产油微藻的筛选与评价、高产油策略及其差异性研究
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摘要
产油微藻是生物柴油生产的理想原料，随着能源危机的加剧，它逐渐成为各国政府和藻类学家关注的焦点。实现微藻生物柴油产业化的关键在于如何提高油脂产量，受藻种性能和培养条件所限，许多已开展的研究工作并不具备实际指导意义，而且对于“天然产油微藻(非模式微藻)”生理生化特性的研究也较少，而这些研究工作正是解决理论瓶颈、提高油脂产量的关键。本论文主要开展了“产油微藻的筛选”、“光强、初始氮供应量和氮源形式三种策略提高微藻油脂产量的可行性”、“两株不同类型天然产油微藻生理生化特性及其差异性研究”、“产油小球藻脂类积累过程中的生长、碳流分配以及光合特性研究”、“产油微藻扩大培养的产能评价”等五个方面的研究工作，主要研究结果如下：
1.对实验室保藏的20株微藻进行了评价(真眼点藻纲2株，硅藻纲1株，绿藻门17
株)，根据生物质浓度、总脂含量、β-胡萝卜素含量以及总脂收获量等参数的高低，筛选出6株总脂收获量超过2.0gL~(-1)的微藻，它们是韦氏真眼点藻(Eustigamatos vischeri)、荒漠栅藻(Scenedesmus deserticola)、麻织绿球藻(Chlorococcum tatrense)、眼点拟微绿球藻(Nannochloropsis oculata)、椭圆绿球藻(Chlorococcum ellipsoideum)和雪绿球藻(Chlorococcum nivale)，经“生物柴油质量预测模型”的验证，6种微藻生物柴油的质量均符合中国(BD100)、美国(ASTM6751)和欧盟(EN14214)的标准；在6株潜力微藻中，韦氏真眼点藻(E.vischeri)的总脂收获量可达4.0gL~(-1)，同时该藻还可以积累2.39%DW的β-胡萝卜素，具有极高的开发利用价值。
2.通过测定生物质浓度、总脂含量和中性脂含量，探索了“光强”、“初始氮供应量”、
“氮源形式”三种策略，对6株产油微藻生长和中性脂积累的影响，结果表明：三种策略均可以提高6株产油微藻的中性脂产量，但存在明显的种间差异和因素交叉影响；6株产油微藻优化后的中性脂产量分别为：韦氏真眼点藻(4.9gL~(-1),高光强+尿素+低氮供应)、荒漠栅藻(4.1gL~(-1),高光强+硝酸钠+高氮供应)、椭圆绿球藻(3.8gL~(-1),高光强+尿素+低氮供应)、眼点拟微绿球藻(3.0gL~(-1),高光强+硝酸钠+高氮供应)、雪绿球藻(2.7gL~(-1),高光强+硝酸钠+低氮供应)、麻织绿球藻(2.7gL~(-1),高光强+硝酸钠+高氮供应)。根据产油微藻对起始氮供应量的响应规律，将产油微藻分为3种类型：类型-I，低氮供应降低了生物质浓度而提高了中性脂含量；类型-II，低氮供应同时降低生物质浓度和中性脂含量；类型-III，低氮供应同时提高了生物质浓度和中性脂含量，类型-III更具产业化价值。
3.本论文研究了3.5mM、5.9mM、8.8mM和17.6mM四种初始氮浓度条件下，两株不同类型的天然产油微藻––荒漠栅藻S.deserticola(类型I)和韦氏真眼点藻E.vischeri(类型III)，在细胞生长、生化组成、氮吸收利用、光合作用及呼吸作用等方面的变化规律及差异性，主要研究结果为：
(1)生长特性：有利于两株微藻细胞分裂的氮浓度均为8.8mM，而有利于两株微藻生物质积累的氮浓度为17.6mM(荒漠栅藻)和5.9mM(韦氏真眼点藻)；随初始氮供应量的降低，荒漠栅藻的单位细胞重量降低，而韦氏真眼点藻的单位细胞重量增加；
(2)氮的吸收利用：两株微藻具有相似的NO3-的吸收规律，但韦氏真眼点藻具有比荒漠栅藻更高蛋白质合成速率和合成量；
(3)生化组成：培养过程中，两株微藻均表现出“碳水化合物和蛋白质含量降低、总脂含量升高”的趋势；高浓度氮供应有利于两株微藻蛋白质的合成，而低浓度氮供应有利于碳水化合物和脂类的合成；荒漠栅藻中性脂的积累速率和积累量显著高于韦氏真眼点藻，而韦氏真眼点藻的碳水化合物含量高于荒漠栅藻；荒漠栅藻在培养中后期大量积累角黄素，其积累量与氮浓度呈负相关，韦氏真眼点藻在培养中后期大量积累β-胡萝卜素，其积累量与氮浓度呈正相关；两株微藻其余光合色素的含量在培养过程中均显降低趋势，降低幅度与初始氮供应量呈负相关；
(4)光合作用和呼吸作用：培养过程中，两株微藻的光合效率(F_v/F_m,rETR和P_n)和呼吸速率均呈现降低趋势，韦氏真眼点藻具有较低的热耗散效率和高的光能利用效率。
4.以分离自广东某热带湖泊的产油微藻JNU13为实验材料，通过18SrRNA基因序列分析和形态学观察，初步确定该藻株为普通小球藻JNU13(Chlorella vulgarisJNU13)，研究了该藻在脂类积累过程中的细胞生长、碳流分配及光合特性，(1)生长与脂类积累：培养前期，普通小球藻JNU13的细胞分裂与胞内脂类积累可以同步进行，培养中后期，细胞分裂停止，胞内脂类积累成为生物量浓度增加的主要原因；(2)碳流分配：培养前期，普通小球藻JNU13固定的碳主要用于碳水化合物的合成，培养中后期，主要用于脂类的合成；(3)光合特性：光合效率的降低对普通小球藻JNU13脂类积累存在反馈抑制作用，脂类积累过程中伴随光系统能量分配的改变，光系统I能量比例降低，光系统II能量比例相对稳定，与氧气释放相关的Z型电子传递链活性降低，可变电子传递链可以为产油微藻JNU13的生长提供能量，高光合效率使高氮供给组获得了比低氮供给组更高的总脂含量。
