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Oil productivity of the tropical marine diatom Thalassiosira sp.
Bioresource Technology 108 (2012) 240–244
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Oil productivity of the tropical marine diatom Thalassiosira sp. Zeily Nurachman a,⇑, Hartati a, Syahfitri Anita a, Etsuroyya Ewidyasari Anward a, Gestria Novirani a, Bill Mangindaan b, Suryo Gandasasmita b, Yana Maolana Syah c, Lily Maria Goretty Panggabean d, Gede Suantika e a
Biochemistry Division, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Jl. Ganesha 10, Bandung 40132, Indonesia Analytical Chemistry Division, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Jl. Ganesha 10, Bandung 40132, Indonesia Organic Chemistry Division, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Jl. Ganesha 10, Bandung 40132, Indonesia d Research Center for Oceanography, Indonesian Institute of Sciences, Jl. Pasir Putih I, Ancol Timur, Jakarta 14430, Indonesia e Ecology and Biosystematics Research Group, School of Life Sciences and Technology, Institut Teknologi Bandung, Jl. Ganesha 10, Bandung 40132, Indonesia b c
a r t i c l e
i n f o
Article history: Received 14 September 2011 Received in revised form 16 December 2011 Accepted 16 December 2011 Available online 23 December 2011 Keywords: Biodiesel Oil productivity TAG Thalassiosira sp. Tropical marine microalgae
a b s t r a c t To understand the potential of cultivating tropical marine diatom Thalassiosira sp. to produce biofuel, biodiesel product properties and growth characteristics of Thalassiosira sp. in three different media were investigated. After medium evaluation, significant Thalassiosira sp. cell growth was observed in both Walne and enriched seawater media, but not in plain seawater medium. The microalgae grew well in alkaline condition (pH range of 8.0–8.8). The average biomass density cultured in Walne and enriched seawater media on the 6th day was 4.36 and 2.50 g L 1, respectively. Based on ESI-IT-MS spectra, the TAGs of algal oil were identified as POP, POO, and SOO, and the FAMEs as oleic acid methyl ester. The oil productivity of Thalassiosira sp. cultured in Walne and enriched seawater media were 150 and 290 lL L 1 d 1, respectively. The density and kinematic viscosity of Thalassiosira sp. biodiesel were 0.857 g mL 1 and 1.151 mm2 s 1. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Most oil-producing countries including Indonesia are both located adjacent to the tectonic plates and close to the sea. It is believed that the fossil fuel produced in Indonesia is not derived from wood-plant fossils (because the geological age of Indonesia is very young), but comes from aquatic plants including microalgae e.g. Botryococcus braunii (Banerjee et al., 2002; Metzger and Largeau, 2005). This is based on the fact that the original territory of Indonesia comes from the sea where tectonic plates shift and volcanoes raise the ground. The movement of tectonic plates through tectonic earthquakes then traps and presses seawater and everything in the seawater including marine microalgae into the soil to form a layer of oil. In the laboratory, this can be demonstrated by squeezing microalgae cells to push oil out. Microalgae occupy almost all layers of the sea in a huge amount. Both living microalgae and their dead bodies are subject to becoming sources of fossil fuel. The process of fossil fuel formation itself is, of course, very complicated and takes a long time. The first step to prove that microalgae contributes to fossil fuel formation is to identify as many types of microalgae from the ocean as possible and then measure their oil productivity. ⇑ Corresponding author. Tel.: +62 22 250 2103; fax: +62 22 250 4154. E-mail address: [email protected] (Z. Nurachman). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.12.082
Many reports and reviews on biodiesel from microalgae have been published elsewhere (Sheehan et al., 1998; Brennan and Owende, 2010; Wijffels and Barbosa, 2010; Gong and Jiang, 2011). Microalgae are microscopic plants that grow in water, can produce high oil content in a range of 20–50% dry mass of biomass and are capable of doubling their biomass in a period of 3.5 h (Chisti, 2007). Microalgae can convert 3–8% solar energy to biomass through photosynthesis (Li et al., 2008). Since microalgal cells grow in aqueous suspension, they have a larger surface area to access water, carbon dioxide and minerals for photosynthesis. Hence, the oil productivity of microalgae is essentially much higher than that of terrestrial plants (Chisti, 2008). Tropical marine microalgae are very interesting subjects of investigation as they produce various types of natural oils (triacylglycerols, TAGs; and fatty acids, FA either saturated, unsaturated, or polyunsaturated) for biodiesel (fatty acid methyl ester, FAME), chlorophyll, and carotenoid compounds. Among microalgae, diatoms (Bacillariophyta) are a very promising source for producing biofuel because these microalgae accumulate carbon in the form of natural oils and dominate the phytoplankton of the seawaters (Sheehan et al., 1998). Diatoms inhabit most bodies of water and can be found in the littoral and pelagic regions. Individually, diatoms do not appear in a highly visible color, but in groups they can be seen due to the presence of photosynthetic plastids. The culturable tropical marine diatom Thalassiosira sp. has been identified
Z. Nurachman et al. / Bioresource Technology 108 (2012) 240–244
as capable of producing biodiesel (Nurachman, 2011). This paper reports further investigation on the growth medium and oil productivity of Thalassiosira sp. as well as biodiesel characteristics.
