- Email: [email protected]
Bio-diesel production directly from the microalgae biomass of Nannochloropsis by microwave and ultrasound radiation
Bioresource Technology 102 (2011) 4265–4269
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Bio-diesel production directly from the microalgae biomass of Nannochloropsis by microwave and ultrasound radiation Miri Koberg a, Moshe Cohen b, Ami Ben-Amotz b, Aharon Gedanken a,⇑ a b
Department of Chemistry and Kanbar Laboratory for Nanomaterials, Bar-Ilan University, Center for Advanced Materials and Nanotechnology, Ramat-Gan 52900, Israel Seambiotic Ltd., Tel Aviv 67021, Israel
a r t i c l e
i n f o
Article history: Received 7 September 2010 Received in revised form 29 November 2010 Accepted 1 December 2010 Available online 17 December 2010 Keywords: Bio-diesel Nannochloropsis Microwave irradiation Sonication Catalyst
a b s t r a c t This work offers an optimized method for the direct conversion of harvested Nannochloropsis algae into bio-diesel using two novel techniques. The first is a unique bio-technology-based environmental system utilizing flue gas from coal burning power stations for microalgae cultivation. This method reduces considerably the cost of algae production. The second technique is the direct transesterification (a one-stage method) of the Nannochloropsis biomass to bio-diesel production using microwave and ultrasound radiation with the aid of a SrO catalyst. These two techniques were tested and compared to identify the most effective bio-diesel production method. Based on our results, it is concluded that the microwave oven method appears to be the most simple and efficient method for the one-stage direct transesterification of the as-harvested Nannochloropsis algae. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Bio-diesel, which is made from renewable biological sources, has gained importance as an alternative energy source. When considering the pollution caused by the combustion of conventional petroleum-based diesel (McKendry, 2002; Reijnders, 2006), a shift to non-conventional sources such as bio-diesel is inevitable. Biodiesel, which consists of fatty acid methyl esters (FAME), has lower CO, CO2 and hydrocarbon emissions; in addition, it is non-toxic and biodegradable (Barnard et al., 2007; Valente et al., 2010). The production of bio-diesel using various materials, such as plants, microalgae, and animal fat, has been attempted as an alternative energy source (Patil et al., 2010). Microalgae have certain advantages compared to other energy crops: (1) Microalgae do not require fresh water and can grow in salt water or even contaminated water, at sea or in ponds, thus preserving fresh water that is becoming an ever more rare and valuable natural resource. (2) Furthermore, algae can be cultivated on land that is unsuitable for food production. The cultivation of maize as a biomass fuel source has caused the significant erosion of arable land, whereas microalgae cultivation does not require arable land, and thus does not displace food crops. (3) Moreover, compared to other biofuel crops, microalgae grow much faster and contain more energy per unit weight. Microalgae can generate as much as 40 times more oil ⇑ Corresponding author. Tel.: +972 3 5318315; fax: +972 3 7384053. E-mail address: [email protected] (A. Gedanken). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.12.004
per acre than other plants used for biofuels (Schenk et al., 2008). (4) In addition, microalgae can produce both biofuels and valuable co-products such as omega 3 and several metabolites of economic interest, such as carotenoids (e.g., astaxanthin, lutein), vitamin E (alpha–tocopherol), and poly unsaturated fatty acids (arachidonic and c linoleic) (Scott et al., 2010). Thus, they have a large potential for feed, food, cosmetics, and pharmaceutical industries, which makes their conversion to bio-diesel cost efficient. In the current work we explore the optimization of bio-diesel production, probing the combination of two techniques: (1) A revolutionizing ecologically-based environmental system utilizing flue gas from coal burning power stations for microalgae cultivation. Seambiotic Ltd. has developed a unique bio-technology process for reducing significantly the cost of algae production. (2) Bio-diesel production by the transesterification process directly from the crude dried solid microalgae (without the initial lipid extraction step) using microwave and sonochemical methods. Various methods have already been used for bio-diesel production from microalgae biomass. All methods consist of two separate stages: (1) Lipid extraction using different technique such as Soxhlet extraction (with n-hexane as solvent), the Bligh and Dyer method with a mixture of chloroform and methanol as solvents, a microwave oven technique, supercritical fluid extraction, ultrasound-assisted extraction, and pressurized fluid extraction (Ranjan et al., 2010; Lee et al., 2010). (2) Bio-diesel production by the transesterification of algal oil using either acid or alkali as a catalyst (Ross et al., 2010). Recently, a one-stage method has been
