蛋白核小球藻发酵产油脂的研究

从5种不同来源的小球藻中筛选到1株油脂产量较高的蛋白核小球藻Chlorella pyrenoidosa No.2。研究了培养基组成及培养条件对其细胞生长和油脂积累的影响。结果表明, 最适培养基组成为(g/L)：葡萄糖 20, 甘氨酸 0.08, MgSO4·7H2O 0.4, K2HPO4 1.0, FeSO4·7H2O 0.004; 适宜的培养温度、初始pH、摇床转速和光照强度分别为28℃、6.0、130 r/min和 650 Lux。在上述优化条件下培养7 d, Chlorella pyrenoidosa No.2的生物量和油脂含量分别由优化前的3.73 g/L 和 40.15%提高到6.56 g/L和59.90%, 油脂产量提高了162%。Chlorella pyrenoidosa No.2能以木糖为碳源产油脂, 可望用于以木质纤维素等可再生生物质资源为原料生产油脂。气相色谱分析表明该油脂的脂肪酸组成与植物油相似, 不饱和脂肪酸含量达71％左右, 可作为生产生物柴油的原料。     

重要理化因子对小球藻生长和油脂产量的影响

本文采用通气培养的方法研究了N、P、Fe3 、盐度、光照强度、温度对小球藻(Chlorella sp. XQ-200419)生长速率、生物量和油脂产量的影响。主要结果如下：N浓度对小球藻的生长和油脂产量均有显著的影响,在KNO3浓度0.05—0.3g/L范围内,小球藻生长速率随N浓度的增加而提高,并积累更多的生物量,而油脂含量随之递减,KNO3浓度为0.3g/L时,油脂产量最高。小球藻对P浓度变化的适应范围很大,K2HPO4浓度在10—160mg/L范围内,对小球藻的生长和油脂产量都没有显著影响。在小球藻培养后期补加不同浓度Fe3 对其生长速率没有显著影响,总脂含量随着Fe3 浓度升高呈现上升的趋势,均比对照有极显著提高,Fe3 浓度为0.75mmol/L时油脂产量最高。盐度对小球藻的生长有一定的抑制作用;油脂含量先随着盐度的增大而提高,当NaCl浓度达到0.6mol/L, 油脂含量又显著降低;油脂含量和油脂产量均在盐度为0.2mol/L时最高。光照强度对处于生长后期的小球藻的生长影响不大,但影响其油脂积累,小球藻的油脂含量和产量随光照强度的增大而显著提高,当光照强度增至280μmolm-2s-1时,油脂含量和油脂产量最高。温度对小球藻的生长速率、生物量、油脂含量和油脂产量都有显著的影响,在15-40℃范围内,随着培养温度的升高,生长速率、生物量、油脂含量和油脂产量都经历了一个先上升然后下降的过程,适合小球藻生长、积累油脂的温度范围是20-35℃,30-35℃时油脂产量最高,40℃时生物量、油脂含量和产量都最低。理化因子对生长和油脂含量的影响分为两种情况：1. 温度、光强、铁浓度和盐度的影响表现为在适宜生长的条件下提高油脂含量,这种模式可以称为“适宜模式”;2. 氮浓度的影响表现为在不利于生长的条件下提高油脂含量,这种模式可以称为“胁迫模式”。两种模式都可以提高油脂含量,但是,只有适宜模式才可以提高油脂产量。在筛选小球藻优良产油藻种时要注意,只有在适宜的培养条件下油脂含量高的藻种才具有高产油潜力。     

小球藻高密度培养及油脂提取条件的优化

【目的】高密度培养小球藻及优化油脂提取条件。【方法】通过进行单因素实验研究不同培养基组成及环境因子对其细胞生长影响,并采用超声波提取法进行正交实验对藻粉油脂提取条件进行研究。【结果】对椭圆小球藻Y4进行异养培养,最适培养条件为:葡萄糖50 g/L,硝酸钾2 g/L,适宜的培养温度、摇床转速和接种量分别为29°C、180 r/min和20%。在此基础上,进行了1 L发酵罐培养实验,获得了干重18.25 g/L的生物量。通过对油脂提取条件进行优化,Y4的油脂提取率由优化前的25.0%提高到60.2%,提高了35.2%。【结论】优化了小球藻的培养条件及油脂提取条件,促进了小球藻的开发和利用。     

Fe~(3+)对小球藻的生长及油脂含量的影响

传统化石能源储量日益减少,生物柴油因其环保可再生性成为优质的石化柴油替代品。利用小球藻生产生物柴油速度快、油脂含量高,受到了广泛关注。为进一步提高小球藻生产生物柴油效率,分别探究了Fe3+的浓度及添加时间对自养和异养小球藻生长及产油的影响,获得最优Fe3+培养条件为:自养小球藻延滞期添加10-3 g/L Fe3+,生物量及油脂含量达2.80 g/L及30.90%;异养小球藻指数期添加10-5 g/L Fe3+,生物量及油脂含量达3.30 g/L及29.05%。经脂肪酸分析,以上条件获得的微藻油脂均可作为生物柴油生产原料。     

