵母菌耐热调控机理提供了依据。 糖蜜是一种大规模工业生产乙醇的理想原料，本研究利用选育高浓度乙醇发酵菌株结合配套的发酵稳定剂，研究了糖蜜高浓度乙醇发酵情况。结果表明采用冷酸沉淀预处理糖蜜溶液，采用分批补料的发酵方式，乙醇浓度最高达到了10.26% (w/w)，发酵时间为42 h。同时观察到在糖蜜发酵中，乙醛含量与乙醇浓度存在一定的相关性。 快速乙醇发酵对于缩短乙醇生产周期、降低乙醇生产成本、减少原料腐烂损失具有重要意义。本研究诱变和筛选得到了一株快速乙醇发酵菌株10232B。在优化后的发酵条件下，采用10L 发酵罐进行分批乙醇发酵，经过18h,乙醇的最终浓度达到88.5g/L,发酵效率93.6%，平均乙醇生产速度达到4.92 g/L/h。此菌株在保持较高乙醇生产浓度的同时，拥有快速生产乙醇的能力，适合作为快速乙醇发酵生产菌种。 由于鲜甘薯具有粘度大的特点，传统液化糖化处理很难在短时间内充分糖化原料；高粘度的醪液也难以进行管道输送，容易堵塞管路；同时，也会降低后续的乙醇发酵效率。 本文采用了快速粘度分析法对鲜甘薯糊化粘度特性进行了分析，进而对预处理条件进行了研究，在最佳预处理条件下，糖化2h 后，醪液葡萄糖值最高可达99.3，粘度4.5×104 mPa.s，而采用传统糖化工艺，醪液DE 值仅为85.8，粘度大于1.0×105 mPa.s。 此预处理方法也可用于快速糖化不加水的醪液。后续的乙醇发酵试验表明，通过此预处理方法获得的糖化醪液对乙醇发酵无负面影响。 在前期已实现了实验室水平的鲜甘薯燃料乙醇快速乙醇发酵基础上，进一步将发酵规模扩大到500L，在中试水平上对甘薯乙醇发酵进行了研究。结果表明在500L 中试规模，采用边糖化边发酵（SSF）工艺，在料液比为3∶1，发酵醪液最高粘度为6×104mPa.s 条件下，发酵37h，乙醇浓度达到了12.7%（v/v），发酵效率91%，发酵强度为2.7 g/（L•h）。与目前国内的薯类乙醇发酵生产技术水平具有明显的优越性。 为研究甘薯、木薯乙醇发酵中残糖的组成，采用了高效液相色谱—蒸发光散射检测法，对乙醇发酵残糖进行了分析。结果表明，甘薯、木薯乙醇发酵残糖均为寡聚糖，主要由葡萄糖、木糖、半乳糖、阿拉伯糖和甘露糖构成。随着发酵时间延长，寡聚糖中的葡萄糖、半乳糖、甘露糖可被缓慢的水解释放。提高糖化酶量仅在一定程度上降低残糖，过量的糖化酶反而会导致残糖增加。同时发现3, 5-二硝基水杨酸法不能准确测定甘薯、木薯乙醇发酵中的残总糖含量。进一步筛选了两株残糖降解菌株，对甘薯乙醇发酵残糖的降解利用率均达到了40%以上，而且还能显著降低发酵醪液粘度。经形态学和rRNA ITS 序列分析，确定这两株菌分别属于为木霉属和曲霉属黑曲霉组。 通过对以甘薯原料为代表的非粮原料发酵技术研究开发，以期形成乙醇转化率高，能耗低，生产效率高、季节适应性好，原料适应性广，经济性强，符合清洁生产机制的燃料乙醇高效转化技术，为具有我国特色的燃料乙醇发展模式提供技术支持。 Sweet potato is one of the major feedstock for the fuel ethanol production in China. The planting area and the yield in China take 90% of the world. Sweet potato is an efficient kind of energy crops. The energy outcome per area is higher than corn or wheat. And the manufacture cost of ethanol is the lowest, compared with corn and wheat. So sweet potato is the favorable crop for the bioethanol production in China. However, the low-level fermentation technology restricts the development of ethanol production by sweet potato, including slow ethanol production rate, low ethanol concentration and high energy cost. To solve these problems, we conducted research on the strain breeding, pretreatment, pilot fermentation test and residual saccharides analysis. To study the impact of hyperbaric condition at bottom of the large fermentor on yeast fermentation, high pressure carbon dioxide (CO2) was adopted to simulate the situation. The results showed that the fermentation was prolonged with the increasing pressure. The pressure of CO2 had little impact on the ethanol yield which could reach 90% under the pressure below 0.3 MPa. The inhibition was combined by the high pressure and CO2. Under the high CO2 pressure, the extracellular and important intracellular enzyme activities were different from those under normal state. The changes under 0.1 MPa and 0.2 MPa were similar. The changes under 0.3 MPa were closer to those under 0.4 MPa. The application of thermotolerance yeast could solve the problem of the inconsistent temperature between fermentation and saccharificaton and fulfill the real simultaneous saccharification and fermentation. And it could reduce the cooling cost. A thermotolerance strain Y-H1 was isolated in our research. It gave high ethanol concentration of 10.7%（w/w）at 40 ℃ for 33 h. The ethanol yield efficiency was over 90%. At 40 ℃, the extracellular and important intracellular enzyme activities of Y-H1 showed the difference with normal state, which may indicate its physiological changes at the high temperature. Molasses is another feedstock for industrial ethanol production. By our ethanol-tolerance strain and the regulation reagents, the fermentation with high ethanol concentration