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reported that provides direct transesterification of a microalgae biomass using conventional heating at 90 °C for 40 min with sulfuric acid as a catalyst (Johnson and Wen, 2009). The current work illustrates the optimization of a one-stage method whereby the microalgae of Nannochloropsis (containing a high lipid content of above 30% of dry biomass) is converted to bio-diesel using the direct transesterification by microwave or sonochemical irradiation, and SrO as a catalyst. Microwave and sonochemical radiation accelerate the disruption of the microalgae cells, and as a result the release of oil is made easier. SrO as a base catalyst can be separated from the reaction mixture and reused as a catalyst. Chronologically, our first efforts were directed towards the extraction of the lipid phase from the algae, and the transesterification of the lipid using MW radiation SrO as a catalyst. Later, we attempted to conduct the transesterification directly on the as-harvested algae. The current report follows these steps, and will first report on obtaining bio-diesel from the lipid phase extracted from the Nannochloropsis algae. 2. Methods 2.1. Methods Crude dried solid Nannochloropsis was obtained from the Seambiotic Company. Methanol, chloroform (Bio Lab), and strontium oxide (99.5%, Alfa Aesar) were used as obtained. Two methods have been used for bio-diesel production: sonication (Sonics and Materials, VC-600, 20 kHz, 0.5 in. Ti horn, at 40% amplitude) and microwave irradiation. A domestic microwave oven (DMO, SHARP R-390F) operating at 2.45 GHz was used for the transesterification reactions, in a batch mode under atmospheric pressure. The output of the domestic microwave reactor was 1100 W. Its modification to accommodate a distillation column has been described elsewhere (Groisman and Gedanken, 2008). This modification, passing a distillation column through the MW oven, is aimed at enhancing safe operation by preventing the liquid from splashing. The MW oven was also modified to stir the reaction cell using a magnetic stirrer. The modification was performed by replacing the bottom part of the oven with a rounded aluminum plate. The plate was carefully attached to the framework to allow magnetic stirring, as described elsewhere (Klan et al., 2001). 2.2. Nannochloropsis cultivation and harvest A culture of the marine Eustigmatophyte Nannochloropsis sp. was inoculated in outdoor raceway ponds at a concentration of less than 0.25 g/L, grown for 7 days to a concentration of 0.5 g/L. The cultures were supplied with N & P and C inorganic nutrients, where the C was supplied by coal burning scrubbed flue gas containing 13% CO2. The culture was harvested using a continuous centrifuge (GEA Westfalia) and the paste was dried with a spray dryer (Anhydro) to obtain a dry powder. 2.3. Lipid extraction and the transesterification process As mentioned above, this research was started by extracting the lipid phase from the microalgae, and only later was it found that this process can be avoided and a direct transesterification on the harvested microalgae can be performed. We will present the results of the two-stage process, namely, extraction followed by transesterification, and will emphasize the one step which is the direct transesterification of the a-harvested microalgae. Crude dried Nannochloropsis (1 g) was mixed with methanol– chloroform (1:2 v/v) for lipid extraction using two extraction methods as follows: (1) The first method is sonication. A sonicator (Sonics and Materials, VC-600, 20 kHz, 0.5 in. Ti horn, at 40% ampli-
tude) was employed for 5 min. (2) The second extraction technique was performed using a microwave oven which was also operated for 5 min at 70% power (cycle mode of 21 s on and 9 s off). This cycle mode is a function provided by the manufacturer of the microwave oven. It is not related to the reflux and/or to the distillation column added to the oven. After the lipid extraction the chloroform–methanol phase that contains the extracted lipids was separated from the microalgae powder by filtration using a funnel with slight suction, followed by the evaporation of the solvent. The mass of the lipid obtained from each sample was determined gravimetrically. For the transesterification of microalgae lipids, a mixture of methanol–chloroform (1:2 v/v) and SrO (0.3 g) was added to the microalgae lipids, and the reaction occurred using two methods, sonication and microwave irradiation. Each of these processes lasted for 2 min. After the reaction was completed, the samples were centrifuged and filtered to separate the methanol–chloroform phase that contained the FAME (fatty acid methyl esters) from the glycerol and the catalyst. The solution of methanol–chloroform was evaporated and the mass of bio-diesel (FAME) was determined gravimetrically. 