Journal of Microbiological Methods 125 (2016) 28–32

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Journal of Microbiological Methods

journal homepage: www.elsevier.com/locate/jmicmeth

A quick colorimetric method for total lipid quantification in microalgae

Avinesh R. Byreddy, Adarsha Gupta, Colin J. Barrow, Munish Puri ⁎
Centre for Chemistry and Biotechnology, School of Life and Environmental Sciences, Waurn Ponds, Geelong, Deakin University, Victoria 3217, Australia

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

Article history: Discovering microalgae with high lipid productivity are among the key milestones for achieving sustainable bio-
Received 2 March 2016 diesel production. Current methods of lipid quantification are time intensive and costly. A rapid colorimetric
Received in revised form 1 April 2016 method based on sulfo-phospho-vanillin (SPV) reaction was developed for the quantification of microbial lipids
Accepted 1 April 2016
to facilitate screening for lipid producing microalgae. This method was successfully tested on marine
Available online 2 April 2016
thraustochytrid strains and vegetable oils. The colorimetric method results correlated well with gravimetric
Keywords:
method estimates. The new method was less time consuming than gravimetric analysis and is quantitative for
Algae lipid determination, even in the presence of carbohydrates, proteins and glycerol.
Biodiesel © 2016 Elsevier B.V. All rights reserved.
Industrial biotechnology
Esterification
Vanillin

1. Introduction Bauen, 2013). Glucose can constitute 80% of total medium cost and up
to a third of total fermentation production costs (Li et al., 2007). To re-
Biodiesel, which refers to long chain alkyl fatty acid esters, is a re- duce these process costs and assist in the commercialization of biodiesel
newable fuel that has attracted more attention in recent years production from heterotrophic microalgae, sustainable low-cost carbon
(Sadeghinezhad et al., 2014, Puri et al., 2013). First generation biodiesel sources are required. One approach is to couple microalgae cultivation
was produced from biomass, vegetable oils, animal fats, and wasting oils with the use of inexpensive carbon sources such as carbon rich waste
by transesterification (van Eijck et al., 2014). The use of edible oils for water, or use of glycerol derived from the biodiesel industry (Di
first-generation biodiesel has resulted in the undesired effect of increas- Caprio et al., 2015, Quiroz Arita et al., 2015). Production costs can also
ing global competition for food crops (Kumar and Sharma, 2015, Pinzi be decreased by discovering new microalgae with improved lipid pro-
et al., 2014). Second-generation biofuels utilize non-competing crops ductivity (Ahmad et al., 2015). Existing techniques used for the screen-
such as jatropha, karanja, jojoba, mahua as well as the use of waste ing and quantification of microbial lipids are gravimetric,
cooking oil, grease, and animal fats. While the use of “inedible” crops re- chromatographic and fluorescence dye methods (Bligh and Dyer,
duces competition with food crops, second-generation biofuels still 1959, Han et al., 2011). Analysis of microbial lipids by gravimetric
compete for arable land, which ultimately negatively impacts the method is usually effective, but has limitations. The gravimetric method
world food supply and can contribute to the destruction of soil re- is time-intensive, requires the use of toxic chemicals and utilizes more
sources (Kiss and Bildea, 2012). In addition, these feedstocks cannot biomass. Spectrophotometric approaches can use a fluorescent dye,
meet current energy demands (Nautiyal et al., 2014, Zhu et al., 2014). Nile Red, for lipid quantification. However, the sensitivity of this method
Sustainable alternatives must be found that can provide the large fuel is poor and microbial cell components such as proteins, pigments and
volumes required, while not compromising the use of agricultural some other environmental factors may interfere with lipid quantifica-
land for food production. Microalgae have attracted attention as an al- tion using these methods (Ren et al., 2015). A disadvantage of using
ternative feedstock for biodiesel production (Bahadar and Bilal Khan, fluorescent dye is it can photo-bleach during exposure to light (Cheng
2013, Mallick et al., 2012). Microalgae are easy to cultivate perhaps bet- et al., 2011). Microbial lipid quantification using triethanol-amine cop-
ter on non-arable land. They also accumulate more lipids as a percent- per salts is an alternative method, however, analyses of fatty acids in
age of their biomass than can plants (Challagulla et al., 2015). the ultraviolet region results without any colour development and sa-
Production of biodiesel from heterotrophic microalgae requires ex- ponification and fatty acids extraction required prior quantification
pensive nutrients particularly glucose as a carbon source (Slade and (Wawrik and Harriman, 2010).
Thraustochytrids, marine heterokonts and classified as oleaginous
microorganism, have been identified as a potential candidate for feed-
⁎ Corresponding author at: Bioprocessing Laboratory, Centre for Chemistry and
stock in the heterotrophic production of lipids (Gupta et al., 2016). Ex-
Biotechnology, Waurn Ponds, Geelong, Deakin University, Victoria 3217, Australia. tensive screening of marine habitats led to the discovery of strains
E-mail address: [email protected] (M. Puri). which produce more than 50% of their biomass as lipids (Chen et al.,

