红发夫酵母发酵虾青素的研究

红发夫酵母分离于北美西部高山地区和日本一些岛屿上落叶树的渗出液中，因其所产主要色素为在水产养殖、食品和医药工业有广阔应用前景的虾青素而成为研究的热点。本论文对红发夫酵母Phaffia rhodozyma 的生长特性、培养参数与培养基组分对生长和虾青素积累的影响及其优化、虾青素合成的调节控制、虾青素的提取测定及红发夫酵母耐高温菌种的诱变进行了系统的研究。 虾青素是红发夫酵母的胞内色素，要对其进行分析首先要对红发夫酵母进行破壁处理，实验发现二甲亚砜是最有效的破壁溶剂，用氯仿和丙酮可以有效地把类胡萝卜素从二甲亚砜破壁后的红发夫酵母细胞中提取出来。 在固定摇床转速为200 rpm，温度为20 ℃的条件下，当种龄为36 h，以10％的接种量接入装液量为30 mL的250 mL三角瓶，初始pH为5.5时最有利于红发夫酵母的生长及类胡萝卜素的合成。 本实验中红发夫酵母最佳利用碳、氮源分别为蔗糖和蛋白胨，但蛋白胨价格昂贵，不适宜作单一氮源，因此使用硫酸铵和酵母膏作为复合氮源。 本论文采用了BP神经网络结合遗传算法的方法来优化红发夫酵母的发酵培养基，得到红发夫酵母发酵培养基的最佳配比为：蔗糖45.10 g/L、硫酸铵3.00 g/L、硫酸镁0.80 g/L、磷酸二氢钾1.40 g/L、酵母膏3.00 g/L、氯化钙0.50 g/L，使用优化后的培养基发酵类胡萝卜素产量达到8.20 mg/L，干重达到9.47 g/L，类胡萝卜素的产量比起始培养基提高了95.90%，干重提高了89.40%。 从代谢途径出发对红发夫酵母合成虾青素调控调控，选择谷氨酸、乙醇、VB1作为添加剂，通过正交试验设计得出三者添加水平分别为0.2 g/L，0.1% (V/V)，10 mg/L时，类胡萝卜素产量提高了25.73%，达到了10.31mg/L。 通过上述优化培养，本论文中红发夫酵母的虾青素产量从1.33 mg/L提高到9.12 mg/L，产量提高了6.86倍；总类胡萝卜素产量从4.23 mg/L提高到10.31 mg/L，产量提高了2.44倍；细胞干重从5.00 g/L提高到11.35 g/L，提高了2.27倍，总体提高效果显著。 红发夫酵母属于中低温菌，本论文采用紫外复合诱变的方式，通过高温筛选，得到一株能在35 ℃下能生长的突变株，但所产类胡萝卜素中虾青素所占比例很小，可能是诱变改变了红发夫酵母的代谢途径，阻断了虾青素的合成。 Phaffia rhodozyma is a heterobasidiomyceteous yeast that was originally isolated from the slime fluxes of brich tree wounds in mountain regions of northern Japan and southern Alaska. Phaffia rhodozyma produces astaxanthin as its principal carotenoid pigment, which has potential applications in acquaculture, food and pharmaceutical industry. This paper researched ways to break cell, analysis of astaxanthin, characteristics of growth, culture parameters and the effects of components of medium on growth and astaxanthin formation , optimization of culture medium, control of astaxanthin synthesis and mutagenesis of Phaffia rhodozyma. It is necessary to disrupt the yeast cell for extracting astaxanthin considering the yeast accumulating carotenoids in cell. Dimethyisulphoxide was the most effective solvent for breaking the yeast cell; acetone and chloroform were effective to extract carotenoids out of the disrupted cell. The optimum pH for growth and carotenoids synthesis is 5.5, the optimum medium volume is 30 mL (in 250 mL flask), the optimum culture time of inoculum is 36 h, the optimum inoculum concentration is 10%. The research on culture medium showed: sucrose is the best one of 6 carbon sources for growth and astaxanthin synthesis. Peptone is the best nitrogen source for growth and astaxanthin synthesis. Uniform Design was used for trial design of the formula medium components, then back-propagation neural network was established to modeling the relationships between the carotenoid yield and the concentration of medium components. Genetic algorithm (GA) was used for global optimization of the model. The optimum combination of the medium was obtained: sucrose 45.10 g/L, ammonium sulfate 3.00 g/L, magnesium sulfate 0.80 g/L, potassium dihydrogen phosphate 1.40 g/L, yeast extract 3.00 g/L, calcium chloride 0.50 g/L. The yield of carotenoid reached 8.20 mg/L, which was 95.90% higher than that of the original medium. Glu, VB1 and ethanol were selected as fermentation addictives, after Orthogonal Test, the carotenoid contents increased by 25.73% when adding 0.16 g/L Glu, VB1 10 mg/L and ethanol 0.1% (V/V). After the above optimization, the astaxanthin content increased 6.86 folds, which is 9.12 mg/L. The carotenoids content increased 2.44 folds, which is 10.31 mg/L. The biomass increased 2.27 folds, which is 11.35 g/L. Phaffia rhodozyma grows in the mild temperature range of 0 to 27 ℃, in this work, a thermotolerant mutant was selected through UV-irradiation. It can grows at 35 ℃, and showed increased carotenoid content. The optimal growth temperature for this mutant is 30 ℃. But the mutant can only produce carotenoids with little astaxanthin accumulation.

