微拟球藻富油藻株筛选及柱状光生物反应器培养评价研究

研究以亲脂性荧光染料BODIPY505/515和流式细胞仪为基础, 从多株诱变海洋微拟球藻(Nannochloropsis oceanica)中筛选到4株候选富油藻株(MT-1,2,3,4), 并利用柱状光生物反应器对诱变株的产油能力进行了综合评价。结果表明, 藻株筛选时最佳BODIPY505/515使用浓度为0.87 μg/mL, 染色时间为10min; 4株诱变株产油性能较野生株有较大提高, 其中MT-4油脂积累达到了干重的66%, 油脂产率比野生型藻株提高了45%, 达到了27.32 mg/(L·d)。4株诱变株的脂肪酸组成合适, 其中C16和C18之和占78%以上, 且主要以饱和脂肪酸和单不饱和脂肪酸为主; 多不饱和脂肪酸只占总脂肪酸的6%—8%, 非常适合生物柴油生产。研究提供了一种针对海洋微拟球藻富油藻株快速、有效的筛选方法, 并以此为基础筛选得到4株极具生物柴油生产潜力的候选藻株, 有望用于规模化生产。     

微藻细胞油脂含量的快速检测方法

以斜生栅藻(Scenedesmus obliquus JNU20)为实验材料,采用尼罗红(NR)荧光光谱法和傅里叶变换红外光谱法(FTIR)测定该藻细胞中的油脂含量。研究结果表明NR的最佳染色条件为:染色前微波处理40 s,二甲基亚砜体积分数1%, NR最终质量浓度1.5 μg/ml,染色时间5 min,染色温度40℃。比较了NR荧光光谱法、FTIR与传统重量法测定的该藻在不同时相的油脂积累情况,利用激光共聚焦显微镜观察了细胞中油体形成的动态过程。实验结果表明NR荧光光谱法、FTIR与传统重量法测定的结果显著相关(R²=0.9258, R²=0.9844),但NR荧光光谱法和FTIR更简便快速,研究结果为规模化筛选高含油量藻株及跟踪产油微藻油脂积累过程奠定了基础。     

基于尼罗红染色分析金藻总脂动态积累

通过尼罗红荧光染色对一株富油微藻金藻Isochrysis sp. CCMM5001建立和完善了一种方便快捷且能准确定量金藻油脂含量的方法,利用该方法探索了不同培养条件对金藻生长和总脂积累的影响。结果表明:经尼罗红染色后,金藻的单细胞荧光强度与其总脂含量呈良好的线性关系;金藻最适生长的氮浓度、光照强度和温度分别为1323 mol/L、148.0 mol/（m2s）、25℃;最适总脂积累的氮浓度、光照强度和温度分别为441 mol/L、92.5 mol/（m2s）、15℃;优化培养条件并采用两阶段培养法后总脂含量和油脂产率都有大幅提高,可分别高达63.3%和22 mg/（Ld）。       

尼罗红荧光优化法快速检测微拟球藻细胞内油脂含量

【目的】利用尼罗红荧光染色法快速检测微拟球藻细胞内的油脂含量。【方法】系统性地调整激发波长与发射波长,确定最佳二甲基亚砜(DMSO)浓度、尼罗红终浓度、染色时间和细胞密度的范围,比较重量法和Triolein标准品的油脂含量分别与荧光强度之间的关系。【结果】分析得出了尼罗红荧光强度与微拟球藻细胞油脂含量的关系,确定优化后染色条件为:二甲基亚砜浓度为5%,尼罗红终浓度为1 mg/L,染色时间为6 min,且细胞密度为(0.5-3.0)×10~6 cells/m L的范围内,激发波长和发射波长分别为515 nm和570 nm。重量法和Triolein标准品的油脂含量分别与尼罗红染色荧光强度之间的关联性,表明尼罗红荧光染色方法可以用来快速准确地检测细胞内的油脂含量,且油脂含量与荧光强度之间正相关,相关系数R~2为0.997 3。尼罗红染色优化后油脂的检测下限达到2μg,大大减少了测定油脂含量所需细胞量。【结论】针对不同种属系统性地确认了荧光激发波长和发射波长,并优化验证得到了最佳尼罗红荧光染色条件,可以快速准确地检测微量微拟球藻细胞内的油脂含量,便于大规模筛选高产油突变藻株。     

