Chen Wei, Zhang Chengwu, Song Lirong, Sommerfeld Milton, Hu Qiang
Department of Applied Biological Sciences, Arizona State University, Mesa, AZ 85212, USA.
J Microbiol Methods. 2009 Apr;77(1):41-7. doi: 10.1016/j.mimet.2009.01.001. Epub 2009 Jan 6.
Isolation of high neutral lipid-containing microalgae is key to the commercial success of microalgae-based biofuel production. The Nile red fluorescence method has been successfully applied to the determination of lipids in certain microalgae, but has been unsuccessful in many others, particularly those with thick, rigid cell walls that prevent the penetration of the fluorescence dye. The conventional "one sample at a time" method was also time-consuming. In this study, the solvent dimethyl sulfoxide (DMSO) was introduced to microalgal samples as the stain carrier at an elevated temperature. The cellular neutral lipids were determined and quantified using a 96-well plate on a fluorescence spectrophotometer with an excitation wavelength of 530 nm and an emission wavelength of 575 nm. An optimized procedure yielded a high correlation coefficient (R(2)=0.998) with the lipid standard triolein and repeated measurements of replicates. Application of the improved method to several green algal strains gave very reproducible results with relative standard errors of 8.5%, 3.9% and 8.6%, 4.5% for repeatability and reproducibility at two concentration levels (2.0 microg/mL and 20 microg/mL), respectively. Moreover, the detection and quantification limits of the improved Nile red staining method were 0.8 microg/mL and 2.0 microg/mL for the neutral lipid standard triolein, respectively. The modified method and a conventional gravimetric determination method provided similar results on replicate samples. The 96-well plate-based Nile red method can be used as a high throughput technique for rapid screening of a broader spectrum of naturally-occurring and genetically-modified algal strains and mutants for high neutral lipid/oil production.
分离出富含高中性脂质的微藻是基于微藻的生物燃料生产取得商业成功的关键。尼罗红荧光法已成功应用于某些微藻脂质的测定，但在许多其他微藻中却未成功，特别是那些具有厚而坚硬细胞壁的微藻，这些细胞壁会阻止荧光染料的渗透。传统的“一次一个样品”方法也很耗时。在本研究中，将溶剂二甲基亚砜（DMSO）在高温下作为染色载体引入微藻样品中。使用96孔板在荧光分光光度计上测定并定量细胞中的中性脂质，激发波长为530 nm，发射波长为575 nm。优化后的程序与脂质标准品三油酸甘油酯具有很高的相关系数（R(2)=0.998），并且对重复样品进行了重复测量。将改进后的方法应用于几种绿藻菌株，在两个浓度水平（2.0 μg/mL和20 μg/mL）下，重复性和再现性的相对标准误差分别为8.5%、3.9%和8.6%、4.5%，结果具有很好的可重复性。此外，改进后的尼罗红染色法对中性脂质标准品三油酸甘油酯的检测限和定量限分别为0.8 μg/mL和2.0 μg/mL。改进后的方法与传统的重量测定法对重复样品的检测结果相似。基于96孔板的尼罗红方法可作为一种高通量技术，用于快速筛选更广泛的天然和转基因藻类菌株及突变体，以获取高中性脂质/油脂产量。
