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Advances in bioconversion of microalgae with high biomass and lipid productivity
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Advances in bioconversion of microalgae with high biomass and lipid productivity Yu-Tzu Huang a,∗, Chung-Wei Lai a, Bo-Wei Wu a, Kuen-Song Lin b, Jeffrey C.S. Wu c, Md Shahriar A Hossain d,e, Yusuke Yamauchi d,e, Kevin C.-W. Wu c a
Department of Environmental Engineering and Research Center for Analysis and Identification, Chung Yuan Christian University, Taoyuan 32023, Taiwan, ROC Department of Chemical Engineering and Materials Science, Yuan Ze University, Taoyuan 32003, Taiwan, ROC c Department of Chemical Engineering, National Taiwan University, Taipei 10617, Taiwan, ROC d Australian Institute for Innovative Materials, University of Wollongong, Squires Way, Innovation Campus, North Wollongong, New South Wales 2519, Australia e International Center for Materials Nanoarchitectonics (MANA), National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan b
a r t i c l e
i n f o
Article history: Received 16 November 2016 Revised 13 May 2017 Accepted 25 May 2017 Available online xxx Keywords: Bioconversion Fatty acid methyl esters Illumination Microalgae
a b s t r a c t Biomass energy is considered a clean and sustainable energy source that can reduce the amount of greenhouse gases. Among the different varieties of biomass, the algae can provide a number of different biofuel sources and reduce carbon dioxide (CO2 ) emissions. Botryococcus braunii is especially rich in lipids, which can be converted into bioenergy, but it typically grows more slowly. The aim of this study was to optimize the cultivation conditions in order to obtain high growth rates, biomass productivity, and lipid productivity. The effects of illumination and CO2 were studied in 21-day intervals. The cultured B. braunii in this work reached the maximum specific growth rate of 0.553 d−1 , and can tolerate CO2 concentrations of up to 10%. An illumination intensity of 60 0 0 lux was identified as the optimum for both biomass and lipid productivities. Compared to the results of other studies, the major components of fatty acid methyl esters (FAMEs) obtained in this study had shorter carbon chains. The percentage of C14:0 and C16:0 in the FAMEs was greater than 70%, indicating potential applications for biojet fuel. © 2017 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
1. Introduction The effects of global warming have become increasingly serious in recent years, not only permanently changing the global climate, but also affecting the international economic situation [1]. Finding solutions to ease the damage caused by global warming and seeking international cooperation are essential. A prior problem to be addressed is how to reduce the amounts of greenhouse gases (GHGs), especially CO2 [2], which is produced by the combustion of fossil fuels. On the other hand, fossil fuels cannot be regenerated, so these resources will be exhausted in the future. As a result, developing clean and renewable energy resources to replace traditional fossil fuels is imperative [3]. Many types of natural energy resources such as water, wind, and solar energy have been studied in the twentieth century, but these resources are still limited by the natural environment and the rate of energy transformation.
∗
Abbreviations: CO2 , carbon dioxide; FAMEs, fatty acid methyl esters. Corresponding author. E-mail address: [email protected] (Y.-T. Huang).
Another renewable resource that has received attention is biomass energy. Because biomass energy is derived from photosynthesis in plants, it is considered a clean energy source [4]. The reduction of GHG by using biomass-based jet fuels is under study [5]. Biofuel production so far has been divided into four generations. The first generation involves fermenting crops directly, which turns glucose or starches into ethanol, but using food as the feedstock can cause other social problems. The second generation involves collecting non-food plants or inedible parts of grains to produce biofuels, including biodiesel and bioethanol, but growing these plants requires large space and much time, therefore limiting production [6]. The third generation involves algae that are able to synthesize abundant amounts of lipids and hydrocarbons by photosynthesis [7–9]. Furthermore, the algae do not need much physical space for culturing before they are ready to be harvested. The fourth generation is currently under development, as scientists are trying to incubate bacteria that can produce hydrocarbons or lipids by genomic techniques. The products of the first and second stages have been commercialized, and although the problems of rooted plants have not been overcome, they can be used directly as
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Please cite this article as: Y.-T. Huang et al., Advances in bioconversion of microalgae with high biomass and lipid productivity, Journal of the Taiwan Institute of Chemical Engineers (2017), http://dx.doi.org/10.1016/j.jtice.2017.05.026
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fuel or mixed with fossil fuels [10]. Algae need little space to grow and have high expected growth rates, so they present an ideal solution [11]. Furthermore, algae can provide different kinds of biofuel sources and reduce CO2 emissions [12,13]. Algae also contain rich nonpolar lipids such as triacylglycerides [14], polar lipids such as phospholipids, hydrocarbons [15], and valuable chemicals [16,17]. These lipids can be converted to biodiesel via transesterification, and the gasoline can be collected after distillation [18]. Fatty acid methyl esters (FAMEs) are the primary components of biodiesel. Fatty acids, which can be converted into FAMEs through the transesterification, are rich in microalgae [19]. The purposes of FAMEs can be categorized according to their carbon chain lengths. For example, FAMEs with fewer than six carbon atoms in their carbon chains are processed to produce liquefied petroleum gas; those with six to twelve carbon atoms are processed for vehicular gasoline; and mixtures with twelve to fifteen carbons are suitable for jet fuel [20]. Biomass energy has actually been used in jet fuel. Air Canada produced biomass fuel from recycled cooking oil, and the first flight with biojet fuel took off on June 25, 2012. This might not only provide a promising outlook for producing biomass energy, but also contributes to reducing GHGs. Biodiesel is not only renewable, but also clean [21]. Among the reported producers of biodiesel, microalgae are significant due to their promising characteristics [22]. Botryococcus braunii, a species of green colonial oil-rich microalgae, may have a hydrocarbon content as high as 50 wt% [23]. Although B. braunii is richer in lipids compared to other species of green microalgae, this species usually grows slowly [24]. Some literatures have studied different cultivation conditions such as nutrient, light intensity, salinity, and CO2 concentration for the improvement of growth rate [25–29]. Our previous study optimized the cultivation conditions and successfully increased the specific growth rate and lipid productivity of B. braunii [30]. In this study we investigate the effects of different light intensities and increasing CO2 concentrations on the growth of B. braunii in 21-day intervals. The compositions of FAMEs in this study consisted mainly of those with short carbon chains, which indicated that the biochemical cultivation parameters are suitable for the production of biojet fuel.