http://dx.doi.org/10.1016/j.mimet.2016.04.002
0167-7012/© 2016 Elsevier B.V. All rights reserved.
 A.R. Byreddy et al. / Journal of Microbiological Methods 125 (2016) 28–32 29

2015). In this study, we developed a colorimetric sulfo-phospho-

vanillin (SPV) method (initially developed for the serum lipids quantifi-
cation (Charbol and Charonnat, 1937)) for quantifying of lipids in
thraustochytrids and other oils containing materials. The accuracy of
the SPV method was compared with gravimetric and gas chromatogra-
phy analysis. This method is useful for rapid screening of microalgal
strains for biodiesel production.

2. Materials and methods

2.1. Chemicals used

All chemicals and medium components used in this study were pro-
cured from Sigma-Aldrich, Australia. Instant ocean sea salts were pro- Fig. 1. The increase in colour intensity was observed on increasing lipid concentration.
cured from Aquarium Systems Inc., USA. Schizochytrium oil (batch
number 11,071,602 J) was obtained from Ocean Nutrition Canada,
Canada. for 10 min and cooled for 5 min in an ice bath. Vanillin-phosphoric
acid reagent (5 ml) was added to each tube and incubated in a shaker
2.2. Cultivation of thraustochytrids incubator at 200 rpm at 37 °C for 15 min. Absorbance was measured
at 530 nm.
A total of four thraustochytrids, Schizochytrium sp. S31 (ATCC
20888), Thraustochytrium sp. PRA 296, Thraustochytrium sp. AMCQS5– 2.5. Lipid extraction
5 and Schizochytrium sp. DT3 were selected for the current study.
Thraustochytrium S31 and PRA 296 were procured from the ATCC and Lipid was extracted and quantified from freeze dried biomass every
used as standard cultures, whereas AMCQS5–5 (JX993841) and DT3 48 h according to a previously published protocol with some modifica-
(KF682125) were isolated from Australian marine samples (Gupta tions (Lewis et al., 2000). Freeze-dried biomass (10 mg) was added to
et al., 2013, Gupta et al. 2016). Seed culture was prepared in GYP me- 600 μL solvent (chloroform and methanol in 2:1 ratio) and the sample
dium containing: Glucose 0.5%, yeast extract 0.2%, peptone 0.2% in 50% vortexed for 2 min, followed by centrifugation at 13,000 rpm for
artificial seawater (Instant Ocean sea salts) and incubated at 150 rpm, 15 min. This extraction process was repeated three times. The three su-
25 °C for 48 h. The inoculum (5%) was later transferred into production pernatants were collected and dried at 50 °C. The lipid percentage was
media containing glycerol (1%), yeast extract (0.2%) and peptone (0.2%) measured gravimetrically.
and incubated at 150 rpm, 25 °C for 120 h. The cultures were harvested All the experiments were performed in triplicates. Results are repre-
by centrifugation at 8000 rpm for 10 min, freeze dried and stored at sented as mean values and standard deviation is b10%.
−20 °C for further use.
3. Results and discussion
2.3. Standard curve preparation
3.1. Biomass and lipid production
For preparing standard curve, commercial Schizochytrium oil was
dissolved in (10 mg in 10 mL) chloroform (final concentration Production of biodiesel from inexpensive carbon sources will reduce
1 mg mL−1). For the preparation of standard curve, different concentra- oil production costs. Glycerol has been suggested as one of the econom-
tions (15–90 μg) of lipid samples were taken in clean glass tubes. The ical carbon source in microbial fermentation and is used extensively for
tubes were incubated at 60 °C for 10 min to evaporate the chloroform. growing thraustochytrids (Leite et al., 2015, Scott et al., 2011). After
100 μL of distilled water was added to each tube. Lipid quantification 5 days, the biomass obtained were 0.93 g L−1 d − 1 in DT3,
was done by SPV reaction. 0.95 g L−1 d −1 in S31, 0.74 g L−1 d −1 in AMCQS5–5 and 0.25 g L−1 d
−1
in PRA-296 (Table 1). Schizochytrium sp. S31 and DT3 produced sim-
2.4. Quantification of thraustochytrid lipids by SPV ilar biomass yields whereas, Thraustochytrium sp. AMCQS5–5 and PRA-