5.利用体积为24L的室内平板式光生物反应器(光径为4.0cm)，对荒漠栅藻(S.deserticola)和眼点拟微绿球藻(N.oculata)进行了扩大培养及产能评价，结果表明：两株微藻在平板式光生物反应器中生物质浓度、总脂含量以及总脂收获量均显著低于0.3L柱式光生物反应器(光径3.0cm)的培养结果；荒漠栅藻和眼点拟微绿球藻的单位面积生物质产率分别为16.43gm~2day~(-1)和17.08gm~2day~(-1)，总脂产率为7.64gm~2day~(-1)和5.48gm~2day~(-1)；荒漠栅藻藻粉成本为1868.2元/千克，眼点拟微绿球藻藻粉成本为1949.9元/千克，室内培养条件下，高通气搅拌成本和光照成本是藻粉成本过高的主要原因。
Oleaginous microalgae are considered as an optimal feedstock for biodiesel production. With the growing energy crisis, research on biodiesel production from microalgae has been the concern of governments and researchers worldwide. To enhance lipid productivity is one of the most critical problems to realize microalgal biodiesel industrialization. Due to the limitation of both of oleaginous microalgal species and experimental condition, some of previous studies have less guiding significance. Moreover, studies on natural oleaginous microalgae (non-model microalgae) are limited. To overcome these shortcomings are exactly the key solving steps for theoretical bottleneck and lipid productivity improvement. This study mainly focused on "oleaginous microalgal screening","effects of high light intensity, initial nitrogen supply, urea on the growth and neutral lipid accumulation of oleaginous microalgae","regulations and differences in physiology and biochemistry of two natural microalgal species under various intial nitrogen supply","growth, carbon flow distribution, and photosynthetic performance of oleaginous microalgae in the process of lipid accumulationand","assessment, and economic analyses of oleaginous microalgae in large-scale cultivation". The main results are listed as follows:
1. Twenty strains of freshwater and marine microalgae, including17strains of Chlorophytes,2strains of Eustigmatophytes, and1strain of Bacillariophytes, were investigated in this study. By comparing their biomass concentration, total lipid content, β-carotene content and total lipid yield, six strains of oleaginous microalgae which had over2.0g L-1of lipid yield were selected. These were E. vischeri, S. deserticola, C. tatrense, N. oculata, C. ellipsoideum, and C. nivale. After testing by prediction model of the quality of biodiesel, their lipids complied with the standards set by China (BD100), USA (ASTM6751), and EU (EN14214). Among them, the lipid yield of E. vischeri could reach up to4.0g L-1. Simultaneously, it could accumulate2.39%DW β-carotene. This microalga was considered to have extremely high developing value.
