In the wake of global warming and rapid fossil fuel depletion, microalgae emerge as promising feedstocks for sustainable biofuel production. Nile red staining acts as a rapid diagnostic tool to measure the amount of biodiesel-convertible lipid that the cells accumulate. There is a need for the development of a more uniform staining procedure. In its first phase, this study examined the dependence of microalgal Nile red fluorescence (
The rapid depletion of fossil fuels together with the effect of greenhouse gas emissions on global climate change has led to an increased commercial interest in biodiesel. Fatty acid methyl ester (FAME) or biodiesel is commercially produced via alkaline-catalyzed transesterification of lipids with methanol [
There are, however, various technological and economic obstacles that need to be overcome before large-scale production of microalgal biodiesel can take place. The identification of lipid-rich microalgal species and the optimization of cultivation conditions that lead to maximal lipid content are of critical importance [
The traditional method used for microalgal lipid determination involves organic solvent extraction of the intracellular lipid and subsequent gravimetric measurement of the extracted lipid [
Nile red staining has frequently been applied as an alternative method for microalgal lipid determination due to its rapidity, simplicity, and low requirement of biomass [
Even though Nile red staining has been successfully applied for lipid quantification on a number of different microalgal species belonging to Bacillariophyceae and Chlorophyceae classes, the variables that affect the fluorescence intensity have not been investigated at a quantitative level and no kinetic model is yet to be developed [
Factorial experimental design is a powerful tool to determine optimal experimental conditions. Unlike a traditional experimental strategy whereby only one independent variable is changed from one experiment to another, factorial experimental design allows for multiple independent variables to be simultaneously modified from one experiment to another and takes into account all possible combinations of the assigned values of the independent variables. Such a design evaluates the direct and interactive effects of the independent variables on the response variable [
Previous microalgal studies investigating the use of Nile red staining for microalgal application have customized their staining procedures according to their individual purpose [
This study had dual objectives. The first one was to create a mathematical model that described microalgal Nile red fluorescence as a function of variables of the staining procedure (cell concentration, ultrasonication power, incubation temperature, and Nile red dye concentration). The model would enable the identification of significant variables and elucidate some of the fundamental mechanisms governing lipid/dye interaction. To generate the model, response surface methodology was applied on the results of factorial experiments. The second objective of the study was to develop a simple set of guidelines for the optimization of Nile red staining across various microalgal species. The guidelines would improve the robustness of the staining method across the different species and facilitate a more reliable diagnostic tool for the quantification of biodiesel-convertible lipid in microalgal cells. To create the standardized guidelines, we reviewed findings from previous studies on microalgal Nile red staining and unified them with the insights revealed by our modelling exercise.
The microalgal strain used in this study,
Nile red dye (9-(diethylamino)benzo[a]phenoxazin-5(5H)-one) was purchased from Sigma-Aldrich Pty. Ltd. (Australia). Organic solvents (acetone, isopropanol, and n-hexane) were obtained from commercial suppliers. Nile red dye (0.01 g) was dissolved in 40 mL of acetone to produce a Nile red stock solution (0.25 mg/mL).
Microalgal culture (a total of 2.1 L for all experiments in Sections
A portion of the microalgal culture was dewatered using a bench-top centrifuge (Heraeus Multifuge 3 S-R, Kendro, Germany) at 4500 rpm for 10 min. The supernatant was discarded and the microalgal paste was rinsed with deionised water to remove residual salts. The resuspended microalgal culture was recentrifuged and the resulting microalgal paste was dried at 60°C in the oven (Model UNE 500 PA, Memmert GmbH + Co., Germany) for 16 h. From weighing the dried biomass, microalgal cell concentration of the culture used for the above staining was determined to be
A Soxhlet apparatus was used for complete lipid extraction from the biomass. Description of a typical Soxhlet operation can be found elsewhere [
A factorial experimental design was used to evaluate the effect of four independent staining variables (cell concentration, ultrasonication power, incubation temperature, and Nile red dye concentration) on Nile red fluorescence. The reasons for the selection of these variables are elaborated in Section
Real and coded values of the independent variables investigated in the factorial experimental design.