不同培养基及组成对2种小球藻生长和油脂的影响

研究了4种培养基及组成对蛋白核小球藻F-9和普通小球藻HYS-2的生长、油脂积累和脂肪酸组成的影响。结果发现knop、Provasoli、f/2、MAV 4种培养基中,f/2培养基更有利于小球藻的快速生长,而MAV培养基更适合油脂积累。在f/2培养基中F-9和HYS-2相对生长速率分别为0.156和0.171,培养9 d细胞干重为0.188 g/L和0.195 g/L。而在MAV培养基中F-9油脂含量最高可达19.67%,HYS-2油脂含量最高为21.91%,脂肪酸最高分别占干重的5.11%和8.71%。N/P为16∶1时小球藻生长最快,培养9 d后F-9和HYS-2的相对生长速率分别为0.23和0.239,最终细胞干重分别为0.107 g/L和0.143 g/L。而F-9和HYS-2在N/P为1∶1条件下积累油脂和脂肪酸含量最高,总脂含量分别占干重的为20.40%和27.39%,总脂肪酸占藻粉干重的含量为12.52%和16.94%。     

不同光质对小球藻光自养培养积累油脂的影响

研究了5种光质对小球藻(Chlorella vulgaris)M209256生长和产油的影响。结果表明:蓝光为小球藻的最适生长和产油光质;与其他光质相比,蓝光培养的小球藻生物量和油脂含量均较高,为2.40×107个/mL和28%;红光培养的小球藻生长最慢且油脂含量最低,为1.32×107个/mL和15.13%,表现出明显的"红降"现象。在GCMS分析的基础上,对油脂甲酯化后的十六烷值进行评估,结果发现:蓝光的十六烷值最高;5种光质培养的小球藻所产油脂,甲酯十六烷值均在47以上。因此,小球藻油脂所制备的生物柴油具有较好的燃烧性能。     

小球藻与固氮菌Mesorhizobium sp.共培养对小球藻生长和油脂积累的促进效果

微藻油脂不仅可以作为功能油脂,同时也是生产生物柴油的重要原料之一。为解决微藻生长与油脂积累之间的矛盾,利用藻菌共培养技术在缺氮条件下将无菌小球藻与细菌以不同初始比例进行共培养,通过测定藻细胞生物量、油脂含量和脂肪酸比例等来研究藻菌共培养对小球藻生长和油脂积累的影响。结果表明,在小球藻与固氮菌B2. 3 70∶1(V/V)共培养体系中,小球藻的生物量和油脂含量较同样条件下单独培养小球藻有了显著提高。其生物量最高可达1. 68g/L、总脂含量为45. 2%、总脂产率为75. 94 mg/(L·d)、中性脂含量为23. 0%及中性脂产率为38. 65mg/(L·d),其生物量和油脂含量分别较单独小球藻培养时提高了66. 3%和47. 7%。同时细菌的加入显著提高了藻细胞内C18∶1脂肪酸的比例。结论表明,通过藻菌共培养技术能够有效提高微藻生物油脂的质量和产量,具有较好的实际利用价值。     

原始小球藻AP2转录因子的生物信息学分析

为了揭示原始小球藻AP2基因家族编码蛋白的理化特性、分子功能和遗传进化特征,该文对原始小球藻AP2基因家族的蛋白成员进行了详细的生物信息学预测和分析,并利用PrtoParam、Pfam 3.20、Protscale等在线工具分析了原始小球藻AP2家族所包含的8个蛋白成员。结果表明:该家族成员含有的氨基酸数为247(XP_011395646.1)到715(XP_011401904.1),理论等电点最大为9.39(XP_011396011.1)、最小为5.81(XP_011398158.1); 家族中各个成员所含有的保守结构域的位置和数量都各不相同; 所有蛋白成员均无信号肽,均不包含跨膜螺旋,不具有跨膜区域; 蛋白成员中最大亲水性值为-3.222(XP_011398158.1),最大疏水性值为2.333(XP_011401904.1),且所有成员的平均亲疏水性值均小于0; 成员中有多个磷酸化位点值远超标准值0.5,最大磷酸化位点值为0.998; 该基因家族的蛋白成员二级结构的组分含量从大到小排序均为无规则卷曲>α-螺旋>延伸链>β-转角,推测α-螺旋和无规则卷曲是其二级结构的主要方式,延伸链和β-转角则分散在所有蛋白成员的氨基酸链中; 所有成员的三级结构分析中均能观察到α-螺旋、β-折叠、β-转角以及N端和C端; 系统进化分析结果表明,在其余15个物种中,与原始小球藻亲缘关系最远的是金牛介球菌(Ostreococcus tauri),亲缘关系最接近的是小球藻(Chlorella variabilis NC64A)和螺旋孢子虫(Helicosporidium),较接近的是苦参3号(Picochlorum sp. SENEW3)。该研究较系统地分析了原始小球藻AP2蛋白家族的理化特性、保守基序及系统进化关系,为进一步研究原始小球藻AP2转录因子功能和互作关系提供一定参考依据。     