2. By analyzing the level of biomass concentration, neutral lipid content, and neutral lipid yield, we investigated effects of high light intensity, initial nitrogen supply, and urea on growth and lipid accumulation in six strains of oleaginous microalgae. The results indicated that these three kinds of strategies of high-yield oil (high light intensity, initial nitrogen supply, and urea) could improve the neutral lipid yield, but a close relationship exists between lipid production, microalgal species, and individual strategies. The neutral lipid yield of microalgae after optimization were listed follows:E. vischeri(4.9g L-1, high light+urea+low nitrogen supply), S. deserticola (4.1g L-1, high light+nitrate+high nitrogen supply), C. ellipsoideum (3.8g L-1, high light+urea+low nitrogen supply), N. oculata (3.0g L-1, high light+nitrate+high nitrogen supply), C. nivale (2.7g L-1, high light+nitrate+low nitrogen supply), C. tatrense(2.1g L-1, high light+nitrate+high nitrogen supply). Considering the changes in biomass concentration and neutral lipid content, we found three patterns of response to low nitrogen supply. These were type-I (decrease in biomass concentration and increase in lipid content), type-Ⅱ (reduction in both biomass concentration and lipid content), and type-Ⅲ (enhancement of both biomass concentration and lipid content). Type-Ⅲ microalgae could be the potential candidate for the large-scale oil production.
3. The growth, biochemical composition, nitrogen uptake, photosynthesis performance and respiration of two kinds of natural oleaginous microalgae, S. deserticola (type-I) and R. vischeri (type-Ⅲ), under3.5mM,5.9mM,8.8mM, and17.6mM of initial nitrogen supply were studied. The main results were listed as follows:
(1) Growth characteristic:the optimal nitrogen concentration for cell division of two microalgal strains was8.8mM. However, the optimal nitrogen concentration for biomass accumulation was17.6mM(S. deserticola) and5.9mM(E. vischeri), respectively. With the initial nitrogen supply decreased (17.6mM→3.5mM), cell weight of S. deserticola reduced, while cell weight of E. vischeriincreased.
(2) NO3-uptake and utilization:S. deserticola and E. vischeri had the similar nitrogen uptake rate. Nevertheless, the protein synthesis rate and yield in E. vischeri were higher than that of S. deserticola, respectively.
(3) Biochemical composition:with the cultivation time, the carbohydrate and protein content in S. deserticola and E. vischeri showed a decreased tendency, while total lipid content showed an increased tendency. High nitrogen supply could accelerate protein synthesis, but low nitrogen supply was beneficial to the synthesis of carbohydrate and lipid in S. deserticola and E. vischeri. The synthesis rate of neutral lipid and content in S. deserticola was significant higher than that of E. vischeri. However, E. vischeri obtained higher carbohydrate content than S. deserticola. In the middle and later periods of culture, S. deserticola could accumulate large amount of canthaxanthin, the content of which was negative positive correlation with nitrogen concentration. However, K vischeri could accumulate large amount of P-carotene, the content of which was positive correlation with nitrogen concentration. All of other pigments, including chrolophyll and carotenoid, showed a great decrease tendency in content.