Microalgal cell concentration | Real value (g dried microalgae/L culture) | 0 | 0.11 | ||
Coded value | 0 | 1 | |||
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Ultrasonication power | Real value (W) | 0 | 65 | 130 | |
Coded value | −1 | 0 | 1 | ||
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Incubation temperature | Real value (°C) | 20 | 30 | 40 | |
Coded value | −1 | 0 | 1 | ||
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Nile red dye concentration | Real value ( |
0 | 2.5 | 6.25 | |
Coded value | −1 | 0 | 1.5 |
Factorial experimental design matrix and values of the observed fluorescence intensity (also referred to as experimental results). Fluorescence intensity was determined at excitation wavelength of 530 nm, emission wavelength of 607 nm, and 15 min poststaining time.
Experiment number | Microalgal cell concentration | Ultrasonication power | Incubation temperature | Nile red dye concentration | Observed fluorescence intensity (afu) |
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1 | 0 | −1 | −1 | −1 | — |
2 | 0 | −1 | −1 | 0 | 62.07 |
3 | 0 | −1 | −1 | 1.5 | 72.41 |
4 | 0 | −1 | 0 | −1 | — |
5 | 0 | −1 | 0 | 0 | 63.52 |
6 | 0 | −1 | 0 | 1.5 | 90.99 |
7 | 0 | −1 | 1 | −1 | — |
8 | 0 | −1 | 1 | 0 | 90.60 |
9 | 0 | −1 | 1 | 1.5 | 152.14 |
10 | 0 | 0 | −1 | −1 | — |
11 | 0 | 0 | −1 | 0 | 44.83 |
12 | 0 | 0 | −1 | 1.5 | 70.69 |
13 | 0 | 0 | 0 | −1 | — |
14 | 0 | 0 | 0 | 0 | 70.69 |
15 | 0 | 0 | 0 | 1.5 | 100.00 |
16 | 0 | 0 | 1 | −1 | — |
17 | 0 | 0 | 1 | 0 | 41.38 |
18 | 0 | 0 | 1 | 1.5 | 110.34 |
19 | 0 | 1 | −1 | −1 | — |
20 | 0 | 1 | −1 | 0 | 51.50 |
21 | 0 | 1 | −1 | 1.5 | 72.10 |
22 | 0 | 1 | 0 | −1 | — |
23 | 0 | 1 | 0 | 0 | 63.79 |
24 | 0 | 1 | 0 | 1.5 | 120.69 |
25 | 0 | 1 | 1 | −1 | — |
26 | 0 | 1 | 1 | 0 | — |
27 | 0 | 1 | 1 | 1.5 | — |
28 | 1 | −1 | −1 | −1 | 18.88 |
29 | 1 | −1 | −1 | 0 | 200.86 |
30 | 1 | −1 | −1 | 1.5 | 137.34 |
31 | 1 | −1 | 0 | −1 | 8.58 |
32 | 1 | −1 | 0 | 0 | 123.61 |
33 | 1 | −1 | 0 | 1.5 | 140.77 |
34 | 1 | −1 | 1 | −1 | 17.24 |
35 | 1 | −1 | 1 | 0 | 177.59 |
36 | 1 | −1 | 1 | 1.5 | 153.45 |
37 | 1 | 0 | −1 | −1 | 17.24 |
38 | 1 | 0 | −1 | 0 | 122.41 |
39 | 1 | 0 | −1 | 1.5 | 131.03 |
40 | 1 | 0 | 0 | −1 | 15.52 |
41 | 1 | 0 | 0 | 0 | 106.90 |
42 | 1 | 0 | 0 | 1.5 | 148.28 |
43 | 1 | 0 | 1 | −1 | 20.69 |
44 | 1 | 0 | 1 | 0 | 148.28 |
45 | 1 | 0 | 1 | 1.5 | 158.62 |
46 | 1 | 1 | −1 | −1 | 17.17 |
47 | 1 | 1 | −1 | 0 | 78.97 |
48 | 1 | 1 | −1 | 1.5 | 101.29 |
49 | 1 | 1 | 0 | −1 | 27.59 |
50 | 1 | 1 | 0 | 0 | 79.31 |
51 | 1 | 1 | 0 | 1.5 | 131.03 |
52 | 1 | 1 | 1 | −1 | — |
53 | 1 | 1 | 1 | 0 | — |
54 | 1 | 1 | 1 | 1.5 | — |
The culture collected in Section
To investigate the effect of ultrasonication power on Nile red staining efficiency, the experimental design evaluated the difference in fluorescence intensity between stained mixtures with intensely presonicated feed mixtures (ultrasonication power = 130 W), mildly presonicated feed mixtures (ultrasonication power = 65 W), or unsonicated feed mixtures (ultrasonication power = 0 W). 125 mL of feed mixture (either microalgal culture or growth medium) was processed with an ultrasonicator (Vibra-Cell Model VCX134PB) manufactured by Sonics & Materials, Inc. (USA). The probe of the ultrasonicator (6 mm in diameter and 113 mm in length) was immersed into the feed mixture to deliver ultrasound at a frequency of 40 kHz and selected acoustic power level (0, 65, or 130 W) for 20 min.
