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Increasing Lipid Accumulation of Chlorella vulgaris Using Spirulina platensis in Flat Plate Reactor for Synthesizing Biodiesel
Available online at www.sciencedirect.com
ScienceDirect Energy Procedia 65 (2015) 58 – 66
Conference and Exhibition Indonesia - New, Renewable Energy and Energy Conservation (The 3rd Indo-EBTKE ConEx 2014)
Increasing Lipid Accumulation of Chlorella vulgaris using Spirulina platensis in Flat Plate Reactor for Synthesizing Biodiesel Dianursantia*, Albert Santosoa a
Kampus UI Depok, Universitas Indonesia, Depok, Jawa Barat 16424, Indonesia
Abstract
Lipid accumulation in potential C. vulgaris could be induced by controlling nitrogen concentration in optimum level. In this study, C. vulgaris and S. platensis were cultured in BG-11 medium in flat plate photobioreactor to assess biomass and lipid productivity. Colony composition 3 : 2 (40 %wt S. platensis) gives highest lipid yield (6.72 %) in continuous illumination 3 000 lx compare to control sample (5.13 %). This confirms that the existence of cyanobacteria induces lipid accumulation in C. vulgaris. Further study is needed to understand better biosynthesis in colony. © 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license © 2015 Dianursanti, A. Santoso. Published by Elsevier Ltd. (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the Scientific Committee of EBTKE ConEx 2014. Peer-review under responsibility of the Scientific Committee of EBTKE ConEx 2014 Keywords: biodiesel, C. vulgaris, lipid accumulation, nitrogen , S.platensis
Nomenclature OD μ X
optical density growth rate dry weight cell
h lx %wt
hour lux = 1 lm · m2 % weight
* Corresponding author. Tel.: +62 811 192 1565 E-mail address: [email protected]; [email protected]
1876-6102 © 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the Scientific Committee of EBTKE ConEx 2014 doi:10.1016/j.egypro.2015.01.032
Dianursanti and Albert Santoso / Energy Procedia 65 (2015) 58 – 66
1. Introduction Method fossil based energy reserve is currently at the verge of crisis [1]. As countries face significant growth of population, they need renewable energy as strategy to cope with increasing demand. Many alternatives are proposed, specifically is algae based energy reserves. Algae is well known for its rapid growth and fast production, reaching 7 up to 31 times of crude palm oil in same amount of space and time [2,3], with higher emission saving. Its cultivation also does not heat up the controversial debate regarding food versus energy war. Chlorella sp. especially is prominent among other algae due to their abundance in nature, thus it is easier to adapt in wider scale, high lipid content (reaching 35 % to 40 % lipid content) [3,4], and quality of lipid (higher saturated lipid content comparing to Neochlorosis oleoabundans; [5]). Looking at these advantages, there is clear incentive to stimulate plant scale production of microalgal cultivation in order to solve current global problem. In using algae as energy source, lipid content becomes vital component since it is the main raw material that is turned into biodiesel through chemical reaction such as trans-esterification or hydrocracking. Its accumulation determines quantity yielded each batch, thus it is within our interest to increase the amount of lipid content. Current study shows that lipid accumulation increases along with nitrogen deficiency, yet its cell growth is significantly inhibited [4,6]. Increasing nitrogen content, on the other hand makes development on plant scale algae cultivation based on bio-waste and commercial fertilizer medium not commercially accessible since it would only increase cell growth but not add lipid quantity in total. Thus, cultivating microalgae in commercial fertilizer medium or bio-waste requires us to have a controller to ensure nitrogen concentration on the optimum level. Cyanobacteria are proposed in this experiment due to their ability in fixating nitrogen and to synergize positively with green algae [6]. Their ability to fixate both organic and inorganic nitrogen enables them to control concentration level not exceeding the ceiling and not falling below the floor set. This is promising to assist in ensuring higher lipid yield in microalgae. Although these advantages of cyanobacteria, there is not enough report about utilizing cyanobacteria to help the development of biodiesel production by being symbiosis agent in increasing the lipid accumulation in microalgae. In this work, it is aimed to suggest more yield of lipid in plant scale level of biodiesel synthesis based on microalgae. To our knowledge, this is the unique report about preliminary study on influential parameters in symbiosis of well-known cyanobacteria S. platensis and C. vulgaris in BG-11 medium. 