Bioresource Technology 162 (2014) 38–44
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Enhancing growth and lipid production of marine microalgae for biodiesel production via the use of different LED wavelengths Chee Loong Teo a, Madiha Atta a, Attaullah Bukhari a, Mohamad Taisir b, Afendi M. Yusuf b, Ani Idris a,⇑ a Department of Bioprocess Engineering, Faculty of Chemical Engineering, c/o Institute of Bioproduct Development (IBD), Universiti Teknologi Malaysia, 81310 UTM Johor Bahru, Johor, Malaysia b Faculty of Mechanical Engineering, Universiti Teknologi Malaysia, c/o B.M. Nagano Industries Sdn. Bhd., Jalan Keluli 3, Kawasan Perindustrian Pasir Gudang, 81700 Johor, Malaysia
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Different LED wavelengths were used
for the cultivation of marine microalgae. LED wavelengths used were blue (457 nm), red (660 nm) and red mix blue. The results were also compared to white fluorescent lamp. Microalgae showed highest growth rate and lipid production under blue LED wavelength. Analysis showed major FAME components were palmitic acid and stearic acid.
a r t i c l e
i n f o
Article history: Received 20 January 2014 Received in revised form 17 March 2014 Accepted 21 March 2014 Available online 1 April 2014 Keywords: Nannochloropsis sp. Tetraselmis sp. LED Wavelength Biodiesel
a b s t r a c t Wavelength of light is a crucial factor which renders microalgae as the potential biodiesel. In this study, Tetraselmis sp. and Nannochloropsis sp. as famous targets were selected. The effect of different light wavelengths on growth rate and lipid production was studied. Microalgae were cultivated for 14 days as under blue, red, red-blue LED and white fluorescent light. The growth rate of microalgae was analyzed by spectrophotometer and cell counting while oil production under improved Nile red method. Optical density result showed the microalgae exhibited better growth curve under blue wavelength. Besides, Tetraselmis sp. and Nannochloropsis sp. under blue wavelength showed the higher growth rate (1.47 and 1.64 day1) and oil production (102.954 and 702.366 a.u.). Gas chromatography analysis also showed that palmitic acid and stearic acid which were compulsory components for biodiesel contribute around 49–51% of total FAME from Nannochloropsis sp. and 81–83% of total FAME from Tetraselmis sp. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction The insufficient petroleum reserves, extreme dependence on foreign energy resource, increasing crude oil prices and current environmental concerns have encouraged scientists to find for alternative environmentally friendly and renewable energy. (Geor⇑ Corresponding author. Tel.: +60 7 5535603; fax: +60 7 5588166. E-mail address: [email protected] (A. Idris). http://dx.doi.org/10.1016/j.biortech.2014.03.113 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
gogianni et al., 2008; Meher et al., 2006; Narasimharao et al., 2007). Biodiesel is seen as the alternative renewable energy because it is sustainable with less harm to the environment (McKendry, 2002; Reijnders, 2006). Biodiesel from microalgae which is considered as the 3rd generation renewable biofuels is believed to be capable of meeting the whole world demand for transport fuels (Chisti, 2007) because marine microalgae can generate as much as 40 times crude oil than other plants per acre of land area (Schenk et al., 2008).
C.L. Teo et al. / Bioresource Technology 162 (2014) 38–44
Marine microalgae are photoautotrophic microorganisms thus light is a very essential factor to growth rate (Adir et al., 2003; Ragni et al., 2008). Light spectral quality and light intensity (quantity) must be considered when choosing the light source for microalgae cultivation. Spectral quality is defined by the absorption spectrum (light wavelengths) of the chlorophyll and other photosynthetically active pigments in microalgae such as phycobilins and carotenoids (Lee, 1999). Energy absorption by photosynthetic organisms is thus dependant on the chemical nature of their constitutive pigments (Carvalho et al., 2011). All chlorophylls (green pigments) have two major absorption bands: blue or blue-green (450–475 nm) and red (630–675 nm). The growth of marine microalgae will be improved by using either red light or blue light (Korbee et al., 2005). Chlorophyll a is the core of reaction pigment, while accessory pigments chlorophyll b, c and d extend the range of light absorption (Richmond, 2004). Light intensity should be delivered evenly over the illuminating surface of culture vessel and with adequate amount of photosynthetically active radiation (PAR) to enable photons reach the cell in culture (Lee, 1999). Excessive intensity may lead to photooxidation and photoinhibition, while low light levels will become growth-limiting (Loera-Quezada et al., 2011). Due to this, the light intensity at which culture growth becomes saturated is an important factor in determining the light utilization efficiency. In general, the growth of many photosynthetic microorganisms gets saturated at about 200 lmol m2 s1, which is about 1/10 of the maximum light intensity in outdoor summer (2000 lmol m2 s1) (Torzillo et al., 2003). However, exploitation of sunlight as the light source faced certain drawbacks such as changing weather, day and night cycles even seasonal changes. This fluctuation in irradiance level could be precluded by artificial light source-LED (Cuaresma et al., 2009, 2011). LED (Light Emitting Diode) is used as the light source in this research due to several advantages: (i) generates lower heat when supplying light to marine microalgae (ii) has longer life-expectancy compared to fluorescence lamp (iii) higher conversion efficiency (Chen et al., 2011). According to Carvalho et al. (2011) the intensity (W/m2) and life time of the LED are 500% and 941% longer respectively than ordinary lamps. LED have the ability to distribute light uniformly in the bioreactor (Posten, 2009) but the light intensity must be sufficiently high to penetrate through the culture. In addition, commercially available LEDs exhibit a PAR efficiency of 1.91 lmol-phs1 W1 and is continuously being improved. Thus LED could the suitable light source for the cultivation of microalgae (Blanken et al., 2013). Recently, a number of researchers have reported on photoperiods and intensity but work on the wavelength of light for Nannochloropsis sp. and Tetraselmis sp. have never been reported yet. Wahidin et al. (2013) reported