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乳酸被广泛应用于食品、医药、化妆、纺织和化工等领域，近年来生物可降解材料聚乳酸的生产，加大了工业乳酸的需求量。微生物发酵法是工业生产乳酸的主要方法，但原料成本及发酵工艺是制约生物合成乳酸规模化生产的主要因素。本研究以嗜热凝结芽胞杆菌（Bacillus coagulans）IPE22为出发菌株，以非粮生物质为原料，对开放式发酵生产乳酸的规律及机理进行研究。（1）针对淀粉质生物质，首先以可溶性淀粉做碳源研究B. coagulans IPE22开放式发酵合成乳酸的机制，发现IPE22菌株可以分泌淀粉酶（29.47 mU/mg）并合成乳酸，但乳酸生产强度较低（0.80 g/（L·h））。进而，针对传统工艺中以淀粉为原料合成乳酸时存在的最适酶解温度与发酵温度不一致导致的操作繁琐问题，利用多种淀粉酶开展不同的酶解策略研究，发现添加中温α-淀粉酶和糖化酶的一步法同步液化糖化发酵（SLSF）工艺可以高效合成乳酸，淀粉转化率、乳酸得率和生产强度分别可达89.77%、0.99 g/g和1.72 g/（L·h）。最后，以富含淀粉的非粮生物质（木薯粉和高粱粉）代替可溶性淀粉开展一步开放式发酵合成乳酸实验，发现乳酸发酵性能与可溶性淀粉为原料时无明显差异。同时，研究还发现在不添加外源酶以淀粉做碳源开放式发酵合成乳酸过程，偶尔会出现乳酸得率下降及副产物大量合成的异常现象，经分析发现该过程主要是污染了B. licheniformis和B. thermoamylovorans两种杂菌。为避免开放式发酵过程染菌的发生，对开放式发酵体系进行了系统的研究，进而开发了不同的污染防控策略。结果表明在开放式发酵初始额外添加葡萄糖或乳酸钠（10 g/L）可以分别减少45.15%和60.81%副产物的生成，而利用新分离的B. thermoamylovorans 48-3和B. coagulans IPE22进行混菌发酵不仅有效避免了杂菌污染（副产物减少95.90%），且与IPE22单菌发酵（0.81 g/g）相比乳酸得率（0.90 g/g）提高了11.11%。（2）虽然采用淀粉质非粮生物质可以一步法开放式发酵合成乳酸，但一些传统被认为是非粮作物的原料目前也被开发用作食品，因此寻求其它非淀粉质非粮生物质做碳源十分必要。木质纤维素原料近年来受到了全世界的广泛关注，但不同原料的水解液中己糖（hexose，如葡萄糖）与戊糖（pentose，包括木糖和阿拉伯糖等）的比例（H:P）差异很大（0.08~6.50），然而H:P的比例对乳酸生产的影响尚不清楚。因此，本文首先研究IPE22菌株利用60 g/L单糖（葡萄糖、木糖、阿拉伯糖）的发酵性能，发现葡萄糖倾向于促进乳酸合成，木糖有益于强化细胞的生长。进而，研究IPE22菌株利用不同H:P混合糖模拟液（60 g/L）的发酵性能，发现随着戊糖含量的降低，最大菌体浓度（OD620）随之减小（从12.50到7.61），生产强度相应下降（3.08到2.05 g/（L·h）），但乳酸产量却始终保持在大约50 g/L。最后，以总糖含量为60 g/L的玉米芯水解液为底物进行发酵，发现其生产强度（2.97 g/（L·h））高于单独以葡萄糖为碳源（2.34 g/（L·h））的发酵过程。因此，不同来源的木质纤维素非粮生物质均可被用做原料生产乳酸, 是一种良好的潜在发酵底物。另外，酸预处理后的玉米芯水解液经氢氧化钠中和后含有大量硫酸钠（2%），对发酵菌株的生长有抑制作用。因此，本文研究将利用双极膜电渗析技术对中和后的玉米芯酸水解液进行脱盐，硫酸根和钠离子的去除率分别为79.07%-86.31%和84.63%-90.90%。利用双极膜电渗析技术脱盐后，回收所得酸溶液用于玉米芯的酸水解实现循环利用，经过四个循环后，共节省了37.02%的硫酸。所以，利用双极膜电渗析技术不仅可以有效去除木质纤维素水解液中的无机盐抑制物，还可以将此无机盐转化为酸碱并进行循环利用，从而减少整个过程中废弃物的产生和资源的浪费。综上所示，本论文针对淀粉质生物质做碳源时建立的一步法同步液化糖化开放式发酵生产乳酸工艺，和针对纤维素源生物质建立的开放式发酵及酸水解液综合利用工艺对开放式发酵合成乳酸具有重要意义。;Lactic acid (LA) was widely used in food, pharmaceutical, cosmetic, textile and chemical areas, and in recent years the production of biodegradable polylactic acid has increased the demand for industrial LA. Microbial fermentation was the main method for industrial LA production, but the cost of raw materials and fermentation process were the main factors that restricted the large-scale biosynthesis of LA. In this study, Bacillus coagulans IPE22 was used as the starting strain, and inedible biomass was used as raw material to study the law and mechanism of open LA fermentation.(1) For starchy biomass, the mechanism of LA fermentation by B. coagulans IPE22 was firstly studied using soluble starch as carbon source. It was found that IPE22 strain can secrete amylase (29.47 mU/mg) and synthesize LA though the productivity is low (0.80 g/(L·h)). Furthermore, aiming at the cumbersome operation caused by the inconsistent temperature of enzymatic hydrolysis and fermentation in the conventional LA production process, various amylases were adopted to carry out fermentations with different enzymatic hydrolysis strategies. It was found that one step simultaneous liquefaction, saccharification and fermentation (SLSF) with the addition of mesothermal α-amylase and glucoamylase was the optimal mode with conversion rate, yield and productivity of 89.77%, 0.99 g/g and 1.72 g/(L·h) respectively. Finally, inedible starchy biomass (cassava and sorghum flours) were used instead of soluble starch to carry out one-step open LA fermentation, and no significant difference was found.At the same