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

Abstract

BackgroundWithin the context of microalgal lipid production for biofuels and bulk chemical applications, specialized higher throughput devices for small scale parallelized cultivation are expected to boost the time efficiency of phototrophic bioprocess development. However, the increasing number of possible experiments is directly coupled to the demand for lipid quantification protocols that enable reliably measuring large sets of samples within short time and that can deal with the reduced sample volume typically generated at screening scale. To meet these demands, a dye based assay was established using a liquid handling robot to provide reproducible high throughput quantification of lipids with minimized hands-on-time.ResultsLipid production was monitored using the fluorescent dye Nile red with dimethyl sulfoxide as solvent facilitating dye permeation. The staining kinetics of cells at different concentrations and physiological states were investigated to successfully down-scale the assay to 96 well microtiter plates. Gravimetric calibration against a well-established extractive protocol enabled absolute quantification of intracellular lipids improving precision from ±8 to ±2 % on average. Implementation into an automated liquid handling platform allows for measuring up to 48 samples within 6.5 h, reducing hands-on-time to a third compared to manual operation. Moreover, it was shown that automation enhances accuracy and precision compared to manual preparation. It was revealed that established protocols relying on optical density or cell number for biomass adjustion prior to staining may suffer from errors due to significant changes of the cells’ optical and physiological properties during cultivation. Alternatively, the biovolume was used as a measure for biomass concentration so that errors from morphological changes can be excluded.ConclusionsThe newly established assay proved to be applicable for absolute quantification of algal lipids avoiding limitations of currently established protocols, namely biomass adjustment and limited throughput. Automation was shown to improve data reliability, as well as experimental throughput simultaneously minimizing the needed hands-on-time to a third. Thereby, the presented protocol meets the demands for the analysis of samples generated by the upcoming generation of devices for higher throughput phototrophic cultivation and thereby contributes to boosting the time efficiency for setting up algae lipid production processes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0433-7) contains supplementary material, which is available to authorized users.

Design and validation of a parallelized micro‐photobioreactor enabling phototrophic bioprocess development at elevated throughput

“…Taking account of the increased sample load and the simultaneously decreased volumes obtained from cultivation at 1 mL scale, a specially tailored robotic assay was used for the quantification of the intracellular neutral lipid content (nLC) (Morschett et al, ). Moreover, the cell dry weight (CDW) was determined at harvest to obtain the nitrate specific biomass yield ( Y X/S ), as well as the overall volumetric productivity of the individual processes (Fig.…”

“…Intracellular lipids were quantified via an automated high throughput Nile red staining assay according to Morschett et al (2016b).…”

Combinatorial impact of Sec signal peptides from Bacillus subtilis and bioprocess conditions on heterologous cutinase secretion by Corynebacterium glutamicum

“…To account for the volume fraction of the cultivation suspension, which is occupied by the cells, an empirical conversion factor was determined to deduce the biovolume from cell dry weight. Biovolume was measured in technical triplicates using a Multisizer 3 (Beckman Coulter, Krefeld, Germany) particle counter (Morschett, Wiechert, & Oldiges, ) for each of the four samples obtained from four different bioprocesses. The samples were adjusted to an optical density (OD 600 ) of approximately 0.1, and 500 µL were diluted in 9.5 ml CASYton Buffer (Schärfe Systems, Reutlingen, Germany).…”

Development of Novel Bioreactor Control Systems Based on Smart Sensors and Actuators

“…Monitoring important metabolites such as lipids, proteins, and starch inside the cells is an urgent requirement in bioprocess development and large-scale production. Morschett et al (2016) developed an automatic Nile red staining assay that enabled the high-throughput quantification of microalgal lipid production for bioprocess development. Several groups also reported the implementation of non-invasive Raman spectroscopy for the monitoring of mAb and lipid production during bioprocess development (He et al, 2017;Santos et al, 2019).…”