自养与异养小球藻脂溶性化合物对比研究

应用细胞培养技术获绿色自养小球藻与乳黄色异养小球藻细胞。异养藻细胞脂溶性化合物占细胞干重的７２％,是自养藻的４倍多。两类细胞烃类化合物的分布特征发生了显变化,自养藻以正十七烷占绝对优势;异养藻富含长链正烷烃,以正二十五烷烃占优势。两类细胞脂溶性色素化合物和苯洗脱组分化合物以及Ｃ、Ｈ、Ｏ、Ｎ１４种有机元素相对含量也差异显。对比实验结果对于异养小球藻的代谢研究和油脂化合物的开发利用具有重要意义。     

植物激素在小球藻异养培养中的作用

本文研究了几种植物激素对小球藻异养培养的影响。结果表明,IAA、IBA及6-BA三种植物激素均不同程度地促进了小球藻的异养生长,培养≤36h时,IAA或IBA以20mg／L的促进小球藻异养生长的效应最大,100mg／L IAA或IBA则抑制了藻的生长;>36h时,100mg／L IAA或IBA表现出促进小球藻生长的效应,并最终获最大净A540增长量;6-BA以0.1 mg／L的促进作用最大。IBA与6-BA组合同样表现出促进小球藻异养生长的效应,但并非IBA和6-BA简单的加合效应,5 mg／LIBA与6-BA组合的效应维持6-BA单因子的作用趋势,20mg／LIBA与lmg/L6-BA组合的效应大于与0.1mg／L6-BA组合的,100mg／LIBA与0.1mg／L6-BA组合的效应在≤36h时大于与1mg／L6-BA组合的,>36h时则相反。另外,高浓度IBA(≥20mg／L)与6-BA组合抑制了前中期异养藻对葡萄糖的吸收,但加速了中后期葡萄的吸收。再者,IBA与6-BA组合加速了异养小球藻对No-3的吸收。     

小球藻细胞的异养转化

总结了异养转化后小球藻细胞在结构、生物化学成分和可能的代谢机制方面的研究进展,并对其应用潜力提出了初步看法。     

植物激素在小球藻异养培养中的作用

本文研究了几种植物激素对小球藻异养培养的影响。结果表明 ,IAA、IBA及 6_BA三种植物激素均不同程度地促进了小球藻的异养生长 ,培养≤ 36h时 ,IAA或IBA以 2 0mg/L的促进小球藻异养生长的效应最大 ,1 0 0mg/LIAA或IBA则抑制了藻的生长 ;>36h时 ,1 0 0mg/LIAA或IBA表现出促进小球藻生长的效应 ,并最终获最大净A540 增长量 ;6_BA以 0 1mg/L的促进作用最大。IBA与 6_BA组合同样表现出促进小球藻异养生长的效应 ,但并非IBA和 6_BA简单的加合效应 ,5mg/LIBA与 6_BA组合的效应维持 6_BA单因子的作用趋势 ,2 0mg/LIBA与 1mg/L 6_BA组合的效应大于与 0 .1mg/L 6_BA组合的 ,1 0 0mg/LIBA与 0 .1mg/L 6_BA组合的效应在≤ 36h时大于与 1mg/L 6_BA组合的 ,>36h时则相反。另外 ,高浓度IBA(≥ 2 0mg/L)与 6_BA组合抑制了前中期异养藻对葡萄糖的吸收 ,但加速了中后期葡萄的吸收。再者 ,IBA与 6_BA组合加速了异养小球藻对NO_3 的吸收。     

以异养细胞作为种子的椭圆小球藻产油脂光自养培养优化

异养细胞种子/光自养培养方法是一种可异养培养的能源微藻培养的有效方法,但已有文献尚未从工艺优化角度考察其发展潜力。为了获得较高细胞密度的用于光自养培养的种子和提高光自养培养的细胞密度与油脂产率,对异养细胞种子/光自养培养的培养基和培养条件进行了优化。结果表明,采用优化后的培养基,椭圆小球藻在摇瓶中异养培养的最高藻细胞密度可达11.04 g/L,比在初始培养基条件下提高了28.0%,在5 L发酵罐中异养培养的藻细胞密度达到73.89 g/L;在2 L柱式光生物反应器中光自养培养的藻细胞密度、油脂含量和油脂产率分别达1.62 g/L、36.34%和6.1 mg/(L·h),油脂成分主要为含C16-C18碳链的脂肪酸,是制备生物柴油的理想原料。经过优化,异养细胞种子/光自养培养这一方法能够显著地提高椭圆小球藻产油脂的能力,这进一步表明异养细胞种子/光自养培养方法有望成为可异养的能源微藻的高效培养方式。     

小球藻的异养培养及应用前景

介绍了小球藻异养培养的营养需求,条件控制以及应用前景。     

谷氨酸对异养培养小球藻生长的影响

在不添加其他氮源的小球藻异养培养基中,谷氨酸可促进小球藻的生长,但基本不增加叶绿素的合成;在以铵盐为氮源时,谷氨酸可明显促进小球藻对铵盐的利用,促进小球藻生物量增加和叶绿素合成;在以硝酸盐为氮源时,谷氨酸可增加小球藻的生物量,对叶绿素含量无明显影响。     