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FAMEs in both oil and biodiesel were analyzed by Electron Spray Ionization-Ion Trap-Mass Spectrometry (ESI-IT-MS). 2.4. Analytical procedures
2. Methods 2.1. Microalgae and culture maintenance The original specimen of Thalassiosira sp. was obtained from the School of Life Sciences and Technology, Institut Teknologi Bandung, Indonesia. Cultures were maintained in Walne artificial seawater medium containing (g L 1): NaNO3, 100; Na2EDTA, 45; NaSiO35H2O, 40; H3BO3, 33.6; NaH2PO42H2O, 20.0; FeCl36H2O, 13.0; MnCl24H2O, 0.36; vitamin B12; vitamin B1, 0.01, 0.2; ZnCl2, 0.021; CoCl2, 0.02; CuSO45H2O, 0.02; and (NH4)6Mo7O2424H2O, 0.009 (Andersen, 2005). All solutions were prepared in sterilized water under aseptic conditions. The simple air-lift photobioreactors were made using transparent glass bottles with a height of 25 cm, an external diameter of 9 cm and a working volume of 800 mL. To maintain slow growth of algal stock culture, cells of Thalassiosira sp. were grown in air-lift photobioreactors and continuously illuminated under a light intensity of 40.5 lmol m 2 s 1 at room temperature, salinity 25 ppt and free air bubbling. The cells had to be periodically regenerated to stay in the growth phase. 2.2. Medium search Two other kinds of growth medium were tested: plain seawater and enriched seawater. The enriched seawater medium was a seawater medium supplemented with additional basic elements containing 300 mg urea, 45 mg NaH2PO42H2O, and 190 mg Na2SiO35H2O, 70 mg Na2EDTA, and 6 mg FeCl3 per 1 L medium. For biomass production, cultures with an initial density of 1 105 cell mL 1 were illuminated under a light intensity of 81 lmol m 2 s 1 at room temperature, photoperiod of light:dark = 12:12, salinity 25 ppt, and free air bubbling. Changes in cell numbers and pH was observed daily. Cell numbers were counted under a microscope with an improved Neubauer hemocytometer. All data were subjected to one-way analysis of variance (ANOVA) and the significance of differences among medium compositions on the yield of biomass was tested using the F test (P < 0.01). 2.3. Oil extraction and biodiesel conversion Oils were extracted using a modification of the methods of Bligh and Dyer (1959). Cultures of Thalassiosira sp. at the start of the stationary phase were harvested by centrifugation at 6000g for 15 min at 4 °C, and its wet biomass was then weighted. The wet biomass (10.1 g obtained from the Walne medium culture or 7.0 g obtained from the enriched seawater culture) was suspended in 70 mL chloroform/methanol (1:1 v/v) and then sonicated for 15 min at 50 Hz frequency, followed by Soxhlet extraction with the same solvent at 60 °C for 12 h. The solvent was removed by rotavaporation, and the volume of oils was measured. Oil productivity of Thalassiosira sp. is defined by the total amount of oils (lL) obtained from 1 L of microalgal culture until the beginning of the stationary phase at experimental conditions, or by the following formula: Oil productivity = Obtained oil (lL g 1 wet biomass) biomass density (g L 1 culture) time of growth (d). For conversion into biodiesel, transesterification of the oils was performed by refluxing microalgal TAGs, methanol, and KOH at a ratio of 10:2.3:0.123 (w/w/w) at 70 °C for 1 h. Biodiesel was separated by centrifugation at 8000g for 5 min at room temperature, followed by rotavaporation to remove the solvent. TAGs and
Mass spectrometry of biodiesels was performed on an ESI-ITMS HCT Bruker-Daltonic GmbH instrument with AgNO3 used as coordination ionization agent (Sandra et al., 2002). A simple flame test was performed on the biodiesels. Kinematic viscosity of biodiesels was determined through the method of ASTM D 445. Biodiesel density was also determined. For comparison, commercial (B40) and palm oil biodiesels were also analyzed. 3. Results and discussion 3.1. Algal characteristics and growth The marine diatom Thalassiosira sp. isolated from waters in the Java sea is a neritic planktonic species that belongs to the order Centricae. Its cells are similar to those of Coscinodiscus, usually drum or disc-shaped, joined in flexible chains by a cytoplasmic or gelatinous thread. The microalgae was yellowish brown, had a rectangular-like shape, and was able to form biosilica cell walls. Cell size was approximately 10 lm in diameter during culture period (smaller than its actual diameter size of 16–20 lm). For this study, culture condition was kept in the tolerance range of microalgae allowing permanent asexual reproduction. Starting from 1 105 cell mL 1 culture in Walne medium, hemocytometer counts for intact cells showed a maximum Thalassiosira sp. density at the late logarithmic phase (on the 6th day) of 9.1 106 cell L 1 culture, with a specific growth rate of 7.2 10 9/d (Fig. 1A). The average wet biomass of Thalassiosira sp. obtained on the 6th day was 4.36 g L 1. Unlike B. braunii cells, which are resistant towards high CO2 concentrations (Ge et al., 2011), Thalassiosira sp. cell was very sensitive to high CO2 concentration, as a decrease of pH of the medium poisoned the cell growth (Nurachman et al., 2010). Hence, in this study only carbon dioxide from free air was used as the carbon source for photosynthesis for the biomass production of Thalassiosira sp. 3.2. Evaluation of growth medium To obtain a simple and inexpensive growth medium as well as high biomass production, plain seawater and enriched seawater media were evaluated for algal culture. A significant growth of Thalassiosira sp. cells in plain seawater medium until 9th day was not observed under the conditions of the experiment (Fig. 1A). This suggested that the plain seawater medium was insufficient to grow Thalassiosira sp. However, in Walne and enriched seawater media, the Thalassiosira sp. cells needed only a day to adapt in the medium, then grew well until reaching the late logarithmic phase on the 6th day, and afterwards fell (Fig. 1A). The time course of Thalassiosira sp. growth in enriched seawater medium was similar to that of in Walne medium. The cell density in enriched seawater medium in the late logarithmic phase (on the 6th day) increased more than 10-fold from the initial cell density of 1 105 cell mL 1. Specific grow rate of Thalassiosira sp. in the late logarithmic phase was 9.0 10 10/d. Upon further assessment of enriched seawater medium, the addition of essential nutrients such as phosphorus, nitrogen, and silicon important for the formation of the biosilica cell walls of Thalassiosira sp. was optimized with the starting medium condition as described in Section 2. In all experiments, the marine diatom Thalassiosira sp. grew well in an alkaline environment at a range of pH 8.0–8.8 (Fig. 1B–D). The effects of adding phosphate with a
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Fig. 1. Time course of Thalassiosira sp. growth. Thalassiosira sp. cell is cultured in three different media (A). Effects of essential nutrient concentrations of urea, phosphate, and silicate in the enriched seawater medium on biomass density of Thalassiosira sp. are shown in (B), (C), and (D), respectively.
