Improvement of the Nile Red fluorescence assay for determination of total lipid content in microalgae independent of chlorophyll content
2015
Orr, Valerie | Rehmann, Lars
Rapid determination of the specific lipid content of microalgae can accelerate screening and development of algae fuel application. Until recently, lipid content was commonly measured using gravimetric solvent extraction. Such methodology is time consuming and requires relatively large sample volumes. The Nile Red lipid assay has recently gained acceptance as it requires minute sample sizes. However, the current methodology is highly biased through interference of chlorophyll. Chlorophyll content can vary significantly between species, cultivation conditions, and culture age, remaining a serious but accepted problem. The assay was improved in this study in two ways. The cell dry weight determination was generalized by chemical chlorophyll removal prior to optical cell density measurements. Algae samples with different chlorophyll content were used to correlate optical measurements to cell dry weight and the correlation was substantially improved compared to non-treated samples (97 % decrease in sum of squared residuals). Degradation of chlorophyll was also found to increase the emission intensity of Nile Red, presumably by removing a source of molecular absorption of red light, potentially increasing the detection threshold. Secondly, milk fat was used as a standard for correlating lipid concentration to Nile Red fluorescence. It was cheaper, more precise, easier to accurately prepare, and linearly correlated to lipid concentration over a broader range than triolein, largely due to the fact that it forms a stable lipid/water micro-emulsion. The improved methodology for the Nile Red lipid assay was found to significantly increase the working range and accuracy of the assay.
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