聚乳酸单体L-乳酸由米根霉固定化发酵玉米秸秆水解物的生产研究
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摘要
生物材料聚乳酸是受到广泛关注的生物相容性良好和全生物降解材料，由乳酸聚合而成。如果聚乳酸单体乳酸能够从廉价的生物质资源通过生物炼制转化而来，则无论对医学发展、环境保护还是资源节约都具有重要意义。乳酸是基本的有机酸之一，是一种重要的平台化合物，实现生物炼制生产也是未来生物基经济代替石油基经济的重要产业组成。要实现生物炼制生产乳酸目前尚有一些技术瓶颈需要被攻克，最主要的有两点，一是将木质纤维素原料低成本、高效率的转化成可生物利用的小分子糖类；二是生物发酵纤维素来源的糖类高效转化生产乳酸。目前该领域的技术积累还不能完全满足大规模的工业化生产，相关产业的经济效益还不能与以石油为基础的产业相比。本研究将针对这两大技术瓶颈，围绕农作物秸秆木质纤维素的高效糖化技术、米根霉固定化发酵生产乳酸等内容展开研究。
我国是农业大国，农作物秸秆资源丰富，在各类秸秆资源中尤其以玉米秸秆最为丰富。如何提高玉米秸秆的糖化效率是利用该资源进行生物炼制，用于生产平台化合物的关键。在利用纤维素酶水解玉米秸秆纤维素生产葡萄糖的过程中，秸秆的致密结构是酶解β-1,4-糖苷键的主要障碍，本研究中我们采用蒸汽爆破技术对木质纤维素结构进行破坏，以提高纤维素酶解效率。
在蒸汽爆破预处理玉米秸秆的研究中，以酶解还原糖产率为响应值，采用均匀实验设计得到最佳汽爆工艺条件，即汽爆压力2.2MPa、液固比1:1、维压时间9min、物料颗粒度40-60目。在此工艺条件下酶解还原糖产率是未处理原料的1.97倍。化学组分分析显示，蒸汽爆破处理后秸秆物料中纤维素含量及木质素含量相对于原料分别增加了29.73%和19.65%，说明通过蒸汽爆破预处理能够提高秸秆物料中纤维素的相对含量。SEM观察可见，蒸汽爆破处理后其结构破坏明显，表面出现褶皱，结构变的蓬松，比表面积增大。
以优化工艺对玉米秸秆进行蒸汽爆破预处理，然后进行水/醇抽提处理，将纤维素、半纤维素和木质素等组分进行分离，进一步提高纤维素酶的可及性。对底物浓度、纤维素酶用量、酶解反应时间等因数采用单因素分析和响应面分析实验进行工艺优化。以酶解还原糖产率为响应值，对回归方程求解得到的最佳工艺条件为：底物浓度53.28g/L，纤维素酶用量53.32FPU/g，酶解时间60.45h，此工艺条件下还原糖产率的最大预测值为694.11mg/g。经过试验验证，得到酶解后还原糖产率为672.36mg/g，与模型预测结果接近。在相同酶解工艺条件下，经过蒸汽爆破-水/醇处理后物料酶解还原糖产率较原料提高了170.46%，较单纯蒸汽爆破后物料提高了28.97%。化学组分分析显示，水/醇处理能有效分离纤维素、半纤维以及木质素，进一步提高物料中的纤维素含量，SEM图谱也显示水醇处理使秸秆物料的结构变的更加的蓬松，这些因素可有效增加纤维素酶对纤维素的可及性，提高了酶解效率。
为了克服丝状真菌发酵过程中细胞形态不易控制，机械运动易造成菌丝断裂损伤；会形成大的菌丝团，阻碍氧气和营养物质的传递等不利生产的特点。对米根酶固定化发酵技术进行了研究，包括固定界面的选择、固定支架的设计以及固定化效果的评价。并利用玉米秸秆水解物为主要碳源，筛选出适合利用水解物发酵产酸的米根霉菌种，采用固定化发酵技术进行了摇瓶和反应器发酵生产乳酸的研究，优化了发酵工艺。
通过多种米根霉和乳酸杆菌发酵产乳酸的比较，筛选出米根霉As3.3462菌种是适合发酵玉米秸秆酶解物生产乳酸的菌种。为了制作出适合米根霉固定化发酵的固定支架，试验筛选出棉布是用于米根霉固定的良好纤维表面，六角星形载体菌丝层层固定后仍具有较大的表面积，多棱角的结构使菌丝层内部具有较好的物质传输特性，适合菌体生长和产酸。通过孢子固定动力学的研究发现，米根霉孢子能完全固定在载体上，随着菌丝的生长，吸附率急剧上升。通过对孢子固定动力曲线的数学拟合，发现孢子的吸附符合一级反应动力学特征。载体尺寸、数量与反应器容量以及孢子接种量间对于菌体生长和乳酸生产存在最佳比例关系。通过100mL的摇瓶实验，直径2.5cm、3个载体、1×106/mL的孢子接种浓度对菌体生长和乳酸生产最为有利。
进一步研究As3.3462米根霉固定化发酵玉米秸秆水解物生产L-乳酸。为了了解米根霉对玉米秸秆水解物的代谢特性，分别研究米根霉对葡萄糖和木糖的代谢，揭示木糖代谢的磷酸戊糖途径乳酸转化率低，但能有效提高生物量，以玉米秸秆水解物发酵生产乳酸主要通过葡萄糖代谢转化。通过葡糖糖和木糖混合模拟玉米秸秆水解物，从糖消耗的动力学分析，米根霉优先消耗葡萄糖，对木糖的消耗需要经历一段时间的“适应性”调整，乳酸生成主要在葡萄糖消耗阶段。玉米秸秆水解物包含22.8%的葡萄糖和27.2%的木糖，其发酵动力学特点与1:1的混糖模拟类似。随着葡萄糖被迅速消耗，木糖的消耗速度逐渐提高，发酵176h时乳酸生产水平达到最大15g/L，转化率约30%，整个过程伴有乙醇的生成。在单因素分析的基础上，通过均匀设计优化了同步糖化发酵，使糖化和发酵过程良好耦合，秸秆转化率接近15%。