concentration between 5 and 45 mg L 1 to the enriched seawater medium showed that the growth of Thalassiosira sp. increased gradually with a lag phase of 2 d followed by the exponential phase. At the beginning of the stationary phase (8th day), cell growth reached maximum cell density at a range of 7.3– 8.3 106 cell mL 1 (Fig. 1B). The value of F test among tested medium cultures was 0.18 (less than the critical value of F at the 99% confidence level of 2.34), indicating no significant difference in biomass production obtained from different phosphate concentrations in the culture medium. The effect of urea concentration in a range of 25–300 mg L 1 showed that Thalassiosira sp. needed 1 d for adaptation (Fig. 1C). Maximum cell density in a range of 4.6–6.6 106 cell mL 1 was observed at the end of exponential phase (6th day). The F test value of 0.26 was less than Fcrit value at the 99% confidence level of 2.45, indicating no significant difference in biomass production obtained from different urea concentrations in the culture medium. The effect of silicate concentration in a range of 10–190 mg L 1 showed that Thalassiosira sp. needed a day to adapt and reached maximum growth to a cell density of between 7.6 and 9.2 106 cell mL 1 at 7th day (Fig. 1D). The F test value of 1.05 was less than Fcrit value at the 99% confidence level of 2.34, indicating no significant difference in biomass production obtained from different silicate concentrations in the medium culture. Thus, the enriched seawater medium with the lowest added amount of phosphate, urea, and silicate: 5 mg NaH2PO42H2O, 25 mg urea, 10 mg Na2SiO35H2O, 70 mg Na2EDTA, and 6 mg FeCl3/L was chosen for the next experiments. The average wet Thalassiosira sp. biomass obtained from 1 L culture in the enriched seawater harvested at the beginning of the stationary phase (at 6th) d was 2.50 g. The ratio of total N/P/Si in the enriched medium was 16:1:1.2 (w/w/w), meanwhile in Walne medium it was 5:1:2.5 (w/w/w), indicating
that the growth of Thalassiosira sp. required the addition of more nitrogenous nutrients. For commercial production of microalgae, the production and energy costs (e.g. use nutrients for culture, light source, cell harvesting and oil extraction) should be reduced to be as low as possible. Excluding the cost of infrastructure, when biomass production of Thalassiosira sp. is conducted using air-lift photobioreactors above the sea, the critical cost for culturing algae such as seawater for the medium, sunlight for photosynthesis and free air for the carbon source are free of charge. Furthermore, these basic requirements for generating algal biomass are available all year long. In terms of additional nutrient cost/L medium, the enriched seawater medium cost was IDR 116, compared to the Walne medium which was IDR 591. (Note: IDR 1 roughly equals USD 0.0001.) Thus, the enriched seawater medium greatly reduced costs; it costs 80% less than the Walne medium. 3.3. Oil yield and biodiesel characteristics Based on knowledge, lipid accumulation in oil bodies of algae occurs during growth rate decrease, thus harvesting of Thalassiosira sp. biomass for biofuel was carried out at the beginning of the stationary phase (on the 6th day). Oils obtained from microalgae cultured in both Walne and enriched seawater media were yellow (Table 1). Shades of green oil were also observed when the oil contained chlorophyll pigments. The amount of oil extracted from 10.1 g wet Thalassiosira sp. biomass (cultured in Walne medium) was 2.1 mL, whereas from 7.0 g wet biomass (cultured in enriched seawater medium) was 4.9 mL. Therefore, the oil productivity of Thalassiosira sp. cell cultured in Walne medium and enriched seawater medium was 150 and 290 lL d-1 L-1 culture, respectively. This means that algal culture in a volume of 545 m3 (about 20%
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Z. Nurachman et al. / Bioresource Technology 108 (2012) 240–244 Table 1 Some characteristics of biodiesel from varied sources. Biodiesel
Thalassiosira sp.