biomass (Formula 2). For determining the microalgal cell number per unit volume, samples were serially diluted, which was followed by counting cells using a hemocytometer (Marienfeld, Lauda-Königshofen, Germany).
2. Material and methods
3. Results and discussion
2.1. Strain and culture medium of microalgae
3.1. The growth rate of B. braunii
The strain of microalgae used in this study was B. braunii NIES2199 (National Institute for Environmental Studies, Tsukuba, Japan). Cultivation of algae was conducted at room temperature (25 °C– 27 °C) in 50 0–20 0 0 mL Erlenmeyer flasks in a modified Chu 13 medium [31]. The details of the culturing procedure were reported in our previous study [30].
Table 1 shows the net specific growth rates (μnet ) of B. braunii cultured under varying conditions. The maximum μnet values for 10 days and 21 days of culturing, when calculated based on biomass, were 0.205 d−1 (12,0 0 0 lux, 5% CO2 ) and 0.138 d−1 (60 0 0 lux, 0.04% CO2 ), respectively. These results show that the B. braunii used in this study grew more efficiently in the first 10 days. In addition, the maximum μmax calculated based on biomass was 0.553 d−1 (60 0 0 lux, 5% CO2 ), indicating that increasing the concentration of CO2 up to 5% may facilitate the growth of B. braunii. When we calculated the growth rate based on cell number, the results show the same trend. The maximum μnet values for 10 days and 21 days of culturing were 0.208 d−1 (60 0 0 lux, 0.04% CO2 ) and 0.130 d−1 (60 0 0 lux, 5% CO2 ), respectively, Regardless of the CO2 concentration, the data showed that culturing at 60 0 0 lux or 12,0 0 0 lux resulted in a greater specific growth rate than at 30 0 0 lux.
2.2. Culturing conditions: light intensity and CO2 concentration The algae were cultured under light intensities of 30 0 0, 60 0 0, and 12,0 0 0 lux (42, 84, and 168 micromole photons m−2 s−1 ) to investigate the effects of light intensity. A lux meter, TM 50,0 0 0 (TOMEI, Tokyo, Japan), was used for measuring light intensities. The influent gas was a mixture of air and pure CO2 , and the resultant CO2 concentrations (0.04%, 5%, and 10%) were culturing parameters for studying the effects of CO2 concentration. The aeration rate was kept at 0.5 volumes per minute. Data was measured in triplicate over 21 days in 12- to 48-h intervals. 2.3. Calculation of growth rate The specific growth rate (μ) was calculated based on the following formulas regarding the cell number (Formula 1) or
μ(d−1 ) = [1n (cell number )2 − 1n (cell number )1 ]/1(t2 − t1 ) (1) We measured cellular dry weight (DW) as to find the biomass of the microalgae. 10 mL of algal culture was filtered through a pre-weighed filter paper and then washed with distilled water, which was followed by drying in an oven at 100 °C until there was no change in the weight. The DW per 10 mL was determined as the total weight minus the weight of the filter paper.
μ(d−1 ) = [1n (biomass )2 − 1n (cell number )1 ]/(t2 − t1 )
(2)
2.4. Determination of lipid content and FAMEs Lipids were extracted from the dried algae powder by using an organic solvent (1:2: v/v chloroform/methanol mixture) with ultrasonication. The lipids dissolved in the chloroform layer were collected, dried, and weighed to determine the total lipid content [32]. The FAMEs products were analyzed by gas chromatography (GC; Perkin–Elmer, Clarus 500) equipped with a flame ionization detector (FID) and SPۛ-2560 capillary column (100 m × 0.25 mm × 0.2 μm film thickness, Supelco Analytical). The programming temperature of the FID and injector was 220 °C, and the temperature of the oven was increased to 175 °C at the rate of 15 °C min–1 . The standards were manufactured by Sigma (nc-glc-67), consisting of a mixture of C14:0 , C16:0 , C18:0 , C18:1 , C18:2 , C20:1 , and C22:0 . The FAMEs composition was identified by the different time peak signals. 2.5. Analysis of chlorophyll-a The chlorophyll-a was extracted from the dried algae powder, and the concentration was measured in triplicate according to NIEA E508.00B (Environmental Analysis Laboratory, EPA, Executive Yuan, Taiwan, 2002).