采用本室自行设计制作的鼓泡式生物反应器，对米根霉固定化发酵的工艺进行了放大研究。考察了反应器中孢子的吸附和萌发状况，结果显示，通气对孢子固定具有干扰作用，在0.5vvm的通气量下需要10h萌发的孢子才能全部固定。通过对菌丝层的通气特性分析显示，菌丝层厚与氧气传输速率间非线性，超过15mm的临界厚度，菌丝层会严重阻碍氧的传输，提示固定发酵的菌丝层厚要控制在15mm的范围内。通过比较固定发酵和游离发酵，前者乳酸转化率可以达到72%，而后者只有40.4%，固定发酵显著优于游离发酵。同时进行了重复批次发酵和同步糖化发酵的研究，重复批次发酵可以连续发酵6批次，乳酸转化率保持在40%以上，同步糖化发酵可以达到57%的转化率。
综上所述，为了有效的将玉米秸秆纤维素转化成L-乳酸，我们对玉米秸秆的物理和化学的预处理技术、生物酶解糖化技术进行了系统研究，建立了一种成本低、环境污染小、效率高的玉米秸秆木质纤维素糖化技术，为玉米秸秆资源用于生物炼制奠定了基础。接着以米根霉发酵玉米秸秆水解物生产L-乳酸为目的，筛选出了适合发酵产酸的米根霉菌种As3.3462，通过比较发现米根霉对葡萄糖和木糖转化途径的差异性，为有效利用秸秆资源提出了新思路。通过不同性质的材料对比筛选出适合米根霉固定的纤维材料，以增大表面积、提高菌团内部物质传输为目的，设计出了一种具有优化几何形状的固定化载体，并用于玉米秸秆水解物的固定化发酵研究。根据该固定化特点，设计制作了鼓泡式生物反应器用于工艺放大研究，通过建立气窜实验模型，分析了玉米秸秸秆水解物发酵液中的溶氧特性和菌丝层中溶氧传输特性，确定了反应器通气控制。利用优化的工艺进行补料分批发酵和同步糖化发酵试验，证实该固定化发酵技术具有较好的稳定性和较高的转化效率，以此研究为基础有望开发出满足产业化的玉米秸秆转化生产L-乳酸的工艺和设备。
Biomaterial polylactide has been widely paid attention in the world. Polylactide, abiocompatible and well-degraded polymeric material, is polymerized from the lacticacid. If the lactic acid can be produced from cheap biomass, that has great significancein medicine development, environment protection and resources saving. The lactic acidis basic organic acid and platform chemical. The biorefineries production of lactic acidwill be important industrial components of bio-based economies. To achievebiorefineries of lactic acid, several technical bottlenecks need to be overcome. One isthe low-cost and high-efficiency transformation of lignocellulose into small-moleculecarbohydrates, and the other is the conversion of carbohydrates into platform chemicalswith high-conversion ratio. Currently available technologies cannot fully meet theserequirements for large-scale industrial production and generate considerable economicbenefits. This research focused on the efficient sugar hydrolysis process andimmobilized fermentation of Rhizopus oryzae for L-lactic acid production.