Palm oil
Commercial B40
0.857 1.151
0.851 4.565
0.840 3.434
Flame of biodiesel
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1
)
of the volume of an olympic-size swimming pool) is capable of generating biomass feedstock for producing one barrel algal oil/d. Algal oils for biofuel should contain free FAs and TAGs. Our previous analysis of free FA in Thalassiosira sp. oils using GC–MS showed values of 41.7% palmitic acid, 9.8% stearic acid, and 47.4% oleic acid (Nurachman et al., 2010). Further TAG analysis of Thalassiosira sp. oil was carried out by electron spray ionization ion trap mass spectrometry (ESI-IT-MS). The oils were analyzed in positive ion mode to either [M + Ag]+ or [M + AgNO3 + Ag]+ adduct ions in a mass range of 200–1400 Da. Saturated fatty acids and fats containing all-saturated fatty acid chains could not be identified by this method because they were not able to form ion complexes with Ag+ (Sandra et al., 2002). Through this method, the mass spectral data of the oils showed the dominant [M + Ag]+ ions at m/z 939.81, m/z 965.77, and m/z 993.75, and these corresponded to the TAGs POP, POO, and SOO, respectively (Supplementary Fig. S5A). [M + AgNO3 + Ag]+ adduct ions of POP, POO, and SOO were also observed at m/z 1108.47, m/z 1134.51, and m/z 1162.50, respectively. To become biodiesel, a high density of TAGs in algae oil processing should be converted to FAMEs. Chemical-based biodiesel synthesis was carried out by transesterification between TAGs and methanol using KOH as a catalyst. The key to success for a complete transesterification reaction was that the amount of mole methanol used at least 6-fold higher than mole TAGs. Supplementary Fig. S5B showed that almost all TAGs from Thalassiosira sp. oil had been converted into FAMEs. These were observed in the mass spectra with the disappearance of the peaks of POP, POO, and SOO, and biodiesel product identified as oleic acid methyl ester (m/z 403.56). Neither palmitic acid nor stearic acid methyl ester was detected by this ESI-IT-MS (Sandra et al., 2002). Thalassiosira sp. biodiesel showed a bright and clean flame, whereas palm oil and commercial biodiesels (B40) displayed more yellow flame with little bits of black smoke (Table 1). Moreover, Thalassiosira sp. biodiesel density at 25 °C was 0.857 g mL 1. This density characteristic was not significantly different from the densities of palm oil and commercial (B40) biodiesel. Xu et al. (2006) reported a density of biodiesel from the microalgae Chlorella protothecoides of 0.864 g mL 1. According to the National Standardization Agency of Indonesia (SNI 04-7182-2006), the density of a biodiesel should be 0.85–0.89 g mL 1 at 40 °C. A more interesting property of Thalassiosira sp. biodiesel was its kinematic viscosity of 1.151 mm2 s 1, whereas the kinematic viscosity of the other biodiesels derived from palm oil and commercial (B40) biodiesel were higher. This is understandable, since Thalassiosira sp. biodiesels
(algal FAMEs) contain more unsaturated fatty acid content, such as oleic acid. A cis double bond in the unsaturated fatty acid structure results in a bend in the hydrocarbon chain, and this bend interferes with the highly ordered packing of fatty acid chains that enhances the fluidity, or reduces the viscosity, of the biodiesel. These algal biodiesel characteristics may be suitable for use as household fuel such as substituting kerosene for stoves and heaters, or for engine fuel with some adjustments. In conclusion, the result of this study showed that the production of tropical marine diatom Thalassiosira sp. biomass in enriched seawater medium can be done simply and inexpensively. Its algae biomass productivity (2.50 g L 1 culture) and algal oil productivity (290 lL d-1 L-1 culture) as well as its biodiesel characteristics are essential to develop the microalgae for biofuel production. Further effort on improving microalgae productivity through genetic engineering is worth studying. Acknowledgements The financial support from the Southeast Asian Regional Center for Graduate Study and Research in Agriculture – Seed Fund for Strategic Research and Training Program and from the Ministry of National Education of the Republic of Indonesia through the Beasiswa Unggulan Program are gratefully acknowledged. We also thank Mr. Tubagus Andhika Nugraha for critical reading of this manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biortech.2011.12.082. References Andersen, R.A. (Ed.), 2005. Algal Culturing Techniques. Elsevier Academic Press, USA. Banerjee, A., Sharma, R., Chisti, Y., Banerjee, U.C., 2002. Botryococcus braunii: a renewable source of hydrocarbons and other chemicals. Crit. Rev. Biotechnol. 22, 245–279. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917. Brennan, L., Owende, P., 2010. Biofuels from microalgae – a review of technologies for production, processing, and extractions of biofiuels and co-products. Renew. Sustain. Energy Rev. 14, 557–577. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25, 294–306. Chisti, Y., 2008. Biodiesel from microalgae beats bioethanol. Trends Biotechnol. 26, 126–131.
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Ge, Y., Liu, J., Tian, G., 2011. Growth characteristics of Botryococcus braunii 765 under high CO2 concentration in photobioreactor. Bioresour. Technol. 102, 130–134. Gong, Y., Jiang, M., 2011. Biodiesel production with microalgae as feedstock: from strains to biodiesel. Biotechnol. Lett. 33, 1269–1284. Li, Y., Horsman, M., Wu, N., Lan, C.Q., Dubois-Calero, N., 2008. Biofuels from microalgae. Biotechnol. Prog. 24, 815–820. Metzger, P., Largeau, C., 2005. Botryococcus braunii: a rich hydrocarbons and related ether lipids. Appl. Microbiol. Biotechnol. 66, 486–496. Nurachman, Z., 2011. Tropical marine microalgae for biodiesel production. SEAMEO-SEARCA, Policy Brief Series 3. Nurachman, Z., Panggabean, L.M.G., Anita, S., 2010. Screening of local marine microalgae for biodiesel production. SEAMEO-SEARCA, SEARCA Agriculture and Development Paper Series 3.