水稻RubisCO的纯化及其与烟草RubisCO性质的比较

利用蔗糖密度梯度离心和DEAE-Sepharose fast flow柱层析等步骤从水稻叶片中纯化了RubisCO。此法不仅快速,而且酶的收得率高,酶的比活达1.15 μmol CO_2 min~(-1) mg~(-1)。 水稻RubisCO的热稳定性比烟草酶差,在活化时对Mg~(2 )较敏感。水稻和烟草RubisCO钝化态时总巯基数和表面巯基数相同,然而当酶活化后,水稻酶表面巯基数增加,而烟草酶则减少。当这些表面巯基被修饰后,水稻酶活性损失60％而烟草酶活力仅损失15％。水稻和烟草RubisCO的远紫外CD光谱有明显的区别,这显示了两者在二级结构、酶比活和性质上的重大差别。     

Large-scale biodiesel production from microalga Chlorella protothecoides through heterotrophic cultivation in bioreactors

An integrated approach of biodiesel production from heterotrophic Chlorella protothecoides focused on scaling up fermentation in bioreactors was reported in this study. Through substrate feeding and fermentation process controls, the cell density of C. protothecoides achieved 15.5 g L(-1) in 5 L, 12.8 g L(-1) in 750 L, and 14.2 g L(-1) in 11,000 L bioreactors, respectively. Resulted from heterotrophic metabolism, the lipid content reached 46.1%, 48.7%, and 44.3% of cell dry weight in samples from 5 L, 750 L, and 11,000 L bioreactors, respectively. Transesterification of the microalgal oil was catalyzed by immobilized lipase from Candidia sp. 99-125. With 75% lipase (12,000 U g(-1), based on lipid quantity) and 3:1 molar ratio of methanol to oil batch-fed at three times, 98.15% of the oil was converted to monoalkyl esters of fatty acids in 12 h. The expanded biodiesel production rates were 7.02 g L(-1), 6.12 g L(-1), and 6.24 g L(-1) in 5 L, 750 L, and 11,000 L bioreactors, respectively. The properties of biodiesel from Chlorella were comparable to conventional diesel fuel and comply with the US Standard for Biodiesel (ASTM 6751). These results suggest that it is feasible to expand heterotrophic Chlorella fermentation for biodiesel production at the industry level.     

Enhanced production of lutein in heterotrophic Chlorella protothecoides by oxidative stress

The fast growing unicellular green microalgae Chlorella protothecoides has attracted interest as a promising organism for commercial production of a high-value carotenoid, lutein, by heterotrophic fermentation. Effects of two oxidant-forming reactive oxygen species (ROS) on the biomass concen-tration, and yield and content of lutein in batch culture of heterotrophic Chlorella protothecoides were investigated in this study. The addition of 0.1 mmol/L H2O2 and 0.01 mmol/L NaClO plus 0.5 mmol/L Fe2 to the culture led to the generation of ·OH and enhanced the lutein content from 1.75 to 1.90 and 1.95 mg/g, respectively. The lutein content further increased to 1.98 mg/g when 0.01 mmol/L H2O2 and 0.5 mmol/L NaClO were added to generate 1O2. The maximum yield of lutein (28.5, 29.8 and 31.4 mg/L) and a high biomass concentration (15.0, 15.3 and 15.9 g/L) were also achieved through the above treatments. The results indicated that 1O2 could promote lutein formation and enhance lutein production in hetero-trophic Chlorella protothecoides. Moreover, 1O2 produced from the reaction of H2O2 and NaClO was more effective in enhancing lutein production and reducing biomass loss than ·OH from the reaction of H2O2 or NaClO plus Fe2 .     

Compaitive Study on Liposoluble Compoands in Autotrophic and Heterotrophic Chlorella protothecoides

Both autotrophically and heterotrophically grown Chlorella protothecoides cells have been obtained in cell cultures. The content of liposoluble compounds in the cells of heterotrophic algae occupied 72% of the total cells in dry weight, which was more than 4 times as high as that in the autotrophic algal cells. There existed remarkbly different distribution patterns of the hydrocarbons in thesetwo kinds of cells. The hydrocarbons in autotrophic cells were characterised by the predominance of C17 normal alkanes, wheraes the heterotrophic cells were rich in normal alkanes of higher molecular weight or longer carbon chain with C25 as the dominant carbon. The structure of the compounds in benzene fraction is not quite clear, but the compounds in autotrophis sample may be related to the degeneration of the pigments. The compounds in heterotrophic sample probably come from lipid acids. The visible--ultraviolet absorption spectrum of the pigment compounds demonstrated the absorption peaks of the acetone extract from the autotrophic cells at 432.5, 451.5, 472.5 and 661.5 nm, reflecting the existence of chlorophyll and carotenoid, both with a rather high concentration. However, the acetone extract from the hetertrophic algal cells only showed absorption peaks at 427.4, 450.8 and 477.5 nm. The absorption peaks of the original green cells completely disappeared at 432.5 and 661.5 nm, reflecting the disappearance of chlorophyll in cells on the whole; the remaining absorption peaks only reflected the existence of carotenoid, but its concentration had already been greatly reduced. The resuls from comparative experiments were of essential significance on the study of physiological metabolism in heterotrophically grown C. protothecoides and on the exploration and application of the lipid compounds in this kind of algae.     

