【作者】 颜日明；

【导师】 储炬； 朱笃；

【作者基本信息】 华东理工大学 ， 发酵工程， 2011， 博士

【摘要】 微藻及其代谢产物在食品和医药工业、能源供应和环境保护等领域有广泛的应用前景,但目前大规模培养微藻仍是一个难题,如何提高微藻的培养密度和光能利用效率是其走向应用的关键技术之一。聚球藻7942(Synechococcus sp.PCC7942)是一个优良的光自养模式蓝藻,其在微藻光合作用机理和光能利用等研究方面具有重要的地位。本文以聚球藻7942作为研究对象,分别从生长动力学、叶绿素荧光诱导动力学和代谢流分析等角度对其在不同培养条件下的生长特性和能量利用进行了较为深入的研究。研究结果为揭示微藻光自养生长的本征动力学规律和提高微藻光能利用效率奠定了重要基础。在1.5 L气升式光生物反应器上对聚球藻7942的光自养生长特征进行了研究。结果表明,藻细胞在光自养培养下基本呈线性生长特征,而且随着入射光强的增加,聚球藻7942的线性生长速率和最大比生长速率不断增加,但叶绿素α含量会逐渐下降。同时,分析了培养过程中光传递对藻细胞生长的影响,结果表明,利用平均光强作为聚球藻7942的限制因子更能反映培养过程本质,以此为基础建立了基于平均光强的生长动力学模型,并初步估算得聚球藻7942光自养条件下的最大比生长速率μm=1.53 d-1。利用连续培养技术,在2.5 L平板式光生物反应器上对不同光强下聚球藻7942的光能吸收与利用进行了分析,结果表明聚球藻7942细胞的最大光能得率为1.15×10-2 g/kJ,相应的最大生物能量得率为24.23%。利用叶绿素荧光技术,分析了光自养培养中氮、磷浓度对于细胞的生长和光合作用.的影响。结果表明适当提高BG-11中的NaN03和K2HP04浓度可以促进聚球藻7942的生长,并且能够增加细胞内蛋白质和叶绿素α的含量。BG-11培养基中NaN03和K2HP04的浓度分别为1.5 g/L和60 mg/L时具有较高光合效率。研究还发现,在氮、磷限制的条件下,聚球藻7942细胞的光合系统Ⅱ(PSⅡ)的光合反应中心会发生可逆性失活。根据微藻光合作用机理,结合叶绿素荧光参数,建立了一个新的光能利用模型（？）和m。分别表示藻细胞基于吸收光能的实际最大细胞生长得率系数和实际光能维持系数)。并根据该模型,获得了聚球藻7942在光自养条件下的本征动力学参数,藻细胞的实际最大光能得率系数(Ymaxθ)和实际光能维持系数(mΘ分别为,0.0119 g/kJ和0.705 kJ/(g·h)。这个新模型解决了微藻光自养生长动力学与传统生长动力学之间的矛盾。建立了聚球藻7942光自养培养代谢网络,并通过代谢通量方法分析了不同入射光强下的碳代谢流分布和能量代谢。结果表明光合磷酸化是ATP和NADPH的主要产生途径,而将CO2固定的Calvin循环则是代谢能量和还原力消耗的主要途径。在一定光强范围,基于ATP生成的细胞得率和最大细胞得率基本维持不变,分别约为2.80 g/mol ATP和2.95 g/mol ATP,而相应的实际光能转化效率分别为13.65%,10.02%和7.86%。聚球藻7942能在以葡萄糖和乙酸为有机碳源的培养基上进行光混养培养。4.0 g/L的葡萄糖和2.0 g/L乙酸均对藻细胞的的生长有明显促进作用,并且能改变藻细胞中碳水化合物、脂类和叶绿素α的含量。运用代谢通量分析方法对聚球藻7942在混养条件下的碳代谢特征进行研究表明,葡萄糖和乙酸能明显促进糖酵解途径和TCA循环的代谢流量,同时发现混养条件下细胞得率和能量转化率均要高于光自养。本研究不仅从宏观的细胞生长的角度,而且还从光合作用机制和代谢的微观角度上探讨了聚球藻7942细胞光自养生长的本征动力学特征和光能利用规律。这为实现微藻的大规模培养奠定了理论基础。更多还原

