Automation of a Nile red staining assayenables high throughput quantification of microalgal lipid production

[摘要] BackgroundWithin the context of microalgal lipid production for biofuels andbulk chemical applications, specialized higher throughput devices for smallscale parallelized cultivation are expected to boost the time efficiency ofphototrophic bioprocess development. However, the increasing number of possibleexperiments is directly coupled to the demand for lipid quantification protocolsthat enable reliably measuring large sets of samples within short time and thatcan deal with the reduced sample volume typically generated at screening scale.To meet these demands, a dye based assay was established using a liquid handlingrobot to provide reproducible high throughput quantification of lipids withminimized hands-on-time.ResultsLipid production was monitored using the fluorescent dye Nile redwith dimethyl sulfoxide as solvent facilitating dye permeation. The stainingkinetics of cells at different concentrations and physiological states wereinvestigated to successfully down-scale the assay to 96 well microtiter plates.Gravimetric calibration against a well-established extractive protocol enabledabsolute quantification of intracellular lipids improving precision from ±8to ±2 % on average. Implementation into an automated liquid handling platformallows for measuring up to 48 samples within 6.5 h, reducing hands-on-time to athird compared to manual operation. Moreover, it was shown that automationenhances accuracy and precision compared to manual preparation. It was revealedthat established protocols relying on optical density or cell number for biomassadjustion prior to staining may suffer from errors due to significant changes ofthe cells’ optical and physiological properties during cultivation.Alternatively, the biovolume was used as a measure for biomass concentration sothat errors from morphological changes can be excluded.ConclusionsThe newly established assay proved to be applicable for absolutequantification of algal lipids avoiding limitations of currently establishedprotocols, namely biomass adjustment and limited throughput. Automation wasshown to improve data reliability, as well as experimental throughputsimultaneously minimizing the needed hands-on-time to a third. Thereby, thepresented protocol meets the demands for the analysis of samples generated bythe upcoming generation of devices for higher throughput phototrophiccultivation and thereby contributes to boosting the time efficiency for settingup algae lipid production processes.

[发布日期] 2016-02-09

[关键词] Chlorella vulgaris;High throughput;Laboratory automation;Lipid quantification;Liquid handling robot;Nile red