under the fluorescent light intensity of 100 lmol m2 s1 and photoperiod of 18 h light: 6 h dark cycle, Nannochloropsis sp. was found to grow favorably with a maximum cell concentration of 6.5 107 cells mL1. Shu et al. (2012) also reported that the high oil content (88 mg/L) and biomass (745 mg/L) of mixed culture of Chlorella sp. and Saccharomyces cerevisiae was obtained under blue LED light intensity of 1000 lux at a temperature of 28 °C for the 24:00 h light and dark cycles. Cheng and Zheng (2013) presented the Chlorella sp. optimal parameters were moderate light intensity 350 lmol m2 s1 with middle photoperiod 14 h light: 10 h dark. However the optimum wavelength for Nannochloropsis sp. and Tetraselmis sp. has not yet been reported anywhere. Thus the aim of this study is to investigate the effect of light wavelength on the growth and lipid content of marine microalgae microalgae, Nannochloropsis sp. and Tetraselmis sp. These two species were selected as objects of the study because they are known to have high lipid content (Chisti, 2007; Teo et al., 2014) and are easily available in our region. In addition the lipids were
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then transesterified to biodiesel and the fatty acid methyl esters (FAMEs) were identified. 2. Methods 2.1. Microalgae cultures Nannochloropsis sp. and Tetraselmis sp. strains were originally obtained from the culture collection of Borneo Marine Research institute (BMRI), Universiti Malaysia Sabah, Malaysia. The Nannochloropsis sp. and Tetraselmis sp. cells were cultured in sterilized seawater enriched with Walne’s medium which contains: 100 g NaNO3, 1.3 g FeCl36H2O; 0.36 g MnCl24H2O; 33.6 g H3BO3; 45 g Na2EDTA; 20 g NaH2PO42H2O; 2.1 g ZnCl2; 2 g CoCl26H2O; 0.9 g (NH4)6Mo7O244H2O; 2 g CuSO45H2O; 0.001 g Vitamin B12; 0.001 g Vitamin B1 and 0.2 lg Biotin per liter. 500 ml of Nannochloropsis sp. and Tetraselmis sp. were cultivated in 1000 mL flasks at 23 ± 0.5 °C, pH 8 ± 0.2 under a light intensity of 100 lmol m2 s1. Each of the flasks was then placed under different LED wavelengths (i) red (660 nm), (ii) blue (457 nm) (iii) red mix blue and (iv) white fluorescence lamp with a 24:00 light–dark cycle for 14 days. The schematic diagram of the experimental set up plan view is illustrated in Fig. 1. The light intensity was measured with a quantum sensor connected to Light Scout Dual solar quantum light meter. Each experiment was performed in duplicates so as to ensure reproducibility of results. 2.2. Growth analysis 2.2.1. Optical density (OD) method The marine microalgae growths were measured using the optical density (OD) method. Samples were taken every 2 days for 14 days where OD readings were taken using a UV–vis spectrophotometer (Shimadzu UVmini-1240) at 540 nm (Rocha et al., 2003). 2.2.2. Cell count The cultures were sampled at a 24 h interval and microalgae growth was monitored by counting the cell number. The cell concentration was determined by a direct microscopic count with a 0.1-mm-deep Neubauer haemocytometer (BOECO, Hamburg, Germany) and a light microscope (Olympus CX21, Japan). The specific growth rate was calculated from the Eq. (1):
l¼
lnðN2 N1 Þ t2 t1
ð1Þ
where N2 and N1 are the cell number concentration at time t2 and t1, respectively. The time required to duplicate the cell number division rate (k), was calculated according to the Eq. (2).
j¼
l ln 2
ð2Þ
2.2.3. Nile red staining method to measure the lipid by fluorescent spectrophotometer The amount of lipid in Tetraselmis sp. and Nannochloropsis sp. were measured rapidly using improved Nile red staining method (Chen et al., 2011) by employing Perkin Elmer LS-55 fluorescence spectrophotometer. For the Nile red staining, 1 ml of sample was added to 50 lL of Nile red (9-diethylamino-5H-benzo[a]phenoxazine-5-one; Sigma, USA) in acetone representing a concentration of 0.1 mg ml1. The mixtures were pretreated using a microwave oven for 1 min. The excitation and emission wavelengths for the fluorescence in this research were 490 nm and 585 nm, respectively.
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Fig. 1. Plan view of the schematic experimental set-up.
2.3. Lipid extraction
2.5. Gas chromatography (GC) analysis of fatty acid methyl esters (FAME)
Wet microalgae biomass (500 ml) was harvested by centrifugation at 8000 rpm for 15 min. Concentrated wet microalgae was mixed with methanol–chloroform (1:2 v/v) and then placed in the microwave at 65 °C for 5 min at the microwave power of 300 W. During the extraction process, a condenser was used in order to prevent loss of solvent due to vaporization. The mixture was then left to cool to room temperature. After the lipid extraction the methanol–chloroform phase that contains the extracted lipids was separated using a separating funnel. Finally, the solvent containing lipid extracted was evaporated by a rotary evaporator (Sharif et al., 2012).
Separation and identification of FAME were performed and analyzed in a gas chromatography (GC) (Agilent Technologies, 7820A) and HP-88 capillary column (60 m 0.25 mm 0.2 lm), using hydrogen gas as the carrier gas at 40 ml/min. The column temperature was set at 220 °C as maximum temperature. Both the injector and flame ionization detector (FID) temperature were set at 220 °C. The back inlet was set at splitless mode and initial temperature was 220 °C. The column initial temperature was set at 80oC during 2 min; the thermal gradient to 220 °C was at a rate of 13 °C/min, post temperature at 50 °C in 2 min.
2.4. Alkali-based transesterification
3. Results and discussions
Methanol was mixed with 0.5 g of sodium hydroxide (NaOH) and stirred for 20 min at 400 rpm at approximately 65 °C. The ratio of methanol to oil in the mixture was kept to 6:1. The mixture of catalyst and methanol was then poured into the conical flask containing the algae oil so as to initiate the transesterification process. The conical flask was stirred continuously for 3 h at 300 rpm and then allowed to settle for 16 h so as to obtain 2 separate layers; the supernatant layer (biodiesel) and sediment layers (methanol). The biodiesel was separated carefully from the sediment layer by a flask separator and washed using 5% water until the entire methanol is removed. The biodiesel was dried using a dryer and kept under running fan for 12 h (Yoo et al., 2010). The biodiesel obtained was stored and the amount of fatty acid methyl esters (FAMEs) were then determined using a gas chromatography (GC).