In a large agricultural country such as China, crop straw especially corn stover iswidely distributed and serves as a renewable resource. The efficiency of thesaccharification hydrolysis of corn stover must be enhanced for bio-refineries toproduce high-quality fuels and platform chemicals. In the process of the enzymatichydrolysis of corn stover cellulose to produce glucose, the supra-molecular structure ofcellulose crystalline regions is a major obstacle for the enzymatic hydrolysis of theβ-1,4-glycosidic bond. This study uses steam explosion technology and water/alcoholtreatment technology to destroy this structure and increase the efficiency of enzymaticcellulose hydrolysis.
The steam explosion pretreatment of corn stalk was optimized with uniform design.The structures of untreated/treated corn stalk were detected by Scanning ElectronMicroscope (SEM) and X-ray diffraction (XRD), while their chemical composition wasalso investigated. Comparing with the untreated corn stalk, reducing sugar yield oftreated corn stalk was increased by97%after hydrolyzing. The relative content ofhemicelluloses and solvend decreased, while cellulose and lignin increased by29.73%and19.65%, respectively; the compact structure of corn stalk was remarkably destroyed.The optimum steam exploded process condition: operating pressure2.2MPa,solid-liquid ratio1:1(mL/g), reaction time9min and materials size40-60meshes.
The enzymatic hydrolysis of cellulose treated by water/ethanol extraction aftersteam explosion was investigated. The substrate concentration, cellulase dosage, andreaction time were optimized with single-factor experiments and the response surfacemethodology. The reducing sugar yield reached672.36mg/g when the substrateconcentration was53.8g/L, cellulase dosage was53.32FPU/g, and reaction time was60.45h. Compared with the untreated and steam-exploded corn stover, the reducingsugar yield increased by170.46%and28.97%, respectively. The chemical compositionsof the untreated/treated corn stover were investigated and their structures werecharacterized by SEM. The results showed that the relative content of cellulose in cornstover treated by water/ethanol extraction increased significantly and the structurebecame fluffy due to the improved cellulose hydrolysis.
To overcome such shortcomings as mycelium fracture injury and limited transferof oxygen and nutrients, the immobilization fermentation of Rhizopus oryzae wasinvestigated, including the choice of immobilization interface material, mountingbracket design, and the evaluation of the effectiveness of immobilization. Fermentationwith corn stover hydrolyzate as the main carbon source was conducted to screening theapplicable R. oryzae. Immobilized fermentation was performed in a shake flask and thereactor fermentation production of lactic acid was also studied to optimize thefermentation process.
Comparison with a variety of R. oryzae-and Lactobacillus-fermented lactic acidproduction revealed that Rhizopus strain3.3462was the optimal fermentation strain. ForR. oryzae immobilized fermentation, the new support matrix was developed with thefiber material and geometry of the support matrix optimized. A comparative analysis ofthe fixing effects, cell growth, and L-lactid acid production showed that cotton fabricwas the optimal fiber surface for the support matrix and the hexagram w the mostsuitable geometry. Spore fixing kinetic studies revealed that R. oryzae spores cancompletely fix on the carrier, and that the adsorption rate sharply increased followingmycelium growth. By the mathematical fitting of the kinetic curves of spore adsorption,spore adsorption was found to follow first-order reaction kinetics. The optimal matrixsize, matrix number, reactor capacity, and spore inoculum density were determined by100mL shake flask experiments. The best culture condition was as follows: matrixdiameter of2.5cm, three matrices, and106spores/mL.