Vanillin phosphoric acid reagent was prepared by dissolving 120 mg

of vanillin in 20 mL of distilled water and the final volume was adjusted
to 100 mL with 85% phosphoric acid. This reagent was stored in dark
conditions until further use. SPV reagent was prepared freshly, which
resulted in high activity with lipid samples. A known amount of
thraustochytrid dried biomass was suspended in distilled water and
added to each test tube (0.2–1 mg). Tubes were incubated at 100 °C

Table 1
Biomass and lipid production in glycerol medium by thraustochytrids.

Item DT3 S31 AMCQS5–5 PRA-296

Biomass (g L−1) 4.69 ± 0.09 4.75 ± 0.1 3.7 ± 0.01 1.28 ± 0.005
Biomass productivity 0.93 ± 0.19 0.95 ± 0.02 0.74 ± 0.002 0.25 ± 0.001
(g L−1 d −1)
Average lipid content 1.43 ± 0.09 1.13 ± 0.06 0.79 ± 0.04 0.25 ± 15
(g L−1)
Lipid productivity 0.28 ± 0.02 0.22 ± 0.01 0.15 ± 0.01 0.05 ± 0.004
Fig. 2. Comparison of various lipids other compounds with sulfo-phospho-vanillin
(g L−1 d −1)
reaction.
30 A.R. Byreddy et al. / Journal of Microbiological Methods 125 (2016) 28–32

section. Lipid content was measured by plotting absorbance at 530 nm

and lipid concentration. A high correlation coefficient was obtained
with R2 value 0.9939 (Fig. 1). This demonstrates the high accuracy of
the SPV method in quantifying lipids in aqueous samples. Interference
by other cellular and medium components such as protein (BSA), carbo-
hydrate (Glucose) and glycerol could not react with SPV reagent (Fig. 2),
which is in consistent with a similar study (Izard and Limberger, 2003).
These medium compounds were used as false positives. Thus it can be
said that other compounds in the medium showed no effect on the ab-
sorbance value.
The basic principle underlying the SPV method is a three step reac-
tion. When the lipids are heated in the presence of sulphuric acid at boil-
ing temperature (low temperature leads to lower reaction rates), a
carbonium ion is formed. An aromatic phosphate ester is formed from
the reaction of vanillin and phosphoric acid. The carbonyl group of
phospho-vanillin reagent forms a stable pink product on reaction with
the carbonium ions (Knight et al., 1972). The product formation de-
Fig. 3. Colorimetric detection of thraustochytrid whole cell lipids. Footnote: Relationship pends mainly on the molecular structure (carbon–carbon double
between the concentration of biomass and absorbance at 530 nm for DT3 (R2 = 0.992), bond) of fatty acids present in the lipids. Thus the molecules which do
S31 (R2 = 0.999), AMCQS5–5 (R2 = 0.993) and PRA-296 (R2 = 0.998).
not possess double bonds did not detectably react with sulfo-
phospho-vanillin reagent. However, colour intensity of one lipid to the
296 exhibited lower biomass productivity dependent on how efficiently other varies due to the difference in fatty acids. It was observed that
the strain utilized the carbon source to produce biomass. However, lipid the colour intensity of the triolein was greater than the polyunsaturated
productivity was documented to be higher in DT3 at 0.28 g L−1 d −1 fatty acids, EPA and DHA (Fig. 2). Presence of multiple double bonds in
when compared to other strains. The next highest biomass was S31 EPA and DHA did not yield more colour due to steric hindrance of their
(0.23 g L−1 d −1) followed by AMCQS5–5 (0.16 g L−1 d −1) and PRA- structure. Saturated fatty acids such as palmitate and stearate were not
296 (0.05 g L−1 d −1) (Table 1). able to react with sulfo-phospho-vanillin reagent (Frings and Dunn,
1970, Mishra et al., 2014). Among the plant oils tested in this study,
3.2. Microalgae oil quantification by SPV method olive oil was observed for maximum colour formation than other oils
such as canola and sunflower oil. The reason may be the presence of
The colorimetric method was first validated to generate the calibra- more than 70% oleic acid content in olive oil. Presence of more unsatu-
tion curve with known quantities of Schizochytrium oil. A known quan- rated fatty acid molecules will lead to the higher absorbance, although
tity of lipid standard in the range of 15–90 μg was taken in clean test the presence of multiple double bonds in a single fatty acid (polyunsat-
tubes and placed in a hot air oven at 60 °C to evaporate chloroform, urated) will not lead to the formation of intense colour. This observation
followed by the SPV reaction protocol described in the experiment stipulates that the reaction is specific to the concentration and the