3.2. The biomass productivity and accumulation of chlorophyll-a Algae were cultured for 21 days and harvested after 10 and 21 days. The DWs of B. braunii cultured at different CO2 concentrations and light intensities were estimated, and the productivities of the algal biomass were reported in our previous study [30]. The
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Fig. 1. The compositions of FAMEs under different culturing conditions. Table 1 The growth rates of algae under different culturing conditions. Culturing days
Light intensity (lux)
CO2 concentration (%)
μnet (d−1 )
μmax a (d−1 )
10
30 0 0
0.04 5 10 0.04 5 10 0.04 5 10
0.126 ± 0.005 0.036 ± 0.002 0.041 ± 0.004 0.195 ± 0.004 0.187 ± 0.009 0.058 ± 0.045 0.204 ± 0.006 0.205 ± 0.006 0.192 ± 0.006
0.238 0.372 0.396 0.484 0.553 0.139 0.452 0.454 0.437
0.04 5 10 0.04 5 10 0.04 5 10
0.080 ± 0.009 0.053 ± 0.001 0.063 ± 0.003 0.138 ± 0.005 0.115 ± 0.062 0.068 ± 0.023 0.130 ± 0.032 0.110 ± 0.027 0.127 ± 0.005
0.090 0.149 0.257 0.166 0.287 0.124 0.072 0.061 0.096
60 0 0
12,0 0 0
21
30 0 0
60 0 0
12,0 0 0
a The μ max calculated based on biomass accumulation from the first day to the tenth day, and from the eleventh day to the final day.
results showed that algae cultured at 30 0 0 lux produced 0.279– 0.500 g L−1 biomass after 10 days, and 0.601–0.850 g L−1 biomass after 21 days. Culturing at 60 0 0 lux produced 1.084–1.304 g L−1 biomass after 10 days, and 2.167–2.950 g L−1 biomass after 21 days, while culturing at 12,0 0 0 lux produced 0.988–1.086 g L−1 biomass after 10 days, and 1.674–2.053 g L−1 biomass after 21 days. These results demonstrated that 60 0 0 lux was the best illumination intensity for the accumulation of algal biomass, and the maximum biomass productivity was 140.46 mg L−1 d−1 at 60 0 0 lux with 0.04% CO2 . The effect of CO2 concentration on biomass was minor compared to the effect of illumination. The biomass productivities under the cultivating conditions of 0.04%, 5%, and 10% CO2 were 40.48–140.46 mg L−1 d−1 , 27.90–120.45 mg L−1 d−1 , and 29.20–108.40 mg L−1 d−1 , respectively. At the optimum illumination intensity of 60 0 0 lux, 9.86– 11.32 mg L−1 chlorophyll-a and 6.19–14.40 mg L−1 chlorophyll-a
were accumulated at 10 days and 21 days, respectively (Appendix Table A1). At 10 days, B. braunii accumulated 8.35–10.10 mg L−1 , 2.98–11.32 mg L−1 , and 2.76–9.86 mg L−1 of chlorophyll-a at 0.04%, 5%, and 10% CO2 , respectively. At 21 days, B. braunii accumulated 2.18–13.45 mg L−1 , 3.63–14.40 mg L−1 , and 2.49–6.19 mg L−1 of chlorophyll-a at 0.04%, 5%, and 10% CO2 , respectively. The results showed that 5% CO2 may increase the chlorophyll-a concentration at 60 0 0 lux, which may be a factor promoting better biomass and lipid productivity. 3.3. The composition and productivity of FAMEs The lipids were extracted from dried algae powder and transesterized into FAMEs, which were analyzed by GC. The ratios of different FAMEs are shown in Fig. 1. The major FAMEs component was C14:0 (24.97–68.61%), particularly at low illumination inten-
Please cite this article as: Y.-T. Huang et al., Advances in bioconversion of microalgae with high biomass and lipid productivity, Journal of the Taiwan Institute of Chemical Engineers (2017), http://dx.doi.org/10.1016/j.jtice.2017.05.026
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Y.-T. Huang et al. / Journal of the Taiwan Institute of Chemical Engineers 000 (2017) 1–6 Table 2 The lipid contents and productivities of FAMEs with different carbon numbers. Culturing days
Light intensity (lux)
CO2 concentration (%)
Lipid contenta (%)
Lipid productivitya (mg L−1 d−1 )
Lipid contentb (%)
Lipid productivityb (mg L−1 d−1 )
10
30 0 0
0.04 5 10 0.04 5 10 0.04 5 10
14.29 ± 1.69 13.83 ± 2.31 11.82 ± 0.65 9.12 ± 0.24 11.69 ± 0.71 8.17 ± 0.47 7.75 ± 1.09 18.14 ± 0.25 20.82 ± 1.88
7.15 ± 1.19 3.86 ± 0.92 3.45 ± 0.27 11.89 ± 0.44 14.08 ± 1.21 8.86 ± 0.72 8.05 ± 1.60 19.70 ± 0.38 20.57 ± 2.63
6.54 ± 0.77 7.15 ± 1.20 8.99 ± 0.49 16.63 ± 0.44 21.62 ± 1.31 24.51 ± 1.40 11.76 ± 1.66 14.67 ± 0.20 11.41 ± 1.03
3.27 ± 0.55 1.99 ± 0.47 2.63 ± 0.20 25.92 ± 2.22 26.04 ± 2.23 26.57 ± 2.15 12.22 ± 2.44 15.93 ± 0.31 11.27 ± 1.44
0.04 5 10 0.04 5 10 0.04 5 10
18.22 ± 7.53 20.98 ± 1.24 12.42 ± 1.10 10.40 ± 0.45 13.31 ± 0.60 8.55 ± 1.58 17.48 ± 2.05 15.79 ± 2.55 22.28 ± 3.00
