【作者】 王永红；

【导师】 张嗣良；

【作者基本信息】 华东理工大学 ， 生物化工， 2000， 博士

【摘要】 蓝藻基因工程的发展迫切要求用封闭式光反应器进行蓝藻的高密度培养。集胞藻6803是光合作用机理和分子生物学研究的重要材料,也是藻类基因工程的常用宿主系统,其营养模式在蓝藻中具有代表性。它的营养模式特点为,除能进行光自养生长外,只有在有光照时才能利用有机物生长,不能进行暗异养生长。对于这一类型的蓝藻,在混合营养培养系统中最有可能实现高密度培养。迄今为止,有关集胞藻6803混合营养培养最佳条件及其在封闭式光生物反应器中的混合营养培养的研究尚未见报道,集胞藻6803混合营养培养的生理生化特性的研究在文献中也未见专门报道。 本文首先对影响集胞藻6803混合营养生长的主要因素进行了较系统的研究,在此基础上开展了2.5L封闭式光生物反应器集胞藻6803混合营养生长的特征和动力学研究,并针对研究过程中发现的与光有关的现象,研究了集胞藻6803混合营养细胞的生理生化特性,最后探讨了集胞藻6803利用葡萄糖生长的生理机制。 有机碳源和光是影响集胞藻6803混合营养生长的两个重要因素。葡萄糖、蔗糖和果糖对集胞藻6803混合营养生长的影响表明,葡萄糖是集胞藻6803混合营养生长的最佳碳源,葡萄糖的最佳浓度范围为1—3g/L。集胞藻6803混合营养生长存在光饱和点,摇瓶和封闭式光生物反应器混合营养培养的饱和光强分别为47.3μEm^-2S^-1和78.8μEm^-2S^-1。当光照强度大于饱和光强时,混合营养生长速率会有所下降。混合营养生长的饱和光强与光自养生长相同。但在相同光照强度下,集胞藻6803混合营养生长的生长速率、最终藻细胞密度和能量得率都高于光自养生长。 光在光自养藻液中的衰减比在混合营养培养藻液中快。对于同一反应器,当入射光强和藻细胞密度相同时,混合营养藻液中的细胞实际得到的光照比光自养藻液中的细胞多。 在反应器内表面的入射光强范围为9.1—111.6μEm^-2S^-1的2.5L封闭式光生物反应器上进行了集胞藻6803混合营养培养研究。实验结果表明,集胞藻6803的混合营养培养系统明显优于光自养培养系统。在封闭式光生物反应器中,混合营养培养更多还原

【Abstract】 The development of cyanobacterial genetic engineering makes it an increasing necessary to develop high cell density cultivation of cyanobacteria in the enclosed photobioreactors. Synechocystis sp. PCC6803, which is important material for studies on the mechanism of photosynthesis and molecular biology as well as the most popular host system for cyanobacterial genetic engineering, has special and representative growth modes in cyanobacteria. It can grow under photoautotrophic and photoheterotrophic but heterotrophic conditions. It is full of promise to obtain high cell density cultivation by mixotrophy, especially for cyanobacteria like Synechocystis sp. PCC6803. Up to now, few papers have been published on the mixotrophic cultivation of Synechocystis sp. PCC6803 in an enclosed photobioreactor. In this thesis, the factors affecting the mixotrophic growth of Synechocystis sp. PCC6803, especially in an enclosed photobioreactor, and the physiologic characteristics of cells grown under mixotrophic conditions were studied and the mechanism of Synechocystis sp. PCC6803 utilizing glucose for growth was also discussed.Organic carbon source and light were two most important factors affecting mixortophic growth of Synechocystis sp. PCC6803. In this study, glucose was the most suitable organic carbon source, and its optimal concentration for mixotrophic growth was about 1-3g/L. The saturated light intensities of mixotrophic growth of Synechocystis sp. PCC6803 in shake flasks and a 2.5L enclosed photobioreactor were 47.3 μ Em^-2s^-1 and 78.8 μ Em^-2s^-1, respectively. When the light intensity was higher than the saturated level, the mixotrophic growth rates would decrease. The saturated level of light intensity in mixotrophic culture was almost the same as that in photoautotrophic culture. At the same level of light intensity, the growth rate, cell density and energy efficiency in mixotriophic conditions were higher than those in photoautotrophic conditions.The light attenuation inside photoautotrophic culture was faster than that in mixortophic culture. In a given bioreactor, when incident light intensity and biomasswere identical, the cells inside mixotrophic culture would receive more light than that inside photoautotrophic culture.Studies on mixotrophic batch cultivation were carried out in a 2.5L enclosed photobioreactor, and its incident light intensities were from 9.1 U Em’Y1 to 111.6 u Em’V ’. It was concluded that mixotrophic culture system was better than photoautotrophic culture system for Synechocystis sp. PCC6803. In the enclosed photobioreactor, after 58.5h mixotrophic cultivation, 2.50g/L cell density, l.Og/L.d productivity, 15.3 u g/ml chlorophyll concentration and 25.3% energy efficiency were achieved, which were respectively 8.9 times, lltimes, 2.3 times and 4.9 times as much as those in photoautotrophic culture.In the 2.5L enclosed photobioreactor, the growth of Synechocystis sp. PCC6803 under photoautotrophic conditions was linear after one-day delayed phase. The mixotrophic growth process of Synechocystis sp. PCC 6803 could be divided into two phases: the first, from beginning to 48h or so, was the exponential growth phase, and the second, from 48h or so to the culture time when residual glucose concentration deceased to zero, was the linear growth phase. The exponential growth phase was only influenced by incident light intensity and could be described by the Andrews model. The linear phase was effected by both of incident light intensity and glucose concentration. Both incident light intensity and glucose concentration could make effects on synthetic rate of chlorophyll, which had close relation to the growth of Synechocystis sp. PCC6803. The variation of pH in the mixotrophic cultivation process was the results from the balance between photoautotrophic metablism and heterotrophic metabolism.The physiologic characteristics of Synechocystis sp. PCC6803 were investigated and it was observed that the oxygen evolution rate, respiratory rate, photosystem II actual photochemical efficiency （ <t> II ） and the slow phase of millisecond delayed light emission of mixotrophic cells were higher than those of photoautotrophic cells. On the basis of the above results and the influences of inhibitors of photosynthetic electron transport DCMU, DBMIB on the mixotrophic growth of Synechocystis sp. PCC6803, it would be concluded that there were two reasons for the higher growth rate of Synechocysis sp. PCC 6803 under mixotrophic conditions than that under photoautotrophic conditions. The first reason was that glucose would not inhibited butaccelerated the photoautrophic growth in mixotrophic cells, and the second reason was the conversion of energy was promoted in photosynthetic system, which increased the anabolism of cells.The above results are useful for the cultivation of transgenic cyanobacteria in enclosed photobioreactors, and would promote the application of mixotrophic cultivation system in microalgae cultivation. Besides, they are also useful to understand the interrelation between photoautotrophy and heterotrophy in cyanobacteria.更多还原