Sandra, P., Medvedovici, A., Zhao, Y., David, F., 2002. Characterization of triglycerides in vegetable oils by silver-ion packed-column supercritical fluid chromatography coupled to mass spectroscopy with atmospheric pressure chemical ionization and coordination ion spray. J. Chromatogr. A 974, 231–241. Sheehan, J., Dunahay, T., Beneman, J., Roessler, P., 1998. A look back at the US Department of Energy’s Aquatic Species Program: biodiesel from algae. US Department of Energy. Wijffels, R.H., Barbosa, M.J., 2010. An outlook on microalgal biofuels. Science 329, 796–799. Xu, H., Miao, X., Wu, Q., 2006. High quality biodiesel production from a microalgae Chlorella protothecoides by heterotrophic growth in fermenters. J. Biotechnol. 126, 499–507.
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Oil productivity of the tropical marine diatom Thalassiosira sp. Zeily Nurachman a,⇑, Hartati a, Syahfitri Anita a, Etsuroyya Ewidyasari Anward a, Gestria Novirani a, Bill Mangindaan b, Suryo Gandasasmita b, Yana Maolana Syah c, Lily Maria Goretty Panggabean d, Gede Suantika e a
Biochemistry Division, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Jl. Ganesha 10, Bandung 40132, Indonesia Analytical Chemistry Division, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Jl. Ganesha 10, Bandung 40132, Indonesia Organic Chemistry Division, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Jl. Ganesha 10, Bandung 40132, Indonesia d Research Center for Oceanography, Indonesian Institute of Sciences, Jl. Pasir Putih I, Ancol Timur, Jakarta 14430, Indonesia e Ecology and Biosystematics Research Group, School of Life Sciences and Technology, Institut Teknologi Bandung, Jl. Ganesha 10, Bandung 40132, Indonesia b c
a r t i c l e
i n f o
Article history: Received 14 September 2011 Received in revised form 16 December 2011 Accepted 16 December 2011 Available online 23 December 2011 Keywords: Biodiesel Oil productivity TAG Thalassiosira sp. Tropical marine microalgae
a b s t r a c t To understand the potential of cultivating tropical marine diatom Thalassiosira sp. to produce biofuel, biodiesel product properties and growth characteristics of Thalassiosira sp. in three different media were investigated. After medium evaluation, significant Thalassiosira sp. cell growth was observed in both Walne and enriched seawater media, but not in plain seawater medium. The microalgae grew well in alkaline condition (pH range of 8.0–8.8). The average biomass density cultured in Walne and enriched seawater media on the 6th day was 4.36 and 2.50 g L 1, respectively. Based on ESI-IT-MS spectra, the TAGs of algal oil were identified as POP, POO, and SOO, and the FAMEs as oleic acid methyl ester. The oil productivity of Thalassiosira sp. cultured in Walne and enriched seawater media were 150 and 290 lL L 1 d 1, respectively. The density and kinematic viscosity of Thalassiosira sp. biodiesel were 0.857 g mL 1 and 1.151 mm2 s 1. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Most oil-producing countries including Indonesia are both located adjacent to the tectonic plates and close to the sea. It is believed that the fossil fuel produced in Indonesia is not derived from wood-plant fossils (because the geological age of Indonesia is very young), but comes from aquatic plants including microalgae e.g. Botryococcus braunii (Banerjee et al., 2002; Metzger and Largeau, 2005). This is based on the fact that the original territory of Indonesia comes from the sea where tectonic plates shift and volcanoes raise the ground. The movement of tectonic plates through tectonic earthquakes then traps and presses seawater and everything in the seawater including marine microalgae into the soil to form a layer of oil. In the laboratory, this can be demonstrated by squeezing microalgae cells to push oil out. Microalgae occupy almost all layers of the sea in a huge amount. Both living microalgae and their dead bodies are subject to becoming sources of fossil fuel. The process of fossil fuel formation itself is, of course, very complicated and takes a long time. The first step to prove that microalgae contributes to fossil fuel formation is to identify as many types of microalgae from the ocean as possible and then measure their oil productivity. ⇑ Corresponding author. Tel.: +62 22 250 2103; fax: +62 22 250 4154. E-mail address: [email protected] (Z. Nurachman). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.12.082
Many reports and reviews on biodiesel from microalgae have been published elsewhere (Sheehan et al., 1998; Brennan and Owende, 2010; Wijffels and Barbosa, 2010; Gong and Jiang, 2011). Microalgae are microscopic plants that grow in water, can produce high oil content in a range of 20–50% dry mass of biomass and are capable of doubling their biomass in a period of 3.5 h (Chisti, 2007). Microalgae can convert 3–8% solar energy to biomass through photosynthesis (Li et al., 2008). Since microalgal cells grow in aqueous suspension, they have a larger surface area to access water, carbon dioxide and minerals for photosynthesis. Hence, the oil productivity of microalgae is essentially much higher than that of terrestrial plants (Chisti, 2008). Tropical marine microalgae are very interesting subjects of investigation as they produce various types of natural oils (triacylglycerols, TAGs; and fatty acids, FA either saturated, unsaturated, or polyunsaturated) for biodiesel (fatty acid methyl ester, FAME), chlorophyll, and carotenoid compounds. Among microalgae, diatoms (Bacillariophyta) are a very promising source for producing biofuel because these microalgae accumulate carbon in the form of natural oils and dominate the phytoplankton of the seawaters (Sheehan et al., 1998). Diatoms inhabit most bodies of water and can be found in the littoral and pelagic regions. Individually, diatoms do not appear in a highly visible color, but in groups they can be seen due to the presence of photosynthetic plastids. The culturable tropical marine diatom Thalassiosira sp. has been identified
Z. Nurachman et al. / Bioresource Technology 108 (2012) 240–244
as capable of producing biodiesel (Nurachman, 2011). This paper reports further investigation on the growth medium and oil productivity of Thalassiosira sp. as well as biodiesel characteristics.
