Algal Research 7 (2015) 11–15

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Algal Research
journal homepage: www.elsevier.com/locate/algal

Short communication

Improved Nile red staining of Scenedesmus sp. by combining ultrasonic

treatment and three-dimensional excitation emission matrix
fluorescence spectroscopy
Hong-Yu Ren, Bing-Feng Liu ⁎, Fanying Kong, Lei Zhao, Nan-Qi Ren ⁎
State Key Laboratory of Urban Water Resource and Environment, School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150090, China

a r t i c l e i n f o a b s t r a c t

Article history: To solve the quantitative limitations of the gravimetric method and the traditional Nile red method, a modified
Received 13 May 2014 method combining ultrasonic treatment with three-dimensional excitation emission matrix (3D EEM) fluores-
Received in revised form 8 November 2014 cence spectroscopy was used to select the optimal excitation/emission wavelength (Ex/Em) and exactly detect
Accepted 16 November 2014
the lipid content of microalgae. Compared with the traditional Nile red method, this modified ultrasound-
Available online xxxx
assisted method can effectively disrupt cell walls and significantly improve staining efficiency. EEM analysis re-
Keywords:
vealed that the fluorescence intensity peak appeared at an Ex/Em of 530/568 nm for Scenedesmus sp. The fluores-
Excitation emission matrix cence intensity was increased 4.3 fold from 210.2 to 895.4 a.u. at an optimal ultrasonic power of 104 W, time of
Lipid 5.0 min and Nile red concentration of 1.5 mg L−1. A high correlation (R2: 0.9957) between the relative fluores-
Microalga cence intensity and lipid concentration was obtained. These results demonstrated the feasibility of the modified
Nile red method, exhibiting especially significant effectiveness in determining the lipid content of microalgae with rigid
Ultrasound and thick cell walls.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction 5-one) method to achieve this purpose; however, the gravimetric meth-
od is complicated and time-consuming (approximately 3–4 days) [13,
Currently, the world's energy and fuels are mostly obtained from 14]. This method is unsuitable for the rapid screening of oleaginous
fossil-based natural resources such as petroleum, coal and natural gas microalgal strains. Nile red is a hydrophobic fluorescent dye frequently
[1,2]. The use of these finite resources has caused increasing environ- used in the determination of microalgal lipid content because the fluo-
mental pollution and severe climate change [3,4], driving an intensi- rescence signal associated with Nile red is linearly correlated with the
fying demand for sustainable and renewable energy sources that can triglyceride (TAG) content in microalgae [13,15,16]. It has been success-
reduce the dependency on fossil fuel [5,6]. Biodiesel, especially fully applied in some algal species (Table 1). However, in some green
microalgae-based biodiesel, has drawn more and more attention as microalgae with rigid and thick cell walls, the Nile red can be prevented
an alternative and environmentally friendly fuel [7,8]. Microalgae from binding the intracellular lipids, resulting in a much lower estimat-
are widely known as a promising feedstock for biodiesel production ed lipid content than the actual value [11]. Therefore, the Nile red meth-
due to their rapid growth rate, high lipid content (more than 50% dry od should be modified through pre-treatment procedures. Dimethyl
weight) and high area-specific yield relative to agricultural oleaginous sulfoxide (DMSO) and microwaves have been successfully used as pre-
crops [9,10]. treatment procedures in determining the lipid content of Chlorella
The quantification of microalgal lipid content is critical to the selec- vulgaris and Pseudochlorococcum sp., which cannot be stained by the tra-
tion of microalgal strains with high biomass and a large amount of neu- ditional Nile red method [11,18]. However, these modified Nile red
tral lipids for the commercial production of renewable biodiesel [11,12]. methods are still ineffective in staining green algae with thicker cell
Therefore, it is essential to create rapid and effective methods to screen walls like Scenedesmus sp. R-16. Ultrasound is the use of sound waves
for lipid-rich algal strains. Previous studies used the gravimetric method with frequencies higher than human hearing range to cause high-
and the Nile red (9-diethylamino-5H-benzo[α]phenoxa-phenoxazine- energy chemical reactions [27]. The shock waves due to acoustic cavita-
tion have been found to result in cellular disruption of microalgae [28].
Ultrasound is able to disrupt the cells at a low temperature with less
⁎ Corresponding authors at: State Key Laboratory of Urban Water Resource and thermal protein denaturation compared to microwaves and autoclaving
Environment, School of Municipal and Environmental Engineering, Harbin Institute of
[29]. Moreover, it can disrupt cells with less energy loss compared to
Technology, P. O. Box 2614, 73 Huanghe Road, Harbin 150090, China.
E-mail addresses: [email protected] (H.-Y. Ren), [email protected] (B.-F. Liu), high-shear force methods [30]. More importantly, ultrasonic treatment
[email protected] (N.-Q. Ren). can be performed without the addition of chemicals or beads, which

http://dx.doi.org/10.1016/j.algal.2014.11.007
2211-9264/© 2014 Elsevier B.V. All rights reserved.
12 H.-Y. Ren et al. / Algal Research 7 (2015) 11–15