The immobilization technique was used for R. oryzae3.3462L-lactic acidproduction from corn stalk hydrolyzate. To couple the saccharification and fermentation process, the simultaneous saccharification and fermentation technology was studied.Fermentation kinetics curve analysis showed that glucose depletion occurred after48hof fermentation, the lactic acid concentration reached51g/L, and the ethanol productionduring the entire fermentation process was low. Using the xylose of the stoverhydrolyzate as the carbon source resulted in a very low xylose consumption rate; thus,xylose was not suitable as the sole carbon source. Using the corn stover hydrolyzate asthe carbon source, R. oryzae underwent a48h adaptation period, after which glucoseand xylose were rapidly consumed and the maximum level of production of lactate was176h. The entire process was generated byproducts such as ethanol and fumaric acid.The simultaneous saccharification and fermentation process suggested that the initialsaccharification time and cellulase were the key factors. The uniform design showedthat saccharification for4h and45FPU/g cellulase dosage yielded better simultaneoussaccharification and fermentation results in shake flask experiments.
An airlift bioreactor designed by our group was used to study the amplificationprocess of L-lactic acid production by immobilized R. oryzae from corn stalkhydrolyzate. The curve of absorption and germination of the spores in the reactorshowed that all spores germinated after6h under ventilation. However, ventilationinterfered with spore absorption and needed10h. Oxygen transmission becomeschallenging after amplifying the culture medium volume. The gassing out measurementshowed that the oxygen uptake and transmission rates of the corn stalkhydrolyzate-containing medium were much larger than those of the glucose-containingmedium. Thus, the corn stalk hydrolyzate-containing medium can be used for R. oryzaefermentation in the airlift bioreactor. A comparison between immobilized and freefermentation revealed that the lactic acid conversion rate reached72%in the former andonly40.4%in the latter, which suggested that immobilized fermentation wassignificantly advantageous over free fermentation. To test the stability of immobilizedfermentation, a second batch fermentation as well as simultaneous saccharification andfermentation were conducted. Repeated batch fermentation can produce nineconsecutive batches, and the lactic acid conversion rate remained above40%to70%. Insimultaneous saccharification and fermentation, the lactic acid conversion rate reached57%.
In summary, to transform the cellulose of corn stover into L-lactic acid, thephysical and chemical pretreatment of corn stover, as well as bio-enzymaticsaccharification were systematically studied. The technology for producing reducing sugar was established based on steam explosion, water/alcohol treatment, and cellulasehydrolysis. For the purpose of R. oryzae fermentation for L-lactic acid production fromcorn stover hydrolysis, a Rhizopus strain with a high lactic acid production capacity wasscreened. A support matrix for immobilized fermentation was then designed and thefermentation technology optimized. A higher L-lactic acid yield was obtained and thetechnology was found be stable for repeated batch fermentations as well assimultaneous saccharification and fermentation.