豌豆核酮糖1,5-二磷酸羧化酶/加氧酶亚基结合蛋白的纯化及特性

用~(35)S-Met在照光下与豌豆完整叶绿体保温,显示新合成的标记的RubisCO大亚基与结合蛋白形成一复合物,经ATP处理后解离为结合蛋白亚基,同时释放出的标记的RubisCO大亚基参与了RubisCO的装配。豌豆叶片提取液经热处理,硫酸铵分部,DEAE-Sepharose fast flow和Sephacryl S-300柱层析在ND-PAGE,SDS-PAGE上显示为一条带,估计纯度达90％以上,得率比以前报道的高12倍。纯化的结合蛋白表面巯基数经测定为12±1个,总巯基数为36±1个。远紫外CD光谱具有典型的α-螺旋结构的光谱特性,α-螺旋度为39％。此外,以纯化的豌豆结合蛋白制备了多克隆抗体。琼脂糖双扩散实验显示,结合蛋白的抗体与结合蛋白产生一条沉淀线,而与豌豆的RubisCO无沉淀反应,这表明所得到的抗体是高度专一的。     

氯化钠处理对原壳小球藻生长及油脂积累的影响

采用Basal培养基,通过光学显微镜、电子显微镜、激光共聚焦显微镜以及尼罗红染色定量等方法研究了不同浓度氯化钠(0、150、300、600 mmol/L)对小球藻属原壳小球藻的生长状态、脂滴分布、总脂含量的影响。结果表明,添加不同浓度的氯化钠对原壳小球藻的生长有明显的影响,随着氯化钠浓度的增加,小球藻的生长速度受到明显的抑制,600 mmol/L氯化钠处理时生长几乎完全被抑制。在显微镜下观察,可见氯化钠浓度的增加会导致小球藻聚集成团,这种现象在150 mmol/L和300 mmol/L氯化钠培养下比较明显;通过电子显微镜下观察,可以发现培养初期,随着氯化钠浓度的增加,小球藻细胞壁增厚,脂滴增多。通过尼罗红染色对脂含量进行定量,处理初期脂滴的合成量在600 mmol/L时最高,但到后期,随着藻生物量的增加,150 mmol/L和300 mmol/L处理下脂合成量逐渐升高,而对照小球藻脂合成量基本不变。稳定期后,从生物量(干重)和脂总量来看,300 mmol/L氯化钠培养处理的小球藻虽然生物量只有对照的73.55%,但是总脂含量却是对照的2.22倍,可见一定浓度的氯化钠处理一定时间可显著提高原壳小球藻的油脂含量。     

不同营养条件下原始小球藻对蒽的富集和降解研究

研究了自养与异养条件下原始小球藻对蒽的降解和富集能力 .结果表明 ,自养条件下 ,浓度为1 0mg·L-1的蒽有 48.18%被降解 ,其中 2 8.81%属于自然光降解 ,仅有 19.37%被原始小球藻降解 .而异养条件下的原始小球藻对浓度为 2 .5mg·L-1的蒽降解率达到 33.5 3%,说明异养原始小球藻不仅能耐受高浓度蒽 ,而且表现出比自养原始小球藻更强的蒽降解能力 .两种条件下 ,80 %以上残留的蒽都被富集到藻细胞中 .虽然自养条件下原始小球藻对蒽的生物富集系数达 90 6 4,远大于异养条件下的生物富集系数(1899) ,但异养条件下藻对蒽的绝对富集量 (2 0 2 .2 9μg)远远高于自养条件下的 6 9.6 87μg .     

Studies on RubisCO Characteristics During Flag Leaf Aging in Hybrid Wheat and Its Parent

The RuBPcase content and activity, and the RuBPoase activity of the flag leaf of wheat (Triticum aestivum L.) reached the highest values at leaf full expansion or at the 10th day after leaf full expansion, then gradually reduced. There was evident of heterosis on the above mentioned parameters during life span of the flag leaf especially the late phase of leaf aging in the tested hybrid wheat as compared with those in its parents. The RuBPcase specific activity of the flag leaf changed slightly during the relatively steady phase of the chlorophyll content, then gradually decreased during the sharp fall phase of the chlorophyll content. Both RuBPcase and RuBPoase activity in the tested hybrid wheat were higher than in its parents, showing that hybrid wheat had higher photosynthetic carboxylation function, accompanied with photorespiration. All these results were in accordance with the measurements of kinetic constants Km (CO2) and Km (O2) of the RubisCO of flag leaf.