7.37 ± 4.31 6.00 ± 0.50 4.33 ± 0.55 14.60 ± 0.89 15.61 ± 1.00 8.82 ± 2.31 17.09 ± 2.84 12.59 ± 2.88 20.25 ± 3.85
8.34 ± 3.45 10.86 ± 0.64 9.44 ± 0.84 18.97 ± 0.82 24.61 ± 1.12 25.64 ± 4.75 26.56 ± 3.12 12.76 ± 2.07 12.21 ± 1.64
3.38 ± 1.98 3.11 ± 0.26 3.29 ± 0.41 28.72 ± 1.84 28.85 ± 1.85 26.45 ± 6.93 25.96 ± 4.31 10.17 ± 2.33 11.09 ± 2.11
60 0 0
12,0 0 0
21
30 0 0
60 0 0
12,0 0 0
a b
Lipid content and productivity of C14 . Lipid content and productivity of C16 – 22 . Table 3 The ratios of FAMEs reported in other references. Added CO2 (%)
Lipid % (ࣚC14 )
Lipid % (C16 )
Lipid % (C18 )
Lipid % (ࣛC20 )
Reference
0
0.77 – 68.61 35.39 39.75 – – 0.62 2.52 65.86 35.13 55.29 56.80 24.97 64.63
9.42 24.79 25.87 37.19 56.16 41.49 38.84 6.83 18 27.01 36.57 34.66 40.09 44.76 29.17
64.10 58.80 3.62 15.49 1.49 47.70 43.05 85 53.84 4.49 13.83 7.84 1.58 15.62 3.76
25.66 15.73 1.89 11.93 2.60 9.83 17.97 5.88 25.64 2.64 12.32 2.12 1.53 14.64 2.43
[33]a [34]b This studyc This studyd This studye [34]b [34]b [35]f [35]f This studyc This studyd This studye This studyc This studyd This studye
1 2 2.5 2.5 5
10
a b c d e f
30 μE m– 2 s– 1 and 16/8 light dark cycle. 1.2 ± 0.2 klux and 16/8 h light dark cycle. 3 klux. 6 klux. 12 klux. 170 μE m– 2 s– 1 and 12/12 light dark cycle.
sity (56.80–68.61% at 30 0 0 lux) (Tables 2 and 3). As light intensity increased, the amount of C16:0 increased up to 56.16% of the total analyzed FAMEs. The optimum cultivation conditions for lipid production (37.92% lipid content, 44.46 mg L−1 d−1 lipid productivity) were 60 0 0 lux, 5% CO2 , and 21 days of culturing, resulting in 35.13% of C14:0 and 36.57% of C16:0 in the FAMEs (Fig. 2). Table 2 shows the productivity for the FAMEs under different culturing conditions. The productivity of C14:0 was relatively high compared to the other FAMEs at low illumination intensity. At the optimum illumination intensity of 60 0 0 lux for the accumulation of biomass and lipids, the productivities of C14:0 and C16:0 were at the similar level of 8.82–16.27 mg L−1 d−1 . Culturing under 60 0 0 lux also enhanced the production of C18:1 and C22:0 , regardless of the concentration of CO2 used in this study. The results show the possibility of producing different FAME compositions by culturing microalgae at various illumination intensities. Compared to other studies, our work presents evidence that short carbon chain products (ࣚ C14) were more abundant than the others (Table 3). In the study by Ashokkumar and Rengasamy [33], 64.10% of the FAMEs had a carbon chain length of 18. Nearly 90% of the FAMEs had carbon chain lengths of 16 or 18 in the study by
Rao et al. [34]. The parameters of this study can reach a high short carbon chain (ࣚ C14) accumulation up to near 70%; however, only 0.77% and 0.62–2.52% of those can be accumulated in the study by Ashokkumar and Rengasamy [33] and Cabanelas et al. [35], respectively. Therefore, results of our work can help to produce FAMEs with shorter carbon chains, which may be suitable for biojet fuel.
4. Conclusions This study found the optimum cultivation conditions for the accumulation of lipids that undergo transesterification to FAMEs of different compositions. The Botryococcus braunii cultured in this work can tolerate CO2 concentrations of up to 10%, with a high maximum specific growth rate of 0.682 d−1 . 60 0 0 lux was identified as the optimum illumination intensity for both biomass and lipid productivities. Compared to the results of other studies, the major components of the FAMEs obtained in this study had shorter carbon chains (C14:0 and C16:0 ). The percentage of C14:0 in the FAMEs was greater than 24.97%, and using low illumination intensity can further increase this percentage to 68.61%.
Please cite this article as: Y.-T. Huang et al., Advances in bioconversion of microalgae with high biomass and lipid productivity, Journal of the Taiwan Institute of Chemical Engineers (2017), http://dx.doi.org/10.1016/j.jtice.2017.05.026
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Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.jtice.2017.05.026. References
Fig. 2. Lipid contents and productivities (a) lipid content at 10th cultured day, (b) lipid content at 21st cultured day, (c) lipid productivity at 10th cultured day, (d) lipid productivity at 21st cultured day.