2. Method 2.1. Pre culture conditions of C. vulgaris and S. Platensis C. vulgaris and S. platensis were used in the study, as provided by Universitas Indonesia, Chemical Engineering Laboratories from the current culture collection. The algae were cultured in 6 L flat plate reactor (0.4 m × 0.25 m × 0.1 m) [4] with bubble purging through cylindrical modified pipe in constant temperature (29 ºC) and ambient pressure (1 atm) containing 600 ml of BG-11 fermentation medium. Concentration of nutrients in the media are NaNO3(17.65 mM), KH2PO4 (0.18 mM), MgSO4.7H2O (0.30 mM), CaCl2.2H2O (0.25 mM), citric acid (0.029 mM), ferric ammonium citrate (0.030 mM), titriplex (0.003 mM), Na2CO3 (0.19 mM), H3BO3 (46 μM), CuSO4.5H2O (0.17 μM), MnCl2.4H2O (9.2 μM), ZnSO4.7H2O (0.77 μM) and Na2MoO4.2H2O (1.6 μM). Aeration flow rate is controlled by gas flow meter (Kofloc) at 5 L · min-1 and 5 % of CO2 concentration. The reactor was incubated under continuous illumination 2 000 lx and 3 000 lx from single front face (0.4 m × 0.25 m) for 8 d. The source of illumination is six parallel Philip Halogen 20W/12V/50Hz. 2.2. Lipid accumulation Three series of reactor containing both C. vulgaris and Spirulina sp. were prepared on different proportion of concentration. The reactors were then observed in constant illumination to get highest lipid quantity, before then the proportion used to evaluate lighting intensity. Biomass density was evaluated using spectrophotometer UV-Vis (LaboMed Inc.) in 600 nm wavelength for C. vulgaris and 480 nm wavelength for S. platensis. Cells were obtained through vortex centrifuge before then disrupted using sonicator and microwave radiation. Lipid was extracted using Bligh Dryer method, then measured by mass difference.
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3. Result and discussion Fig. 1 shows growth profile of C. vulgaris and S. platensis on wavelength 480 nm. Analysis of cell density is approached using spectrophotometry UV-vis. Optical density (OD) is gained to measure cell density, and is converted using calibration curve shown in Fig. 2. Length of lag phase of colony shows no significant difference with control sample in hour unit. It shows positive possibility of symbiosis with no detected effect on adaptation process. They both share conserved photosynthetic electron transfer and phosphorylation mechanisms, though the apparatus and localization is different [7,8]. Contrasting to predicted competition, the existence of S. platensis fasten significant log phase. Colony composition 3 : 2 shows fastest trend with less than 70 h of cultivation, significantly higher compares to control sample that doubles in 72 h. Cell reproducibility is presumably enhanced with the existence of S. platensis as it changes the nutrient concentration into more organic and ready to be consumed through secretion of metabolic enzyme. Cyanobacteria exert nitrate reductase, an enzyme used to convert nitrate ions to more preferable ammonium ion [9]. The ammonium ion is then used together to construct mainly chloroplast and other cell apparatus [10,11]. However, under lower illumination intensity, the more the amount of cyanobacteria is, the less effective the mechanism works. Colony composition 3 : 2 fails to show faster significant growth compares to 3 : 1. It is suggested that cyanobacteria’s rate of consumption of nitrogenous ions is prevailing over green microalgae’s, thus confirming the ability of cyanobacteria to adjust better in more extreme condition, especially under light deficiency [9,12]. Further study is needed to understand better the complex interplay shift in metabolism pathway of the colony.