【Abstract】 Microalgal cells and their metabolites were widely applied in food and pharmaceutical industry as well as energy supply and environmental protection. However, so far the large-scale cultivation of microalgae is still difficult and it is crucial to increase the cell density and improve the light energy efficiency for its practical industrial application. Synechococcus sp. PCC7942 is a superior typical cyanobacterium for autophototrophic cultivation and it holds a special place in the development of the photosynthesis mechanisms and light energy utilization of microalgae. In the present work, we investigated the growth characteristics and energy utilization of Synechococcus sp. PCC7942 under different culture conditions by the application of growth kinetics, chlorophyll fluorescence kinetics and metabolic flux analysis. This study will lay a foundation for revealing the intrinsic growth kinetics and improving the efficiency of light energy conversion of microalgae in autophototrophic culture.Synechococcus sp. PCC7942 was cultivated in a 1.5 L airlift photobioreactor and the characteristics of cell growth were investigated. The results showed that the growth curve of Synechococcus sp. PCC7942 cell was linear in the autotrophic culture. The linear growth rate and the maximum specific growth rate both increased while the content of chlorophyll a decreased with the elevation of incident light intensity. Furthermore, the effect of light transmission on cell growth was also studied and it was found that the average light intensity was more appropriate to serve as a key factor for cell growth. Therefore, a growth kinetic model was setup based on the conception of average light intensity and the maximum specific growth rate of Synechococcus sp. PCC7942 under autophototrophic condition was estimated to be 1.53 d-1.Continuous culture was conducted in a 2.5 L flat photobioreactor and the light absorption and utilization of Synechococcus sp. PCC7942 cell were discussed. Results demonstrated that the cell jgrowth yield and the maximum-bioenergy efficiency based on the absorbed light energy were evaluated to be 1.15×10-2 g/kJ and 24.23%, respectively.The effect of concentration of nitrogen and phosphorus on cell growth and photosynthesis were also investigated with application of the chlorophyll fluorescence analysis in the present studies.It was suggested that the appropriate increase of the concentration of NaNO3 and K2HPO4 in BG-11 medium can promote the cell growth and enhance the content of protein and chlorophyll a of Synechococcus sp. PCC7942 and high photosynthesis efficiency was achieved at the concentration of 1.5 g/L NaN03 and 60 mg/L K2HPO4.’Moreover, the photochemical reaction center of photosynthetic system (PSII) would inactivate reversibility under condition of nitrogen or phosphorus limitation.On the basis of the mechanism of photosynthesis and chlorophyll fluorescence analysis, a new light utilization model which express as Eabθ=μ/Ymaxθ+mθ(here Ymaxθand mθwere the actual maximum cell growth yield and the actual maintenance coefficient respectively) was established. According to the model, the intrinsic parameters of cell growth in autotrophic culture were obtained. The actual maximum cell growth yield (Ymaxθ) and the actual maintenance coefficient (mθ) of Synechococcus sp. PCC7942 was 0.0119 g/kJ and 0.705 kJ/(g-h). The new model can well resolve the conflict between the autophototrophic growth kinetics and the traditional growth kinetics.A comprehensive network structure for the autotrophic growth of Synechococcus sp. PCC7942 was proposed, and the carbon and energetic metabolism under different incident light intensity was investigated based on metabolic flux analysis in this paper. Results showed that the photophosphorylation and Calvin cycle were the main producer and trapper of ATP and NADPH.The values of cell growth yield and the maximum cell growth yield based on ATP generation were more stable which were 2.80 g/mol-ATP and 2.95 g/mol-ATP respectively under given conditions. The corresponding actual light conversion efficiencies were 13.65%,10.02% and 7.86%, respectively.Synechococcus sp. PCC7942 can grow in the mixotrophic culture in the presence of glucose and acetate.4.0 g/L glucose or 2.0 g/L acetate can improve the cell growth and alter the contents of carbohydrates, lipids and chlorophyll a of cell. Furthermore, the glucose acetate can also enhance the metabolic flux through glycolysis pathway and TCA cycle. On the basis of metabolic flux analysis, the cell growth yield and energy conversion efficiency under the mixotrophic conditions was higher than that in the photoautotrophic condition.In the present work, the intrinsic growth kinetics and light utilization were discussed based on the cell growth characteristics, photosynthetic mechanism and metabolic flux analysis. The results will establish a solid foundation for microalgae mass cultivation.更多还原