Acknowledgments Authors would like to thank the Ministry of Science and Technology (MOST) of Taiwan for supporting this research (101-2623E-033-001-ET, 102-2623-E-033-001-ET, 104-2622-E-155-013-CC2). This work was partially supported by the Australian Institute for Innovative Materials (AIIM) Gold/2017 grant. The authors would like to thank Dr Macs Bio-Pharma Private Limited for helpful suggestions and discussions.
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Advances in bioconversion of microalgae with high biomass and lipid productivity Yu-Tzu Huang a,∗, Chung-Wei Lai a, Bo-Wei Wu a, Kuen-Song Lin b, Jeffrey C.S. Wu c, Md Shahriar A Hossain d,e, Yusuke Yamauchi d,e, Kevin C.-W. Wu c a
Department of Environmental Engineering and Research Center for Analysis and Identification, Chung Yuan Christian University, Taoyuan 32023, Taiwan, ROC Department of Chemical Engineering and Materials Science, Yuan Ze University, Taoyuan 32003, Taiwan, ROC c Department of Chemical Engineering, National Taiwan University, Taipei 10617, Taiwan, ROC d Australian Institute for Innovative Materials, University of Wollongong, Squires Way, Innovation Campus, North Wollongong, New South Wales 2519, Australia e International Center for Materials Nanoarchitectonics (MANA), National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan b
a r t i c l e
i n f o
Article history: Received 16 November 2016 Revised 13 May 2017 Accepted 25 May 2017 Available online xxx Keywords: Bioconversion Fatty acid methyl esters Illumination Microalgae
a b s t r a c t Biomass energy is considered a clean and sustainable energy source that can reduce the amount of greenhouse gases. Among the different varieties of biomass, the algae can provide a number of different biofuel sources and reduce carbon dioxide (CO2 ) emissions. Botryococcus braunii is especially rich in lipids, which can be converted into bioenergy, but it typically grows more slowly. The aim of this study was to optimize the cultivation conditions in order to obtain high growth rates, biomass productivity, and lipid productivity. The effects of illumination and CO2 were studied in 21-day intervals. The cultured B. braunii in this work reached the maximum specific growth rate of 0.553 d−1 , and can tolerate CO2 concentrations of up to 10%. An illumination intensity of 60 0 0 lux was identified as the optimum for both biomass and lipid productivities. Compared to the results of other studies, the major components of fatty acid methyl esters (FAMEs) obtained in this study had shorter carbon chains. The percentage of C14:0 and C16:0 in the FAMEs was greater than 70%, indicating potential applications for biojet fuel. © 2017 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
1. Introduction The effects of global warming have become increasingly serious in recent years, not only permanently changing the global climate, but also affecting the international economic situation [1]. Finding solutions to ease the damage caused by global warming and seeking international cooperation are essential. A prior problem to be addressed is how to reduce the amounts of greenhouse gases (GHGs), especially CO2 [2], which is produced by the combustion of fossil fuels. On the other hand, fossil fuels cannot be regenerated, so these resources will be exhausted in the future. As a result, developing clean and renewable energy resources to replace traditional fossil fuels is imperative [3]. Many types of natural energy resources such as water, wind, and solar energy have been studied in the twentieth century, but these resources are still limited by the natural environment and the rate of energy transformation.
∗
Abbreviations: CO2 , carbon dioxide; FAMEs, fatty acid methyl esters. Corresponding author. E-mail address: [email protected] (Y.-T. Huang).
Another renewable resource that has received attention is biomass energy. Because biomass energy is derived from photosynthesis in plants, it is considered a clean energy source [4]. The reduction of GHG by using biomass-based jet fuels is under study [5]. Biofuel production so far has been divided into four generations. The first generation involves fermenting crops directly, which turns glucose or starches into ethanol, but using food as the feedstock can cause other social problems. The second generation involves collecting non-food plants or inedible parts of grains to produce biofuels, including biodiesel and bioethanol, but growing these plants requires large space and much time, therefore limiting production [6]. The third generation involves algae that are able to synthesize abundant amounts of lipids and hydrocarbons by photosynthesis [7–9]. Furthermore, the algae do not need much physical space for culturing before they are ready to be harvested. The fourth generation is currently under development, as scientists are trying to incubate bacteria that can produce hydrocarbons or lipids by genomic techniques. The products of the first and second stages have been commercialized, and although the problems of rooted plants have not been overcome, they can be used directly as
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fuel or mixed with fossil fuels [10]. Algae need little space to grow and have high expected growth rates, so they present an ideal solution [11]. Furthermore, algae can provide different kinds of biofuel sources and reduce CO2 emissions [12,13]. Algae also contain rich nonpolar lipids such as triacylglycerides [14], polar lipids such as phospholipids, hydrocarbons [15], and valuable chemicals [16,17]. These lipids can be converted to biodiesel via transesterification, and the gasoline can be collected after distillation [18]. Fatty acid methyl esters (FAMEs) are the primary components of biodiesel. Fatty acids, which can be converted into FAMEs through the transesterification, are rich in microalgae [19]. The purposes of FAMEs can be categorized according to their carbon chain lengths. For example, FAMEs with fewer than six carbon atoms in their carbon chains are processed to produce liquefied petroleum gas; those with six to twelve carbon atoms are processed for vehicular gasoline; and mixtures with twelve to fifteen carbons are suitable for jet fuel [20]. Biomass energy has actually been used in jet fuel. Air Canada produced biomass fuel from recycled cooking oil, and the first flight with biojet fuel took off on June 25, 2012. This might not only provide a promising outlook for producing biomass energy, but also contributes to reducing GHGs. Biodiesel is not only renewable, but also clean [21]. Among the reported producers of biodiesel, microalgae are significant due to their promising characteristics [22]. Botryococcus braunii, a species of green colonial oil-rich microalgae, may have a hydrocarbon content as high as 50 wt% [23]. Although B. braunii is richer in lipids compared to other species of green microalgae, this species usually grows slowly [24]. Some literatures have studied different cultivation conditions such as nutrient, light intensity, salinity, and CO2 concentration for the improvement of growth rate [25–29]. Our previous study optimized the cultivation conditions and successfully increased the specific growth rate and lipid productivity of B. braunii [30]. In this study we investigate the effects of different light intensities and increasing CO2 concentrations on the growth of B. braunii in 21-day intervals. The compositions of FAMEs in this study consisted mainly of those with short carbon chains, which indicated that the biochemical cultivation parameters are suitable for the production of biojet fuel.