我国是农业大国，农作物秸秆资源丰富，在各类秸秆资源中尤其以玉米秸秆最为丰富。如何提高玉米秸秆的糖化效率是利用该资源进行生物炼制，用于生产平台化合物的关键。在利用纤维素酶水解玉米秸秆纤维素生产葡萄糖的过程中，秸秆的致密结构是酶解β-1,4-糖苷键的主要障碍，本研究中我们采用蒸汽爆破技术对木质纤维素结构进行破坏，以提高纤维素酶解效率。
在蒸汽爆破预处理玉米秸秆的研究中，以酶解还原糖产率为响应值，采用均匀实验设计得到最佳汽爆工艺条件，即汽爆压力2.2MPa、液固比1:1、维压时间9min、物料颗粒度40-60目。在此工艺条件下酶解还原糖产率是未处理原料的1.97倍。化学组分分析显示，蒸汽爆破处理后秸秆物料中纤维素含量及木质素含量相对于原料分别增加了29.73%和19.65%，说明通过蒸汽爆破预处理能够提高秸秆物料中纤维素的相对含量。SEM观察可见，蒸汽爆破处理后其结构破坏明显，表面出现褶皱，结构变的蓬松，比表面积增大。
以优化工艺对玉米秸秆进行蒸汽爆破预处理，然后进行水/醇抽提处理，将纤维素、半纤维素和木质素等组分进行分离，进一步提高纤维素酶的可及性。对底物浓度、纤维素酶用量、酶解反应时间等因数采用单因素分析和响应面分析实验进行工艺优化。以酶解还原糖产率为响应值，对回归方程求解得到的最佳工艺条件为：底物浓度53.28g/L，纤维素酶用量53.32FPU/g，酶解时间60.45h，此工艺条件下还原糖产率的最大预测值为694.11mg/g。经过试验验证，得到酶解后还原糖产率为672.36mg/g，与模型预测结果接近。在相同酶解工艺条件下，经过蒸汽爆破-水/醇处理后物料酶解还原糖产率较原料提高了170.46%，较单纯蒸汽爆破后物料提高了28.97%。化学组分分析显示，水/醇处理能有效分离纤维素、半纤维以及木质素，进一步提高物料中的纤维素含量，SEM图谱也显示水醇处理使秸秆物料的结构变的更加的蓬松，这些因素可有效增加纤维素酶对纤维素的可及性，提高了酶解效率。
为了克服丝状真菌发酵过程中细胞形态不易控制，机械运动易造成菌丝断裂损伤；会形成大的菌丝团，阻碍氧气和营养物质的传递等不利生产的特点。对米根酶固定化发酵技术进行了研究，包括固定界面的选择、固定支架的设计以及固定化效果的评价。并利用玉米秸秆水解物为主要碳源，筛选出适合利用水解物发酵产酸的米根霉菌种，采用固定化发酵技术进行了摇瓶和反应器发酵生产乳酸的研究，优化了发酵工艺。
通过多种米根霉和乳酸杆菌发酵产乳酸的比较，筛选出米根霉As3.3462菌种是适合发酵玉米秸秆酶解物生产乳酸的菌种。为了制作出适合米根霉固定化发酵的固定支架，试验筛选出棉布是用于米根霉固定的良好纤维表面，六角星形载体菌丝层层固定后仍具有较大的表面积，多棱角的结构使菌丝层内部具有较好的物质传输特性，适合菌体生长和产酸。通过孢子固定动力学的研究发现，米根霉孢子能完全固定在载体上，随着菌丝的生长，吸附率急剧上升。通过对孢子固定动力曲线的数学拟合，发现孢子的吸附符合一级反应动力学特征。载体尺寸、数量与反应器容量以及孢子接种量间对于菌体生长和乳酸生产存在最佳比例关系。通过100mL的摇瓶实验，直径2.5cm、3个载体、1×106/mL的孢子接种浓度对菌体生长和乳酸生产最为有利。
进一步研究As3.3462米根霉固定化发酵玉米秸秆水解物生产L-乳酸。为了了解米根霉对玉米秸秆水解物的代谢特性，分别研究米根霉对葡萄糖和木糖的代谢，揭示木糖代谢的磷酸戊糖途径乳酸转化率低，但能有效提高生物量，以玉米秸秆水解物发酵生产乳酸主要通过葡萄糖代谢转化。通过葡糖糖和木糖混合模拟玉米秸秆水解物，从糖消耗的动力学分析，米根霉优先消耗葡萄糖，对木糖的消耗需要经历一段时间的“适应性”调整，乳酸生成主要在葡萄糖消耗阶段。玉米秸秆水解物包含22.8%的葡萄糖和27.2%的木糖，其发酵动力学特点与1:1的混糖模拟类似。随着葡萄糖被迅速消耗，木糖的消耗速度逐渐提高，发酵176h时乳酸生产水平达到最大15g/L，转化率约30%，整个过程伴有乙醇的生成。在单因素分析的基础上，通过均匀设计优化了同步糖化发酵，使糖化和发酵过程良好耦合，秸秆转化率接近15%。
采用本室自行设计制作的鼓泡式生物反应器，对米根霉固定化发酵的工艺进行了放大研究。考察了反应器中孢子的吸附和萌发状况，结果显示，通气对孢子固定具有干扰作用，在0.5vvm的通气量下需要10h萌发的孢子才能全部固定。通过对菌丝层的通气特性分析显示，菌丝层厚与氧气传输速率间非线性，超过15mm的临界厚度，菌丝层会严重阻碍氧的传输，提示固定发酵的菌丝层厚要控制在15mm的范围内。通过比较固定发酵和游离发酵，前者乳酸转化率可以达到72%，而后者只有40.4%，固定发酵显著优于游离发酵。同时进行了重复批次发酵和同步糖化发酵的研究，重复批次发酵可以连续发酵6批次，乳酸转化率保持在40%以上，同步糖化发酵可以达到57%的转化率。
综上所述，为了有效的将玉米秸秆纤维素转化成L-乳酸，我们对玉米秸秆的物理和化学的预处理技术、生物酶解糖化技术进行了系统研究，建立了一种成本低、环境污染小、效率高的玉米秸秆木质纤维素糖化技术，为玉米秸秆资源用于生物炼制奠定了基础。接着以米根霉发酵玉米秸秆水解物生产L-乳酸为目的，筛选出了适合发酵产酸的米根霉菌种As3.3462，通过比较发现米根霉对葡萄糖和木糖转化途径的差异性，为有效利用秸秆资源提出了新思路。通过不同性质的材料对比筛选出适合米根霉固定的纤维材料，以增大表面积、提高菌团内部物质传输为目的，设计出了一种具有优化几何形状的固定化载体，并用于玉米秸秆水解物的固定化发酵研究。根据该固定化特点，设计制作了鼓泡式生物反应器用于工艺放大研究，通过建立气窜实验模型，分析了玉米秸秸秆水解物发酵液中的溶氧特性和菌丝层中溶氧传输特性，确定了反应器通气控制。利用优化的工艺进行补料分批发酵和同步糖化发酵试验，证实该固定化发酵技术具有较好的稳定性和较高的转化效率，以此研究为基础有望开发出满足产业化的玉米秸秆转化生产L-乳酸的工艺和设备。
