Monitoring oil production for biobased feedstock in the microalga Nannochloropsis 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.
KeywordsMicroalgae Nitrogen starvation Lipid quantification Biofuels Fluorescent dyes
The authors acknowledge the Fundação para a Ciência e a Tecnologia (FCT), Portuguese Agency for Scientific Research, for the PhD research grants to TE, VTA, and TFGGC SFRH/BD/81385/2011, SFRH/BD/48269/2008, and SFRH/BD/95459/2013. The authors are also grateful for support from “The Coimbra Chemistry Centre” which is funded by the FCT, through the projects FCT UID/QUI/UI0313/2013 and COMPETE Programme (Operational Programme for Competitiveness).
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