Effects of Temperature and pH on Cell Growth of a Flocculating Microalga (Chlorella vulgaris) JSC-7

微藻是可广泛用于健康食品及水产养殖的饵料,同时,微藻细胞内积累的油脂可作为可再生生物燃料,因此微藻的生长和代谢受到广泛关注。温度和pH对微藻的生物量积累有很大影响,考察不同温度和pH条件下微藻细胞的生长有助于寻找最佳的条件进行微藻的培养。自絮凝小球藻JSC-7（Chlorella vulgaris JSC-7）可实现自沉降采收,有利于降低微藻生产成本,优化其生长条件对更好地利用该微藻具有重要意义。考察了温度（22∽40℃）及pH（6.0∽10.0）对其细胞生长、叶绿素含量和油脂产量的影响。在所选取的温度及pH范围内,JSC-7细胞均可生长,显示该藻种可以适应广泛的温度和pH条件。适合细胞生长的温度依次为31℃〉28℃〉35℃〉25℃,pH依次为7.0〉8.0〉6.0。pH 8.0时生物量和油脂的积累量最多,说明该藻株在弱碱条件下更适合生长和产油。当温度为31℃、pH为7.0时,可获得最高的生长量（OD690=0.941）、叶绿素含量（19 mg/L）及油脂产量（39.07%/克干重）。     

尼罗红荧光法快速检测培养过程中湛江等鞭金藻的中性脂

研究一种快速准确测定微藻中中性脂的方法。湛江等鞭金藻是一种中性脂含量高且具有开发潜力的能源微藻。以湛江等鞭金藻为实验对象,首先优化尼罗红染色的条件。当二甲基亚砜体积分数为2.0%、尼罗红质量浓度为1.00μg/m L、细胞密度为1.0×106个/m L、激发波长为480 nm、检测波长为580 nm时,优化的染色时间为10min。其次测定了背景荧光对检测的影响。结果表明,在不同细胞状态下,背景荧光强度大约是微藻内荧光强度的20%左右,可以忽略。最后比较了尼罗红荧光法和重量法。结果表明,荧光强度与中性脂含量的相关系数R2=0.946 8,虽然两者相关性并不十分高,但作为一种快速测定微藻中中性脂的方法,尼罗红荧光法依然是研究微藻培养过程中中性脂含量变化的有效方法。     

低场核磁共振技术快速检测小球藻油脂含量及其在高通量选育中的应用

为了建立一个高效的高产油微藻诱变育种流程,微藻中油脂含量快速和准确的测定在其中具有重要作用。在本研究中,首先利用低场核磁共振技术,建立了直接检测干藻粉和培养液中小球藻油脂含量的方法,其信号强度与细胞中油脂含量存在特异的线性关系,干藻粉和藻液中油脂含量与信号值拟合的R2均高于0.99,说明该方法用于小球藻油脂含量的检测是准确和可行的。同时该方法与传统油脂测量方法相比,具有快速、简便和准确等优点。但其通量不及尼罗红染色法,因此,我们开发了将尼罗红染色法用于初筛,低场核磁共振技术用于复筛的新型高通量藻种复合筛选方法,并将此筛选方法应用于一种异养高产油原壳小球藻的诱变育种过程中。首先从3 098株诱变藻种中初筛得到108株具有较高油脂含量的藻株,然后利用低场核磁共振技术复筛得到9株高产油性能的藻株,其中一株甘油三酯含量超过20%,比原始藻株提高1倍,培养168 h后培养液油脂浓度达到5 g/L,证明此诱变育种流程不仅提高了筛选的效率还可靠且可行。     