3.1. Effect of the growth rate in different wavelength
Optical Density (540nm)
Optical Density (540nm)
Fig. 2(a) shows Tetraselmis sp. growth curve under different wavelengths during the 14 days cultivation. Generally, the optical density of batch cultures increased from day 1 to 14. The cultures were sampled at 48 h interval with starting optical density reading around 0.045. From the overall trend of growth rates, Tetraselmis sp. grows better under blue light compared to red light. Higher growth rate observed for Tetraselmis sp. Fig. 2(a) clearly shows that the blue illumination condition results in higher biomass starting from the 8th day when compared to the other light conditions. After day 14 the growth tends to slow down and remained stationary with time due to nutrient depletion and wastes start to accumulate in the culture medium.
Time (day)
(a)
Time (day)
(b)
Fig. 2. Growth curve in different wavelengths (a) Tetraselmis sp. (b) Nannochloropsis sp.
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The results revealed that both microalgae species tend to grow better under the blue LED light. It was reported that blue light tend to promote gene transcription and enhance the regulation of activated enzymes (Ruyters, 1984). This explains for higher growth rates achieved when the micro algae were cultivated under blue light. It was also observed that both of the microalgae species do not grow very well under red light. Studies have reported that cells were damaged by red light thus probably explained for the low growth obtained. It was also reported that when the cells were damaged by the red light, the cells can be repaired by exposure to low intensity of blue light. Thus, the growth rate and biomass production of marine microalgae were very much related to the type of light wavelength (Shu et al., 2012). Table 1 summarizes the effect of wavelengths on Tetraselmis sp. and Nannochloropsis sp. specific growth rate, division rate and cell density. The highest specific growth rate (1.47 day1) for Tetraselmis sp. was achieved under blue light condition with the division rate of 2.09 day1. The specific growth and division rates for Tetraselmis sp. cultivated under red-blue and white light are similar (1.44 day1) and (2.07 day1), respectively although the maximum cell density are different. As can be observed the specific growth, division rates and maximum cell concentration of Nannochloropsis sp. were much higher compared to Tetraselmis sp. for all the different wavelengths. The highest the specific growth rate was attained under blue light which reached 1.64 day1 with the division rate of 2.37 day1. Table 2 compares the growth rate of the current study with other species of microalgae. The results are close to that reported by Atta et al. (2013) when cultivating Chlorella vulgaris under the intensity of blue LED. The intensity of the blue LED enables deep penetration through the batch culture thus enhancing the cell division and growth rates of microalgae. Atta et al. (2013) reported that C. vulgaris showed better growth rate in the blue light compared with white light. The growth rates for other microalgae species grown under white light are also depicted in Table 2.
cell conc. (x 107 cells/ml)
cell conc. (x 10 7 cells/ml)
Fig. 2(b) shows a similar trend for Nannochloropsis sp. which grows better under blue light compared to red light. Rapid growth rate was observed for Nannochloropsis sp. grown under blue illumination. The biomass in the batch culture also increased steadily from day 1 until day 14 with starting optical density reading around 0.057. Unlike Tetraselmis sp., the growth profile for Nannochloropsis sp. is distinct; exhibiting the lag phase (2 days) and the exponential phase after day 2. After day 10, the growth rates tend to slow down and remained stationary with time due to nutrient depletion and wastes start to accumulate in the culture medium. Cell counting is a quantitative method to calculate the growth rate of microorganisms according to cell number using hemocytometer. Fig. 3 illustrates the concentration of cell number of Tetraselmis sp. and Nannochloropsis sp. respectively for blue, red, blue mix red and white light. Tetraselmis sp. reached maximum cell concentration during day 10. Blue light showed highest value (0.256 107 cell mL1) while red, red-blue and white light only obtained 0.205 107 cell mL1, 0.23 107 cell mL1, 0.244 107 cell mL1, respectively. After day 10, Tetraselmis sp. cell concentration starts to decrease upon reaching the death phase. A visible change in the color of the culture was observed from green to yellow brownish. In the case of Nannochloropsis sp. maximum cell concentration under blue light occurred on day 14 (see Fig. 3(b)). It was observed that the cell concentrations during the initial 6 day period were almost the same for the different wavelengths but on day 8, Nannochloropsis sp. cultivated under the blue light exhibited higher cell concentration compared to the others. On day 10, the cell concentration value of Nannochloropsis sp. under blue light reached 1.23 107 cell mL1, a value higher than the other conditions (red, red-blue and white) which 1.1 107 cell mL1, 1.11 107 cell mL1 and 0.931 107 cell mL1, respectively. Nannochloropsis sp. under blue light continued to grow aggressively until day 14 achieving maximum cell concentration value of 1.45 107 cell mL1.
Time (day)
Time (day)
(a)
(b)
Fig. 3. Growth curve in different wavelength (cell count) (a) Tetraselmis sp. (b) Nannochloropsis sp.
Table 1 Maximum cell density, specific growth rate (l) and division times (k) of Tetraselmis sp. and Nannochloropsis sp. cultivated at different wavelengths. Microalgae species
Tetraselmis sp
Type of wavelength
Max. cell density, (107 cell/ml)
l (day1)
k (day1)
Nannochloropsis sp Max. cell density, (107 cell/ml)
l (day1)
k (day1)
Blue (457 nm) Red (660 nm) Red Blue White
0.256 0.205 0.230 0.244
1.47 1.43 1.44 1.44
2.09 2.06 2.07 2.07
1.45 1.11 1.10 0.931
1.64 1.61 1.61 1.59
2.37 2.32 2.32 2.29
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Table 2 Comparison the specific growth, l (day1) with other species of microalgae. Type of wavelength
Specific growth rate; l (day1)
Microalgae species
Light intensity (lmol m2 s1)
Photoperiod (L:D) (h)
Blue (457 nm)
White
Red (660 nm)
Red Blue
References
Tetraselmis sp. Nannochloropsis sp. C. vulgaris
100 100 200 200 200 100 80–110 100 100 100 100 100
24:0 24:0 12:12 16:08 24:00 24:00 24:00 16:08 16:08 16:08 16:08 16:08
1.47 1.64 1.26 1.24 1.22 – – – – – – –
1.44 1.59 1.15 1.20 1.18 0.777 0.13 0.564 0.175 0.451 0.677 0.745
1.43 1.61 – – – – – – – – – –
1.44 1.61 – – – – – – – – – –
Current work Current work Atta et al. (2013)
Chlorella sp. C. minutissima Thalassiosira pseudonana Odontella aurita Isochrysis galbana Chromulina ochromonoides Dunaliella tertiolecta
3.2. Influence of different wavelengths on the lipid content
blue LED, Isochrysis galbana produced maximum lipid content (155 mg/L). Ribulose bisphosphate carboxylase/oxygenase (Rubisco) and carbonic anhydrase are the enzymes that affect the carbon dioxide rates in microalgae. These enzymes are basically under the control of blue light. The higher the enzyme activity, the higher the accumulation of triglycerides under the blue LED light (Roscher and Zetsche, 1986). Light is a crucial factor for formation of triacylglycerides and light wavelength required for different species vary from one another. In addition, fatty acid composition is different for different marine microalgae in response to different light wavelength exposures. Khotimchenko and Yakovleva (2005) also stated that light will stimulate triacylglycerides synthesis, formation of particularly chloroplast membranes and growth. Therefore, morphological change of marine microalgae will influence the overall lipid content (Wahidin et al., 2013).