2.4. Transesterification process directly from the crude dried solid microalgae Crude dried Nannochloropsis (1 g) was mixed with methanol– chloroform (1:2 v/v) and a SrO catalyst (0.3 g). The reaction mixture was heated using two different methods, sonication and microwave irradiation, as described above, for 5 min. After the completion of the reaction, the samples were centrifuged and filtered to separate the methanol–chloroform phase that contained the bio-diesel (FAME) from the microalgae powder, the glycerol and the catalyst. The solution of methanol–chloroform was evaporated, and the mass of bio-diesel (FAME) was determined gravimetrically. During the sonication process the temperature was measured by placing a digital thermocouple in the reaction cell. The temperature was found to be 50 °C. The temperature in the microwave reaction was measured by a pyrometer (M.R.C. Ltd.) as soon as the reaction was completed. The temperature was found to be 60 °C for the direct transesterification conducted by microwave. We have therefore conducted also the transesterification at 60 °C by regular reflux using the conventional protocol. The comparison between the results is presented below. 2.5. Characterization The yield of bio-diesel was evaluated by its weight relative to the weight of the microalgae biomass. A bio-diesel product was analyzed by 1H NMR spectroscopy (Bruker) and recorded on a 200 or 300 MHz spectrometer. The chemical shifts were referenced to CDCl3. The conversion percentage was calculated directly from the integrated areas of the triglyceride and FAME signals. The composition of FAME contained in the bio-diesel was further analyzed via gas chromatography (Shimadzu GC 2010) with a flame ionization detector (FID) and a DB-23 Agilent column (length: 60 m, ID: 0.25 mm, Film: 0.15 mm). A FAME Mix C4–C24 was used as an external standard. The bio-diesel samples were dissolved in hexane. Carrier gas: helium, flow rate 109 mL/min. Column oven temperature program: initial temp: 50 °C, hold time 1 min, increasing the temperature to 175 °C at a heating rate of 25 °C/min. When the temperature was further increased to 230 °C the heating rate was changed to 4 °C/min, hold time 5 min, total time: 24.75 min. The microalgae biomass was analyzed and characterized before and after the lipid extraction and the transesterification reaction by light microscopy (Apo-Tome AxioImager.z1 microscope), and Scanning Electron Microscopy (SEM, JSM-840, JEOL).
M. Koberg et al. / Bioresource Technology 102 (2011) 4265–4269
3. Results and discussion 3.1. Nannochloropsis cultivation by Seambiotic Under the limitations of current technology, algae can theoretically convert up to 15% of the photosynthetically available solar irradiation (PAR), or roughly 6% of the total incident radiation, into new cell mass. From the technology for gas transfer and cleaning, command control of its concentration in cultivation ponds, and its absorption in algae for energy-rich products, high-yield, oil-rich algae strains that grow in open-pond systems have been developed. Carbon dioxide feed is the biggest cost item in the long run of algal cultivation. Nutrients are other key requirements for microalgal growth. Seambiotic maintains a 1000-m2 site with eight open raceway ponds that can produce approximately 20 g/m2/ day of algae. The algae ponds are situated several 100 m from the power plant smokestacks. For carbon, Seambiotic uses flue gas from a power plant that combusts coal and contains 13% of free of charge CO2. The flue gas is undergoing a cleaning process and is then mobilized to the ponds. The carbon dioxide and nitrogen from the flue gas are consumed by the microalgae. Carbon is a key requirement, as the composition of microalgae is about 45% carbon. This is generally supplied as CO2. Theoretically, for each gram of microalgae about 2 g of CO2 are required, based on a mass balance. This provides both a source of carbon for enhancing algal growth and a means for capturing CO2 before it is released to the atmosphere, thus leading to net greenhouse gas (GHG) emission reduction. Seambiotic uses seawater that the power station utilizes for cooling the turbines. Scheme 1 outlines the stages in the cultivation process of Seambiotic.