Table 1 microalgal lipid by three-dimensional excitation emission matrix (3D

Excitation and emission wavelengths used in microalgal lipid determination by Nile red EEM) fluorescence spectroscopy are quite limited.
staining.
Therefore, the green microalga Scenedesmus sp. R-16, which has a
Microalga Excitation Emission Reference thick cell wall structure, was chosen as the model algal strain. EEM
wavelength wavelength fluorescence spectroscopy was used to identify the optimum excitation
(nm) (nm)
and emission wavelengths for this microalgal species. Using the optimal
Alexandrium minutum CBA5 547 580 [17] Ex/Em, an ultrasound-assisted Nile red method was developed to im-
Ankistrodemus pseudobraunii 530 575 [18]
prove the staining efficiency. Finally, the relationship between the rela-
Botryococcus braunii FACHB-357 480 580 [19]
Botryococcus braunii UTEX 572 490 585 [20] tive fluorescence intensity and lipid concentration was confirmed.
Chaetoceros calcitrans 1085/3 480 590 [14]
Chaetoceros socialis CBA2 547 580 [17] 2. Material and methods
Chlorella protothecoides 488 570 [21]
Chlorella pyrenoidosa 480 580 [19]
Chlorella saccharophila 500 578 [22]
2.1. Alga and culture condition
Chlorella vulgaris 530 570 [23]
Chlorella vulgaris 530 575 [18] The microalga Scenedesmus sp. R-16 was cultured in BG11 medium
Chlorella vulgaris C7 480 580 [24] as described previously [32]. After adjusting the pH to 7.0 using HCl
Chlorella vulgaris CCAP211/1113 480 590 [14]
(1 mol L−1) and NaOH (1 mol L−1), the medium was sterilized at 121
Chlorella zofingiensis 490 580 [11]
Chlorella zofingiensis 530 575 [18] °C for 15 min [33]. After inoculation, the cultures were incubated at
Desmodesmus quadricauda 530 575 [18] 25 ± 1 °C in a shaker at 130 rpm [34].
Dunaliella primolecta 11/34 480 590 [14]
Dunaliella salina 488 580 [16] 2.2. Gravimetric determination
Micractinium pusillum 470 570 [25]
Microcystis aeruginosa 480 580 [19]
Nannochloris sp. 530 575 [18] Cultivated algal cells were harvested and dried. Lipids were extract-
Nannochloropsis sp. 480 575 [13] ed from the dried algal cells (100 mg or more) in accordance with the
Nannochloropsis sp. 480 580 [19] previously described method [19,23,35]. The same process was repeat-
Nitzschia closterium 530 575 [26]
ed three times for the complete extraction of the lipids from the algae.
Palmellococcus miniatus 530 575 [18]
Pseudochlorococcum sp. 490 580 [11]
All samples were measured in triplicate. The lipid concentration was
Skeletonema marinoi CBA4 547 580 [17] determined according to Eq. (1):

Lipid concentrationðmg lipid=L cultureÞ

¼ Cell dry weightðg algae=L cultureÞ
could become secondary waste streams that require further treatment  Algal lipid contentðmg lipid=g algaeÞ: ð1Þ
in the experimental process [29]. Hence, ultrasonic treatment is an
ideal pre-treatment method.
Although the Nile red method has been frequently used to deter- 2.3. EEM measurement
mine the lipid content of various algal species, the excitation and
emission wavelengths vary considerably among different classes of Algal samples were measured in a cuvette (1 cm) by a FP-6500
microalgae (Table 1). Even for the same microalgal species, the excita- fluorescence spectrophotometer (JASCO, Japan). EEM fluorescence
tion and emission wavelengths can be quite different [11,14,18,23,24]. spectroscopy was collected at scanning emission wavelengths at 1 nm
This difference might be caused by the diversity of algal strains and increments by varying the excitation wavelength at 5 nm increments
their fatty acid compositions. Because the sensitivity of the Nile red to determine the optimal excitation and emission wavelengths. The
method is determined by the wavelengths that provide the maximum intensity of EEM fluorescence spectroscopy was represented by con-
fluorescence intensity [31], it is necessary to identify the optimal excita- tour lines with the X-axis representing the emission wavelength at
tion and emission wavelengths of the tested microalgal strain prior 220–660 nm and the Y-axis representing the excitation wavelength
to fluorescence measurement. To date, reports on the observation of at 220–660 nm [36,37].