提高微生物油脂生产能力的研究进展

微生物油脂是生物柴油生产领域具有广阔前景的新油脂资源。然而, 利用产油微生物进行油脂的工业化生产仍存在限氮条件下油脂生产强度不够高、对廉价高氮生物质原料的利用效率低等瓶颈问题。随着近年来发酵工程、生物信息学及分子生物学技术的发展, 国内外研究者利用不同策略优化微生物油脂的生产条件, 并对其油脂积累代谢途径进行改造, 旨在获得适用于工业化生产的产油性能优良的油脂菌。本综述总结了国内外利用生化工程、基因工程以及新兴的转录因子工程策略提高产油微生物油脂生产强度和扩大产油微生物廉价底物利用范围方面的研究进展, 并展望了基于组学研究、模块途径工程以及反向代谢工程的综合策略在理性改造产油微生物以提高其油脂发酵性能中的应用。     

Physiological evaluation of a new Chlorella sorokiniana isolate for its biomass production and lipid accumulation in photoautotrophic and heterotrophic cultures

A novel green unicellular microalgal isolate from the freshwater of the Inner Mongolia Province of China and named as CCTCC M209220, grows between pH 6 and 11 and temperatures of 20-35°C with optimal conditions at pH 9 and 30°C. Morphological features and the phylogenetic analysis for the 18S rRNA gene reveal that the isolate is a Chlorella sorokiniana strain. A nitrogen source test reveals that this strain can grow well with nitrate and urea, but not ammonium. The strain can grow heterotrophically with glucose as the carbon source and accumulates lipid content as high as 56% (w/w) dry weight after 7 days in high glucose concentrations compared to 19% lipids achieved in 30 days of photoautotrophic culture. The relative neutral lipid content as a fraction of the total lipid is also much higher in heterotrophic culture as compared to photoautotrophic culture.     

pH值对沼液培养的普通小球藻生长及油含量积累的影响

以50％的沼液为普通小球藻的全营养培养基,考察培养基的起始pH值对小球藻生长及油脂含量的影响,普通小球藻对不同初始pH的沼液中氮、磷的去除情况。设定了2组实验,一组只调节初始接种培养液的pH,分别为6.0、6.5、7.0、7.5、8.0、8.5;另一组将培养液pH分别固定在6.0、6.5、7.0、7.5、8.0、8.5,pH用稀HCl和NaOH进行调节。研究发现在pH 6.5和pH 7.0的偏酸环境有利于小球藻生长,而pH在7.0~8.5的偏碱性条件下有利于小球藻油脂的积累,因此综合小球藻生长和油脂积累2个因素,得到最适合小球藻生长和油脂积累的pH为7.0。培养结束后沼液中氮磷的去除率分别达到了95％和97％,沼液中的总氮由原来的134.91 mg/L降至4.86 mg/L,总磷由10.19 mg/L降到0.32 mg/L。     

利用叶绿素荧光快速筛选紫外诱变高含油小球藻

本实验利用尼罗红荧光与叶绿素荧光比值表征小球藻的油脂含量,快速筛选出一株紫外诱变高产油藻株。经过叶绿素荧光分析表明,诱变株M2的生长及光合作用没有显著变化。检测诱变株M2总油脂含量32.1%,比野生藻株的23.1%提高了9个百分点。     

一株高蛋白含量小球藻TX的分离鉴定及其诱变育种

【背景】小球藻由于蛋白含量高、营养丰富,在水产养殖上可直接作为鱼、虾、贝类的优质饵料。【目的】对从养殖环境中分离的小球藻进行诱变,选育生长快、蛋白含量高的突变株,为水产养殖天然饵料生产提供优良藻种资源。【方法】以从养殖环境中筛选的生长相对较快且蛋白含量较高的TX作为出发藻株,对该藻株进行分子鉴定,并对该藻株进行紫外诱变、甲基磺酸乙脂(ethyl methyl sulfonate,EMS)诱变和复合诱变,采用96孔板高通量筛选技术和递进式重复筛选方法选育高生物量、高蛋白突变株。【结果】经18SrRNA基因序列分析,TX鉴定为Chlorella sorokiniana,从540个可能的突变株中筛选到8个遗传稳定且生长较快的突变株,其中H10的总蛋白含量达64.2%,可溶性蛋白含量达0.44g/L,干重达0.72g/L,分别较出发藻株提高3.4%、15.8%和26.2%。【结论】突变株H10蛋白含量高且生长较快,可用于天然饵料生产。     