Dianursanti and Albert Santoso / Energy Procedia 65 (2015) 58 – 66
Fig. 1. Profile of cell density versus time under illumination: [A] control C. vulgaris 100 % (%wt), [B] colony C. vulgaris-S. platensis (4 : 1), [C] colony C. vulgaris-S. platensis (3 : 1), [D] colony C. vulgaris-S. platensis (2 : 1), [E] colony C. vulgaris-S. platensis (3 : 2)
Light intensity plays significant role in growth promotion of the colony. Previous experiments show S. platensis and C. vulgaris have different sensitivity on light [4,13–16]. Comparing to cultivation under light intensity 2 000 lx, higher light intensity 3 000 lx presents faster lag phase under higher composed S. platensis within less than 20 cultivation hours, as shown in Fig. 3. It indicates higher light intensity fasten adaptation process of both colony. Fig. 3 also shows under illumination 3 000 lx, growth rate approached using Monod equation [4] of colony 3 : 2 is relatively stable, shows consistent rate in comparison of others. This phenomenon might be the result of light sensitivity of the colony. Higher light intensity might promote cell growth rapidly due to sufficient provision of lights, and metabolic interaction between both strains [4]. Further study, however, is in need to understand more metabolic pathways preferred by colony, in comparison of pure cultures. Furthermore, as microalgal photosynthetic metabolism tends to increase pH [4], it affects C. vulgaris growth that is well under more acidic condition (pH 6 to pH 8) [4,17]. It is shown by Fig. 4 that more S. platensis induces more stable pH, though the difference was relatively small. Beside it is suggested that light intensity may play metabolic role in S. platensis, the main reason might be secretion of important enzyme, such as Indol Acetic acid (IAA) from S. platensis that helps C. vulgaris to grow better under basic condition, and helps in buffering acidity. Moreover, higher light intensity may also enable both algae to compete for nitrogenous compounds, resulting into lower log phase and lower cell density. However, further study is suggested to understand biosynthesis process under the influence of light intensity
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Dianursanti and Albert Santoso / Energy Procedia 65 (2015) 58 – 66
Fig. 2 Calibration curve of OD versus cell mass
Dianursanti and Albert Santoso / Energy Procedia 65 (2015) 58 – 66
Fig. 3. Profile of growth rate versus time under illumination: [A] control C. vulgaris 100 % (%wt), [B] colony C. vulgaris-S. platensis (4 : 1), [C] colony C. vulgaris-S. platensis (3 : 1), [D] colony C. vulgaris-S. platensis (2 : 1), [E] colony C. vulgaris-S. platensis (3 : 2)
Fig. 5 shows the existence of S. platensis generates higher lipid yield compare to control sample. This confirms the hypothesis that existence of cyanobacteria induce higher lipid accumulation due to decrease of consumable nitrogen compound [4,12,18]. Colony composition 3 : 2 yields highest lipid content in cultivation under light intensity 3 000 lx, increasing lipid yield up to 30 % from control sample while under light intensity 2 000 lx, it the colony 3 : 2 increases lipid yield up to 69.4 %. This phenomenon suggests the effect of S. platensis existence towards lipid yield is more significant under illumination 2 000 lx, in comparison of 3 000 lx. Both increments might be the result of kept consumable nitrogen compound concentration on optimum level. Furthermore, this might be the result of better performance of cyanobacteria in controlling nitrogenous compound concentration level, provided sufficient amount of vital light. This phenomenon also suggests that under stronger light intensity, percentage of S. platensis in cell density could be increased to induce higher lipid accumulation. It is important to be noted down that the differences between colony 3 : 1, 2 : 1, and 3 : 2 are relatively insignificant, in comparison to control sample. This phenomenon suggests that too high percentage of S. platensis may have little effect, if not adverse, on lipid accumulation of C. vulgaris.
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Fig. 4. Profile of pH versus time under illumination: [A] control C. vulgaris 100 % (%wt), [B] colony C. vulgaris-S. platensis (4 : 1), [C] colony C. vulgaris-S. platensis (3 : 1), [D] colony C. vulgaris-S. platensis (2 : 1), [E] colony C. vulgaris-S. platensis (3 : 2)
Dianursanti and Albert Santoso / Energy Procedia 65 (2015) 58 – 66
Fig. 5. Lipid yield comparison under various composition of colony: [A] control C. vulgaris 100 % (%wt), [B] colony C. vulgaris-S. platensis (4 : 1), [C] colony C. vulgaris-S. platensis (3 : 1), [D] colony C. vulgaris-S. platensis (2 : 1), [E] colony C. vulgaris-S. platensis (3 : 2)
4. Conclusion This study constitutes first report regarding the use of cyanobacteria to increase lipid accumulation in potential C. vulgaris. Positive trend of symbiosis in this experiment confirms the hypothesis that cyanobacteria could be used to increase lipid accumulation inside C. vulgaris. This study also highlights the complexity in biosynthesis of lipid in colony with light sensitivity, an area that requires further investigations. Acknowledgements The authors would like to acknowledge the funding of this work by DIKTI (Indonesian General Directory of University) through PKM-P (Student Creativity Program – Research Category) and PUPT (University Quality Research). Authors would also like to acknowledge Pijar Religia, Marina, Ihsan Wiratama, Maryam Mardiyyah, Setia Bakti, and Maria Regina for their contributions in this research. References [1] Huang G, Chen F, Wei D, Zhang X, Chen G. Biodiesel production by microalgal biotechnology. Appl Energy. Elsevier Ltd; 2010;87(1): 38–46. [2] Demirbas A, Fatih Demirbas M. Importance of algae oil as a source of biodiesel. Energy Conversion Management. 2011 Jan;52(1):163–70. [3] Kaiwan-arporn P, Hai PD, Thu NT, Annachhatre AP. Cultivation of cyanobacteria for extraction of lipids. Biomass and Bioenergy. 2012 Sep;44:142–9.
