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栅藻(Scenedesmus obliquus)藻菌共生体系的构建及调控
王瑞民
学位类型博士
导师丛威
2015-10
学位授予单位中国科学院研究生院
学位授予地点北京
学位专业生物化工
关键词产油微藻 藻菌共生体系 胞外多聚物 微生物群落 开放培养
其他摘要

由于光合效率高、生长速度快、含油量高等特性,微藻被认为是最有希望替代化石能源的生物质能源。开放池培养是实现微藻规模化培养的主要方式,但如何提高产油微藻在开放体系下培养的稳定性,增强抵御外界物种侵扰的能力以及进一步提高微藻生物量产率成为限制微藻生物柴油产业化的瓶颈。微藻与微生物的共生体系可以为微藻的生长提供良好的微环境,但目前有关微藻-微生物共生体系对微藻生物量和油脂积累能力的影响的研究仍然比较缺乏。本论文以以一株产油微藻斜生栅藻(Scenedesmus obliquus)为研究对象,通过人工构建藻菌共生体系,研究了该体系在自养条件下对栅藻的生长和油脂积累特性的影响及机制,并进一步探索了藻菌共培养体系在开放条件下的稳定性和抵御外界物种入侵的能力。 本论文构建了栅藻与真核生物的共生体系(即共培养体系)。当假丝酵母Candida tropicalis和酿酒酵母Saccharomyces cerevisiae分别与纯化后的斜生栅藻共培养时,只有前者可以增加栅藻的生物量、光合活性和油脂含量,它们分别比纯栅藻体系提高了30.3%、61%和22.5%。相反,酿酒酵母则无显著效应。不同初始接种比例的研究结果表明,栅藻和C. tropicalis的接种比例为3:1时,C. tropicalis对栅藻生长的促进效应最显著。C. tropicalis的存在可以消耗上清液中的胞外多聚物(EPS)和溶解氧,从而减轻它们对微藻生长的抑制作用。此外,栅藻和C. tropicalis的共培养仅在无杂菌的封闭式光反应器中可以促进微藻生长并提高其油脂积累能力,在开放条件下,该促进效应并不显著。 本论文构建了栅藻与原核生物的共培养体系。对未纯化的栅藻S. obliquus在正常培养过程中细菌菌群的分析表明,有益菌群和有害菌群共同存在于培养体系中。从栅藻培养液中共筛选到5株与栅藻共生的有益菌菌株,它们分别属于不同的种属,包括Brevundimonas aurantiaca (菌株2-1), Rhizobium sp. (菌株2-2), Pseudomonas sp. (菌株3-4), Acidovorax facilis (菌株3-10)和Diaphorobacter sp. (菌株3-11)。将5株菌株分别与纯的栅藻共培养10天后发现,它们均可以显著提高栅藻的生物量。其中菌株3-10的效果最好,使栅藻生物量增加了24.8%。同时,细菌的存在提高了栅藻油脂的含量,增加了饱和脂肪酸和十八烯酸的比例,降低了不饱和脂肪酸的比例。扫描电镜的结果表明在藻菌共培养体系中,细菌附着在栅藻的表面,这更有利于二者的物质交换。EPS的分析结果进一步证实,细菌可以降解EPS,改变EPS中蛋白质和多糖的组分及其含量,进而促进栅藻的生长和油脂积累。 对已构建的微藻-细菌共培养体系进行调控,研究初始接种比例、外源金属离子浓度、缺氮条件以及细菌菌株组合对栅藻生物量、油脂含量和EPS含量的影响。当初始藻菌比例为3:1时,细菌对栅藻生长的促进效果最显著。EPS是藻菌体系调控的核心,外源添加CaCl2的浓度为100 mg/L时,显著提高了藻菌体系EPS中蛋白质和多糖的含量,并且CaCl2的添加并不影响栅藻的生长和油脂含量。在缺氮诱导的条件下,藻菌体系中栅藻的油脂含量和产率最高比纯栅藻体系分别提高了33.3%和73.9%。不同菌株的组合结果表明,多种菌株与栅藻共培养时也可以促进后者的生长及油脂积累;菌株2-2,3-4,3-10和3-11的组合效果最显著,可以使栅藻的生物量和油脂含量分别增加28.5%和14.6%。 利用Applikon微型反应器研究了栅藻与细菌之间的气体交换和物质交换,揭示藻菌体系的共生机制。证实细菌的参与可以降低培养系统中的溶解氧,促进栅藻生长,后者又进一步消耗二氧化碳,释放氧气,利于细菌生长,从而形成栅藻与细菌之间气体交换的循环。证实细菌可以降低EPS(包括结合型和游离型)中大分子量化合物以及总有机碳的含量,增加体系中总无机碳的含量,为栅藻的光合作用提供原料。共生体系中栅藻胞外微环境的改变也促使栅藻将胞内的总糖转化为油脂,这些最终促使了栅藻油脂含量的提高。 最后,本论文考察了实验室开放条件下栅藻-细菌共生体系中栅藻的生物量和油脂积累情况以及该体系的稳定性。在开放条件下连续培养16天后,藻菌体系中栅藻的生物量比封闭条件的纯栅藻体系提高了17.1%,比开放条件的纯栅藻体系提高了95%,同时,藻菌体系中栅藻的油脂含量比纯栅藻封闭体系也增加了24%。微生物群落的分析结果证实,藻菌体系中的细菌菌群保持稳定,初始接种的菌种在培养过程中(12天)始终是体系中的优势菌群,其丰度为总菌群的74-82.9%;小丰度(1%以下)细菌菌种的丰度维持在10%左右。相反,纯栅藻体系在开放条件下其细菌菌群发生了明显改变,小丰度菌种的比例在培养后期增加到40%。开放条件下的培养后期,藻菌体系和纯栅藻体系中均检测到多种真核生物群落,但是,仅在纯栅藻的开放培养体系中检测到有害微型动物,Colpodea(肾形虫属)和Platyophrya(匙口虫属)。可见,藻菌共培养可以增加细菌菌群的稳定性,减少有害物种的入侵,增强体系在开放条件下的稳定性。

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As a source of third-generation biodiesel, microalgae have attracted a great deal of attentions because of their excellent properties including high growth rate, high photosynthetic efficiency and high lipid content. Therefore, microalgae are considered to be ideal organisms for developing highly productive and robust strains that are essential for economically viable biodiesel production. Large-scale cultivation in open ponds is the main mode for microalgal lipid production, but it is hindered by low biomass productivity and instability during cultivation resulted from invasions by pathogens. The consortium of microalgae and microorganisms can provide a comfortable micro-enviroment for microalgal growth. To date, the effects of consortium system on the microalgal growth and lipid production are still unclear. In this study, the artificial consortia of Scenedesmus obliquus and microorganisms were constructed to study their effects on the microalgal growth and lipid production. Moreover, the syntrophic machenism between microalgae