was investigated. In fed-batch mode combined with cold acid deposition, the highest ethanol concentration was 10.26% (w/w) for 42h. The aldehyde concentration in fermentation was found to be related to ethanol concentration. The development of a rapid ethanol fermentation strain of Zymomonas mobilis is essential for reducing the cost of ethanol production and for the timely utilization of fresh material that is easily decayed in the Chinese bioethanol industry. A mutant Z. mobilis strain, 10232B, was generated by UV mutagenesis. Under these optimized conditions, fermentation of the mutant Z. mobilis 10232B strain was completed in just 18 h with a high ethanol production rate, at an average of 4.92 gL-1h-1 per batch. The final maximum ethanol concentration was 88.5 gL-1, with an ethanol yield efficiency of 93.6%. This result illustrated the potential use of the mutant Z. mobilis 10232B strain in rapid ethanol fermentation in order to help reduce the cost of industrial ethanol production. As fresh sweet potato syrup shows high viscosity, it is hard to be fully converted to glucose by enzymes in the traditional saccharification process. The high-viscosity syrup is difficult to be transmitted in pipes, which may be easily blocked. Meanwhile it could also reduce the later ethanol fermentation efficiency. To solve these problems, effects of the pretreatment conditions were investigated. The highest dextrose equivalent value of 99.3 and the lowest viscosity of 4.5×104 mPa.s were obtained by the most favorable pretreatment conditions, while those of 85.8 and over 1.0×105 mPa.s was produced by traditional treatment conditions. The pretreatment could also be applied on the material syrup without adding water. The later experiments showed that the pretreated syrup had no negative effect on the ethanol fermentation and exhibited lower viscosity. The fuel ethanol rapid production from fresh sweet potato was enlarged in the 500L pilot scale after its fulfillment on the laboratory level. The optimal ratio of material to water was 3 to 1 in 500L fermentor. With low-temperature-cooking (85 ℃) using SSF, the Saccharomyces cerevisiae was able to produce ethanol 97.44 g/kg for 37h, which reached 92% of theoretical yield. The average ethanol production rate was 4.06 g/kg/h. And the maximum viscosity of syrup reached 6×104mPa.s. The results showed its superiority over current industrial ethanol fermentation. The compositions of the residual saccharides in the ethanol fermentation by sweet potato and cassava were analyzed by high performance liquid chromatography coupled with evaporative light-scattering detector. The results showed that all the residual saccharides were oligosaccharides, mainly composed of glucose, xylose, galactose, arabinose and mannose. The glucose, galactose and mannose could be slowly hydrolyzed from oligosaccharides in syrup during a long period. To increase the glucoamylase dosage could lower the residual saccharides to a certain extent. However, excess glucoamylase dosage led to more residual saccharides. And the method of 3, 5-dinitrosalicylic acid could not accurately quantify the residual total saccharides content. Two residual saccharides degrading strains were isolated, which could utilize 40% of total residual saccharide and lower the syrup viscosity. With the analysis of morphology and internal transcribed spacer sequence, they were finally identified as species of Trichoderma and Aspergillus niger.