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。     

基于尼罗红染色分析金藻总脂动态积累简

通过尼罗红荧光染色对一株富油微藻金藻Isochrysis sp.CCMM5001建立和完善了一种方便快捷且能准确定量金藻油脂含量的方法,利用该方法探索了不同培养条件对金藻生长和总脂积累的影响。结果表明:经尼罗红染色后,金藻的单细胞荧光强度与其总脂含量呈良好的线性关系;金藻最适生长的氮浓度、光照强度和温度分别为1323μmol/L、148.0μmol/(m2·s)、25℃;最适总脂积累的氮浓度、光照强度和温度分别为441μmol/L、92.5μmol/(m2·s)、15℃;优化培养条件并采用两阶段培养法后总脂含量和油脂产率都有大幅提高,可分别高达63.3%和22 mg/(L·d)。     

温度及pH对自絮凝小球藻JSC-7生长的影响（英文）

微藻是可广泛用于健康食品及水产养殖的饵料,同时,微藻细胞内积累的油脂可作为可再生生物燃料,因此微藻的生长和代谢受到广泛关注。温度和pH对微藻的生物量积累有很大影响,考察不同温度和pH条件下微藻细胞的生长有助于寻找最佳的条件进行微藻的培养。自絮凝小球藻JSC-7(Chlorella vulgaris JSC-7)可实现自沉降采收,有利于降低微藻生产成本,优化其生长条件对更好地利用该微藻具有重要意义。考察了温度(22~40℃)及pH(6.0~10.0)对其细胞生长、叶绿素含量和油脂产量的影响。在所选取的温度及pH范围内,JSC-7细胞均可生长,显示该藻种可以适应广泛的温度和pH条件。适合细胞生长的温度依次为31℃28℃35℃25℃,pH依次为7.08.06.0。pH 8.0时生物量和油脂的积累量最多,说明该藻株在弱碱条件下更适合生长和产油。当温度为31℃、pH为7.0时,可获得最高的生长量(OD690=0.941)、叶绿素含量(19 mg/L)及油脂产量(39.07%/克干重)。     

Microwave-assisted Nile red method for in vivo quantification of neutral lipids in microalgae

In vivo determination of neutral lipids with Nile red fluorescence has been used as a rapid screening method for certain types of microalgae, but has been unsuccessful in others, particularly those with thick, rigid cell walls that prevent penetration of the fluorescence dye into the cell. To solve the problem, a microwave-assisted Nile red staining method for microalgal lipid determination was developed. In a two-step staining protocol, 50 and 60 s were selected as the optimal microwave times for the pretreatment and staining process, respectively. Moreover, several calibration methods for quantitative analysis of neutral lipids in microalgae were investigated and compared with conventional gravimetric methods. Factors that affected the in vivo quantification of cellular neutral lipids were also investigated. Application of the new method for detection and quantification of neutral lipids in a number of green microalgae was demonstrated.     

Enhancement of BODIPY505/515 lipid fluorescence method for applications in biofuel-directed microalgae production

This paper describes a microalgal cell lipid fluorescence enhancement method using BODIPY(505/515), which can be used to screen for lipids in wild-type microalgae and to monitor lipid content within microalgae production processes to determine optimal harvesting time. The study was based on four microalgae species (Dunaliella teteriolecta, Tetraselmis suecica, Nannochloropsis oculata, and Nannochloris atomus) selected because of their inherent high lipid content. An extended analysis was carried out with N. oculata due to the depressed fluorescence observed when compared with the other experimental strains. BODIPY(505/515) lipid fluorescence was determined for two solvent pre-treatment methods (DMSO and glycerol) and four staining condition parameters (analysis time, staining temperature, dye concentration, and algal cell concentration). It was found that lipid fluorescence of thick cell-walled microalgae, such as N. oculata, is significantly enhanced by both the pre-treatment methods and staining condition parameters, thereby significantly enhancing lipid fluorescence by ca. 800 times the base autofluorescence. The lipid fluorescence enhancement method provides a quick and simple index for in vivo Flow Cytometry quantification of total lipid contents for purposes of species screening or whole culture monitoring in biofuel-directed microalgae production.     