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FAMEs in both oil and biodiesel were analyzed by Electron Spray Ionization-Ion Trap-Mass Spectrometry (ESI-IT-MS). 2.4. Analytical procedures
2. Methods 2.1. Microalgae and culture maintenance The original specimen of Thalassiosira sp. was obtained from the School of Life Sciences and Technology, Institut Teknologi Bandung, Indonesia. Cultures were maintained in Walne artificial seawater medium containing (g L 1): NaNO3, 100; Na2EDTA, 45; NaSiO35H2O, 40; H3BO3, 33.6; NaH2PO42H2O, 20.0; FeCl36H2O, 13.0; MnCl24H2O, 0.36; vitamin B12; vitamin B1, 0.01, 0.2; ZnCl2, 0.021; CoCl2, 0.02; CuSO45H2O, 0.02; and (NH4)6Mo7O2424H2O, 0.009 (Andersen, 2005). All solutions were prepared in sterilized water under aseptic conditions. The simple air-lift photobioreactors were made using transparent glass bottles with a height of 25 cm, an external diameter of 9 cm and a working volume of 800 mL. To maintain slow growth of algal stock culture, cells of Thalassiosira sp. were grown in air-lift photobioreactors and continuously illuminated under a light intensity of 40.5 lmol m 2 s 1 at room temperature, salinity 25 ppt and free air bubbling. The cells had to be periodically regenerated to stay in the growth phase. 2.2. Medium search Two other kinds of growth medium were tested: plain seawater and enriched seawater. The enriched seawater medium was a seawater medium supplemented with additional basic elements containing 300 mg urea, 45 mg NaH2PO42H2O, and 190 mg Na2SiO35H2O, 70 mg Na2EDTA, and 6 mg FeCl3 per 1 L medium. For biomass production, cultures with an initial density of 1 105 cell mL 1 were illuminated under a light intensity of 81 lmol m 2 s 1 at room temperature, photoperiod of light:dark = 12:12, salinity 25 ppt, and free air bubbling. Changes in cell numbers and pH was observed daily. Cell numbers were counted under a microscope with an improved Neubauer hemocytometer. All data were subjected to one-way analysis of variance (ANOVA) and the significance of differences among medium compositions on the yield of biomass was tested using the F test (P < 0.01). 2.3. Oil extraction and biodiesel conversion Oils were extracted using a modification of the methods of Bligh and Dyer (1959). Cultures of Thalassiosira sp. at the start of the stationary phase were harvested by centrifugation at 6000g for 15 min at 4 °C, and its wet biomass was then weighted. The wet biomass (10.1 g obtained from the Walne medium culture or 7.0 g obtained from the enriched seawater culture) was suspended in 70 mL chloroform/methanol (1:1 v/v) and then sonicated for 15 min at 50 Hz frequency, followed by Soxhlet extraction with the same solvent at 60 °C for 12 h. The solvent was removed by rotavaporation, and the volume of oils was measured. Oil productivity of Thalassiosira sp. is defined by the total amount of oils (lL) obtained from 1 L of microalgal culture until the beginning of the stationary phase at experimental conditions, or by the following formula: Oil productivity = Obtained oil (lL g 1 wet biomass) biomass density (g L 1 culture) time of growth (d). For conversion into biodiesel, transesterification of the oils was performed by refluxing microalgal TAGs, methanol, and KOH at a ratio of 10:2.3:0.123 (w/w/w) at 70 °C for 1 h. Biodiesel was separated by centrifugation at 8000g for 5 min at room temperature, followed by rotavaporation to remove the solvent. TAGs and
Mass spectrometry of biodiesels was performed on an ESI-ITMS HCT Bruker-Daltonic GmbH instrument with AgNO3 used as coordination ionization agent (Sandra et al., 2002). A simple flame test was performed on the biodiesels. Kinematic viscosity of biodiesels was determined through the method of ASTM D 445. Biodiesel density was also determined. For comparison, commercial (B40) and palm oil biodiesels were also analyzed. 3. Results and discussion 3.1. Algal characteristics and growth The marine diatom Thalassiosira sp. isolated from waters in the Java sea is a neritic planktonic species that belongs to the order Centricae. Its cells are similar to those of Coscinodiscus, usually drum or disc-shaped, joined in flexible chains by a cytoplasmic or gelatinous thread. The microalgae was yellowish brown, had a rectangular-like shape, and was able to form biosilica cell walls. Cell size was approximately 10 lm in diameter during culture period (smaller than its actual diameter size of 16–20 lm). For this study, culture condition was kept in the tolerance range of microalgae allowing permanent asexual reproduction. Starting from 1 105 cell mL 1 culture in Walne medium, hemocytometer counts for intact cells showed a maximum Thalassiosira sp. density at the late logarithmic phase (on the 6th day) of 9.1 106 cell L 1 culture, with a specific growth rate of 7.2 10 9/d (Fig. 1A). The average wet biomass of Thalassiosira sp. obtained on the 6th day was 4.36 g L 1. Unlike B. braunii cells, which are resistant towards high CO2 concentrations (Ge et al., 2011), Thalassiosira sp. cell was very sensitive to high CO2 concentration, as a decrease of pH of the medium poisoned the cell growth (Nurachman et al., 2010). Hence, in this study only carbon dioxide from free air was used as the carbon source for photosynthesis for the biomass production of Thalassiosira