biomass (Formula 2). For determining the microalgal cell number per unit volume, samples were serially diluted, which was followed by counting cells using a hemocytometer (Marienfeld, Lauda-Königshofen, Germany).
2. Material and methods
3. Results and discussion
2.1. Strain and culture medium of microalgae
3.1. The growth rate of B. braunii
The strain of microalgae used in this study was B. braunii NIES2199 (National Institute for Environmental Studies, Tsukuba, Japan). Cultivation of algae was conducted at room temperature (25 °C– 27 °C) in 50 0–20 0 0 mL Erlenmeyer flasks in a modified Chu 13 medium [31]. The details of the culturing procedure were reported in our previous study [30].
Table 1 shows the net specific growth rates (μnet ) of B. braunii cultured under varying conditions. The maximum μnet values for 10 days and 21 days of culturing, when calculated based on biomass, were 0.205 d−1 (12,0 0 0 lux, 5% CO2 ) and 0.138 d−1 (60 0 0 lux, 0.04% CO2 ), respectively. These results show that the B. braunii used in this study grew more efficiently in the first 10 days. In addition, the maximum μmax calculated based on biomass was 0.553 d−1 (60 0 0 lux, 5% CO2 ), indicating that increasing the concentration of CO2 up to 5% may facilitate the growth of B. braunii. When we calculated the growth rate based on cell number, the results show the same trend. The maximum μnet values for 10 days and 21 days of culturing were 0.208 d−1 (60 0 0 lux, 0.04% CO2 ) and 0.130 d−1 (60 0 0 lux, 5% CO2 ), respectively, Regardless of the CO2 concentration, the data showed that culturing at 60 0 0 lux or 12,0 0 0 lux resulted in a greater specific growth rate than at 30 0 0 lux.
2.2. Culturing conditions: light intensity and CO2 concentration The algae were cultured under light intensities of 30 0 0, 60 0 0, and 12,0 0 0 lux (42, 84, and 168 micromole photons m−2 s−1 ) to investigate the effects of light intensity. A lux meter, TM 50,0 0 0 (TOMEI, Tokyo, Japan), was used for measuring light intensities. The influent gas was a mixture of air and pure CO2 , and the resultant CO2 concentrations (0.04%, 5%, and 10%) were culturing parameters for studying the effects of CO2 concentration. The aeration rate was kept at 0.5 volumes per minute. Data was measured in triplicate over 21 days in 12- to 48-h intervals. 2.3. Calculation of growth rate The specific growth rate (μ) was calculated based on the following formulas regarding the cell number (Formula 1) or
μ(d−1 ) = [1n (cell number )2 − 1n (cell number )1 ]/1(t2 − t1 ) (1) We measured cellular dry weight (DW) as to find the biomass of the microalgae. 10 mL of algal culture was filtered through a pre-weighed filter paper and then washed with distilled water, which was followed by drying in an oven at 100 °C until there was no change in the weight. The DW per 10 mL was determined as the total weight minus the weight of the filter paper.
μ(d−1 ) = [1n (biomass )2 − 1n (cell number )1 ]/(t2 − t1 )
(2)
2.4. Determination of lipid content and FAMEs Lipids were extracted from the dried algae powder by using an organic solvent (1:2: v/v chloroform/methanol mixture) with ultrasonication. The lipids dissolved in the chloroform layer were collected, dried, and weighed to determine the total lipid content [32]. The FAMEs products were analyzed by gas chromatography (GC; Perkin–Elmer, Clarus 500) equipped with a flame ionization detector (FID) and SPۛ-2560 capillary column (100 m × 0.25 mm × 0.2 μm film thickness, Supelco Analytical). The programming temperature of the FID and injector was 220 °C, and the temperature of the oven was increased to 175 °C at the rate of 15 °C min–1 . The standards were manufactured by Sigma (nc-glc-67), consisting of a mixture of C14:0 , C16:0 , C18:0 , C18:1 , C18:2 , C20:1 , and C22:0 . The FAMEs composition was identified by the different time peak signals. 2.5. Analysis of chlorophyll-a The chlorophyll-a was extracted from the dried algae powder, and the concentration was measured in triplicate according to NIEA E508.00B (Environmental Analysis Laboratory, EPA, Executive Yuan, Taiwan, 2002).