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Dianursanti and Albert Santoso / Energy Procedia 65 (2015) 58 – 66 [4] Dianursanti. Pengembangan sistem produksi biomassa C. vulgaris dalam reaktor plat datar melalui optimasi pencahayaan menggunakan teknik filtrasi pada aliran kultur media [Development of Chlorella vulgaris Biomass production system in a flat plate reactor through lighting optimization using filtration technique in culture media flow]. Depok; 2012. [Bahasa Indonesia] [5] Li Q, Du W, Liu D. Perspectives of microbial oils for biodiesel production. Appl Microbiol Biotechnol. 2008 Oct;80(5):749–56. [6] Li Y, Horsman M, Wang B, Wu N, Lan CQ. Effects of nitrogen sources on cell growth and lipid accumulation of green alga Neochloris oleoabundans. Appl Microbiol Biotechnol. 2008 Dec;81(4):629–36. [7] Wijanarko A, Witarto AB, Soemantojo RW. Effect of photoperiodicity on CO 2 fixation by C. vulgaris Buitenzorg in bubble column photobioreactor for food supplement production. Depok 2004 [8] Srirangan K, Pyne ME, Chou CP. Bioresource technology biochemical and genetic engineering strategies to enhance hydrogen production in photosynthetic algae and cyanobacteria. 2011;102:8589–604. [9] Evans CT. Effect of nitrogen source on lipid accumulation in oleaginous yeasts. Hull: 1984;1693–704. [10] Markou G, Georgakakis D. Cultivation of filamentous cyanobacteria (blue-green algae ) in agro-industrial wastes and wastewaters : A review. Athens: 2011;88:3389–401. [11]Graham LE and Wilcox LW. Algae. London: Prentice Hall; 2000. [12]Blankenship RE. Molecular mechanism of photosynthesis. London: Prentice Hall ; 1997. [13]Prihantini NB, Wardhana W, Hendrayanti D, Widyawan A, Ariyani Y, Rianto R. Biodiversitas cyanobacteria dari beberapa situ/danau di kawasan Jakarta-Depok-Bogor, Indonesia. [Biodiversity of Cyanobacteria from several ponds/lakes of Jakarta-Depok-Bogor, Indonesia]. Makara Sains 2008;12(1):44–54.[Bahasa Indonesia]. [14]Gami B, Naik A, Patel B. Cultivation of Spirulina sp. in different liquid media. Bodakdev: 2011;2(3):15–26. [15]Eriksen NT. The technology of microalgal culturing. Biotechnol Lett. 2008 Sep;30(9):1525–36. [16]De Morais MG, Costa JAV. Carbon dioxide fixation by Chlorella kessleri, C. vulgaris, Scenedesmus obliquus and Spirulina sp. cultivated in flasks and vertical tubular photobioreactors. Biotechnol Lett. 2007 Sep;29(9):1349–52. [17]Chiu S-Y, Kao C-Y, Chen C-H, Kuan T-C, Ong S-C, Lin C-S. Reduction of CO2 by a high-density culture of Chlorella sp. in a semicontinuous photobioreactor. Bioresour Technol;99(9):3389–96. [18]Packer M. Algal capture of carbon dioxide ; biomass generation as a tool for greenhouse gas mitigation with reference to New Zealand energy strategy and policy. Energy Policy. Elsevier; 2009;37(9):3428–37.