sp. 3.2. Evaluation of growth medium To obtain a simple and inexpensive growth medium as well as high biomass production, plain seawater and enriched seawater media were evaluated for algal culture. A significant growth of Thalassiosira sp. cells in plain seawater medium until 9th day was not observed under the conditions of the experiment (Fig. 1A). This suggested that the plain seawater medium was insufficient to grow Thalassiosira sp. However, in Walne and enriched seawater media, the Thalassiosira sp. cells needed only a day to adapt in the medium, then grew well until reaching the late logarithmic phase on the 6th day, and afterwards fell (Fig. 1A). The time course of Thalassiosira sp. growth in enriched seawater medium was similar to that of in Walne medium. The cell density in enriched seawater medium in the late logarithmic phase (on the 6th day) increased more than 10-fold from the initial cell density of 1 105 cell mL 1. Specific grow rate of Thalassiosira sp. in the late logarithmic phase was 9.0 10 10/d. Upon further assessment of enriched seawater medium, the addition of essential nutrients such as phosphorus, nitrogen, and silicon important for the formation of the biosilica cell walls of Thalassiosira sp. was optimized with the starting medium condition as described in Section 2. In all experiments, the marine diatom Thalassiosira sp. grew well in an alkaline environment at a range of pH 8.0–8.8 (Fig. 1B–D). The effects of adding phosphate with a
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concentration between 5 and 45 mg L 1 to the enriched seawater medium showed that the growth of Thalassiosira sp. increased gradually with a lag phase of 2 d followed by the exponential phase. At the beginning of the stationary phase (8th day), cell growth reached maximum cell density at a range of 7.3– 8.3 106 cell mL 1 (Fig. 1B). The value of F test among tested medium cultures was 0.18 (less than the critical value of F at the 99% confidence level of 2.34), indicating no significant difference in biomass production obtained from different phosphate concentrations in the culture medium. The effect of urea concentration in a range of 25–300 mg L 1 showed that Thalassiosira sp. needed 1 d for adaptation (Fig. 1C). Maximum cell density in a range of 4.6–6.6 106 cell mL 1 was observed at the end of exponential phase (6th day). The F test value of 0.26 was less than Fcrit value at the 99% confidence level of 2.45, indicating no significant difference in biomass production obtained from different urea concentrations in the culture medium. The effect of silicate concentration in a range of 10–190 mg L 1 showed that Thalassiosira sp. needed a day to adapt and reached maximum growth to a cell density of between 7.6 and 9.2 106 cell mL 1 at 7th day (Fig. 1D). The F test value of 1.05 was less than Fcrit value at the 99% confidence level of 2.34, indicating no significant difference in biomass production obtained from different silicate concentrations in the medium culture. Thus, the enriched seawater medium with the lowest added amount of phosphate, urea, and silicate: 5 mg NaH2PO42H2O, 25 mg urea, 10 mg Na2SiO35H2O, 70 mg Na2EDTA, and 6 mg FeCl3/L was chosen for the next experiments. The average wet Thalassiosira sp. biomass obtained from 1 L culture in the enriched seawater harvested at the beginning of the stationary phase (at 6th) d was 2.50 g. The ratio of total N/P/Si in the enriched medium was 16:1:1.2 (w/w/w), meanwhile in Walne medium it was 5:1:2.5 (w/w/w), indicating
that the growth of Thalassiosira sp. required the addition of more nitrogenous nutrients. For commercial production of microalgae, the production and energy costs (e.g. use nutrients for culture, light source, cell harvesting and oil extraction) should be reduced to be as low as possible. Excluding the cost of infrastructure, when biomass production of Thalassiosira sp. is conducted using air-lift photobioreactors above the sea, the critical cost for culturing algae such as seawater for the medium, sunlight for photosynthesis and free air for the carbon source are free of charge. Furthermore, these basic requirements for generating algal biomass are available all year long. In terms of additional nutrient cost/L medium, the enriched seawater medium cost was IDR 116, compared to the Walne medium which was IDR 591. (Note: IDR 1 roughly equals USD 0.0001.) Thus, the enriched seawater medium greatly reduced costs; it costs 80% less than the Walne medium. 3.3. Oil yield and biodiesel characteristics Based on knowledge, lipid accumulation in oil bodies of algae occurs during growth rate decrease, thus harvesting of Thalassiosira sp. biomass for biofuel was carried out at the beginning of the stationary phase (on the 6th day). Oils obtained from microalgae cultured in both Walne and enriched seawater media were yellow (Table 1). Shades of green oil were also observed when the oil contained chlorophyll pigments. The amount of oil extracted from 10.1 g wet Thalassiosira sp. biomass (cultured in Walne medium) was 2.1 mL, whereas from 7.0 g wet biomass (cultured in enriched seawater medium) was 4.9 mL. Therefore, the oil productivity of Thalassiosira sp. cell cultured in Walne medium and enriched seawater medium was 150 and 290 lL d-1 L-1 culture, respectively. This means that algal culture in a volume of 545 m3 (about 20%