Fig. 1. Three-dimensional excitation emission matrix (3D EEM) fluorescence spectroscopy of Scenedesmus sp. (a) without ultrasonic treatment and (b) with ultrasonic treatment.
 H.-Y. Ren et al. / Algal Research 7 (2015) 11–15 13

stained cells (Fig. 1a). According to previous reports, peak A was located
in the region dominated by protein-like substances, and peak B was
located in the region dominated by soluble microbial by-product-like
materials [38,39]. It should be noted that an obscure region was ob-
served at Ex/Em of 500–600/550–620 nm. However, the fluorescence
was so weak that no obvious peak could be identified. The algal cells
stained by the traditional Nile red method without ultrasonic treatment
were examined through by a microfluorometer. Only a small amount of
the dye can penetrate the cell walls and bind the intracellular neutral
lipids (yellow fluorescence) (Fig. 2). Most algal cells appeared red
(Fig. 2) due to chlorophyll autofluorescence [40]. These results sug-
gested that the traditional Nile red method was not suitable for the
detection of lipids in Scenedesmus sp. R-16.
In contrast to the fluorescence results without ultrasonic treatment,
the ultrasound-assisted staining procedure improved the staining
Fig. 2. Fluorescence micrograph of algal cells stained with the traditional Nile red method. efficiency. After the ultrasonic treatment, a distinct and clear peak
(peak C) centered at Ex/Em of 530/568 nm was observed (Fig. 1b).
Because ultrasonic irradiation can disrupt cell wall structures [30], the
2.4. Ultrasound-assisted Nile red staining intracellular substances (e.g., lipids) in algal cells were easily released
to the aqueous medium [41,42], enhancing the Nile red fluorescence
A suspension of microalgae (5 mL) was centrifuged at 8000 g for (Fig. 3). This implied that EEM fluorescence spectroscopy coupled
10 min and washed with pure water several times. Afterwards, the col- with ultrasonic treatment was a useful and effective method to ascer-
lected algal cells were resuspended and sonicated by an ultrasonic horn tain the optimal Ex/Em. With ultrasonic treatment, the Nile red could
(Sonics VCX130PB, USA) at an ultrasonic frequency of 20 kHz and max- bind algal lipids more easily, which is beneficial to the accurate mea-
imum power output of 130 W. After Nile red solution (0.5 mg mL−1 in surement of lipid content.
acetone) was added, the mixture was gently vortexed for 1 min and fur-
ther incubated in the dark for 15 min. The fluorescence of the samples 3.2. Optimization of ultrasound-assisted Nile red method
was determined using a fluorescence spectrophotometer. The fluores-
cence of the microalgae was measured before and after the addition of Ultrasound has also been employed to increase the efficiency of algal
Nile red to subtract the intrinsic fluorescence of the algae. In addition, lipid extraction in recent studies [28]. It can reduce the reaction time
the fluorescence of Nile red alone was also measured. The relative and enhance mass transfer [42,43] and has been demonstrated to be
fluorescence of Nile red for lipids was obtained after subtraction of effective in disrupting algal cells and improving Nile red staining effi-
both the autofluorescence of microalgae and the self-fluorescence of ciency [28–30,44]. Considering the important role of ultrasound in the
Nile red. The excitation and emission wavelengths used in this ex- Nile red method, the effects of three factors (ultrasonic power, time
periment were 530 and 568 nm, respectively. Each experiment was and Nile red concentration) on fluorescence intensity were explored.
performed in triplicate. An excitation wavelength of 530 nm and emission wavelength of
568 nm were used as described in the above experiments.
3. Results and discussion Different ultrasonic powers (0, 26, 52, 78, 104 and 130 W) were
compared at an ultrasonic irradiation of 20 kHz. The results showed
3.1. Fluorescence properties with and without ultrasonic treatment that the fluorescence intensity increased 4.3-fold, from 210.2 to 895.4
a.u., when the ultrasonic treatment power increased from 0 to 104 W
Fluorescence properties were investigated with and without ultra- (Fig. 4a). However, further increasing the power to 130 W did not
sonic treatment to determine the effect of ultrasound. Without ultra- yield a sustained increase. The fluorescence intensity decreased sharply
sonic treatment, two distinct peaks at Ex/Em of 220–230/330–350 nm to 448.6 a.u., possibly because of lipid degradation by high ultrasonic
(peak A) and 270–280/330–350 nm (peak B) were identified in the power [45]. Considering that the Nile Red fluorescence intensity linearly

Fig. 3. Schematic of ultrasound-assisted Nile red staining.

