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固态发酵具有节能节水、环境友好的优势，是缓解现代发酵工业中废水量大、环境污染严重等瓶颈问题的重要措施。论文首先从固态发酵培养基固体基质物化性质切入，研究其对固态发酵的影响。其次，针对好氧和厌氧等不同微生物特性，研发出适合的固态发酵新工艺。取得的主要研究结果如下：（1）固态发酵培养基中的固体基质多为难以直接利用的有机大分子，适宜的前处理有助于微生物快速生长。论文选用汽爆处理麸皮固体基质，在0.5 MPa、5 min条件下，总游离糖含量从9.83 mg/g增加至30.75 mg/g。论文选用从毛红曲作为实验菌株进行固态发酵酯化酶。与对照组相比，汽爆处理麸皮基质中菌体量由30.5 mg/g增加至63.6 mg/g，酯化酶活力由0.99 U/g 增加至1.72 U/g。此外，汽爆在实现大分子组分预降解的同时还可以起到固体基质灭菌作用，论文对比了物料随蒸汽流动汽爆灭菌和物料的原位汽爆灭菌方式，两种灭菌方式分别在128 oC、5 min和133 oC、10 min达到灭菌效果。物料随蒸汽流动汽爆灭菌方式比原位汽爆灭菌效率高，但是原位汽爆灭菌不会造成固体基质的过度粉碎，因此这两种汽爆灭菌方式适用于不同固体基质的灭菌。（2）从培养基固体基质物理特性出发，研究其对固态发酵过程基质团结收缩和装料系数的影响。论文对麸皮造粒得到了空心圆柱和实心圆柱两种固体基质颗粒。与未造粒原料相比，空心圆柱基质颗粒和实心圆柱基质颗粒的形变量分别减少9.5%和20.7%，表明造粒后的培养基具有更强的抗结团收缩能力。将未造粒培养基和造粒培养基用于固态发酵，在装料体积为100 mL时，未造粒培养基、空心圆柱和实心圆柱固体基质装料量分别为21.2 g，62.7 g，和67.5 g，发酵后总生物量分别为1.60 g，2.97 g和2.22 g，说明造粒能有效提高装料系数，提高总生物量积累。（3）发明两段式气相双动态固态好氧发酵小盾壳霉新工艺。与对照组相比，在空气脉动高压0.2 MPa、20 s，常压维持20 min的周期脉动条件下，气相双动态固态发酵240 h时发酵基质中生物量由98.9 mg/g增加至147 mg/g，表明气相双动态有利于菌体生长。但是，由此导致体系内温度快速积累使得小盾壳霉不分化形成分生孢子器，从根本上抑制小盾壳霉孢子的产生。两阶段固态发酵是指小盾壳酶前期进入周期脉动体系培养，后期转入低温培养。在此工艺下，固态发酵216 h时，气相双动态体系内孢子产量为1.53×1010 /g干基。（4）研究吐温-20、聚乙二醇、木质素磺酸钠等助剂对汽爆玉米秸秆固态酶解影响，其中3%的聚乙二醇和吐温-20都能加速葡聚糖转化，吐温-20辅助酶解体系下72 h葡聚糖转化率达到73.85%。在此基础上构建了描述汽爆玉米秸秆葡聚糖转化率和时间关系的扩散-反应高固酶解动力学模型。基于此动力学模型的计算结果表明，吐温-20酶解体系中相对扩散速率(ε2/ε3)提高7.3%。此外，论文采用低场核磁表征酶解过程固体基质中水的状态变化，结果显示吐温-20辅助酶解体系下固体基质对水的束缚作用减小，为酶的扩散提供了较好的条件。（5）针对固态厌氧发酵存在的氧分压高，CO2积累不利于乙醇固态发酵问题，发明了氮气周期脉动固态厌氧发酵乙醇新方法。在高压0.2 MPa、30 s，常压180 min 条件下，发酵24 h时，氮气周期脉动固态发酵体系中乙醇浓度达到39.9 g/L，而氮气保护组和对照组的乙醇浓度为21.7 g/L和22.2 g/L。论文了检测两种发酵体系CO2浓度变化，氮气周期脉动固态发酵体系中CO2浓度始终维持较低的水平（小于5000 ppm），而对照组CO2浓度持续积累，在21 h达到38080 ppm，证实脉动过程有利于传质过程。氮气为糖化酵母提供了较好的厌氧环境，脉动过程则加速了CO2移除，因而提高了糖化酵母固态厌氧发酵乙醇的效率。;Solid-state fermentation has advantages of energy-saving, water-saving and environment-friendly. It should be the important strategy for releasing the bottleneck problems in current fermentation industry, including large amount water discharge and serious environment pollution. Physico-chemical properties of solid medium in solid-state fermentation were firstly studied, as well as its effect on solid-state fermentation. Secondly, suitable processes were developed based on biological characteristics of aerobic and anaerobic microorganism. The main results were listed as follows:(1) Solid medium in solid-state fermentation medium usually contains organic macromolecule which cannot be directly used by microorganism. Proper pretreatment of the solid medium is benefit to microbial growth. In this thesis, steam explosion was employed to pretreat wheat bran. Results showed that total free sugars increased from 9.83 mg/g to 30.75 mg/g in wheat bran after steam explosion at 0.5 MPa, 5 min. Monascus pilosus was employed to perform solid-state fermentation for esterifying enzyme production. Biomass in steam-exploded wheat bran increased from 30.5 mg/g to 63.6 mg/g while esterifying capability increased from 0.99 U/g to 1.72 U/g. Additionally, steam explosion can also get solid medium sterilized while pre-hydrolyze macromolecule in solid medium. Steam-explosion sterilization where the solid medium moves along with the gas flow and in-situ steam-explosion sterilization were compared. The two methods can sterilize the solid medium at 128 oC, 5 min and 133 oC, 10 min, respectively. In-situ steam-explosion sterilization showed lower efficiency but cannot overgrind the solid medium. Therefore, the two sterilization methods were suitable for different solid medium.