Application of the standard addition method for the absolute quantification of neutral lipids in microalgae using Nile red

Microalgae are considered one of the best candidates for biofuel production due to their high content in neutral lipids, therefore, an accurate quantification of these lipids in microalgae is fundamental for the identification of the better candidates as biodiesel source.Nile red is a fluorescent dye widely employed for the quantification of neutral lipids in microalgae. Usually, the fluorescence intensity of the stained samples is correlated to the neutral lipid content determined with standard methods, in order to draw a standard curve and deduce the neutral lipids concentration of the unknown samples positioning their fluorescence intensity values on the curve.Standard methods used for the neutral lipids determination are laborious and often implying solvent extraction and/or other transformation (i.e. saponification or transesterification) of the sample. These methods are also time consuming and may give rise to an underestimation of the lipid content due to variable extraction yields.The approach described in this paper combines the standard addition method and the fluorometric staining using Nile red, avoiding the association of traditional neutral lipids quantification methods to the fluorometric determination. After optimization of instrument parameters and staining conditions, a linear correlation between the fluorescence intensity of each sample stained with the Nile red and its neutral lipids content deduced with the standard addition method was identified. The obtained curve allowed the direct determination of neutral lipids content maintaining a linearity range from 0.12 to 12 μg of neutral lipids per ml of sample, without need of pre-concentration. This curve was then used in the quantification of the neutral lipids content in culture of Skeletonema marinoi (Bacillariophyceae) at different days from the inoculum. This method was also successfully applied on Chaetoceros socialis (Bacillariophyceae) and Alexandrium minutum (Dinophyceae).     

Monitoring oil production for biobased feedstock in the microalga <Emphasis Type="Italic">Nannochloropsis</Emphasis> sp.: a novel method combining the BODIPY BD-C12 fluorescent probe and simple image processing

A simple reliable method with fast response for lipid detection and quantification is proposed, combining a new highly lipophilic fluorescent probe BODIPY BD-C12 and image analysis to determine the algal lipid content and the lipid production in the microalgae Nannochloropsis sp. Lipid bodies stained with BODIPY BD-C12 have a characteristic multicolor fluorescence, and their volumes were determined using a sphere volume approach. The method developed was applied in the evaluation of lipid accumulation by Nannochloropsis sp. under different cultivation conditions (varying nitrate and salinity concentrations and combined effect of these two variables). The results show an increase of lipid content in Nannochloropsis sp. cultivated in nitrogen replete and depleted conditions, from 9.4 to 40.8 μm³ cell^?1 and 35.5 to 73.5%, respectively. The findings are also compared with conventional methods for determination of neutral lipids and with results obtained from the dyes Nile Red and BODIPY 505/515. A reasonable agreement between neutral lipid production measured by BODIPY BD-C12 and gravimetric methods (correlation coefficient of 0.98) was obtained. The neutral lipids production decreased from 964.6 to 244.8 mg L^?1 and from 809.1 to 396.7 mg L^?1, as the nitrate concentration increased from 0 to 0.3 g L^?1. It is observed that, with the two commercially available dyes, lipid quantification using Nile Red leads to an overestimation of lipids, while the use of BODIPY 505/515 promoted unreliable measures due to rapid bleaching of the chromophore. The method proposed shows excellent potential to become a standard, yet advanced, strategy for rapid evaluation and quantification of intracellular lipids in microalgae, a crucial step of the scaling-up process involved in the production of biobased products.     

Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections: comparison with oil red O

Nile red is a phenoxazone dye that fluoresces intensely, and in varying color, in organic solvents and hydrophobic lipids. However, the fluorescence is fully quenched in water. The dye acts, therefore, as a fluorescent hydrophobic probe. We utilized this novel property of nile red to develop a sensitive fluorescent histochemical stain for tissue lipids. Nile red was prepared by boiling Nile blue A under reflux for 2 hr in 0.5% H2SO4, and extracting the product into xylene. For staining, the purified dye is dissolved in 75% glycerol (1-5 micrograms/ml) and applied to frozen tissue sections. Tissue lipids then fluoresce yellow-gold to red, depending on their relative hydrophobicity. Using sections of liver and aorta from a cholesterol-fed rabbit, we assessed the value of Nile red as a stain for neutral lipids by comparing the staining pattern obtained with that produced by oil red O, a commonly used dye for tissue cholesteryl esters and triacylglycerols. In the cholesterol fatty liver, Nile red staining was comparable to that of oil red O. In contrast, Nile red staining of rabbit aortic atheroma revealed ubiquitous lipid deposits not observed with oil red O staining. These latter results suggest that Nile red can detect neutral lipid deposits, presumably unesterified cholesterol, not usually seen with oil red O or other traditional fat stains.     

Optimization of staining conditions for microalgae with three lipophilic dyes to reduce precipitation and fluorescence variability

When the fluorescence signal of a dye is being quantified, the staining protocol is an important factor in ensuring accuracy and reproducibility. Increasingly, lipophilic dyes are being used to quantify cellular lipids in microalgae. However, there is little discussion about the sensitivity of these dyes to staining conditions. To address this, microalgae were stained with either the lipophilic dyes often used for lipid quantification (Nile Red and BODIPY) or a lipophilic dye commonly used to stain neuronal cell membranes (DiO), and fluorescence was measured using flow cytometry. The concentration of the cells being stained was found not to affect the fluorescence. Conversely, the concentration of dye significantly affected the fluorescence intensity from either insufficient saturation of the cellular lipids or formation of dye precipitate. Precipitates of all three dyes were detected as events by flow cytometry and fluoresced at a similar intensity as the chlorophyll in the microalgae. Prevention of precipitate formation is, therefore, critical to ensure accurate fluorescence measurement with these dyes. It was also observed that the presence of organic solvents, such as acetone and dimethyl sulfoxide (DMSO), were not required to increase penetration of the dyes into cells and that the presence of these solvents resulted in increased cellular debris. Thus, staining conditions affected the fluorescence of all three lipophilic dyes, but Nile Red was found to have a stable fluorescence intensity that was unaffected by the broadest range of conditions and could be correlated to cellular lipid content.     

Nile red: a selective fluorescent stain for intracellular lipid droplets

We report that the dye nile red, 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one, is an excellent vital stain for the detection of intracellular lipid droplets by fluorescence microscopy and flow cytofluorometry. The specificity of the dye for lipid droplets was assessed on cultured aortic smooth muscle cells and on cultured peritoneal macrophages that were incubated with acetylated low density lipoprotein to induce cytoplasmic lipid overloading. Better selectivity for cytoplasmic lipid droplets was obtained when the cells were viewed for yellow-gold fluorescence (excitation, 450-500 nm; emission, greater than 528 nm) rather than red fluorescence (excitation, 515-560 nm; emission, greater than 590 nm). Nile red-stained, lipid droplet-filled macrophages exhibited greater fluorescence intensity than did nile red-stained control macrophages, and the two cell populations could be differentiated and analyzed by flow cytofluorometry. Such analyses could be performed with either yellow-gold or red fluorescence, but when few lipid droplets per cell were present, the yellow-gold fluorescence was more discriminating. Nile red exhibits properties of a near-ideal lysochrome. It is strongly fluorescent, but only in the presence of a hydrophobic environment. The dye is very soluble in the lipids it is intended to show, and it does not interact with any tissue constituent except by solution. Nile red can be applied to cells in an aqueous medium, and it does not dissolve the lipids it is supposed to reveal.