and microorganisms in the consortia was also investigated. Additionally, the stability of S. obliquus and bacterium consortia was studied under open conditions in the lab. The consortia (i.e. co-cultivation) of microalgae and eukaryotic strains, including Candida tropicalis and Saccharomyces cerevisiae, were constructed respectively. In the co-cultures of S.obliquus and C. tropicalis, microalgal biomass concentration, net photosynthetic activity and lipid content increased by 30.3%, 61% and 22.5%, respectively, compared to S. obliquus alone, but no stimulation was observed in the co-culture of S. obliquus and Saccharomyces cerevisiae. It has been demonstrated that microalgae exhibited the highest concentration of biomass and net photosynthetic activity when S. obliquus and C. tropicalis were seeded at the ratio of 3:1, compared to other seeding ratios. In the consortium of microalgae and C. tropicalis, the later could degrade and use the extracellular polymeric substances (EPS) excreted from microalgae, providing a favorable micro-environment for the microalgal growth. The co-cultivation of S. obliquus and C. tropicalis could not increase the microalgal growth under open condition, although it could happen in the closed photobioreactor. The consortia of S. obliquus and bacterial strains were constructed. Based on the analysis of bacterial diversity in culutres of xenic S. obliquus, it was found that both beneficial and harmful bacterial populations were associated with xenic microalgae. The supernatant of axenic microalgal cultures was used to subculture bacterial isolates and 5 favorably syntrophic bacterial strains were obtained, including Brevundimonas aurantiaca (2-1), Rhizobium sp. (2-2), Pseudomonas sp. (3-4), Acidovorax facilis (3-10) and Diaphorobacter sp. (3-11). When the syntrophic bacterial individuals were co-cultivated with axenic S. obliquus respectively, microalgal growth was enhanced in all consortia, among which A. facilis represented the largest biomass concentration increase of 24.8% compared to the axenic culture of S. obliquus. The consortium system also increased microalgal lipid content, lipid productivity, and the proportion of saturated fatty acids and oleic acid. According to scanning electron microscopy (SEM) analysis, the selected syntrophic bacterial strains adhered directly to the S. obliquus cell surface, which was helpful for the material exchange between microalgae and bacterium. Moreover, the participation of bacterial strains could metabolize EPS and change both concentrations and compositions of proteins and polysaccharides dissolved in the co-cultures, contributing to the increased microalgal biomass and lipid production in the consortia. Studies on the regulation of microalgae-bacterium consortia were performed. In the meantime, the effects of different regulation parameters on microalgal growth and lipid production and EPS concentrations during cultivation were investigated, including seeding ratios of microalgae and bacterial strains, concentrations of exogenousmetal ions, nitrogen deficiency and different combinations of syntrophic bacterial strains. The best S. obliquus growth was observed when microalgae and bacterial individuals were seeded in a 3:1 ratio. The EPS concentration, an important regulation parameter, increased significantly in microalgae-bacterium consortia upon addition of 100 mg/L CaCl2; while, the microalgal growth and