The use of fluorescent Nile red and BODIPY for lipid measurement in microalgae

Microalgae are currently emerging as one of the most promising alternative sources for the next generation of food, feed, cosmetics and renewable energy in the form of biofuel. Microalgae constitute a diverse group of microorganisms with advantages like fast and efficient growth. In addition, they do not compete for arable land and offer very high lipid yield potential. Major challenges for the development of this resource are to select lipid-rich strains using high-throughput staining for neutral lipid content in microalgae species. For this purpose, the fluorescent dyes most commonly used to quantify lipids are Nile red and BODIPY 505/515. Their fluorescent staining for lipids offers a rapid and inexpensive analysis tool to measure neutral lipid content, avoiding time-consuming and costly gravimetric analysis. This review collates and presents recent advances in algal lipid staining and focuses on Nile red and BODIPY 505/515 staining characteristics. The available literature addresses the limitations of fluorescent dyes under certain conditions, such as spectral properties, dye concentrations, cell concentrations, temperature and incubation duration. Moreover, the overall conclusion of the present review study gives limitations on the use of fluorochrome for screening of lipid-rich microalgae species and suggests improved protocols for staining recalcitrant microalgae and recommendations for the staining quantification.

Introduction

Microalgae are currently considered as potential actors for the third biofuel generation for several reasons. They can be produced, in a autotrophic manner and on non-agricultural land, be cultivated in seawater or wastewater and offer a higher lipid productivity than first-generation biofuels [1,2]. Reviews on the topic ‘microalgae and energy’ are flourishing to support this promising path [3-8]. Publications on new algae, manuscripts revisiting known algae and teams discovering new paths have become increasingly common. The main bottlenecks of the full chain process have been identified, with claims to contribute to the decrease of the environmental and economic costs of microalgal biofuel [9-12]. However, one of the cornerstones of working with lipid-producing microalgae that has barely been discussed is the necessity to carry out accurate, easy, reliable and repeatable measurements of lipid quantity and quality. This is especially true when triacylglycerols (TAG) are targeted as the neutral lipid storage form in the cell, as these represent a precursor that can be transformed into biodiesel via a transesterification step.

Some reliable techniques exist that require heavy and expensive equipment and take up working time of qualified technicians [13]: in addition to gravimetry measurements [14,15], a large panel of chromatography strategies have been developed, like thin-layer chromatography (TLC), high-pressure liquid chromatography (HPLC) and gas chromatography (GC) coupled with mass spectrometry (MS) [16]. With such an approach, the full process for a single TAG measurement takes a few hours, with a total cost (including equipment, manpower and consumables) above $50 to $100 per sample.

Fluorescent dyes offer an indirect measurement for lipids. They are easier and cheaper to use than the above methods and require a much smaller amount of equipment. These dyes bind specifically to the compounds of interest and have the additional advantage of offering a quick response [17,18]. Vital stains for the detection of intracellular lipid by fluorescence microscopy, spectrofluorometry or flow cytometry [19] need to be used. The most common dyes for lipids are Nile red [19] and BODIPY 505/515 [20]. Nile red and BODIPY 505/515 offer several advantageous characteristics for in situ screening [21]. The initial reason for using these fluorescent dyes was to perform fast screening of potential oleaginous microalgae in order to identify promising sources for commercial biofuel production [1,22]. Nile red was previously used in semi-quantitative techniques [18,23,24] but, with improved methods, is now widely used to quantify lipid level. BODIPY 505/515 has been used more recently as a potential alternative to Nile red. In fact, these dyes do not stain all microalgae successfully, even though methods are constantly improving, as reported in the literature. There is a real need to screen a large number of microalgae using a rapid, accurate and reliable method for detection and quantification of lipids produced [23,25], but caution must be taken concerning the sensitivity of fluorimetric methods and, in particular, the issue of fading (fluorescence extinction). This review provides an updated state of the art on fluorescent dyes and their use in the literature, with a particular focus on Nile red and BODIPY 505/515, which are the two most popular stains used to assess lipid content especially the neutral lipid content of microalgae.

Lipid-staining fluorochromes: Nile red and BODIPY 505/515

Among markers that correlate fluorescence with the lipid content in microalgae cells, Nile red (9-diethylamino-5H-benzo[a]phenoxazine-5-one) is the most commonly used lipophilic stain for intracellular TAG detection in microalgae prior to BODIPY 505/515 (4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene) which is a green lipophilic fluorescent dye recently used as an alternative to Nile red staining (Table 1).