3.3. Analysis of microalgae biodiesel The compositions of triglyceride fatty acid methyl esters (biodiesel) found in Nannochloropsis sp. and Tetraselmis sp. cultivated under blue light are presented in Table 3. Both species have methyl esters in the range of C14–C18. A majority of the FAMEs in Tetraselmis sp. consist of palmitic acid methyl ester (C16:0) and stearic acid methyl ester (C18:0); in fact they contribute to 81–83% of the total FAMEs content. However the composition of FAMEs in Nannochloropsis sp. is rather evenly distributed where palmitic acid methyl ester (C16:0) and stearic acid methyl ester (C18:0) contribute around 49–51% of total FAMEs. The other 50% of FAMEs consists of myristic, elaidic, oleic, linolelaidic and linoleic acid methyl esters; almost equally distributed. The absence of methyl
Fluorescence intensity (a.u.)
Fluorescence intensity (a.u.)
Improved Nile red analysis was used to determine the neutral lipid content in microalgae under different wavelengths and the higher values of fluorescent intensity (a.u.) indicate higher lipid content in the microalgae. Fig. 4(a) shows the fluorescence intensity of Tetraselmis sp. in a variety of wavelengths during the initial 6 days which is still in the lag phase. However after the 6th day, there is sudden increase in the fluorescent intensity for Tetraselmis sp. cultivated in the blue light. The sudden increase in the fluorescent intensity reflects high lipid content obtained in the Tetraselmis sp. cultivated in the blue light condition. A different scenario was observed for the Tetraselmis sp. cultivated in red, red-blue and white light where lower fluorescent intensity was observed which reflected lower lipid content. The results revealed that higher neutral lipid content was obtained in the Tetraselmis sp.’s cultivated in blue light. A similar phenomenon is observed for Nannochloropsis sp. cultivated in different wavelengths where Nannochloropsis sp. cultivated in blue light exhibits higher fluorescence intensity reflecting high neutral lipid content (see Fig. 4(b)). Apparently the lag phase for the Nannochloropsis sp. is only 2–8 days and increase in fluorescence intensity is observed on the tenth day. Blue light promotes Nannochloropsis sp. and Tetraselmis sp. biomass growth and simultaneously produce more neutral lipid than red, red-blue and white light. Fig. 5(a) and (b) clearly indicate that the marine microalgae growth rates are related to lipid content. Tetraselmis sp. and Nannochloropsis sp. cultivated under blue light has the highest growth rate and also lipid content. Yoshioka et al. (2012) found that under
Wensel et al. (2014) Jena et al. (2011) Roleda et al. (2013)
Time (day)
Time (day)
(a)
(b)
Fig. 4. Nile red fluorescence intensity plot over time (Day) (a) Tetraselmis sp. (b) Nannochloropsis sp.
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Fluorescence intensity (a.u.)
growth rate(day-1)
growth rate(day-1)
Fluorescence intensity (a.u.)
C.L. Teo et al. / Bioresource Technology 162 (2014) 38–44
Type of wavelength
(a)
(b)
Fig. 5. Relationship between growth rate of microalgae and lipid content under the light intensity of 100 lmol m2 s1 and different wavelengths (blue, red, red-blue and white light). (a) Tetraselmis sp. (b) Nannochloropsis sp.
Table 3 The compositions of FAMEs in Tetraselmis sp. and Nannochloropsis sp. cultivated under blue light. Microalgae species Fatty acid methyl ester Myristic acid methyl ester Palmitic acid methyl ester Stearic acid methyl ester Elaidic acid methyl ester Oleic acid methyl ester Linolelaidic acid methyl ester Linoleic acid methyl ester
Tetraselmis sp. wt.% (C14:0) 1.47 (C16:0) 53.7 (C18:0) 29.18 (C18:1n9t) 4.79 (C18:1n9c) 4.77 (C18:2n6t) 3.02 (C18:2n6c) 3.07
Nannochloropsis sp. wt.% 11.87 16.49 32.85 10.56 8.97 9.41 9.84
esters with carbon chains >19 carbons guarantee a low viscosity for the microalgae biodiesel.
4. Conclusion Nannochloropsis sp. and Tetraselmis sp. were successfully cultivated under different wavelengths. The blue wavelength (450– 495 nm) stimulates higher growth rate and crude oil production for both Nannochloropsis sp. and Tetraselmis sp. Nannochloropsis sp. has higher growth rate and crude oil production compared to Tetraselmis sp. In directly, the higher FAME production under blue wavelength cultivation of marine microalgae was obtained compared to other wavelength cultivation.
Acknowledgements Financial support from Universiti Teknologi Malaysia (Research University Grant/QJ1300.7125.00H03) for this research is gratefully acknowledged.
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Sharif, D.I., Gallon, J., Smith, C.J., Dudley, E., 2012. Quorum sensing in Cyanobacteria: N-octanoyl-homoserine lactone release and response, by the epilithic colonial cyanobacterium Gloeothece PCC6909. ISME J. 1, 1171–1182. Shu, C.H., Tsai, C.H., Liao, W.H., Chen, K.Y., Huang, H.C., 2012. Effects of light quality on the accumulation of oil in a mixed culture of Chlorella sp. and S. cerevisiae. J. Chem. Technol. Biotechnol. 87, 601–607. Teo, C.L., Jamaluddin, H., Zain, N.A.M., Idris, A., 2014. Biodiesel production via lipase catalysed transesterification of microalgae lipids from Tetraselmis sp. Renew. Energy 68, 1–5. Torzillo, G., Pushparaj, B., Masojidek, J., Vonshak, A., 2003. Biological constraints in algal biotechnology. Biotechnol. Bioprocess Eng. 8, 338–348.