北山及邻区古生代-早中生代增生与碰撞大地构造格局

The Beishan orogenic collage locates at the triple-joint among Xinjiang, Gansu, and Inner Mongolia Provinces, at which the Siberian, Tarim and North China plates join together. It also occupies the central segment of the southern Central Asian Orogenic Belt (CAOB). The main study area in the present suty focused on the southwest part of the Beishan Mountain, which can be subdivided into four units southernward, the Mazhongshan continental block, Huaniushan Arc, Liuyuan suture zone and Shibanshan-Daqishan Arc. 1. The Huaniushan Arc was formed by northernward dipping subduction from the Orcovician to Permian, in which volcanic rocks ranging from basic to acidic with island arc affinity were widely developed. The granitiod intrusions become smaller and younger southward, whichs indicates a southward rollback of slab. The granitiod intrusions are mainly composed of I type granites, and their geochemical compositions suggest that they have affinities of island arc settings. In the early Paleozoic（440Ma-390Ma）. The Shibanshan-Daqishan Arc, however, were produced in the southernward dipping subduction system from Carboniferous to Permian. Volcanic rocks from basic to acidic rocks are typical calcic-alkaline rocks. The granitiod intrusions become smaller and younger northernward, indicating subdution with a northernward rollback. The granitiod intrusions mainly consist of I-type granites, of which geochemical data support they belong to island arc granite. 2. Two series of adakite intrusions and eruptive rocks have been discovered in the southern margin of the Huaniushan Island Arc. The older series formed during Silurian (441.7±2.5Ma) are gneiss granitoid. These adakite granites intruded the early Paleozoic Liuyuan accretionary complex, and have the same age as most of the granite intrusions in the Huanniushan Arc. Their geochemical compostions demonstrate that they were derived from partial melting of the subudcted oceanic slab. These characteristics indicate a young oceanic crust subduction in the early Paleozoic. The late stage adakites with compositons of dacites associate with Nb-enriched basalts, and island arc basalts and dacites. Their geochemistries demonstrate that the adakites are the products of subducted slab melts, whereas the Nb-enriched basalt is products of the mantle wedge which have metasomatized by adakite melts. Such a association indicates the existences of a young ocean slab subduction. 3. The Liuyuan suture zone is composed of late Paleozoic ophiolites and two series of accretionary complexes with age of early Paleozoic. The early Paleozoic accretionary complex extensively intruded by early Palozioc granites is composed of metamorphic clastics, marble, flysch, various metamorphic igneous rocks (ultramafic, mafic and dacite), and eclogite blocks, which are connected by faults. The original compositions of the rocks in this complex are highly varied, including MORB, E-MORB, arc rocks. Geochronological study indicates that they were formed during the Silurian (420.9±2.5Ma and 421.1±4.3Ma). Large-scale granitiods intruded in the accretionary complex suggest a fast growth effect at the south margin of the Huaniushan arc. During late Paleozoic, island arc were developed on this accretionary complex. The late Paleozoic ophiolite has an age of early Permian (285.7±2.2Ma), in which the rock assemblage includes ultra-mafic, gabbros, gabbros veins, massive basalts, pillow basalt, basaltic clastic breccias, and thin layer tuff, with chert on the top.These igneous rocks have both arc and MORB affinities, indicating their belonging to SSZ type ophiolite. Therefore, oceanic basins area were still existed in the Liuyuan area in the early Permian. 4. The mafic-ultramafic complexes are distributed along major faults, and composed of zoned cumulate rocks, in which peridotites are surrounded by pyroxenite, hornblendites, gabbros norite and diorite outward. They have island-arc affinities and are consistent with typical Alaska-type mafic-ultramafic complexes. The geochronological results indicate that they were formed in the early Permian. 5. The Liuyuan A-type granite were formed under post-collisional settings during the late Triassic (230.9±2.5Ma), indicating the persistence of orogenic process till the late Triassic in the study area. Geochronological results suggested that A-type granites become younger southward from the Wulungu A-type granite belt to Liuyuan A-type granite belt, which is in good agreement with the accretionary direction of the CAOB in this area, which indicate that the Liuyuan suture is the final sture of the Paleo-Asin Ocean. 6. Structural geological evidence demonstrate the W-E spreading of main tectonic terrenes. These terrenes had mainly underwent through S-N direction contraction and NE strike-faulting. The study area had experienced a S-N direction compression after the Permian, indicating a collisional event after the Permian. Based on the evidene from sedimentary geology, paleontology, and geomagnetism, our studies indicate that the orogenic process can be subdivided into five stages: (1) the pre-orogenic stage occurred before the Ordovicain; (2) the subduction orogenic stage occurred from the Orcovician to the Permian; (3) the collisional orogenic stage occurred from the late Permian to the late Triassic; (4) the post-collision stage occurred after the Triassic. The Liuyuan areas have a long and complex tectonic evolutional history, and the Liuyuan suture zone is one of the most important sutures. It is the finally suture zone of the paleo-Asian ocean in the Beishan area.