ScienceDirect Energy Procedia 65 (2015) 58 – 66
Conference and Exhibition Indonesia - New, Renewable Energy and Energy Conservation (The 3rd Indo-EBTKE ConEx 2014)
Increasing Lipid Accumulation of Chlorella vulgaris using Spirulina platensis in Flat Plate Reactor for Synthesizing Biodiesel Dianursantia*, Albert Santosoa a
Kampus UI Depok, Universitas Indonesia, Depok, Jawa Barat 16424, Indonesia
Abstract
Lipid accumulation in potential C. vulgaris could be induced by controlling nitrogen concentration in optimum level. In this study, C. vulgaris and S. platensis were cultured in BG-11 medium in flat plate photobioreactor to assess biomass and lipid productivity. Colony composition 3 : 2 (40 %wt S. platensis) gives highest lipid yield (6.72 %) in continuous illumination 3 000 lx compare to control sample (5.13 %). This confirms that the existence of cyanobacteria induces lipid accumulation in C. vulgaris. Further study is needed to understand better biosynthesis in colony. © 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license © 2015 Dianursanti, A. Santoso. Published by Elsevier Ltd. (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the Scientific Committee of EBTKE ConEx 2014. Peer-review under responsibility of the Scientific Committee of EBTKE ConEx 2014 Keywords: biodiesel, C. vulgaris, lipid accumulation, nitrogen , S.platensis
Nomenclature OD μ X
optical density growth rate dry weight cell
h lx %wt
hour lux = 1 lm · m2 % weight
* Corresponding author. Tel.: +62 811 192 1565 E-mail address: [email protected]; [email protected]
1876-6102 © 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the Scientific Committee of EBTKE ConEx 2014 doi:10.1016/j.egypro.2015.01.032
Dianursanti and Albert Santoso / Energy Procedia 65 (2015) 58 – 66
1. Introduction Method fossil based energy reserve is currently at the verge of crisis [1]. As countries face significant growth of population, they need renewable energy as strategy to cope with increasing demand. Many alternatives are proposed, specifically is algae based energy reserves. Algae is well known for its rapid growth and fast production, reaching 7 up to 31 times of crude palm oil in same amount of space and time [2,3], with higher emission saving. Its cultivation also does not heat up the controversial debate regarding food versus energy war. Chlorella sp. especially is prominent among other algae due to their abundance in nature, thus it is easier to adapt in wider scale, high lipid content (reaching 35 % to 40 % lipid content) [3,4], and quality of lipid (higher saturated lipid content comparing to Neochlorosis oleoabundans; [5]). Looking at these advantages, there is clear incentive to stimulate plant scale production of microalgal cultivation in order to solve current global problem. In using algae as energy source, lipid content becomes vital component since it is the main raw material that is turned into biodiesel through chemical reaction such as trans-esterification or hydrocracking. Its accumulation determines quantity yielded each batch, thus it is within our interest to increase the amount of lipid content. Current study shows that lipid accumulation increases along with nitrogen deficiency, yet its cell growth is significantly inhibited [4,6]. Increasing nitrogen content, on the other hand makes development on plant scale algae cultivation based on bio-waste and commercial fertilizer medium not commercially accessible since it would only increase cell growth but not add lipid quantity in total. Thus, cultivating microalgae in commercial fertilizer medium or bio-waste requires us to have a controller to ensure nitrogen concentration on the optimum level. Cyanobacteria are proposed in this experiment due to their ability in fixating nitrogen and to synergize positively with green algae [6]. Their ability to fixate both organic and inorganic nitrogen enables them to control concentration level not exceeding the ceiling and not falling below the floor set. This is promising to assist in ensuring higher lipid yield in microalgae. Although these advantages of cyanobacteria, there is not enough report about utilizing cyanobacteria to help the development of biodiesel production by being symbiosis agent in increasing the lipid accumulation in microalgae. In this work, it is aimed to suggest more yield of lipid in plant scale level of biodiesel synthesis based on microalgae. To our knowledge, this is the unique report about preliminary study on influential parameters in symbiosis of well-known cyanobacteria S. platensis and C. vulgaris in BG-11 medium. 2. Method 2.1. Pre culture conditions of C. vulgaris and S. Platensis C. vulgaris and S. platensis were used in the study, as provided by Universitas Indonesia, Chemical Engineering Laboratories from the current culture collection. The algae were cultured in 6 L flat plate reactor (0.4 m × 0.25 m × 0.1 m) [4] with bubble purging through cylindrical modified pipe in constant temperature (29 ºC) and ambient pressure (1 atm) containing 600 ml of BG-11 fermentation medium. Concentration of nutrients in the media are NaNO3(17.65 mM), KH2PO4 (0.18 mM), MgSO4.7H2O (0.30 mM), CaCl2.2H2O (0.25 mM), citric acid (0.029 mM), ferric ammonium citrate (0.030 mM), titriplex (0.003 mM), Na2CO3 (0.19 mM), H3BO3 (46 μM), CuSO4.5H2O (0.17 μM), MnCl2.4H2O (9.2 μM), ZnSO4.7H2O (0.77 μM) and Na2MoO4.2H2O (1.6 μM). Aeration flow rate is controlled by gas flow meter (Kofloc) at 5 L · min-1 and 5 % of CO2 concentration. The reactor was incubated under continuous illumination 2 000 lx and 3 000 lx from single front face (0.4 m × 0.25 m) for 8 d. The source of illumination is six parallel Philip Halogen 20W/12V/50Hz. 2.2. Lipid accumulation Three series of reactor containing both C. vulgaris and Spirulina sp. were prepared on different proportion of concentration. The reactors were then observed in constant illumination to get highest lipid quantity, before then the proportion used to evaluate lighting intensity. Biomass density was evaluated using spectrophotometer UV-Vis (LaboMed Inc.) in 600 nm wavelength for C. vulgaris and 480 nm wavelength for S. platensis. Cells were obtained through vortex centrifuge before then disrupted using sonicator and microwave radiation. Lipid was extracted using Bligh Dryer method, then measured by mass difference.