time, abnormal phenomenon of decreased LA yield and increased by-products accumulation appeared occasionally during open LA fermentation from starch without the addition of exogenous enzyme, which was mainly caused by the contamination of B. thermoamylovorans and B. licheniformis. In order to avoid the microbial contamination during open fermentation, systematic research was carried out and different contamination prevention and control strategies were developed. The results showed that the addition of glucose or sodium lactate (10 g/L) at the initial of open fermentation reduced the accumulation of by-products by 45.15% and 60.81% respectively. However, the co-fermentation of newly isolated B. thermoamylovorans 48-3 with B. coagulans IPE22 was the most effective strategy, which not only reduced 95.90% by-products formation also increased the LA yield (0.90 g/g) by 11.11% (compared to single strain fermentation (0.81 g/g)).(2) Although starchy inedible biomass could be used to produce LA by one-step open fermentation, some of them traditionally considered inedible have been currently developed as food. Thus, seeking other non-starch inedible biomass was necessary. Lignocellulosic materials attracted widely attention in recent years, however the ratio of hexose (glucose) to pentose (xylose and arabinose) of different lignocellulosic hydrolysates (H:P) varied greatly (0.08~6.50), and their effects on LA production were unclear still. In this study, sugars (glucose, xylose and arabinose) of 60 g/L were utilized as carbon source separately, and results proved that glucose tended to promote LA production while xylose preferred to enhance cell growth. Then, fermentations of simulated sugar mixtures (60 g/L) with various H:P ratios were studied. With the increase of pentose content, maximal cell density (13.10 to 7.61) and LA productivity (3.08 to 2.05 g/(L·h)) decreased, while similar LA concentrations (50 g/L) were obtained. At last, corn cob hydrolysate with 60 g/L sugars was used to produce LA, and its productivity (2.97 g/(L·h)) was higher than that of single glucose utilization (2.34 g/(L·h)). In all, lignocellulosic biomass derived from various sources could be applied to produce LA, and they were good alternatives of fermentation substrates.In addition, corncob acid hydrolysate neutralized by sodium hydroxide contained a large amount of sodium sulfate (2%), which inhibited the growth of B. coagulans. In this study, the hydrolysate was desalted by bipolar membrane electrodialysis (BMED), and the removal rates of sulfate and sodium ions were 79.07%-86.31% and 84.63%-90.90% respectively. The acid solution recovered from desalination by BMED was recycled through acid hydrolysis of corn cob, and a total of 37.02% sulfuric acid was saved after four recycles. Therefore, inorganic salt inhibitors were removed effectively by BMED technology, and the acid and alkali recovered from salt inhibitors were recycled meanwhile, which reduced pollution generation and resource waste in the whole process.In summary, the one step open SLSF process for starchy biomass and the open fermentation and acid hydrolysate integrated utilization process for lignocellulosic biomass were both established in this study, which was of great significance for open LA fermentation.
|王玉珏. 凝结芽胞杆菌利用非粮生物质合成乳酸的研究[D]. 中国科学院大学,2019.|
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