Objective and tools for the choice of fluorochrome

Evaluating the content of lipids in microalgae can be done by several alternative measurement approaches. First, fluorescence microscopy offers the possibility to calculate the volume of lipid vesicles [73]. This measurement can be carried out by staining with Nile red or BODIPY 505/515 but does not allow high-throughput study of microalgae lipid content. The use of a spectrofluorimeter with i) a classic vial or ii) a microplate allows faster measurements. The classic fluorimeter method in a vial requires a large amount of biomass (1 to 4 mL) and is relatively time consuming, as measurement is not automated. The use of a microplate allows a high-throughput method with several advantages in terms of time and does not require large amounts of biomass and solvent for the quantification of neutral lipid content. However, spectrofluorimeter analyses are more appropriate for Nile red staining because of the self-fluorescence of BODIPY 505/515 [44,83]. Then, flow cytometry allows efficient fluorescence measurement suitable for both Nile red and BODIPY 505/515. This high-throughput method also saves time and requires a lower algal biomass. In all cases, measurement sensitivity by fluorescence techniques requires standardization and optimization of instrument parameters [19,23]. Precision of staining conditions is essential for the reliability and robustness of the method.

Current studies show a good efficiency of Nile red staining for many species, but staining with BODIPY 505/515 seems more appropriate for thick-walled rigid microalgae. However, if no previous work is available for a particular species, tests of Nile red and BODIPY 505/515 analysis should be run separately. For each species, the fluorochrome that gives the highest signal-to-noise ratio should be chosen.

Optimization of staining parameters with Nile red and BODIPY 505/515

Dye stock solutions should be stored at controlled temperature and protected from the light, for up to 30 days at −20°C, avoiding thus fluorescence loss and evaporation. A safe option consists in preparing single-use aliquots of Nile red solution and storing these at low temperature and protected from light [84].

The optimal algal and dye concentrations are dependent on the microalgae species and can be determined by testing several dilutions. By setting the right concentration ranges, a linear correlation should be achieved for cell concentration and dye fluorescence intensity. Generally, the final concentration of Nile red in the culture sample ranges between 0.1 and 2 μg mL −1 . In parallel, final concentration of BODIPY 505/515 ranges between 0.04 and 2.6 μg mL −1 . The appropriate type of blank should then be used.

The kinetic of fluorescence intensity over time is species specific. Incubation times usually range between 5 and 15 min for both the Nile red and BODIPY 505/515. Fluorescence kinetics should be experimentally tested for each species in order to assess the optimal incubation. Fluorescence should be measured over intervals from seconds to hours, making sure that photobleaching does not occur between two measurements.

Permeation is a facultative and preliminary step that can be required depending on the cell wall composition. This step can be carried out using DMSO, glycerol or even physical treatments. However, since this is an additional time-consuming step which can moreover increase error in the labelling, its necessity must be carefully evaluated.

Fluorescence reading of fluorochromes

Fluorescence measurement to efficiently assess lipid quantity and quality in microalgae is carried out using optimized excitation and emission wavelengths. Using Nile red staining, information on lipid classes is obtained by the choice of the Ex/Em wavelength pair. These measurements allow information to be obtained on lipid composition of microalgae species via the polar/neutral ratio (Table 3). Care should be taken to ensure the homogeneity of the dye-culture mixture before reading the fluorescence.

Calibration of fluorescent staining

Lastly, for each species, a calibration curve would establish the relationship between fluorescence and cell lipids, as measured gravimetrically, and confirm the correct staining procedure (Table 2).

Conclusion

This review highlights the key steps of a protocol for efficient lipid labelling in microalgae species with Nile red and BODIPY 505/515. It clearly appears that markers used without accurate preliminary work to optimize the protocol may result in quantification errors. The major advantage of lipid-droplet staining in microalgae cells using fluorochromes remains the possibility of high-throughput quantification. These fluorochromes are likely to become standard advanced biotechnology tools in the future and participate in a preliminary step of the scaling-up for the production of biofuels or feed oils. Additional means must be found in order to improve the high-throughput screening of microalgae candidates for various applications. Measurements of lipids per biomass unit (cell, carbon…) require a calibration curve correlating the fluorescence to lipid content, determined analytically. The key issue is that the calibration should be carried out regularly to demonstrate the relevance of protocol for the targeted species and its physiological state. Metabolic stress involving nutrient limitation [40,42,55,63,88,106,107] or enrichment [106] to enhance TAG content must be considered with caution, since the optimal staining protocol may not be the same in cells with different physiological states.

Abbreviations

polyunsaturated fatty acid

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Acknowledgements

This work was financially supported by the Facteur 4 ANR project (ANR-12-BIME-0004).