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3. Result and discussion Fig. 1 shows growth profile of C. vulgaris and S. platensis on wavelength 480 nm. Analysis of cell density is approached using spectrophotometry UV-vis. Optical density (OD) is gained to measure cell density, and is converted using calibration curve shown in Fig. 2. Length of lag phase of colony shows no significant difference with control sample in hour unit. It shows positive possibility of symbiosis with no detected effect on adaptation process. They both share conserved photosynthetic electron transfer and phosphorylation mechanisms, though the apparatus and localization is different [7,8]. Contrasting to predicted competition, the existence of S. platensis fasten significant log phase. Colony composition 3 : 2 shows fastest trend with less than 70 h of cultivation, significantly higher compares to control sample that doubles in 72 h. Cell reproducibility is presumably enhanced with the existence of S. platensis as it changes the nutrient concentration into more organic and ready to be consumed through secretion of metabolic enzyme. Cyanobacteria exert nitrate reductase, an enzyme used to convert nitrate ions to more preferable ammonium ion [9]. The ammonium ion is then used together to construct mainly chloroplast and other cell apparatus [10,11]. However, under lower illumination intensity, the more the amount of cyanobacteria is, the less effective the mechanism works. Colony composition 3 : 2 fails to show faster significant growth compares to 3 : 1. It is suggested that cyanobacteria’s rate of consumption of nitrogenous ions is prevailing over green microalgae’s, thus confirming the ability of cyanobacteria to adjust better in more extreme condition, especially under light deficiency [9,12]. Further study is needed to understand better the complex interplay shift in metabolism pathway of the colony.
Dianursanti and Albert Santoso / Energy Procedia 65 (2015) 58 – 66
Fig. 1. Profile of cell density versus time under illumination: [A] control C. vulgaris 100 % (%wt), [B] colony C. vulgaris-S. platensis (4 : 1), [C] colony C. vulgaris-S. platensis (3 : 1), [D] colony C. vulgaris-S. platensis (2 : 1), [E] colony C. vulgaris-S. platensis (3 : 2)
Light intensity plays significant role in growth promotion of the colony. Previous experiments show S. platensis and C. vulgaris have different sensitivity on light [4,13–16]. Comparing to cultivation under light intensity 2 000 lx, higher light intensity 3 000 lx presents faster lag phase under higher composed S. platensis within less than 20 cultivation hours, as shown in Fig. 3. It indicates higher light intensity fasten adaptation process of both colony. Fig. 3 also shows under illumination 3 000 lx, growth rate approached using Monod equation [4] of colony 3 : 2 is relatively stable, shows consistent rate in comparison of others. This phenomenon might be the result of light sensitivity of the colony. Higher light intensity might promote cell growth rapidly due to sufficient provision of lights, and metabolic interaction between both strains [4]. Further study, however, is in need to understand more metabolic pathways preferred by colony, in comparison of pure cultures. Furthermore, as microalgal photosynthetic metabolism tends to increase pH [4], it affects C. vulgaris growth that is well under more acidic condition (pH 6 to pH 8) [4,17]. It is shown by Fig. 4 that more S. platensis induces more stable pH, though the difference was relatively small. Beside it is suggested that light intensity may play metabolic role in S. platensis, the main reason might be secretion of important enzyme, such as Indol Acetic acid (IAA) from S. platensis that helps C. vulgaris to grow better under basic condition, and helps in buffering acidity. Moreover, higher light intensity may also enable both algae to compete for nitrogenous compounds, resulting into lower log phase and lower cell density. However, further study is suggested to understand biosynthesis process under the influence of light intensity