3.2. Comparison of bio-diesel production methods Our main goal was to explore the possibility of conducting a transesterification of the harvested microalgae without separating the lipid phase. After accomplishing these goals, i.e., the transesterification of the harvested microalgae by sonication and MW radiation, we started the process of the optimization of bio-diesel production from the microalgae. In order to obtain the optimum conditions, various experiments have been conducted (Scheme 2, and Fig. 1). Since this is the first report in which sonochemistry and MW are being used for the transesterification of harvested microalgae, we carried out a comparison among six different reactions. (1) The first reaction was carried out sonochemically and in two steps, namely, extraction and transesterification. (2) These two steps were performed using microwave radiation. (3) A one-step direct transesterification using sonication. (4) A one-step transesterification by microwave. (5) Direct transesterification without the initial extraction step by a regular reflux technique. (6) In addition, a one-step transesterification reaction with identical and same amounts of precursors was conducted for 5 min as a control reaction at room temperature without using microwave or sonication. Fig. 1 shows the bio-diesel yields of the various reactions conducted on the microalgae biomass. The yield of bio-diesel was estimated by its weight relative to the weight of the microalgae biomass. As shown in Fig. 1, the reaction performed by the two separate steps, extraction and transesterification, using sonication, yielded 18.9% bio-diesel, whereas using microwave irradiation resulted in a higher bio-diesel yield of 32.8%. Moreover, a direct
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transesterification reaction using microwave resulted in an even higher bio-diesel yield of 37.1%. The direct transesterification by sonication resulted in a bio-diesel yield of 20.9%. The direct transesterification by reflux yielded 6.95%, and the control reaction marked above as number (6) yielded only a 2.9% conversion to bio-diesel in the one-step direct transesterification. These results reveal the importance of microwave irradiation or sonochemistry in accelerating the transesterification reaction. In addition, Fig. 1 indicates that using direct transesterification is not only a simpler and time saving process, as compared to the two-step process, but also yields higher amounts of bio-diesel. Fig. 1 illustrates the biodiesel conversion of microalgae lipids. The conversion of microalgae lipids to FAME (bio-diesel) was calculated by the integration of 1H NMR signals, as described elsewhere (Meher et al., 2006). The relevant signals chosen for integration were those of methoxy groups in the FAME (3.66 ppm, singlet) and those of the a-methylene protons present in all the triglyceride derivatives (2.3 ppm, triplet) of the microalgae lipids. According to Fig. 1, when using direct transesterification by microwave irradiation, we reach the highest conversion (99.9% conversion) of the triglyceride to biodiesel. The control reaction at room temperature demonstrated only a 10.7% conversion. 3.3. Fatty acid composition The nature of the fatty acid composition in the Nannochloropsis was determined by GC analysis (Table 1). The analysis was conducted on the products of the four (1)–(4) reactions mentioned above. According to Table 1, the major composition of bio-diesel, produced using the illustrated techniques, consists of methyl esters of palmitic (C16:0) and palmitoleic (C16:1) acids. The presence of a high percentage of saturated fatty acids is known to impart good oxidation resistance to the bio-diesel (Kondamudi et al., 2009). Table 1 shows a low percentage of methyl esters having a carbon chain of >18 carbons. This guarantees a low viscosity for the bio-diesel. 3.4. Characteristics of Nannochloropsis as a crude dried solid microalgae Light microscope micrographs of the microalgae biomass before and after bio-diesel production were taken (not shown). The original Nannochloropsis cells are arranged in large clusters. The micrograph of the biomass after the direct transesterification reaction by sonication shows a few broken clusters of biomass. We also observe cells that were disrupted by the sonication. Micrographs of a Nannochloropsis biomass after microwave irradiation show a more severe damage to the cell caused by the MW (not shown). Several cells appear to be unaffected by the MW, but a more careful examination reveals that the number of the cells that remain intact are reduced. The crude dried solid microalgae of Nannochloropsis were also characterized by Scanning Electron Microscopy (SEM). SEM micrograph of a Nannochloropsis biomass before bio-diesel production with various particles with an average size of 13 lm (not shown). Each particle is related to a cluster that is composed of many smaller cells. After the direct transesterification reaction using sonication and microwave, a part of the cell clusters was broken into smaller particles with an average size of 8 lm (related to sonicated biomass), and some with an average size of 6 lm (related to biomass after the microwave irradiation). The figures
Scheme 1. Seambiotic microalgae cultivation process.