Fig. 4. Change of colure after reaction with sulfo-phospho-vanillin reagent. Footnote: Increase of colour represents the correlation between the concentrations of lipid in various
thraustochytrids cells such as (a) DT3, (b) S31, (c) AMCQS5–5 and (d) PRA-296 Footnote: Triolein yielded intense colour compared with other lipid standards. Compounds with no
bonds in their structure fail to react with SPV reagent.
 A.R. Byreddy et al. / Journal of Microbiological Methods 125 (2016) 28–32 31

Table 2 screen the potential lipid producing strains from the environmental
Comparison of thraustochytrid lipid content by SPV and gravimetric methods. samples. In this study, we used a colorimetric method (SPV) to quantify
Thraustochytrid Lipid percentage by Lipid percentage by Difference the total lipids in an aqueous medium containing thraustochytrids, a po-
strains SPV method gravimetric method tential lipid producer. The lowest amount of biomass was used for quan-
DT3 33.3 ± 1.2 30.7 ± 0.59 2.6 tifying the lipid content was 50 μg. This allowed us to quantify the lipids
S31 25.8 ± 0.9 23.9 ± 0.86 1.9 from the thraustochytrids cells by skipping the extraction step. More-
AMCQS5–5 22.8 ± 1.1 21.4 ± 0.78 1.4 over, the colorimetric method not only quantifies lipid content accu-
PRA-296 18.0 ± 1.4 20.2 ± 0.62 2.2
rately but also requires not more than one hour to complete. In
contrast, gravimetric quantification of lipids, extraction and GC analysis
is both more time and labour intensive.
chemical structure of the samples. The reason for this reaction is the for-
mation of a single carbonium ion per molecule and steric hindrance ef- 3.4. Validating the accuracy of the SPV method
fect in polyunsaturated fatty acids (Frings and Dunn 1970).
The presence of various fatty acids in microbial lipids limits the accu- Gravimetric quantification of lipids from the four selected
racy of the method. This can be rectified by selecting suitable oil with thraustochytrids was done to validate the accuracy of the SPV method.
the same fatty acid content as a reference. Previous studies have re- Thraustochytrid lipids were extracted using a conventional solvent ex-
ported the quantification of bacterial and microalgal lipids with refer- traction method with modifications. The total lipid percentages deter-
ence to triolein and canola oil, respectively (Knight et al., 1972, Mishra mined gravimetrically from four thraustochytrids DT3, S31, AMCQS5–
et al., 2014). We used the commercial Schizochytrium oil as reference 5 and PRA-296 were 33.3%, 25.8%, 22.8% and 18%, respectively. Lipid
for precise calibration of lipids in thraustochytrids. percentages determined also by the SPV method showed similar quan-
tities in all thraustochytrids. Lipid contents determined by SPV and
3.3. Lipid quantification in thraustochytrid whole cells via SPV method gravimetric methods were compared to understand the correlation
(Table 2). This result confirms the reliability of the SPV method in quan-
The established colorimetric method was applied to the quantifica- tifying the total lipids from thraustochytrid whole cells. Table 3 summa-
tion of lipids in whole thraustochytrid cells. Fig. 3 shows the coefficient rizes highlights and limitations of different methods reported for rapid
of determination between biomass concentration and absorbance with lipid quantification in oleaginous microorganisms.
R2 N 0.99. All four cultures showed an increase in the intensity of pink Microalgal lipids are a potential primary component in biodiesel
colour formation with respect to the increasing biomass concentration production. Isolation and identification of the potential lipid-
(Fig. 4). Lipid content in all four cultures was quantified by converting producing microalgae is a key step in achieving economically sustain-
the absorbance into mass using a reference curve with a low standard able biodiesel production. Quantification of lipids from microalgae
deviation. Strain development and lipid quantification are major mile- must be performed frequently in microbial screening and other re-
stones on the path to sustainable production of biodiesel from the mi- search areas, such as in the screening of genetically engineered mi-
crobial sources. A rapid lipid quantification technique is required to crobes for high lipid production.

Table 3
Comparison of rapid lipid quantification methods and its limitations.