14 H.-Y. Ren et al. / Algal Research 7 (2015) 11–15

1000
(a) ultrasonic treatment time (5.0 min) can enhance the staining efficiency
of Nile red.
800 To optimize the Nile red concentration, five concentrations (0, 0.5,
1.0, 1.5 and 3.0 mg L− 1) were evaluated at an ultrasonic power of
Intensity (a. u.)

104 W for 5.0 min. When the Nile red concentration increased from 0
600
to 1.5 mg L− 1, the fluorescence intensity gradually increased from
23.0 to 907.3 a.u. (Fig. 4c). However, further increase of the Nile red
400 concentration to 3.0 mg L−1 did not enhance the fluorescence intensity.
To minimize the inhibitory effect of Nile red solution, 1.5 mg L−1 Nile
red was selected for further ultrasound-assisted staining. It is worth
200
noting that the optimal Nile red concentration under our experimental
condition is lower than those used in staining C. vulgaris and Chlorella
0 protothecoides [21,40].
0 26 52 78 104 130
Ultrasonic treatment power (W)
3.3. Relationship between relative fluorescence intensity and
1000 lipid concentration
(b)

800 The relative fluorescence intensity of ultrasound-treated cells

stained with Nile red was calculated and compared to the gravimetric
measurement. There was a strong linear correlation between the rela-
Intensity (a. u.)

600 tive fluorescence intensity and the lipid concentration (Fig. 5). This linear
relationship can be expressed as y = 37.141x + 60.225 (x: lipid concen-
400 tration; y: relative fluorescence intensity; R2: 0.9957). These results
showed that the ultrasound-assisted Nile red staining method was ap-
propriate for the determination of the lipid content in the microalga
200
Scenedesmus sp. R-16. Compared with the gravimetric determination,
the proposed method was easier to perform and can be accomplished
0 in a shorter time. It also eliminated toxic solvents, avoiding potential
0 2.5 5.0 7.5 10.0
harm to researchers and the environment [20].
Ultrasonic treatment time (min)
For microalgae with thick cell walls, the suggested procedure to
1000 determine the algal lipid content is as follows: (1) Identify the optimal
(c) Ex/Em for algae by combining ultrasonic treatment and EEM fluores-
cence spectroscopy, (2) explore the relationship between relative fluo-
800
rescence intensity and lipid concentration and (3) calculate the lipid
content using the quantitative equation.
Intensity (a. u.)

600
4. Conclusions
400
To overcome the drawbacks of the gravimetric method and the
traditional Nile red method, this study reports a new modified Nile
200 red method combining ultrasonic treatment and 3D EEM fluores-
cence spectroscopy to detect the optimal excitation/emission wave-
0 length and then determine the algal lipid content. This procedure
0 0.5 1.0 1.5 3.0 was demonstrated to be theoretically feasible. Compared with tradi-
-1
Nile red concentration (mg L )
tional methods, it was easier, faster and more efficient, especially for
oleaginous algal species with thick and rigid cell walls. Because dif-
Fig. 4. Effects of (a) ultrasonic treatment power, (b) ultrasonic treatment time and (c) Nile
ferent microalgal species or strains may exhibit different excitation–
red concentration on the fluorescence intensity of green alga Scenedesmus sp.

correlated with microalgal lipid levels [17,20,40], a low fluorescence in- 1200
Relative fluorescence intensity (a.u.)

tensity might be obtained when the lipid concentration declined due to y = 37.141x + 60.225
degradation caused by the increased ultrasonic power. The above re- 1000 R2 = 0.9957
sults revealed that 104 W was the optimal power for ultrasound-
assisted irradiation. This power was therefore used in subsequent 800
experiments.
Various time intervals of ultrasonic treatment were used, ranging 600
from 0 to 10.0 min. The results demonstrated that the ultrasonic
treatment time had a dramatic effect on fluorescence intensity. The 400
fluorescence intensity increased more than two times when ultrasonic
200
treatment time was increased from 0 to 5.0 min (Fig. 4b). When it
was more than 5.0 min, the fluorescence intensity decreased markedly
0
to 609.8 a.u. Given that oxidation is the main cause of lipid degradation 0 5 10 15 20 25 30
[46], sonication energy beyond the level needed for algal cell disruption Lipid concentration (mg L-1)
might induce lipid oxidation [29]. These results suggested that long
ultrasonic treatment could lead to the degradation of lipids and unfa- Fig. 5. Linear correlation between relative fluorescence intensity and lipid concentration
vorably affect the Nile red fluorescence intensity. Thus, appropriate determined by the gravimetric method for Scenedesmus sp.
 H.-Y. Ren et al. / Algal Research 7 (2015) 11–15 15

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