Investigation of biomass concentration,lipid production,and cellulose content in Chlorella vulgaris cultures using response surface methodology

The microalgae Chlorella vulgaris produce lipids that after extraction from cells can be converted into biodiesel. However, these lipids cannot be efficiently extracted from cells due to the presence of the microalgae cell wall, which acts as a barrier for lipid removal when traditional extraction methods are employed. Therefore, a microalgae system with high lipid productivity and thinner cell walls could be more suitable for lipid production from microalgae. This study addresses the effect of culture conditions, specifically carbon dioxide and sodium nitrate concentrations, on biomass concentration and the ratio of lipid productivity/cellulose content. Optimization of culture conditions was done by response surface methodology. The empirical model for biomass concentration (R² = 96.0%) led to a predicted maximum of 1123.2 mg dw L^?1 when carbon dioxide and sodium nitrate concentrations were 2.33% (v/v) and 5.77 mM, respectively. For lipid productivity/cellulose content ratio (R² = 95.2%) the maximum predicted value was 0.46 (mg lipid L^?1 day^?1)(mg cellulose mg biomass^?1)^?1 when carbon dioxide concentration was 4.02% (v/v) and sodium nitrate concentration was 3.21 mM. A common optimum point for both variables (biomass concentration and lipid productivity/cellulose content ratio) was also found, predicting a biomass concentration of 1119.7 mg dw L^?1 and lipid productivity/cellulose content ratio of 0.44 (mg lipid L^?1 day^?1)(mg cellulose mg biomass^?1)^?1 for culture conditions of 3.77% (v/v) carbon dioxide and 4.01 mM sodium nitrate. The models were experimentally validated and results supported their accuracy. This study shows that it is possible to improve lipid productivity/cellulose content by manipulation of culture conditions, which may be applicable to any scale of bioreactors. Biotechnol. Bioeng. 2013; 110: 2114–2122. © 2013 Wiley Periodicals, Inc.     

Ozone-induced ultrastructural changes in the plasma membrane of Chlorella sorokiniana

Abstract. Modifications in plasma membrane structure and permeability were observed in Chlorella sorokiniana following exposure to 0.2 gm⁻³(140 p.p.m.) O₃ for 30 min. Sixty-eight per cent of the cells were plasmolysed after 15 min O₃ exposure with disruption of organelles similar to that previously described in higher plants. Freeze-fracture exposed large areas of plasma membrane in 90% of the control cells and those exposed to O₃for short periods. After 20 min O₃ 90% of the cells cross-fracture, which indicates a change in molecular interactions in the membrane exposed to O₃ The earliest observed ultraslructural alteration is an aggregation of particles on the plasma membrane P face, statistically significant after 10 min O₃ Changes in ⁸⁶Rb influx occur during a similar time. After more extended exposure to O³ the plasma membrane P face shows regions of lipid phase transition to the crystalline state.     

Metabolic profiling reveals growth related FAME productivity and quality of Chlorella sorokiniana with different inoculum sizes

Inoculum size strongly affects cell growth and lipid accumulation of microalgae, one of the most potential biodiesel feedstock, however, the metabolic mechanism(s) of the lipid biosynthesis upon inoculum size has not been fully explored yet. The effects of inoculum size on cell growth, lipid accumulation, and metabolic changes of a green microalga Chlorella sorokiniana were investigated. In our experimental range of inoculum size, the productivity and the cetane number (CN) of fatty acid methyl esters (FAME) increased with increasing initial cell density, and the inoculum of 1 × 10(7) cells mL(-1) processed much higher productivity (up to 2.02-fold) and CN (up to 1.19-fold) of the FAME than the others. A significant correlation between the metabolic profile and quantity and quality of lipid production was revealed by partial least-squares to latent structures (PLS) analysis, and 15 key metabolites were identified. Most of those metabolites were involved in the photosynthetically fixed carbon metabolism. Furthermore, light intensity as one of the vital limitation factors for the high inoculum size cultivation was evaluated by illumination assay and the results revealed that increasing light intensity could improve the polyunsaturated fatty acids composition and lipid accumulation of C. sorokiniana. The lipid productivity of the culture was improved by 71.21% with the light intensity of 110 μmol m(-2) s(-1), compared to that under the irradiance of 65 μmol m(-2) s(-1).