Out of the 800 mL of microalgal culture used for the factorial experiments, 400 mL was used as a feed mixture, while 400 mL was filtered as mentioned above to produce the MF growth medium feed mixture. The feed mixture (either microalgal culture or growth medium) was divided into three and ultrasonicated according to the above description (0 W, 65 W, or 130 W). The sonicated feed mixture was pipetted as 4 mL aliquots into clear-sided cuvettes. Each cuvette was incubated at a select temperature (20, 30, or 40°C) for 10 minutes in an oven (Model UNE 500 PA, Memmert GmbH + Co., Germany). The sonicated and incubated feed mixture in the cuvette was then stained with Nile red stock solution. The volume of Nile red stock solution added was appropriately adjusted to produce one of the following Nile red dye concentrations: 0, 2.5, or 6.25
Response surface analysis was applied to the results of the experimental design in Section
In the quadratic model, the values of independent (or input) variables were coded according to the following equation:
The regression coefficients of the model were subjected to
The efficiency of ultrasonication in disrupting microalgal cells was evaluated via reduction in the intact cell counts. A small sample of the microalgal culture (10
Preliminary observation under the microscope indicated that the Nile red dye was able to interact with the intracellular lipid globules within the TS cells. As can be seen in the microscopic images (Figure
Microscopic images of a
Figure
The effect of poststaining time on the fluorescence intensity of stained microalgal culture. Excitation wavelength = 530 nm and emission wavelength = 580 nm.
Table
Our preliminary Nile red staining indicated that emission wavelength in the range of 605–610 nm was likely to be a more optimum choice for TS cells than 580 nm (results not shown). For this reason, we examined the fluorescence emission spectra obtained from the factorial design experiments in Section
Emission spectra of three different experiments from the factorial experimental design (Table
Experiment 29 is the stained microalgal culture (microalgal cell concentration = 0.11 g dried microalgae/L culture, Nile red dye concentration = 2.5
Experiment 28 is the unstained microalgal culture (microalgal cell concentration = 0.11 g dried microalgae/L culture, Nile red dye concentration = 0
Our excitation and emission wavelengths (530/607 nm) fell within the range generally reported to be optimal for microalgal Nile red staining (Table
We now discuss the statistical analysis of the factorial experimental design. Table
Table
Polynomial modelling of fluorescence intensity as a composite function of the four independent variables. Results (observed fluorescence intensities) of factorial experimental design as displayed in Table
Model term | Regression coefficient | Standard error |
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Intercept | 55.52 | 10.57 | n/a |
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1.15 | 8.74 | 0.896 |
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5.80 | 7.95 | 0.472 |
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14.71 | 8.74 | 0.104 |
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7.36 | 7.95 | 0.363 |
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−14.35 | 9.86 | 0.158 |
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−7.56 | 9.86 | 0.450 |
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−19.76 | 10.45 | 0.070 |
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5.69 | 7.50 | 0.455 |
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−0.72 | 4.98 | 0.887 |
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8.37 | 4.98 | 0.105 |
Summary of previous studies on microalgal Nile red staining.