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(a) Nannochloropsis biomass
MW OR Sono
MW OR Sono
Algal lipids
Methanol, Chloroform
Methanol, Chloroform, SrO
Bio-diesel (FAME)
(b) MW OR Sono OR Reflux OR None
Nannochloropsis biomass
Methanol, Chloroform, SrO
Bio-diesel (FAME)
Scheme 2. Bio-diesel production process using the following methods: (a) A two-step reaction, namely, extraction and transesterification, was performed using microwave radiation or sonochemistry. (b) A one-step direct transesterification using microwave or sonochemistry or reflux or control reaction without heating and stirring.
120
120
100
100
80
80
60
60
40
40
20
20
0
Bio-diesel yield (% of dry biomass)
Bio-diesel conversion (% of microalgae lipids)
Bio-diesel conversion (% of microalgae lipids) Bio-diesel yield (% of dry biomass)
0 Ex-trans Sono
Ex-trans MW
Direct Direct MW Reflux Sono
None
Fig. 1. Bio-diesel yield of the Nannochloropsis microalgae and bio-diesel conversion of the microalgae lipids using various techniques. Five techniques used in bio-diesel production: (Ex-trans Sono) extraction and transesterification reaction steps occurred separately using sonication, (Ex-trans MW) extraction and transesterification reaction steps occurred separately using microwave, (Direct Sono) direct transesterification without the initial extraction step using sonication, (Direct MW) direct transesterification without the initial extraction step using microwave, (Reflux) direct transesterification without the initial extraction step using regular reflux technique, and (None) extraction and transesterification reaction occurred without heating and stirring.
Table 1 Fatty acid composition of Nannochloropsis obtained using different methods. Fatty acid
C12:0 C14:0 C15:0 C16:0 C16:1 C17:0 C18:0 C18:1 C18:2 C18:3 n3 C20:3 n6 C20:3 n3 C20:5
Fatty acid composition (wt.%) Ex-trans Sono
Ex-trans MW
Direct Sono
Direct MW
ND 6.6 0.6 42.8 27.3 0.4 1.0 9.1 1.3 0.4 0.3 1.7 4.9
ND 6.2 0.6 40.2 28.3 0.4 1.0 9.6 1.4 0.3 0.3 2 6.5
1.7 7.2 0.6 46.5 27.2 0.4 1.1 9 0.7 ND ND ND 0.4
ND 6.1 0.5 38.4 27.7 0.4 1 8.8 1.2 0.7 0.5 2.1 6.5
ND: not detected.
(not shown) show a greater damage to the clusters occurring upon MW treatment. From the illustrated micrographs, the explanation for the higher yield of bio-diesel obtained by MW is made clear. Obtaining larger amounts of bio-diesel is related to the exposure of more cells to the MW by breaking it into smaller clusters, enabling a more efficient transesterification. The reason that the MW radiation is more effective in the destruction of the cells can be just a temperature effect, i.e., higher temperatures obtained in the MW oven than in the sonochemical reaction. However, this can be the reason only if we assume that the sonochemical reaction is conducted in the bulk and not in-
side the collapsing bubble, and not in the 200 nm ring around the collapsing bubble. Since the temperatures in these regions are 5000 and 2000 K, respectively (Suslick, 1989), if we rule out this possibility and assume that a higher temperature is obtained in the sonochemical reaction, we will attribute the MW effect to the ionic nature of the transition state of the transesterification reaction. This helps to couple the MW to the reacting molecules and cause the acceleration of the chemical reaction. We have compared the yield of the transesterification reaction of the direct reaction under microwave radiation with the regular reflux technique. Both reactions were conducted at 60 °C for 5 min. Using microwave we have obtained a bio-diesel yield of 37.1%, while only 6.9% were obtained using the regular reflux technique. In addition, when using direct transesterification by microwave irradiation, we reach the highest conversion (99.9%) of the triglyceride to bio-diesel. The regular reflux technique demonstrated only a 74.6% conversion. This difference is due to the heating up the reaction mixture in MW, faster than in the conventional reflux. MW radiation is more effective in the destruction of the cells and accelerates better the transesterification reaction. Although the temperature of the sonochemical process is lower than of conventional heating by reflux, the bio-diesel yield and conversion of the triglyceride to bio-diesel of the sonochemical process is much higher than regular reflux. The efficiency of the sonochemical process is due to effect of the collapsing bubbles that causes a local very high temperature leading to the breaking of more cells into smaller clusters, to oil release, and to a fast transesterifcation reaction.