Biomaterial polylactide has been widely paid attention in the world. Polylactide, abiocompatible and well-degraded polymeric material, is polymerized from the lacticacid. If the lactic acid can be produced from cheap biomass, that has great significancein medicine development, environment protection and resources saving. The lactic acidis basic organic acid and platform chemical. The biorefineries production of lactic acidwill be important industrial components of bio-based economies. To achievebiorefineries of lactic acid, several technical bottlenecks need to be overcome. One isthe low-cost and high-efficiency transformation of lignocellulose into small-moleculecarbohydrates, and the other is the conversion of carbohydrates into platform chemicalswith high-conversion ratio. Currently available technologies cannot fully meet theserequirements for large-scale industrial production and generate considerable economicbenefits. This research focused on the efficient sugar hydrolysis process andimmobilized fermentation of Rhizopus oryzae for L-lactic acid production.
In a large agricultural country such as China, crop straw especially corn stover iswidely distributed and serves as a renewable resource. The efficiency of thesaccharification hydrolysis of corn stover must be enhanced for bio-refineries toproduce high-quality fuels and platform chemicals. In the process of the enzymatichydrolysis of corn stover cellulose to produce glucose, the supra-molecular structure ofcellulose crystalline regions is a major obstacle for the enzymatic hydrolysis of theβ-1,4-glycosidic bond. This study uses steam explosion technology and water/alcoholtreatment technology to destroy this structure and increase the efficiency of enzymaticcellulose hydrolysis.
The steam explosion pretreatment of corn stalk was optimized with uniform design.The structures of untreated/treated corn stalk were detected by Scanning ElectronMicroscope (SEM) and X-ray diffraction (XRD), while their chemical composition wasalso investigated. Comparing with the untreated corn stalk, reducing sugar yield oftreated corn stalk was increased by97%after hydrolyzing. The relative content ofhemicelluloses and solvend decreased, while cellulose and lignin increased by29.73%and19.65%, respectively; the compact structure of corn stalk was remarkably destroyed.The optimum steam exploded process condition: operating pressure2.2MPa,solid-liquid ratio1:1(mL/g), reaction time9min and materials size40-60meshes.
The enzymatic hydrolysis of cellulose treated by water/ethanol extraction aftersteam explosion was investigated. The substrate concentration, cellulase dosage, andreaction time were optimized with single-factor experiments and the response surfacemethodology. The reducing sugar yield reached672.36mg/g when the substrateconcentration was53.8g/L, cellulase dosage was53.32FPU/g, and reaction time was60.45h. Compared with the untreated and steam-exploded corn stover, the reducingsugar yield increased by170.46%and28.97%, respectively. The chemical compositionsof the untreated/treated corn stover were investigated and their structures werecharacterized by SEM. The results showed that the relative content of cellulose in cornstover treated by water/ethanol extraction increased significantly and the structurebecame fluffy due to the improved cellulose hydrolysis.