Study | Microalgal species | Excitation and emission wavelength (nm) | Optimum poststaining time (min) | Optimum Nile red dye concentration ( |
Optimum staining temperature (°C) | Main solvent | Cosolvent (use of auxiliary step) |
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This study |
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530 and 607 | 15 | 4.69 | 40 | Acetone | — |
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Bertozzini et al. [ |
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547 and 580 | 6 | 0.25 | Room (20) | Acetone | IPA |
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Chen et al. [ |
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530 and 575 | 10 | 0.50 | 40 | DMSO | — |
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Chen et al. [ |
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490 and 580 | 1 | 1.00 | Not mentioned (assumed to be 40–50) | Acetone | DMSO (microwave-assisted) |
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Cirulis et al. [ |
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488 and 564–606 | 30 | 5–10 | Room (20) | Acetone or DMSO | — |
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Doan and Obbard [ |
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480 and 575 | 5–10 | 0.70 | Room (20) | Acetone | DMSO or glycerol |
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Huang et al. [ |
Various |
480 and 570–590 | 4 | 2.00 | Not mentioned | DMSO | — |
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Lee et al. [ |
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490 and 585 | 10 | 1.00 | Not mentioned | Acetone | — |
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Pick and Rachutin-Zalogin [ |
Various |
488 and 580–625 | 5 | 1.27 | 25 | DMF | Glycerol |
The statistical significance of each independent variable on fluorescence intensity was determined via
Figure
Fluorescence intensity predicted by the quadratic model versus fluorescence intensity observed in the experiments. Refer to Table
3D response surface plots of fluorescence intensity as predicted by the quadratic model. In each plot, the effects of two independent variables (in coded values) on fluorescence intensity are simultaneously demonstrated. For each surface plot, the other independent variables which do not form the plot axes are fixed (in coded values): (a) incubation temperature = 0, Nile red dye concentration = 0; (b) microalgal cell concentration = 1, ultrasonication power = 0. Excitation wavelength = 530 nm, emission wavelength = 607 nm, and poststaining time = 15 min.
As previously discussed, fluorescence intensity in the stained growth medium resulted from background dye/medium interaction, while fluorescence intensity in the stained microalgal culture was the combined outcome of dye/lipid interaction and dye/medium interaction. Observed fluorescence intensity for all experiments that stained growth medium (coded value of microalgal cell concentration = 0, coded value of Nile red dye concentration = 0 or 1.5) averaged out at
The model obtained a positive value (65.95) for the regression coefficient of microalgal cell concentration (Table
In these experiments, we were using ultrasonication power as a proxy for the level of cell disruption. Disrupting microalgal cells releases intracellular lipid to the surrounding medium, thus allowing the Nile red molecule to bypass membrane permeation and to directly interact with the lipid.
Ultrasonication of the microalgal culture (coded value of microalgal cell concentration = 1) at 0 W (coded value of ultrasonication power = −1), 65 W (coded value of ultrasonication power = 0), and 135 W (coded value of ultrasonication power = 1) disrupted 0%, 38.5%, and 95.0% of intact cells, respectively. Based on the relatively high
The lack of correlation between cell rupture and fluorescence intensity suggested that the cell wall/membrane structure of the
The relatively high
We speculated fluorescence intensity to increase with a rise in the dye concentration. The findings from our statistical analysis, however, demonstrated a nonlinear relationship between dye concentration and fluorescence intensity. Because of the negative regression value (−42.62) of the quadratic main effect for Nile red dye concentration (Table
Under our experimental conditions, this optimum dye concentration was found to be 4.69
We carried out a simple staining study of our strain at logarithmic and stationary growth phase to evaluate if the optimized staining procedure can be applied across different physiological states. As they transition through the growth phases (from logarithmic to stationary), microalgal cells experience various biochemical and physiological changes [
Table
Two conclusions can be derived out of the variability observed in Table
Because of the wide interspecies variability in the optimum staining conditions, the use of Nile red staining on a particular microalgal species needs to be carefully refined to take into account the specific requirements of the species. Using a single staining protocol over a wide range of species without catering to the need of each species will lead to inaccurate estimation of their lipid contents. Once optimized, however, the staining method can be used as a simple diagnostic tool for monitoring lipid quantity of the microalgal species under various environmental and growth conditions.
The second conclusion arising from Table
We thus propose the six-step operating framework as shown in Figure
Operating framework for the optimization of Nile red staining of a new microalgal species.