(4) Photosynthetic performance and respiration:with the cultivation time, the respiration rate and photosynthetic efficiency of S. deserticola and K vischeri, expressed as Fv/Fm, rETR and Pn, showed a decreased tendency. E. vischeri exhibited less non-photochemical quenching and higher photosynthetic efficiency than S. deserticola.
5. A strain of oleaginous microalgae JNU13, which was isolated from a tropical lake in Guangdong province, was used as experimental material in this study. By analyzing18S rRNA gene sequence and cellular ultrastructure, this microalgal strain was identified to be Chlorella vulgaris JNU13. The growth, carbon flow distribution and photosynthetic performance were investigated. The main results were listed as follows:(1) in the early stage of cultivation, lipid accumulation and cell division of Chlorella vulgaris JNU13could be synchronized. During the middle and late stages of cultivation, cell division suspended, the increase in lipid content became the main reason for biomass concentration increase;(2) in the early stage of cultivation, most of the CO2fixed by microalgae were used for the synthesis of carbohydrate. However, during the middle and late stages of cultivation, the fixed carbon was mainly used for the synthesis of lipid;(3) the decreased photosynthetic efficiency had obvious feedback inhibition on lipid accumulation of Chlorella vulgaris JNU13. The energy distribution between photosystems has been changed in the process of lipid accumulation (reduce in the energy distribution of photosystem I, no obvious change in the energy distribution of photosystem II). The activity of z-scheme electron transport chain related to oxygen release decreased. Alternative electron transport chain could offere energy for the growth of Chlorella vulgaris JNU13. High photosynthetic efficiency was the main reason that high nitrogen supply treatments could obtain higher lipid content than low nitrogen supply treatment.
4. The large-scale cultivation of S. deserticola and N. oculata were conducted in a self-designed flat glass photobioreactor (24L, light path:4.0cm). Potential lipid productivity and cost of these two strains of microalgae were investigated. The results showed that biomass concentration, lipid content and lipid yield of S. deserticola and N. oculata cultured in flat glass photobioreactor were remarkable low compared to that of bubble column photoreactor (0.3L, light path:3.0cm). The areal biomass productivity of S. deserticola and N. oculata were16.43g m2day-1and17.08g m2day-1, respectively. The areal lipid productivity of S. deserticola and N. oculata were7.64g m2day-1and5.48g m2day-1. The total costs of S. deserticola and N. oculata were Y1868.2kg-1and Y1949.9kg-1, respectively. Under the laboratory conditions, high agitation, aeration and light cost resulted in high total cost of microalgal biomass.
1.对实验室保藏的20株微藻进行了评价(真眼点藻纲2株，硅藻纲1株，绿藻门17