Method Microorganisms Standard Highlights/limitations References

oil/process
time (min)

Sulfo-phospho-vanillin Escherichia coli and Triolein/40 • Suitability of SPV methods for screening mutant bacte- Izard and
Treponema denticola rial strains Limberger (2003)
• Lipid detection was limited to Triolein, tripalmitin and
phospholipids
Triethanol-amine Phaeodactylum tricornutum and Chlorella vulgaris Laurate and • Saponification and fatty acids extraction required prior Wawrik and
copper salts CM2 decanoate/42 to quantification Harriman (2010)
• Assay showed lower signal with PUFAs
Sulfo-phospho-vanillin Chlorella sp. Corn oil/42 • Optimized the method in 96 well plate Cheng et al. (2011)
• Used the extracted and purified lipids for assay
Nile red Ascomycete and Basidiomycete yeast NA/20 • Optimized the parameters which influence fluores- Sitepu et al. (2012)
cence emission
• Inconsistence lipid quantification due to variable dye
diffusion across membranes
• Accuracy of the method is dependent on microalgae
strain and solvent used
• Limited photo-stability and interference with
chlorophyll
BODIPY Chlorella vulgaris, Dunaliella primolecta and NA/10 • BODIPY quantifies range of lipids such as fatty acids, Govender et al.
Chaetoceros calcitrans phospholipids and esters (2012); Rumin
• Exceptional photo-stability et al. (2015)
• Background fluorescence of the dye in the medium and
ii) failure to quantify neutral lipids between rich and
low oil strains.
Sulfo-phospho-vanillin Ettlia sp., Monoraphidium sp., Chlorella vulgaris and Canola/30 • Quantify lipids in microalgae Mishra et al.
Nannochloropsis oceanica • Lipid quantity was measured with standard canola oil (2014)
• Accuracy of the method depends on fatty acid compo-
sition of the oil
Sulfo-phospho-vanillin Schizochytrium sp. S31, Thraustochytrium sp. PRA Schizochytrium • Lipid quantified in different plant oils and PUFAs (DHA This study
296, Thraustochytrium sp. AMCQS5–5 and oil/30 and EPA)
Schizochytrium sp. DT3 • Schizochytrium oil used as standard to quantify lipid
from thraustochytrids
• This method measures fatty acids in oil samples
32 A.R. Byreddy et al. / Journal of Microbiological Methods 125 (2016) 28–32

4. Conclusion Izard, J., Limberger, R.J., 2003. Rapid screening method for quantitation of bacterial cell
lipids from whole cells. J. Microbiol. Methods 55, 411–418.
Kiss, A.A., Bildea, C.S., 2012. A review of biodiesel production by integrated reactive sepa-
This study demonstrated the use of sulfo-phospho-vanillin reaction ration technologies. J. Chem. Technol. Biotechnol. 87, 861–879.
to quantify lipids accumulation in thraustochytrids. The SPV method re- Knight, J.A., Anderson, S., Rawle, J.M., 1972. Chemical basis of the sulfo-phospho-vanillin
reaction for estimating total serum lipids. Clin. Chem. 18, 199–202.
sulted in similar lipid content determination in thraustochytrid whole Kumar, M., Sharma, M.P., 2015. Assessment of potential of oils for biodiesel production.
cells as did gravimetric/gas chromatography analysis. Triolein contains Renew. Sust. Energ. Rev. 44, 814–823.
only mono-unsaturated fatty acids and thus the colour intensity was Leite, G.B., Paranjape, K., Abdelaziz, A.E.M., Hallenbeck, P.C., 2015. Utilization of biodiesel-
derived glycerol or xylose for increased growth and lipid production by indigenous
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Carbohydrates, proteins and glycerol did not interfere in the SPV reac- Lewis, T., Nichols, P.D., McMeekin, T.A., 2000. Evaluation of extraction methods for recov-
tion. SPV is a rapid method that requires low levels of chemicals and ery of fatty acids from lipid-producing microheterotrophs. J. Microbiol. Methods 43,
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so is useful for the screening of microalgal cultures for biodiesel
Li, X., Xu, H., Wu, Q., 2007. Large-scale biodiesel production from microalga Chlorella
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This work was funded by Australia–India strategic research fund Mishra, S.K., Suh, W.I., Farooq, W., Moon, M., Shrivastav, A., Park, M.S., Yang, J.-W., 2014.
(AISRF#BF050044). Avinesh Byreddy and Adarsha Gupta acknowledge Rapid quantification of microalgal lipids in aqueous medium by a simple colorimetric
method. Bioresour. Technol. 155, 330–333.
the PhD scholarship awarded by Deakin University, Australia. Nautiyal, P., Subramanian, K.A., Dastidar, M.G., 2014. Production and characterization of
biodiesel from algae. Fuel Process. Technol. 120, 79–88.
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