Improving the reliability of fluorescence-based neutral lipid content measurements in microalgal cultures

Siegler, Hector De la Hoz; Ayidzoe, W.; Ben-Zvi, Amos; Burrell, Robert E.; McCaffrey, William C.

doi:10.1016/j.algal.2012.07.004

Cited by 47 publications

(22 citation statements)

References 30 publications

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“…Recent improvements to the Nile Red staining method have been reported that bypass the initial shortcomings of the protocol 10,11 . Staining the cells in the presence of a carrier solvent such as DMSO 10 or ethanol 10,11 linearizes the relationship between oil content and absorbance, allowing for reliable quantitative measurements.…”

Section: Introductionmentioning

confidence: 99%

“…Furthermore, by calibrating using representative samples of the desired algal species this method produces relatively precise measurements that are directly interpretable. There exist many protocols outlining methods of staining algae with Nile Red optimized for different strains and applications; the protocol presented here was originally developed by de la Hoz Siegler et al 11 for Auxenochlorella 1. Remove a sample volume from the growing algal culture that will provide at least 200 mg of dry biomass, 400-600 mg is preferable.…”

Section: Introductionmentioning

confidence: 99%

“…Staining the cells in the presence of a carrier solvent such as DMSO 10 or ethanol 10,11 linearizes the relationship between oil content and absorbance, allowing for reliable quantitative measurements. The solvent helps permeabilize the cell membrane so that the Nile Red molecules can pass through.…”

Section: Introductionmentioning

confidence: 99%

“…Nile Red, a dye that fluoresces preferentially in non-polar environments, has been used to identify or quantify lipid bodies in various organisms including nematodes 7 , yeast 8 , bacteria 9 , and algae [10][11][12][13][14][15][16][17][18][19] . Initial techniques involving Nile Red were mostly qualitative or semi-quantitative, combining the stain with single-cuvette spectrophotometry or flow cytometry.…”

Section: Introductionmentioning

confidence: 99%

“…In order to most accurately account for background noise in the measurements, a standard curve correlating fluorescence intensity to oil content is developed using algal cells of known oil composition. The method is adapted from previously published protocols 10,11 . By using a 96-well spectrophotometer, one is able to analyze the same amount of samples in an hour that would take days to monitor by gravimetric methods.…”

Section: Introductionmentioning

confidence: 99%

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A Simple and Rapid Protocol for Measuring Neutral Lipids in Algal Cells Using Fluorescence

Storms

Cameron

Siegler

et al. 2014

JoVE

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Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal fermentation processes and screening for new oil-rich strains requires a fast and reliable protocol for determination of intracellular lipid content. Current practices rely largely on gravimetric methods to determine oil content, techniques developed decades ago that are time consuming and require large sample volumes. In this paper, Nile Red, a fluorescent dye that has been used to identify the presence of lipid bodies in numerous types of organisms, is incorporated into a simple, fast, and reliable protocol for measuring the neutral lipid content of Auxenochlorella protothecoides, a green alga. The method uses ethanol, a relatively mild solvent, to permeabilize the cell membrane before staining and a 96 well micro-plate to increase sample capacity during fluorescence intensity measurements. It has been designed with the specific application of monitoring bioprocess performance. Previously dried samples or live samples from a growing culture can be used in the assay. Video LinkThe video component of this article can be found at

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

A Simple and Rapid Protocol for Measuring Neutral Lipids in Algal Cells Using Fluorescence

Storms

Cameron

Siegler

et al. 2014

JoVE

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The effects of exogenous cerium on photosystem II as probed by in vivo chlorophyll fluorescence and lipid production ofScenedesmus obliquusXJ002

Cheng

Du²,

Long

et al. 2020

Biotechnology and Applied Biochemistry

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Cerium is the most abundant rare earth metal in the earth's crust, and it has deleterious effects on aquatic ecosystems from fertilizer runoff. Scenedesmus obliquus is an oil-rich microalga that grows rapidly and is sensitive to many kinds of toxins. Given that microalgae are useful indicators of eutrophication and toxic stress, it was found that lower concentrations of cerium (0.50-5.00 mg•L −1 ) stimulated algal growth and increased chlorophyll a content, whereas higher concentrations (above 50.00 mg•L −1 ) had an inhibitory effect on algal growth and chlorophyll a content. The algal growth rate and chlorophyll a content peaked at a cerium concentration of 5.00 mg•L −1 . Both the donor and acceptor sides of photosystem II (PSII) reaction centers were sensitive to cerium-induced stress. Specifically, high concentrations of cerium damaged the oxygen evolving complex and PSII reaction center and suppressed electron transport at the donor and receptor side of the reaction center, influencing the absorption, transfer, and application of light energy in S. obliquus XJ002. In addition, we established a simple method to quantify the intracellular lipid content of S. obliquus XJ002, and the optimum staining conditions for Nile red were as follows: volume percentage of dimethyl sulfoxide was 2%, the concentration of Nile red was 2.0 μg•mL −1 , and the staining time of Nile red was 5 min. The addition of cerium resulted in a significant increase in the total lipid content of XJ002. When the concentration of cerium was 50 mg•L −1 , the total lipid content was 16.26% higher than the control group. This information will enhance our ability to utilize microelement fertilizer in biomass accumulation programs and will help to further reveal the key regulatory factors in the lipid metabolism, and would lay the foundation for promoting the research of microalgae bioenergy.

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Current developments in high‐throughput analysis for microalgae cellular contents

Lee

Chang

Wang

2013

Biotechnology Journal

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Microalgae have emerged as one of the most promising feedstocks for biofuels and bio-based chemical production. However, due to the lack of effective tools enabling rapid and high-throughput analysis of the content of microalgae biomass, the efficiency of screening and identification of microalgae with desired functional components from the natural environment is usually quite low. Moreover, the real-time monitoring of the production of target components from microalgae is also difficult. Recently, research efforts focusing on overcoming this limitation have started. In this review, the recent development of high-throughput methods for analyzing microalgae cellular contents is summarized. The future prospects and impacts of these detection methods in microalgae-related processing and industries are also addressed.

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Improving the reliability of fluorescence-based neutral lipid content measurements in microalgal cultures

Cited by 47 publications

References 30 publications

A Simple and Rapid Protocol for Measuring Neutral Lipids in Algal Cells Using Fluorescence

A Simple and Rapid Protocol for Measuring Neutral Lipids in Algal Cells Using Fluorescence

The effects of exogenous cerium on photosystem II as probed by in vivo chlorophyll fluorescence and lipid production ofScenedesmus obliquusXJ002

Current developments in high‐throughput analysis for microalgae cellular contents

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