株)，根据生物质浓度、总脂含量、β-胡萝卜素含量以及总脂收获量等参数的高低，筛选出6株总脂收获量超过2.0gL~(-1)的微藻，它们是韦氏真眼点藻(Eustigamatos vischeri)、荒漠栅藻(Scenedesmus deserticola)、麻织绿球藻(Chlorococcum tatrense)、眼点拟微绿球藻(Nannochloropsis oculata)、椭圆绿球藻(Chlorococcum ellipsoideum)和雪绿球藻(Chlorococcum nivale)，经“生物柴油质量预测模型”的验证，6种微藻生物柴油的质量均符合中国(BD100)、美国(ASTM6751)和欧盟(EN14214)的标准；在6株潜力微藻中，韦氏真眼点藻(E.vischeri)的总脂收获量可达4.0gL~(-1)，同时该藻还可以积累2.39%DW的β-胡萝卜素，具有极高的开发利用价值。
2.通过测定生物质浓度、总脂含量和中性脂含量，探索了“光强”、“初始氮供应量”、
“氮源形式”三种策略，对6株产油微藻生长和中性脂积累的影响，结果表明：三种策略均可以提高6株产油微藻的中性脂产量，但存在明显的种间差异和因素交叉影响；6株产油微藻优化后的中性脂产量分别为：韦氏真眼点藻(4.9gL~(-1),高光强+尿素+低氮供应)、荒漠栅藻(4.1gL~(-1),高光强+硝酸钠+高氮供应)、椭圆绿球藻(3.8gL~(-1),高光强+尿素+低氮供应)、眼点拟微绿球藻(3.0gL~(-1),高光强+硝酸钠+高氮供应)、雪绿球藻(2.7gL~(-1),高光强+硝酸钠+低氮供应)、麻织绿球藻(2.7gL~(-1),高光强+硝酸钠+高氮供应)。根据产油微藻对起始氮供应量的响应规律，将产油微藻分为3种类型：类型-I，低氮供应降低了生物质浓度而提高了中性脂含量；类型-II，低氮供应同时降低生物质浓度和中性脂含量；类型-III，低氮供应同时提高了生物质浓度和中性脂含量，类型-III更具产业化价值。
3.本论文研究了3.5mM、5.9mM、8.8mM和17.6mM四种初始氮浓度条件下，两株不同类型的天然产油微藻––荒漠栅藻S.deserticola(类型I)和韦氏真眼点藻E.vischeri(类型III)，在细胞生长、生化组成、氮吸收利用、光合作用及呼吸作用等方面的变化规律及差异性，主要研究结果为：
(1)生长特性：有利于两株微藻细胞分裂的氮浓度均为8.8mM，而有利于两株微藻生物质积累的氮浓度为17.6mM(荒漠栅藻)和5.9mM(韦氏真眼点藻)；随初始氮供应量的降低，荒漠栅藻的单位细胞重量降低，而韦氏真眼点藻的单位细胞重量增加；
(2)氮的吸收利用：两株微藻具有相似的NO3-的吸收规律，但韦氏真眼点藻具有比荒漠栅藻更高蛋白质合成速率和合成量；
(3)生化组成：培养过程中，两株微藻均表现出“碳水化合物和蛋白质含量降低、总脂含量升高”的趋势；高浓度氮供应有利于两株微藻蛋白质的合成，而低浓度氮供应有利于碳水化合物和脂类的合成；荒漠栅藻中性脂的积累速率和积累量显著高于韦氏真眼点藻，而韦氏真眼点藻的碳水化合物含量高于荒漠栅藻；荒漠栅藻在培养中后期大量积累角黄素，其积累量与氮浓度呈负相关，韦氏真眼点藻在培养中后期大量积累β-胡萝卜素，其积累量与氮浓度呈正相关；两株微藻其余光合色素的含量在培养过程中均显降低趋势，降低幅度与初始氮供应量呈负相关；
(4)光合作用和呼吸作用：培养过程中，两株微藻的光合效率(F_v/F_m,rETR和P_n)和呼吸速率均呈现降低趋势，韦氏真眼点藻具有较低的热耗散效率和高的光能利用效率。
4.以分离自广东某热带湖泊的产油微藻JNU13为实验材料，通过18SrRNA基因序列分析和形态学观察，初步确定该藻株为普通小球藻JNU13(Chlorella vulgarisJNU13)，研究了该藻在脂类积累过程中的细胞生长、碳流分配及光合特性，(1)生长与脂类积累：培养前期，普通小球藻JNU13的细胞分裂与胞内脂类积累可以同步进行，培养中后期，细胞分裂停止，胞内脂类积累成为生物量浓度增加的主要原因；(2)碳流分配：培养前期，普通小球藻JNU13固定的碳主要用于碳水化合物的合成，培养中后期，主要用于脂类的合成；(3)光合特性：光合效率的降低对普通小球藻JNU13脂类积累存在反馈抑制作用，脂类积累过程中伴随光系统能量分配的改变，光系统I能量比例降低，光系统II能量比例相对稳定，与氧气释放相关的Z型电子传递链活性降低，可变电子传递链可以为产油微藻JNU13的生长提供能量，高光合效率使高氮供给组获得了比低氮供给组更高的总脂含量。
5.利用体积为24L的室内平板式光生物反应器(光径为4.0cm)，对荒漠栅藻(S.deserticola)和眼点拟微绿球藻(N.oculata)进行了扩大培养及产能评价，结果表明：两株微藻在平板式光生物反应器中生物质浓度、总脂含量以及总脂收获量均显著低于0.3L柱式光生物反应器(光径3.0cm)的培养结果；荒漠栅藻和眼点拟微绿球藻的单位面积生物质产率分别为16.43gm~2day~(-1)和17.08gm~2day~(-1)，总脂产率为7.64gm~2day~(-1)和5.48gm~2day~(-1)；荒漠栅藻藻粉成本为1868.2元/千克，眼点拟微绿球藻藻粉成本为1949.9元/千克，室内培养条件下，高通气搅拌成本和光照成本是藻粉成本过高的主要原因。
Oleaginous microalgae are considered as an optimal feedstock for biodiesel production. With the growing energy crisis, research on biodiesel production from microalgae has been the concern of governments and researchers worldwide. To enhance lipid productivity is one of the most critical problems to realize microalgal biodiesel industrialization. Due to the limitation of both of oleaginous microalgal species and experimental condition, some of previous studies have less guiding significance. Moreover, studies on natural oleaginous microalgae (non-model microalgae) are limited. To overcome these shortcomings are exactly the key solving steps for