Wahidin, S., Idris, A., Shaleh, S.R.M., 2013. The influence of light intensity and photoperiod on the growth and lipid content of microalgae Nannochloropsis sp. Bioresour. Technol. 129, 7–11. Wensel, P., Helms, G., William, H.B., Davis, C., Kirchhoff, H., Bule, M., Yu, L., Chen, S., 2014. Isolation, characterization, and validation of oleaginous, multi-trophic, and haloalkaline-tolerant microalgae for two-stage cultivation. Algal Res. 10. Yoo, C., Jun, S.Y., Lee, J.Y., Ahn, C.Y., Oh, H.M., 2010. Selection of microalgae for lipid production under high levels carbon dioxide. Bioresour. Technol. 101, 71–74. Yoshioka, M., Yago, T., Yoshie-Stark, Y., Arakawa, H.T., 2012. Effect of high frequency of intermittent light on the growth and fatty acid profile of Isochrysis galbana. Aquaculture 338–341, 111–117.
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Enhancing growth and lipid production of marine microalgae for biodiesel production via the use of different LED wavelengths Chee Loong Teo a, Madiha Atta a, Attaullah Bukhari a, Mohamad Taisir b, Afendi M. Yusuf b, Ani Idris a,⇑ a Department of Bioprocess Engineering, Faculty of Chemical Engineering, c/o Institute of Bioproduct Development (IBD), Universiti Teknologi Malaysia, 81310 UTM Johor Bahru, Johor, Malaysia b Faculty of Mechanical Engineering, Universiti Teknologi Malaysia, c/o B.M. Nagano Industries Sdn. Bhd., Jalan Keluli 3, Kawasan Perindustrian Pasir Gudang, 81700 Johor, Malaysia
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Different LED wavelengths were used
for the cultivation of marine microalgae. LED wavelengths used were blue (457 nm), red (660 nm) and red mix blue. The results were also compared to white fluorescent lamp. Microalgae showed highest growth rate and lipid production under blue LED wavelength. Analysis showed major FAME components were palmitic acid and stearic acid.
a r t i c l e
i n f o
Article history: Received 20 January 2014 Received in revised form 17 March 2014 Accepted 21 March 2014 Available online 1 April 2014 Keywords: Nannochloropsis sp. Tetraselmis sp. LED Wavelength Biodiesel
a b s t r a c t Wavelength of light is a crucial factor which renders microalgae as the potential biodiesel. In this study, Tetraselmis sp. and Nannochloropsis sp. as famous targets were selected. The effect of different light wavelengths on growth rate and lipid production was studied. Microalgae were cultivated for 14 days as under blue, red, red-blue LED and white fluorescent light. The growth rate of microalgae was analyzed by spectrophotometer and cell counting while oil production under improved Nile red method. Optical density result showed the microalgae exhibited better growth curve under blue wavelength. Besides, Tetraselmis sp. and Nannochloropsis sp. under blue wavelength showed the higher growth rate (1.47 and 1.64 day1) and oil production (102.954 and 702.366 a.u.). Gas chromatography analysis also showed that palmitic acid and stearic acid which were compulsory components for biodiesel contribute around 49–51% of total FAME from Nannochloropsis sp. and 81–83% of total FAME from Tetraselmis sp. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction The insufficient petroleum reserves, extreme dependence on foreign energy resource, increasing crude oil prices and current environmental concerns have encouraged scientists to find for alternative environmentally friendly and renewable energy. (Geor⇑ Corresponding author. Tel.: +60 7 5535603; fax: +60 7 5588166. E-mail address: [email protected] (A. Idris). http://dx.doi.org/10.1016/j.biortech.2014.03.113 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
gogianni et al., 2008; Meher et al., 2006; Narasimharao et al., 2007). Biodiesel is seen as the alternative renewable energy because it is sustainable with less harm to the environment (McKendry, 2002; Reijnders, 2006). Biodiesel from microalgae which is considered as the 3rd generation renewable biofuels is believed to be capable of meeting the whole world demand for transport fuels (Chisti, 2007) because marine microalgae can generate as much as 40 times crude oil than other plants per acre of land area (Schenk et al., 2008).
C.L. Teo et al. / Bioresource Technology 162 (2014) 38–44
Marine microalgae are photoautotrophic microorganisms thus light is a very essential factor to growth rate (Adir et al., 2003; Ragni et al., 2008). Light spectral quality and light intensity (quantity) must be considered when choosing the light source for microalgae cultivation. Spectral quality is defined by the absorption spectrum (light wavelengths) of the chlorophyll and other photosynthetically active pigments in microalgae such as phycobilins and carotenoids (Lee, 1999). Energy absorption by photosynthetic organisms is thus dependant on the chemical nature of their constitutive pigments (Carvalho et al., 2011). All chlorophylls (green pigments) have two major absorption bands: blue or blue-green (450–475 nm) and red (630–675 nm). The growth of marine microalgae will be improved by using either red light or blue light (Korbee et al., 2005). Chlorophyll a is the core of reaction pigment, while accessory pigments chlorophyll b, c and d extend the range of light absorption (Richmond, 2004). Light intensity should be delivered evenly over the illuminating surface of culture vessel and with adequate amount of photosynthetically active radiation (PAR) to enable photons reach the cell in culture (Lee, 1999). Excessive intensity may lead to photooxidation and photoinhibition, while low light levels will become growth-limiting (Loera-Quezada et al., 2011). Due to this, the light intensity at which culture growth becomes saturated is an important factor in determining the light utilization efficiency. In general, the growth of many photosynthetic microorganisms gets saturated at about 200 lmol m2 s1, which is about 1/10 of the maximum light intensity in outdoor summer (2000 lmol m2 s1) (Torzillo et al., 2003). However, exploitation of sunlight as the light source faced certain drawbacks such as changing weather, day and night cycles even seasonal changes. This fluctuation in irradiance level could be precluded by artificial light source-LED (Cuaresma et al., 2009, 2011). LED (Light Emitting Diode) is used as the light source in this research due to several advantages: (i) generates lower heat when