TY - JOUR
T1 - A high throughput Nile red method for quantitative measurement of neutral lipids in microalgae
AU - Chen, Wei
AU - Zhang, Chengwu
AU - Song, Lirong
AU - Sommerfeld, Milton
AU - Hu, Qiang
PY - 2009/4
Y1 - 2009/4
N2 - 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 (R2 = 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 μg/mL and 20 μg/mL), respectively. Moreover, the detection and quantification limits of the improved Nile red staining method were 0.8 μg/mL and 2.0 μg/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.
AB - 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 (R2 = 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 μg/mL and 20 μg/mL), respectively. Moreover, the detection and quantification limits of the improved Nile red staining method were 0.8 μg/mL and 2.0 μg/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.
KW - Biofuel
KW - Fluorescence
KW - Green algae
KW - Microalgae
KW - Neutral lipids
KW - Nile red
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U2 - 10.1016/j.mimet.2009.01.001
DO - 10.1016/j.mimet.2009.01.001
M3 - Article
C2 - 19162091
AN - SCOPUS:62349095514
SN - 0167-7012
VL - 77
SP - 41
EP - 47
JO - Journal of Microbiological Methods
JF - Journal of Microbiological Methods
IS - 1
ER -