theoretical bottleneck and lipid productivity improvement. This study mainly focused on "oleaginous microalgal screening","effects of high light intensity, initial nitrogen supply, urea on the growth and neutral lipid accumulation of oleaginous microalgae","regulations and differences in physiology and biochemistry of two natural microalgal species under various intial nitrogen supply","growth, carbon flow distribution, and photosynthetic performance of oleaginous microalgae in the process of lipid accumulationand","assessment, and economic analyses of oleaginous microalgae in large-scale cultivation". The main results are listed as follows:
1. Twenty strains of freshwater and marine microalgae, including17strains of Chlorophytes,2strains of Eustigmatophytes, and1strain of Bacillariophytes, were investigated in this study. By comparing their biomass concentration, total lipid content, β-carotene content and total lipid yield, six strains of oleaginous microalgae which had over2.0g L-1of lipid yield were selected. These were E. vischeri, S. deserticola, C. tatrense, N. oculata, C. ellipsoideum, and C. nivale. After testing by prediction model of the quality of biodiesel, their lipids complied with the standards set by China (BD100), USA (ASTM6751), and EU (EN14214). Among them, the lipid yield of E. vischeri could reach up to4.0g L-1. Simultaneously, it could accumulate2.39%DW β-carotene. This microalga was considered to have extremely high developing value.
2. By analyzing the level of biomass concentration, neutral lipid content, and neutral lipid yield, we investigated effects of high light intensity, initial nitrogen supply, and urea on growth and lipid accumulation in six strains of oleaginous microalgae. The results indicated that these three kinds of strategies of high-yield oil (high light intensity, initial nitrogen supply, and urea) could improve the neutral lipid yield, but a close relationship exists between lipid production, microalgal species, and individual strategies. The neutral lipid yield of microalgae after optimization were listed follows:E. vischeri(4.9g L-1, high light+urea+low nitrogen supply), S. deserticola (4.1g L-1, high light+nitrate+high nitrogen supply), C. ellipsoideum (3.8g L-1, high light+urea+low nitrogen supply), N. oculata (3.0g L-1, high light+nitrate+high nitrogen supply), C. nivale (2.7g L-1, high light+nitrate+low nitrogen supply), C. tatrense(2.1g L-1, high light+nitrate+high nitrogen supply). Considering the changes in biomass concentration and neutral lipid content, we found three patterns of response to low nitrogen supply. These were type-I (decrease in biomass concentration and increase in lipid content), type-Ⅱ (reduction in both biomass concentration and lipid content), and type-Ⅲ (enhancement of both biomass concentration and lipid content). Type-Ⅲ microalgae could be the potential candidate for the large-scale oil production.