(2) Physical characteristics of solid medium were studied, as well as its effect on conglomeration and loading coefficient. Hollow and solid cylindrical solid medium were obtained in granulation experiments of wheat bran. Compared with raw material, displacement of hollow and solid cylindrical solid medium reduced 9.5% and 20.7% respectively, indicting better anti-conglomeration capability of solid medium after granulation. Both of the raw and granulated solid medium was used to perform solids-state fermentation. In 100 mL working volume, loading coefficient of raw matrix, hollow and solid cylindrical solid medium were 21.2 g, 62.7 and 67.5 respectively, and total biomass were 1.60 g, 2.97 g and 2.22 g after solid-state fermentation. The results indicated granulation of wheat bran contributed to improving loading coefficient and biomass accumulation.(3) A two-steps gas double-dynamic solid-state aerobic fermentation process of Conithyrium minitans was developed. Gas double-dynamic solid-state fermentation increased biomass from 98.9 mg/g to 147 mg/g at 240 h while air pressure was set at 0.2 Mpa, 20s and normal pressure was kept for 20 min periodically, indicating gas-double dynamic contributed microbial growth. However, rapid microbial growth led to temperature accumulation, hindering pycnidia differentiation and formation of Conithyrium minitans and inhibiting conidia production radically. The two-steps gas-double dynamic refer to the process that cultivating Conithyrium minitans in gas double-dynamic environment at initial stage and transfer into low temperature when pycnidia began to differentiate. In the two-steps gas double-dynamic solid-state fermentation process, conidia were 1.53×1010 /g dry mass. (4) Effect of tween-20, polyethylene glycol and sodium lignosulfonate on high-solid enzymatic hydrolysis of steam-exploded cornstalk. Results showed that 3% tween-20 and polyethylene glycol both contributed to glucan conversion. At 72 h, glucan conversion ratio reached to 73.85% in tween-20 assisted enzymatic hydrolysis. Additionally, a diffusion-reaction kinetic model which described the relationship between glucan conversion ratio and time was established. The model revealed that tween-20 increased relative diffusion rate by 7.3%. Additionally, low field nuclear magnetic resonance was employed to characterized water variation in enzymatic hydrolysis process of the steam-exploded cornstalk. The result revealed that less water restraint in tween-20 assisted enzymatic hydrolysis which provided a favorable environment for enzyme diffusion.(5) In order to solve the problems of high oxygen partial pressure and CO2 accumulation which go against sold-state anaerobic fermentation, nitrogen periodic pulsation sold-state anaerobic fermentation was invented. 39.9 g/L ethanol was obtained at 24 h while high pressure was set at 0.2 MPa, 20 s and normal pressure was kept for 180 min. 21.7 g/L and 22.2 g/L ethanol was obtained at 24 h in nitrogen protection group and control group. CO2 concentration maintained at a low level(less than 5000 ppm) in nitrogen periodic pulsation sold-state anaerobic fermentation while that in control group accumulated to 38080 ppm at 21 h, indicating enhanced mass transfer by periodic pulsation. Nitrogen provided a desired anaerobic environment and periodic pulsation accelerated CO2 removal, thus improving solid-state anaerobic fermentation ethanol performance of Saccharomyces cerevisiae.
|刘阳. 固态发酵培养基物化特性及其固态发酵新工艺的研究[D]. 中国科学院大学,2019.|
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