3.2. The biomass productivity and accumulation of chlorophyll-a Algae were cultured for 21 days and harvested after 10 and 21 days. The DWs of B. braunii cultured at different CO2 concentrations and light intensities were estimated, and the productivities of the algal biomass were reported in our previous study [30]. The
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Fig. 1. The compositions of FAMEs under different culturing conditions. Table 1 The growth rates of algae under different culturing conditions. Culturing days
Light intensity (lux)
CO2 concentration (%)
μnet (d−1 )
μmax a (d−1 )
10
30 0 0
0.04 5 10 0.04 5 10 0.04 5 10
0.126 ± 0.005 0.036 ± 0.002 0.041 ± 0.004 0.195 ± 0.004 0.187 ± 0.009 0.058 ± 0.045 0.204 ± 0.006 0.205 ± 0.006 0.192 ± 0.006
0.238 0.372 0.396 0.484 0.553 0.139 0.452 0.454 0.437
0.04 5 10 0.04 5 10 0.04 5 10
0.080 ± 0.009 0.053 ± 0.001 0.063 ± 0.003 0.138 ± 0.005 0.115 ± 0.062 0.068 ± 0.023 0.130 ± 0.032 0.110 ± 0.027 0.127 ± 0.005
0.090 0.149 0.257 0.166 0.287 0.124 0.072 0.061 0.096
60 0 0
12,0 0 0
21
30 0 0
60 0 0
12,0 0 0
a The μ max calculated based on biomass accumulation from the first day to the tenth day, and from the eleventh day to the final day.
results showed that algae cultured at 30 0 0 lux produced 0.279– 0.500 g L−1 biomass after 10 days, and 0.601–0.850 g L−1 biomass after 21 days. Culturing at 60 0 0 lux produced 1.084–1.304 g L−1 biomass after 10 days, and 2.167–2.950 g L−1 biomass after 21 days, while culturing at 12,0 0 0 lux produced 0.988–1.086 g L−1 biomass after 10 days, and 1.674–2.053 g L−1 biomass after 21 days. These results demonstrated that 60 0 0 lux was the best illumination intensity for the accumulation of algal biomass, and the maximum biomass productivity was 140.46 mg L−1 d−1 at 60 0 0 lux with 0.04% CO2 . The effect of CO2 concentration on biomass was minor compared to the effect of illumination. The biomass productivities under the cultivating conditions of 0.04%, 5%, and 10% CO2 were 40.48–140.46 mg L−1 d−1 , 27.90–120.45 mg L−1 d−1 , and 29.20–108.40 mg L−1 d−1 , respectively. At the optimum illumination intensity of 60 0 0 lux, 9.86– 11.32 mg L−1 chlorophyll-a and 6.19–14.40 mg L−1 chlorophyll-a
were accumulated at 10 days and 21 days, respectively (Appendix Table A1). At 10 days, B. braunii accumulated 8.35–10.10 mg L−1 , 2.98–11.32 mg L−1 , and 2.76–9.86 mg L−1 of chlorophyll-a at 0.04%, 5%, and 10% CO2 , respectively. At 21 days, B. braunii accumulated 2.18–13.45 mg L−1 , 3.63–14.40 mg L−1 , and 2.49–6.19 mg L−1 of chlorophyll-a at 0.04%, 5%, and 10% CO2 , respectively. The results showed that 5% CO2 may increase the chlorophyll-a concentration at 60 0 0 lux, which may be a factor promoting better biomass and lipid productivity. 3.3. The composition and productivity of FAMEs The lipids were extracted from dried algae powder and transesterized into FAMEs, which were analyzed by GC. The ratios of different FAMEs are shown in Fig. 1. The major FAMEs component was C14:0 (24.97–68.61%), particularly at low illumination inten-
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Y.-T. Huang et al. / Journal of the Taiwan Institute of Chemical Engineers 000 (2017) 1–6 Table 2 The lipid contents and productivities of FAMEs with different carbon numbers. Culturing days
Light intensity (lux)
CO2 concentration (%)
Lipid contenta (%)
Lipid productivitya (mg L−1 d−1 )
Lipid contentb (%)
Lipid productivityb (mg L−1 d−1 )
10
30 0 0
0.04 5 10 0.04 5 10 0.04 5 10
14.29 ± 1.69 13.83 ± 2.31 11.82 ± 0.65 9.12 ± 0.24 11.69 ± 0.71 8.17 ± 0.47 7.75 ± 1.09 18.14 ± 0.25 20.82 ± 1.88
7.15 ± 1.19 3.86 ± 0.92 3.45 ± 0.27 11.89 ± 0.44 14.08 ± 1.21 8.86 ± 0.72 8.05 ± 1.60 19.70 ± 0.38 20.57 ± 2.63
6.54 ± 0.77 7.15 ± 1.20 8.99 ± 0.49 16.63 ± 0.44 21.62 ± 1.31 24.51 ± 1.40 11.76 ± 1.66 14.67 ± 0.20 11.41 ± 1.03
3.27 ± 0.55 1.99 ± 0.47 2.63 ± 0.20 25.92 ± 2.22 26.04 ± 2.23 26.57 ± 2.15 12.22 ± 2.44 15.93 ± 0.31 11.27 ± 1.44
0.04 5 10 0.04 5 10 0.04 5 10
18.22 ± 7.53 20.98 ± 1.24 12.42 ± 1.10 10.40 ± 0.45 13.31 ± 0.60 8.55 ± 1.58 17.48 ± 2.05 15.79 ± 2.55 22.28 ± 3.00
7.37 ± 4.31 6.00 ± 0.50 4.33 ± 0.55 14.60 ± 0.89 15.61 ± 1.00 8.82 ± 2.31 17.09 ± 2.84 12.59 ± 2.88 20.25 ± 3.85
8.34 ± 3.45 10.86 ± 0.64 9.44 ± 0.84 18.97 ± 0.82 24.61 ± 1.12 25.64 ± 4.75 26.56 ± 3.12 12.76 ± 2.07 12.21 ± 1.64
3.38 ± 1.98 3.11 ± 0.26 3.29 ± 0.41 28.72 ± 1.84 28.85 ± 1.85 26.45 ± 6.93 25.96 ± 4.31 10.17 ± 2.33 11.09 ± 2.11
60 0 0