3. The growth, biochemical composition, nitrogen uptake, photosynthesis performance and respiration of two kinds of natural oleaginous microalgae, S. deserticola (type-I) and R. vischeri (type-Ⅲ), under3.5mM,5.9mM,8.8mM, and17.6mM of initial nitrogen supply were studied. The main results were listed as follows:
(1) Growth characteristic:the optimal nitrogen concentration for cell division of two microalgal strains was8.8mM. However, the optimal nitrogen concentration for biomass accumulation was17.6mM(S. deserticola) and5.9mM(E. vischeri), respectively. With the initial nitrogen supply decreased (17.6mM→3.5mM), cell weight of S. deserticola reduced, while cell weight of E. vischeriincreased.
(2) NO3-uptake and utilization:S. deserticola and E. vischeri had the similar nitrogen uptake rate. Nevertheless, the protein synthesis rate and yield in E. vischeri were higher than that of S. deserticola, respectively.
(3) Biochemical composition:with the cultivation time, the carbohydrate and protein content in S. deserticola and E. vischeri showed a decreased tendency, while total lipid content showed an increased tendency. High nitrogen supply could accelerate protein synthesis, but low nitrogen supply was beneficial to the synthesis of carbohydrate and lipid in S. deserticola and E. vischeri. The synthesis rate of neutral lipid and content in S. deserticola was significant higher than that of E. vischeri. However, E. vischeri obtained higher carbohydrate content than S. deserticola. In the middle and later periods of culture, S. deserticola could accumulate large amount of canthaxanthin, the content of which was negative positive correlation with nitrogen concentration. However, K vischeri could accumulate large amount of P-carotene, the content of which was positive correlation with nitrogen concentration. All of other pigments, including chrolophyll and carotenoid, showed a great decrease tendency in content.
(4) Photosynthetic performance and respiration:with the cultivation time, the respiration rate and photosynthetic efficiency of S. deserticola and K vischeri, expressed as Fv/Fm, rETR and Pn, showed a decreased tendency. E. vischeri exhibited less non-photochemical quenching and higher photosynthetic efficiency than S. deserticola.
5. A strain of oleaginous microalgae JNU13, which was isolated from a tropical lake in Guangdong province, was used as experimental material in this study. By analyzing18S rRNA gene sequence and cellular ultrastructure, this microalgal strain was identified to be Chlorella vulgaris JNU13. The growth, carbon flow distribution and photosynthetic performance were investigated. The main results were listed as follows:(1) in the early stage of cultivation, lipid accumulation and cell division of Chlorella vulgaris JNU13could be synchronized. During the middle and late stages of cultivation, cell division suspended, the increase in lipid content became the main reason for biomass concentration increase;(2) in the early stage of cultivation, most of the CO2fixed by microalgae were used for the synthesis of carbohydrate. However, during the middle and late stages of cultivation, the fixed carbon was mainly used for the synthesis of lipid;(3) the decreased photosynthetic efficiency had obvious feedback inhibition on lipid accumulation of Chlorella vulgaris JNU13. The energy distribution between photosystems has been changed in the process of lipid accumulation (reduce in the energy distribution of photosystem I, no obvious change in the energy distribution of photosystem II). The activity of z-scheme electron transport chain related to oxygen release decreased. Alternative electron transport chain could offere energy for the growth of Chlorella vulgaris JNU13. High photosynthetic efficiency was the main reason that high nitrogen supply treatments could obtain higher lipid content than low nitrogen supply treatment.
4. The large-scale cultivation of S. deserticola and N. oculata were conducted in a self-designed flat glass photobioreactor (24L, light path:4.0cm). Potential lipid productivity and cost of these two strains of microalgae were investigated. The results showed that biomass concentration, lipid content and lipid yield of S. deserticola and N. oculata cultured in flat glass photobioreactor were remarkable low compared to that of bubble column photoreactor (0.3L, light path:3.0cm). The areal biomass productivity of S. deserticola and N. oculata were16.43g m2day-1and17.08g m2day-1, respectively. The areal lipid productivity of S. deserticola and N. oculata were7.64g m2day-1and5.48g m2day-1. The total costs of S. deserticola and N. oculata were Y1868.2kg-1and Y1949.9kg-1, respectively. Under the laboratory conditions, high agitation, aeration and light cost resulted in high total cost of microalgal biomass.
引文
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