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Commercial B40
0.857 1.151
0.851 4.565
0.840 3.434
Flame of biodiesel
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of the volume of an olympic-size swimming pool) is capable of generating biomass feedstock for producing one barrel algal oil/d. Algal oils for biofuel should contain free FAs and TAGs. Our previous analysis of free FA in Thalassiosira sp. oils using GC–MS showed values of 41.7% palmitic acid, 9.8% stearic acid, and 47.4% oleic acid (Nurachman et al., 2010). Further TAG analysis of Thalassiosira sp. oil was carried out by electron spray ionization ion trap mass spectrometry (ESI-IT-MS). The oils were analyzed in positive ion mode to either [M + Ag]+ or [M + AgNO3 + Ag]+ adduct ions in a mass range of 200–1400 Da. Saturated fatty acids and fats containing all-saturated fatty acid chains could not be identified by this method because they were not able to form ion complexes with Ag+ (Sandra et al., 2002). Through this method, the mass spectral data of the oils showed the dominant [M + Ag]+ ions at m/z 939.81, m/z 965.77, and m/z 993.75, and these corresponded to the TAGs POP, POO, and SOO, respectively (Supplementary Fig. S5A). [M + AgNO3 + Ag]+ adduct ions of POP, POO, and SOO were also observed at m/z 1108.47, m/z 1134.51, and m/z 1162.50, respectively. To become biodiesel, a high density of TAGs in algae oil processing should be converted to FAMEs. Chemical-based biodiesel synthesis was carried out by transesterification between TAGs and methanol using KOH as a catalyst. The key to success for a complete transesterification reaction was that the amount of mole methanol used at least 6-fold higher than mole TAGs. Supplementary Fig. S5B showed that almost all TAGs from Thalassiosira sp. oil had been converted into FAMEs. These were observed in the mass spectra with the disappearance of the peaks of POP, POO, and SOO, and biodiesel product identified as oleic acid methyl ester (m/z 403.56). Neither palmitic acid nor stearic acid methyl ester was detected by this ESI-IT-MS (Sandra et al., 2002). Thalassiosira sp. biodiesel showed a bright and clean flame, whereas palm oil and commercial biodiesels (B40) displayed more yellow flame with little bits of black smoke (Table 1). Moreover, Thalassiosira sp. biodiesel density at 25 °C was 0.857 g mL 1. This density characteristic was not significantly different from the densities of palm oil and commercial (B40) biodiesel. Xu et al. (2006) reported a density of biodiesel from the microalgae Chlorella protothecoides of 0.864 g mL 1. According to the National Standardization Agency of Indonesia (SNI 04-7182-2006), the density of a biodiesel should be 0.85–0.89 g mL 1 at 40 °C. A more interesting property of Thalassiosira sp. biodiesel was its kinematic viscosity of 1.151 mm2 s 1, whereas the kinematic viscosity of the other biodiesels derived from palm oil and commercial (B40) biodiesel were higher. This is understandable, since Thalassiosira sp. biodiesels
(algal FAMEs) contain more unsaturated fatty acid content, such as oleic acid. A cis double bond in the unsaturated fatty acid structure results in a bend in the hydrocarbon chain, and this bend interferes with the highly ordered packing of fatty acid chains that enhances the fluidity, or reduces the viscosity, of the biodiesel. These algal biodiesel characteristics may be suitable for use as household fuel such as substituting kerosene for stoves and heaters, or for engine fuel with some adjustments. In conclusion, the result of this study showed that the production of tropical marine diatom Thalassiosira sp. biomass in enriched seawater medium can be done simply and inexpensively. Its algae biomass productivity (2.50 g L 1 culture) and algal oil productivity (290 lL d-1 L-1 culture) as well as its biodiesel characteristics are essential to develop the microalgae for biofuel production. Further effort on improving microalgae productivity through genetic engineering is worth studying. Acknowledgements The financial support from the Southeast Asian Regional Center for Graduate Study and Research in Agriculture – Seed Fund for Strategic Research and Training Program and from the Ministry of National Education of the Republic of Indonesia through the Beasiswa Unggulan Program are gratefully acknowledged. We also thank Mr. Tubagus Andhika Nugraha for critical reading of this manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biortech.2011.12.082. References Andersen, R.A. (Ed.), 2005. Algal Culturing Techniques. Elsevier Academic Press, USA. Banerjee, A., Sharma, R., Chisti, Y., Banerjee, U.C., 2002. Botryococcus braunii: a renewable source of hydrocarbons and other chemicals. Crit. Rev. Biotechnol. 22, 245–279. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917. Brennan, L., Owende, P., 2010. Biofuels from microalgae – a review of technologies for production, processing, and extractions of biofiuels and co-products. Renew. Sustain. Energy Rev. 14, 557–577. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25, 294–306. Chisti, Y., 2008. Biodiesel from microalgae beats bioethanol. Trends Biotechnol. 26, 126–131.
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