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Dianursanti and Albert Santoso / Energy Procedia 65 (2015) 58 – 66
Fig. 2 Calibration curve of OD versus cell mass
Dianursanti and Albert Santoso / Energy Procedia 65 (2015) 58 – 66
Fig. 3. Profile of growth rate versus time under illumination: [A] control C. vulgaris 100 % (%wt), [B] colony C. vulgaris-S. platensis (4 : 1), [C] colony C. vulgaris-S. platensis (3 : 1), [D] colony C. vulgaris-S. platensis (2 : 1), [E] colony C. vulgaris-S. platensis (3 : 2)
Fig. 5 shows the existence of S. platensis generates higher lipid yield compare to control sample. This confirms the hypothesis that existence of cyanobacteria induce higher lipid accumulation due to decrease of consumable nitrogen compound [4,12,18]. Colony composition 3 : 2 yields highest lipid content in cultivation under light intensity 3 000 lx, increasing lipid yield up to 30 % from control sample while under light intensity 2 000 lx, it the colony 3 : 2 increases lipid yield up to 69.4 %. This phenomenon suggests the effect of S. platensis existence towards lipid yield is more significant under illumination 2 000 lx, in comparison of 3 000 lx. Both increments might be the result of kept consumable nitrogen compound concentration on optimum level. Furthermore, this might be the result of better performance of cyanobacteria in controlling nitrogenous compound concentration level, provided sufficient amount of vital light. This phenomenon also suggests that under stronger light intensity, percentage of S. platensis in cell density could be increased to induce higher lipid accumulation. It is important to be noted down that the differences between colony 3 : 1, 2 : 1, and 3 : 2 are relatively insignificant, in comparison to control sample. This phenomenon suggests that too high percentage of S. platensis may have little effect, if not adverse, on lipid accumulation of C. vulgaris.
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Fig. 4. Profile of pH versus time under illumination: [A] control C. vulgaris 100 % (%wt), [B] colony C. vulgaris-S. platensis (4 : 1), [C] colony C. vulgaris-S. platensis (3 : 1), [D] colony C. vulgaris-S. platensis (2 : 1), [E] colony C. vulgaris-S. platensis (3 : 2)
Dianursanti and Albert Santoso / Energy Procedia 65 (2015) 58 – 66
Fig. 5. Lipid yield comparison under various composition of colony: [A] control C. vulgaris 100 % (%wt), [B] colony C. vulgaris-S. platensis (4 : 1), [C] colony C. vulgaris-S. platensis (3 : 1), [D] colony C. vulgaris-S. platensis (2 : 1), [E] colony C. vulgaris-S. platensis (3 : 2)
4. Conclusion This study constitutes first report regarding the use of cyanobacteria to increase lipid accumulation in potential C. vulgaris. Positive trend of symbiosis in this experiment confirms the hypothesis that cyanobacteria could be used to increase lipid accumulation inside C. vulgaris. This study also highlights the complexity in biosynthesis of lipid in colony with light sensitivity, an area that requires further investigations. Acknowledgements The authors would like to acknowledge the funding of this work by DIKTI (Indonesian General Directory of University) through PKM-P (Student Creativity Program – Research Category) and PUPT (University Quality Research). Authors would also like to acknowledge Pijar Religia, Marina, Ihsan Wiratama, Maryam Mardiyyah, Setia Bakti, and Maria Regina for their contributions in this research. References [1] Huang G, Chen F, Wei D, Zhang X, Chen G. Biodiesel production by microalgal biotechnology. Appl Energy. Elsevier Ltd; 2010;87(1): 38–46. [2] Demirbas A, Fatih Demirbas M. Importance of algae oil as a source of biodiesel. Energy Conversion Management. 2011 Jan;52(1):163–70. [3] Kaiwan-arporn P, Hai PD, Thu NT, Annachhatre AP. Cultivation of cyanobacteria for extraction of lipids. Biomass and Bioenergy. 2012 Sep;44:142–9.
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