supplying light to marine microalgae (ii) has longer life-expectancy compared to fluorescence lamp (iii) higher conversion efficiency (Chen et al., 2011). According to Carvalho et al. (2011) the intensity (W/m2) and life time of the LED are 500% and 941% longer respectively than ordinary lamps. LED have the ability to distribute light uniformly in the bioreactor (Posten, 2009) but the light intensity must be sufficiently high to penetrate through the culture. In addition, commercially available LEDs exhibit a PAR efficiency of 1.91 lmol-phs1 W1 and is continuously being improved. Thus LED could the suitable light source for the cultivation of microalgae (Blanken et al., 2013). Recently, a number of researchers have reported on photoperiods and intensity but work on the wavelength of light for Nannochloropsis sp. and Tetraselmis sp. have never been reported yet. Wahidin et al. (2013) reported under the fluorescent light intensity of 100 lmol m2 s1 and photoperiod of 18 h light: 6 h dark cycle, Nannochloropsis sp. was found to grow favorably with a maximum cell concentration of 6.5 107 cells mL1. Shu et al. (2012) also reported that the high oil content (88 mg/L) and biomass (745 mg/L) of mixed culture of Chlorella sp. and Saccharomyces cerevisiae was obtained under blue LED light intensity of 1000 lux at a temperature of 28 °C for the 24:00 h light and dark cycles. Cheng and Zheng (2013) presented the Chlorella sp. optimal parameters were moderate light intensity 350 lmol m2 s1 with middle photoperiod 14 h light: 10 h dark. However the optimum wavelength for Nannochloropsis sp. and Tetraselmis sp. has not yet been reported anywhere. Thus the aim of this study is to investigate the effect of light wavelength on the growth and lipid content of marine microalgae microalgae, Nannochloropsis sp. and Tetraselmis sp. These two species were selected as objects of the study because they are known to have high lipid content (Chisti, 2007; Teo et al., 2014) and are easily available in our region. In addition the lipids were
39
then transesterified to biodiesel and the fatty acid methyl esters (FAMEs) were identified. 2. Methods 2.1. Microalgae cultures Nannochloropsis sp. and Tetraselmis sp. strains were originally obtained from the culture collection of Borneo Marine Research institute (BMRI), Universiti Malaysia Sabah, Malaysia. The Nannochloropsis sp. and Tetraselmis sp. cells were cultured in sterilized seawater enriched with Walne’s medium which contains: 100 g NaNO3, 1.3 g FeCl36H2O; 0.36 g MnCl24H2O; 33.6 g H3BO3; 45 g Na2EDTA; 20 g NaH2PO42H2O; 2.1 g ZnCl2; 2 g CoCl26H2O; 0.9 g (NH4)6Mo7O244H2O; 2 g CuSO45H2O; 0.001 g Vitamin B12; 0.001 g Vitamin B1 and 0.2 lg Biotin per liter. 500 ml of Nannochloropsis sp. and Tetraselmis sp. were cultivated in 1000 mL flasks at 23 ± 0.5 °C, pH 8 ± 0.2 under a light intensity of 100 lmol m2 s1. Each of the flasks was then placed under different LED wavelengths (i) red (660 nm), (ii) blue (457 nm) (iii) red mix blue and (iv) white fluorescence lamp with a 24:00 light–dark cycle for 14 days. The schematic diagram of the experimental set up plan view is illustrated in Fig. 1. The light intensity was measured with a quantum sensor connected to Light Scout Dual solar quantum light meter. Each experiment was performed in duplicates so as to ensure reproducibility of results. 2.2. Growth analysis 2.2.1. Optical density (OD) method The marine microalgae growths were measured using the optical density (OD) method. Samples were taken every 2 days for 14 days where OD readings were taken using a UV–vis spectrophotometer (Shimadzu UVmini-1240) at 540 nm (Rocha et al., 2003). 2.2.2. Cell count The cultures were sampled at a 24 h interval and microalgae growth was monitored by counting the cell number. The cell concentration was determined by a direct microscopic count with a 0.1-mm-deep Neubauer haemocytometer (BOECO, Hamburg, Germany) and a light microscope (Olympus CX21, Japan). The specific growth rate was calculated from the Eq. (1):
l¼
lnðN2 N1 Þ t2 t1
ð1Þ
where N2 and N1 are the cell number concentration at time t2 and t1, respectively. The time required to duplicate the cell number division rate (k), was calculated according to the Eq. (2).
j¼
l ln 2
ð2Þ
2.2.3. Nile red staining method to measure the lipid by fluorescent spectrophotometer The amount of lipid in Tetraselmis sp. and Nannochloropsis sp. were measured rapidly using improved Nile red staining method (Chen et al., 2011) by employing Perkin Elmer LS-55 fluorescence spectrophotometer. For the Nile red staining, 1 ml of sample was added to 50 lL of Nile red (9-diethylamino-5H-benzo[a]phenoxazine-5-one; Sigma, USA) in acetone representing a concentration of 0.1 mg ml1. The mixtures were pretreated using a microwave oven for 1 min. The excitation and emission wavelengths for the fluorescence in this research were 490 nm and 585 nm, respectively.
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Fig. 1. Plan view of the schematic experimental set-up.
2.3. Lipid extraction
2.5. Gas chromatography (GC) analysis of fatty acid methyl esters (FAME)
Wet microalgae biomass (500 ml) was harvested by centrifugation at 8000 rpm for 15 min. Concentrated wet microalgae was mixed with methanol–chloroform (1:2 v/v) and then placed in the microwave at 65 °C for 5 min at the microwave power of 300 W. During the extraction process, a condenser was used in order to prevent loss of solvent due to vaporization. The mixture was then left to cool to room temperature. After the lipid extraction the methanol–chloroform phase that contains the extracted lipids was separated using a separating funnel. Finally, the solvent containing lipid extracted was evaporated by a rotary evaporator (Sharif et al., 2012).
Separation and identification of FAME were performed and analyzed in a gas chromatography (GC) (Agilent Technologies, 7820A) and HP-88 capillary column (60 m 0.25 mm 0.2 lm), using hydrogen gas as the carrier gas at 40 ml/min. The column temperature was set at 220 °C as maximum temperature. Both the injector and flame ionization detector (FID) temperature were set at 220 °C. The back inlet was set at splitless mode and initial temperature was 220 °C. The column initial temperature was set at 80oC during 2 min; the thermal gradient to 220 °C was at a rate of 13 °C/min, post temperature at 50 °C in 2 min.