To overcome such shortcomings as mycelium fracture injury and limited transferof oxygen and nutrients, the immobilization fermentation of Rhizopus oryzae wasinvestigated, including the choice of immobilization interface material, mountingbracket design, and the evaluation of the effectiveness of immobilization. Fermentationwith corn stover hydrolyzate as the main carbon source was conducted to screening theapplicable R. oryzae. Immobilized fermentation was performed in a shake flask and thereactor fermentation production of lactic acid was also studied to optimize thefermentation process.
Comparison with a variety of R. oryzae-and Lactobacillus-fermented lactic acidproduction revealed that Rhizopus strain3.3462was the optimal fermentation strain. ForR. oryzae immobilized fermentation, the new support matrix was developed with thefiber material and geometry of the support matrix optimized. A comparative analysis ofthe fixing effects, cell growth, and L-lactid acid production showed that cotton fabricwas the optimal fiber surface for the support matrix and the hexagram w the mostsuitable geometry. Spore fixing kinetic studies revealed that R. oryzae spores cancompletely fix on the carrier, and that the adsorption rate sharply increased followingmycelium growth. By the mathematical fitting of the kinetic curves of spore adsorption,spore adsorption was found to follow first-order reaction kinetics. The optimal matrixsize, matrix number, reactor capacity, and spore inoculum density were determined by100mL shake flask experiments. The best culture condition was as follows: matrixdiameter of2.5cm, three matrices, and106spores/mL.
The immobilization technique was used for R. oryzae3.3462L-lactic acidproduction from corn stalk hydrolyzate. To couple the saccharification and fermentation process, the simultaneous saccharification and fermentation technology was studied.Fermentation kinetics curve analysis showed that glucose depletion occurred after48hof fermentation, the lactic acid concentration reached51g/L, and the ethanol productionduring the entire fermentation process was low. Using the xylose of the stoverhydrolyzate as the carbon source resulted in a very low xylose consumption rate; thus,xylose was not suitable as the sole carbon source. Using the corn stover hydrolyzate asthe carbon source, R. oryzae underwent a48h adaptation period, after which glucoseand xylose were rapidly consumed and the maximum level of production of lactate was176h. The entire process was generated byproducts such as ethanol and fumaric acid.The simultaneous saccharification and fermentation process suggested that the initialsaccharification time and cellulase were the key factors. The uniform design showedthat saccharification for4h and45FPU/g cellulase dosage yielded better simultaneoussaccharification and fermentation results in shake flask experiments.
An airlift bioreactor designed by our group was used to study the amplificationprocess of L-lactic acid production by immobilized R. oryzae from corn stalkhydrolyzate. The curve of absorption and germination of the spores in the reactorshowed that all spores germinated after6h under ventilation. However, ventilationinterfered with spore absorption and needed10h. Oxygen transmission becomeschallenging after amplifying the culture medium volume. The gassing out measurementshowed that the oxygen uptake and transmission rates of the corn stalkhydrolyzate-containing medium were much larger than those of the glucose-containingmedium. Thus, the corn stalk hydrolyzate-containing medium can be used for R. oryzaefermentation in the airlift bioreactor. A comparison between immobilized and freefermentation revealed that the lactic acid conversion rate reached72%in the former andonly40.4%in the latter, which suggested that immobilized fermentation wassignificantly advantageous over free fermentation. To test the stability of immobilizedfermentation, a second batch fermentation as well as simultaneous saccharification andfermentation were conducted. Repeated batch fermentation can produce nineconsecutive batches, and the lactic acid conversion rate remained above40%to70%. Insimultaneous saccharification and fermentation, the lactic acid conversion rate reached57%.
In summary, to transform the cellulose of corn stover into L-lactic acid, thephysical and chemical pretreatment of corn stover, as well as bio-enzymaticsaccharification were systematically studied. The technology for producing reducing sugar was established based on steam explosion, water/alcohol treatment, and cellulasehydrolysis. For the purpose of R. oryzae fermentation for L-lactic acid production fromcorn stover hydrolysis, a Rhizopus strain with a high lactic acid production capacity wasscreened. A support matrix for immobilized fermentation was then designed and thefermentation technology optimized. A higher L-lactic acid yield was obtained and thetechnology was found be stable for repeated batch fermentations as well assimultaneous saccharification and fermentation.
引文
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