lipid production were not affected by CaCl2 at the same concentration. After cultivation under nitrogen source deficiency for 7 days, the lipid content and lipid productivity in the consortia of S. obliquus and syntrophic bacterial strains were 33.3% and 73.9% higher than those in S. obliquus, respectively. Furthermore, the presence of mixed bacterial strains could also enhance microalgal growth and lipid production. Among the different bacterial combinations, the mixture of strain 2-2, 3-4, 3-10 and 3-11 showed the most positive effects, where microalgal biomass concentration and lipid content increased by 28.5% and 14.6% compared to axenic S. obliquus. Thereafter, the gas exchange and carbon exchange between microalgae and bacteria were studied with Applikon microreactor to understand the mechanism by which microalgae-bacterium consortia enhance microalgal growth. The participation of syntrophic bacterial strains utilized the dissolved oxygen, reducing oxygen stress and thus enhancing microalgal growth. The microalgae further assimilated carbon dioxide and released oxygen that could be used by the bacteria. Meanwhile, the syntrophic bacteria degraded compounds with high molecular weight and organic carbon secreted by microalgae, which reduced the concentration of total organic carbon (TOC) and increased the concentration of total inorganic carbon (TIC). The decrease of TOC stress and the increase of TIC were helpful for microalgal photosynthesis and growth. Additionally, the favorable micro-environment due to bacterial presence could enhance the transformation of polysaccharide to lipid in microalgae. In the last, the stability of microalgae-bacterium consortia was examined when the photobioreactors were operated under open conditions in the laboratory. After 16 days of cultivation, the biomass concentration in the consortia under open condition was 17.1% and 95% higher than that in axenic S. obliquus cultures under closed conditions and under open conditions, respectively. Meanwhile, the lipid content was improved by 24% in airtificial consortia under open conditions compared to axenic S. obliquus cultivation under closed conditions. Furthermore, it was demonstrated that the participation of bacterial strains could help to keep the stability of bacterial community in consortia. That is, the species and correspoinding proportion of seeded individual strains were basically stable, which contributed 74-82.9% of all bacterial populations in total, and bacteria with the proportion (no more than 1% in total) accounted for about 10% during 12 days of cultivation. On the contrary, the bacterial community in axenic microalgal cultures changed significantly, and the bacterial strains with the proportion under 1% increased to 40% after 12 days of cultivation under open conditions. In addition, under open conditions, the harmful microfauna including Colpodea and Platyophrya, which lead to reduction of miacroalgal biomass, were detected in axenic S. obliquus cultures, but not found in consortia. These preliminary results imply that the airtificial consortia of S. obliquus and bacteria improved the stability of microalgal cultivation under open conditions. 

语种中文
文献类型学位论文
条目标识符http://ir.ipe.ac.cn/handle/122111/21357
专题研究所(批量导入)
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王瑞民. 栅藻(Scenedesmus obliquus)藻菌共生体系的构建及调控[D]. 北京. 中国科学院研究生院,2015.
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