2.4. Alkali-based transesterification
3. Results and discussions
Methanol was mixed with 0.5 g of sodium hydroxide (NaOH) and stirred for 20 min at 400 rpm at approximately 65 °C. The ratio of methanol to oil in the mixture was kept to 6:1. The mixture of catalyst and methanol was then poured into the conical flask containing the algae oil so as to initiate the transesterification process. The conical flask was stirred continuously for 3 h at 300 rpm and then allowed to settle for 16 h so as to obtain 2 separate layers; the supernatant layer (biodiesel) and sediment layers (methanol). The biodiesel was separated carefully from the sediment layer by a flask separator and washed using 5% water until the entire methanol is removed. The biodiesel was dried using a dryer and kept under running fan for 12 h (Yoo et al., 2010). The biodiesel obtained was stored and the amount of fatty acid methyl esters (FAMEs) were then determined using a gas chromatography (GC).
3.1. Effect of the growth rate in different wavelength
Optical Density (540nm)
Optical Density (540nm)
Fig. 2(a) shows Tetraselmis sp. growth curve under different wavelengths during the 14 days cultivation. Generally, the optical density of batch cultures increased from day 1 to 14. The cultures were sampled at 48 h interval with starting optical density reading around 0.045. From the overall trend of growth rates, Tetraselmis sp. grows better under blue light compared to red light. Higher growth rate observed for Tetraselmis sp. Fig. 2(a) clearly shows that the blue illumination condition results in higher biomass starting from the 8th day when compared to the other light conditions. After day 14 the growth tends to slow down and remained stationary with time due to nutrient depletion and wastes start to accumulate in the culture medium.
Time (day)
(a)
Time (day)
(b)
Fig. 2. Growth curve in different wavelengths (a) Tetraselmis sp. (b) Nannochloropsis sp.
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The results revealed that both microalgae species tend to grow better under the blue LED light. It was reported that blue light tend to promote gene transcription and enhance the regulation of activated enzymes (Ruyters, 1984). This explains for higher growth rates achieved when the micro algae were cultivated under blue light. It was also observed that both of the microalgae species do not grow very well under red light. Studies have reported that cells were damaged by red light thus probably explained for the low growth obtained. It was also reported that when the cells were damaged by the red light, the cells can be repaired by exposure to low intensity of blue light. Thus, the growth rate and biomass production of marine microalgae were very much related to the type of light wavelength (Shu et al., 2012). Table 1 summarizes the effect of wavelengths on Tetraselmis sp. and Nannochloropsis sp. specific growth rate, division rate and cell density. The highest specific growth rate (1.47 day1) for Tetraselmis sp. was achieved under blue light condition with the division rate of 2.09 day1. The specific growth and division rates for Tetraselmis sp. cultivated under red-blue and white light are similar (1.44 day1) and (2.07 day1), respectively although the maximum cell density are different. As can be observed the specific growth, division rates and maximum cell concentration of Nannochloropsis sp. were much higher compared to Tetraselmis sp. for all the different wavelengths. The highest the specific growth rate was attained under blue light which reached 1.64 day1 with the division rate of 2.37 day1. Table 2 compares the growth rate of the current study with other species of microalgae. The results are close to that reported by Atta et al. (2013) when cultivating Chlorella vulgaris under the intensity of blue LED. The intensity of the blue LED enables deep penetration through the batch culture thus enhancing the cell division and growth rates of microalgae. Atta et al. (2013) reported that C. vulgaris showed better growth rate in the blue light compared with white light. The growth rates for other microalgae species grown under white light are also depicted in Table 2.
cell conc. (x 107 cells/ml)
cell conc. (x 10 7 cells/ml)
Fig. 2(b) shows a similar trend for Nannochloropsis sp. which grows better under blue light compared to red light. Rapid growth rate was observed for Nannochloropsis sp. grown under blue illumination. The biomass in the batch culture also increased steadily from day 1 until day 14 with starting optical density reading around 0.057. Unlike Tetraselmis sp., the growth profile for Nannochloropsis sp. is distinct; exhibiting the lag phase (2 days) and the exponential phase after day 2. After day 10, the growth rates tend to slow down and remained stationary with time due to nutrient depletion and wastes start to accumulate in the culture medium. Cell counting is a quantitative method to calculate the growth rate of microorganisms according to cell number using hemocytometer. Fig. 3 illustrates the concentration of cell number of Tetraselmis sp. and Nannochloropsis sp. respectively for blue, red, blue mix red and white light. Tetraselmis sp. reached maximum cell concentration during day 10. Blue light showed highest value (0.256 107 cell mL1) while red, red-blue and white light only obtained 0.205 107 cell mL1, 0.23 107 cell mL1, 0.244 107 cell mL1, respectively. After day 10, Tetraselmis sp. cell concentration starts to decrease upon reaching the death phase. A visible change in the color of the culture was observed from green to yellow brownish. In the case of Nannochloropsis sp. maximum cell concentration under blue light occurred on day 14 (see Fig. 3(b)). It was observed that the cell concentrations during the initial 6 day period were almost the same for the different wavelengths but on day 8, Nannochloropsis sp. cultivated under the blue light exhibited higher cell concentration compared to the others. On day 10, the cell concentration value of Nannochloropsis sp. under blue light reached 1.23 107 cell mL1, a value higher than the other conditions (red, red-blue and white) which 1.1 107 cell mL1, 1.11 107 cell mL1 and 0.931 107 cell mL1, respectively. Nannochloropsis sp. under blue light continued to grow aggressively until day 14 achieving maximum cell concentration value of 1.45 107 cell mL1.
Time (day)
Time (day)
(a)
(b)
Fig. 3. Growth curve in different wavelength (cell count) (a) Tetraselmis sp. (b) Nannochloropsis sp.