Verify the lipid content of the given culture of the microalgal species. A chloroform/methanol/water solvent mixture at 1 : 2 : 0.8 v/v/v [
In order to penetrate into the microalgal cell, Nile red dye needs to be dissolved in a relatively polar solvent that is capable of interacting with the polar components of both the cell wall (cellulose) and the cell membrane (phospholipid) [
As an initial protocol, we recommend staining the microalgal culture with Nile red stock solution in acetone at 100 : 1 v/v at room temperature (20°C) for 10 minutes. Use 0.1 g dried microalgae/L culture as cell concentration. The dye concentration in the stock solution should be 0.25 mg/mL (this equates to 2.5
The interaction of Nile red dye with a specific lipid class emits a distinct fluorescence that peaks at a particular wavelength [
Even though microalgal lipid constitutes various lipid classes, Nile red stained microalgal cells in general emit a single broad peak between the wavelengths of 570 and 630 nm (Figure
As discussed in Section
The kinetic study should be carried out with the carrier solvent and at the optimal emission wavelength obtained from previous steps. The fluorescence intensity is to be recorded at regular intervals for 30 min after staining. Since most of the species in Table
As discussed in Section
Refinement of the dye concentration should be carried out at the optimal emission wavelength and staining time obtained from the previous steps. Given the wide range of optimal dye concentration reported in Table
A linear calibration curve that correlates the volumetric lipid content of the microalgal species (
From Steps
The calibration curve is now ready to be used for lipid quantification of different cultures of the microalgal species. To find the volumetric lipid content of a particular culture, check the OD of the culture and dilute appropriately until it reaches the OD equivalent to cell concentration of 0.1 g dried microalgae/L culture. Stain the diluted culture using the optimized staining protocol and compare its fluorescence value to the calibration curve.
Nile red staining in microalgal cells is a complex phenomenon, the exact mechanism of which is currently still unknown. Based on the findings from this study as well as other previous studies on microalgal Nile red staining, we are able to piece together a possible mechanism for the staining process. We propose the Nile red fluorescence in microalgal cells to be a five-stage process (Figure
A schematic diagram showing the pathway that drives Nile red interaction with microalgal intracellular lipid. Stage
Since Nile red dye is hydrophobic in nature, it needs to be dissolved in a polar carrier solvent to facilitate cell permeation [
The carrier solvent will interact with the polar components of both the cell wall and the cell membrane. This interaction will enable the dye-solvent complex to diffuse across the cell wall/membrane structure and penetrate into the cell.
The dye-solvent complex will now diffuse within the cell to find the lipid globule. For simplification, Figure
The dye will bind with the lipid globule to form a dye-lipid complex. As a result of this interaction, the complex is able to absorb blue-green photons (490–530 nm), reach an excited state, and emit photons of lower energy level between 560 and 635 nm (yellow and red). The stronger the interaction between the dye and the lipid is, the more intense the resulting fluorescence becomes. The strength of dye/lipid interaction is a direct function of the amount of lipid, the ratio of Nile red dye to lipid (or Nile red dye concentration), and the poststaining time [
Residual carrier solvent will now diffuse out of the cell back to the surrounding medium.
In the near future, microalgal biofuel is predicted to play a significant role in the global provision of sustainable energy. A critical understanding of Nile red behaviour as a rapid diagnostic tool for microalgal lipid quantification is needed for the identification of lipid-rich species and the monitoring of lipid content when optimizing growth conditions.
Through statistical modelling, this study provided new insights into the behaviour of microalgal Nile red staining. A quadratic model that described Nile red fluorescence intensity in microalgal cells (
By compiling findings from previous microalgal Nile red research, the study also demonstrated the wide diversity in the staining conditions used across the different species. Using insights gained from this study and previous Nile red studies, we established a simple six-step operating framework for the optimization of Nile red staining procedure across different microalgal strains. The framework enables future studies to accurately and rapidly optimize the staining protocol according to the specific requirements of the species being investigated. This will in turn improve the robustness and reliability of the staining method as a diagnostic tool to measure the amount of accumulated microalgal lipid.
The authors declare that there is no conflict of interests regarding the publication of this paper.