12,0 0 0
21
30 0 0
60 0 0
12,0 0 0
a b
Lipid content and productivity of C14 . Lipid content and productivity of C16 – 22 . Table 3 The ratios of FAMEs reported in other references. Added CO2 (%)
Lipid % (ࣚC14 )
Lipid % (C16 )
Lipid % (C18 )
Lipid % (ࣛC20 )
Reference
0
0.77 – 68.61 35.39 39.75 – – 0.62 2.52 65.86 35.13 55.29 56.80 24.97 64.63
9.42 24.79 25.87 37.19 56.16 41.49 38.84 6.83 18 27.01 36.57 34.66 40.09 44.76 29.17
64.10 58.80 3.62 15.49 1.49 47.70 43.05 85 53.84 4.49 13.83 7.84 1.58 15.62 3.76
25.66 15.73 1.89 11.93 2.60 9.83 17.97 5.88 25.64 2.64 12.32 2.12 1.53 14.64 2.43
[33]a [34]b This studyc This studyd This studye [34]b [34]b [35]f [35]f This studyc This studyd This studye This studyc This studyd This studye
1 2 2.5 2.5 5
10
a b c d e f
30 μE m– 2 s– 1 and 16/8 light dark cycle. 1.2 ± 0.2 klux and 16/8 h light dark cycle. 3 klux. 6 klux. 12 klux. 170 μE m– 2 s– 1 and 12/12 light dark cycle.
sity (56.80–68.61% at 30 0 0 lux) (Tables 2 and 3). As light intensity increased, the amount of C16:0 increased up to 56.16% of the total analyzed FAMEs. The optimum cultivation conditions for lipid production (37.92% lipid content, 44.46 mg L−1 d−1 lipid productivity) were 60 0 0 lux, 5% CO2 , and 21 days of culturing, resulting in 35.13% of C14:0 and 36.57% of C16:0 in the FAMEs (Fig. 2). Table 2 shows the productivity for the FAMEs under different culturing conditions. The productivity of C14:0 was relatively high compared to the other FAMEs at low illumination intensity. At the optimum illumination intensity of 60 0 0 lux for the accumulation of biomass and lipids, the productivities of C14:0 and C16:0 were at the similar level of 8.82–16.27 mg L−1 d−1 . Culturing under 60 0 0 lux also enhanced the production of C18:1 and C22:0 , regardless of the concentration of CO2 used in this study. The results show the possibility of producing different FAME compositions by culturing microalgae at various illumination intensities. Compared to other studies, our work presents evidence that short carbon chain products (ࣚ C14) were more abundant than the others (Table 3). In the study by Ashokkumar and Rengasamy [33], 64.10% of the FAMEs had a carbon chain length of 18. Nearly 90% of the FAMEs had carbon chain lengths of 16 or 18 in the study by
Rao et al. [34]. The parameters of this study can reach a high short carbon chain (ࣚ C14) accumulation up to near 70%; however, only 0.77% and 0.62–2.52% of those can be accumulated in the study by Ashokkumar and Rengasamy [33] and Cabanelas et al. [35], respectively. Therefore, results of our work can help to produce FAMEs with shorter carbon chains, which may be suitable for biojet fuel.
4. Conclusions This study found the optimum cultivation conditions for the accumulation of lipids that undergo transesterification to FAMEs of different compositions. The Botryococcus braunii cultured in this work can tolerate CO2 concentrations of up to 10%, with a high maximum specific growth rate of 0.682 d−1 . 60 0 0 lux was identified as the optimum illumination intensity for both biomass and lipid productivities. Compared to the results of other studies, the major components of the FAMEs obtained in this study had shorter carbon chains (C14:0 and C16:0 ). The percentage of C14:0 in the FAMEs was greater than 24.97%, and using low illumination intensity can further increase this percentage to 68.61%.
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Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.jtice.2017.05.026. References
Fig. 2. Lipid contents and productivities (a) lipid content at 10th cultured day, (b) lipid content at 21st cultured day, (c) lipid productivity at 10th cultured day, (d) lipid productivity at 21st cultured day.
Acknowledgments Authors would like to thank the Ministry of Science and Technology (MOST) of Taiwan for supporting this research (101-2623E-033-001-ET, 102-2623-E-033-001-ET, 104-2622-E-155-013-CC2). This work was partially supported by the Australian Institute for Innovative Materials (AIIM) Gold/2017 grant. The authors would like to thank Dr Macs Bio-Pharma Private Limited for helpful suggestions and discussions.
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Please cite this article as: Y.-T. Huang et al., Advances in bioconversion of microalgae with high biomass and lipid productivity, Journal of the Taiwan Institute of Chemical Engineers (2017), http://dx.doi.org/10.1016/j.jtice.2017.05.026