Table 1 Maximum cell density, specific growth rate (l) and division times (k) of Tetraselmis sp. and Nannochloropsis sp. cultivated at different wavelengths. Microalgae species
Tetraselmis sp
Type of wavelength
Max. cell density, (107 cell/ml)
l (day1)
k (day1)
Nannochloropsis sp Max. cell density, (107 cell/ml)
l (day1)
k (day1)
Blue (457 nm) Red (660 nm) Red Blue White
0.256 0.205 0.230 0.244
1.47 1.43 1.44 1.44
2.09 2.06 2.07 2.07
1.45 1.11 1.10 0.931
1.64 1.61 1.61 1.59
2.37 2.32 2.32 2.29
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Table 2 Comparison the specific growth, l (day1) with other species of microalgae. Type of wavelength
Specific growth rate; l (day1)
Microalgae species
Light intensity (lmol m2 s1)
Photoperiod (L:D) (h)
Blue (457 nm)
White
Red (660 nm)
Red Blue
References
Tetraselmis sp. Nannochloropsis sp. C. vulgaris
100 100 200 200 200 100 80–110 100 100 100 100 100
24:0 24:0 12:12 16:08 24:00 24:00 24:00 16:08 16:08 16:08 16:08 16:08
1.47 1.64 1.26 1.24 1.22 – – – – – – –
1.44 1.59 1.15 1.20 1.18 0.777 0.13 0.564 0.175 0.451 0.677 0.745
1.43 1.61 – – – – – – – – – –
1.44 1.61 – – – – – – – – – –
Current work Current work Atta et al. (2013)
Chlorella sp. C. minutissima Thalassiosira pseudonana Odontella aurita Isochrysis galbana Chromulina ochromonoides Dunaliella tertiolecta
3.2. Influence of different wavelengths on the lipid content
blue LED, Isochrysis galbana produced maximum lipid content (155 mg/L). Ribulose bisphosphate carboxylase/oxygenase (Rubisco) and carbonic anhydrase are the enzymes that affect the carbon dioxide rates in microalgae. These enzymes are basically under the control of blue light. The higher the enzyme activity, the higher the accumulation of triglycerides under the blue LED light (Roscher and Zetsche, 1986). Light is a crucial factor for formation of triacylglycerides and light wavelength required for different species vary from one another. In addition, fatty acid composition is different for different marine microalgae in response to different light wavelength exposures. Khotimchenko and Yakovleva (2005) also stated that light will stimulate triacylglycerides synthesis, formation of particularly chloroplast membranes and growth. Therefore, morphological change of marine microalgae will influence the overall lipid content (Wahidin et al., 2013).
3.3. Analysis of microalgae biodiesel The compositions of triglyceride fatty acid methyl esters (biodiesel) found in Nannochloropsis sp. and Tetraselmis sp. cultivated under blue light are presented in Table 3. Both species have methyl esters in the range of C14–C18. A majority of the FAMEs in Tetraselmis sp. consist of palmitic acid methyl ester (C16:0) and stearic acid methyl ester (C18:0); in fact they contribute to 81–83% of the total FAMEs content. However the composition of FAMEs in Nannochloropsis sp. is rather evenly distributed where palmitic acid methyl ester (C16:0) and stearic acid methyl ester (C18:0) contribute around 49–51% of total FAMEs. The other 50% of FAMEs consists of myristic, elaidic, oleic, linolelaidic and linoleic acid methyl esters; almost equally distributed. The absence of methyl
Fluorescence intensity (a.u.)
Fluorescence intensity (a.u.)
Improved Nile red analysis was used to determine the neutral lipid content in microalgae under different wavelengths and the higher values of fluorescent intensity (a.u.) indicate higher lipid content in the microalgae. Fig. 4(a) shows the fluorescence intensity of Tetraselmis sp. in a variety of wavelengths during the initial 6 days which is still in the lag phase. However after the 6th day, there is sudden increase in the fluorescent intensity for Tetraselmis sp. cultivated in the blue light. The sudden increase in the fluorescent intensity reflects high lipid content obtained in the Tetraselmis sp. cultivated in the blue light condition. A different scenario was observed for the Tetraselmis sp. cultivated in red, red-blue and white light where lower fluorescent intensity was observed which reflected lower lipid content. The results revealed that higher neutral lipid content was obtained in the Tetraselmis sp.’s cultivated in blue light. A similar phenomenon is observed for Nannochloropsis sp. cultivated in different wavelengths where Nannochloropsis sp. cultivated in blue light exhibits higher fluorescence intensity reflecting high neutral lipid content (see Fig. 4(b)). Apparently the lag phase for the Nannochloropsis sp. is only 2–8 days and increase in fluorescence intensity is observed on the tenth day. Blue light promotes Nannochloropsis sp. and Tetraselmis sp. biomass growth and simultaneously produce more neutral lipid than red, red-blue and white light. Fig. 5(a) and (b) clearly indicate that the marine microalgae growth rates are related to lipid content. Tetraselmis sp. and Nannochloropsis sp. cultivated under blue light has the highest growth rate and also lipid content. Yoshioka et al. (2012) found that under
Wensel et al. (2014) Jena et al. (2011) Roleda et al. (2013)
Time (day)
Time (day)
(a)
(b)
Fig. 4. Nile red fluorescence intensity plot over time (Day) (a) Tetraselmis sp. (b) Nannochloropsis sp.
43
Fluorescence intensity (a.u.)
growth rate(day-1)
growth rate(day-1)
Fluorescence intensity (a.u.)
C.L. Teo et al. / Bioresource Technology 162 (2014) 38–44
Type of wavelength
(a)
(b)
Fig. 5. Relationship between growth rate of microalgae and lipid content under the light intensity of 100 lmol m2 s1 and different wavelengths (blue, red, red-blue and white light). (a) Tetraselmis sp. (b) Nannochloropsis sp.
Table 3 The compositions of FAMEs in Tetraselmis sp. and Nannochloropsis sp. cultivated under blue light. Microalgae species Fatty acid methyl ester Myristic acid methyl ester Palmitic acid methyl ester Stearic acid methyl ester Elaidic acid methyl ester Oleic acid methyl ester Linolelaidic acid methyl ester Linoleic acid methyl ester
Tetraselmis sp. wt.% (C14:0) 1.47 (C16:0) 53.7 (C18:0) 29.18 (C18:1n9t) 4.79 (C18:1n9c) 4.77 (C18:2n6t) 3.02 (C18:2n6c) 3.07
Nannochloropsis sp. wt.% 11.87 16.49 32.85 10.56 8.97 9.41 9.84
esters with carbon chains >19 carbons guarantee a low viscosity for the microalgae biodiesel.
4. Conclusion Nannochloropsis sp. and Tetraselmis sp. were successfully cultivated under different wavelengths. The blue wavelength (450– 495 nm) stimulates higher growth rate and crude oil production for both Nannochloropsis sp. and Tetraselmis sp. Nannochloropsis sp. has higher growth rate and crude oil production compared to Tetraselmis sp. In directly, the higher FAME production under blue wavelength cultivation of marine microalgae was obtained compared to other wavelength cultivation.
Acknowledgements Financial support from Universiti Teknologi Malaysia (Research University Grant/QJ1300.7125.00H03) for this research is gratefully acknowledged.
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