4. Conclusions In summary, the current work reports on the capability of producing bio-diesel fuel from Nannochloropsis microalgae. The microalgae are prepared by a unique bio-technology-based environmental system utilizing flue gas from coal burning power stations for microalgae cultivation using seawater. This technique reduces the cost of algae production significantly. Direct transesterification (a onestage process) of the as-harvested Nannochloropsis biomass resulted in a higher bio-diesel yield content than that in a two-stage process. The paper shows that if the one step is done by the MW irradiation technique, very large amounts of bio-diesel can be obtained in 5 min. References Barnard, T.M., Leadbeater, N.E., Boucher, M.B., Stencel, L.M., Wilhite, B.A., 2007. Continuous-flow preparation of biodiesel using microwave heating. Energy Fuels 21, 1777–1781. Groisman, Y., Gedanken, A., 2008. Continuous flow, circulating microwave system and its application in nanoparticle fabrication and biodiesel synthesis. J. Phys. Chem. C 112, 8802–8808. Johnson, M.B., Wen, Z., 2009. Production of biodiesel fuel from the microalga schizochytrium limacinum by direct transesterification of algal biomass. Energy Fuels 23, 5179–5183. Klan, P., Hajek, M., Cirkva, V., 2001. The electrodeless discharge lamp: a prospective tool for photochemistry Part 3. The microwave photochemistry reactor. J. Photochem. Photobiol. A: Chem. 140, 185–189.
M. Koberg et al. / Bioresource Technology 102 (2011) 4265–4269 Kondamudi, N., Strull, J., Misra, M., Mohapatra, S., 2009. A green process for producing biodiesel from feather meal. J. Agric. Food Chem. 57, 6163–6166. Lee, J.Y., Yoo, C., Jun, S.Y., Ahn, C.Y., Oh, H.M., 2010. Comparison of several methods for effective lipid extraction from microalgae. Bioresour. Technol. 101, S75–S77. McKendry, P., 2002. Energy production from biomass (part 1): overview of biomass. Bioresour. Technol. 83, 37–46. Meher, L.C., Sagar, D.V., Naik, S.N., 2006. Technical aspects of biodiesel production by transesterification – a review. Renew. Sust. Energ. Rev. 10, 248–268. Patil, P.D., Gude, V.G., Camacho, L.M., Deng, S., 2010. Microwave-assisted catalytic transesterification of camelina sativa oil. Energy Fuels 24, 1298–1304. Ranjan, A., Patil, C., Moholkar, V.S., 2010. Mechanistic assessment of microalgal lipid extraction. Ind. Eng. Chem. Res. 49, 2979–2985. Reijnders, L., 2006. Conditions for the sustainability of biomass based fuel use. Energy Policy 34, 863–876.
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Ross, A.B., Biller, P., Kubacki, M.L., Li, H., Lea-Langton, A., Jones, J.M., 2010. Hydrothermal processing of microalgae using alkali and organic acids. Fuel 89, 2234–2243. Suslick, K.S., 1989. The chemical effects of ultrasound. Scientific American 260, 80– 86. Schenk, P.M., Thomas-Hall, S.R., Stephens, E., Marx, U.C., Mussgnug, J.H., Posten, C., Kruse, O., Hankamer, B., 2008. Second generation biofuels: high-efficiency microalgae for biodiesel production. Bioenerg. Res. 1, 20–43. Scott, S.A., Davey, M.P., Dennis, J.S., Horst, I., Howe, C.J., Lea-Smith, D.J., Smith, A.G., 2010. Biodiesel from algae: challenges and prospects. Curr. Opin. Biotechnol. 21, 277–286. Valente, O.S., Silva, M.J.D., Pasa, V.M.D., Belchior, C.R.P., Sodreˇ, J.R., 2010. Fuel consumption and emissions from a diesel power generator fuelled with castor oil and soybean biodiesel. Fuel 89, 3637–3642.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Bio-diesel production directly from the microalgae biomass of Nannochloropsis by microwave and ultrasound radiation Miri Koberg a, Moshe Cohen b, Ami Ben-Amotz b, Aharon Gedanken a,⇑ a b
Department of Chemistry and Kanbar Laboratory for Nanomaterials, Bar-Ilan University, Center for Advanced Materials and Nanotechnology, Ramat-Gan 52900, Israel Seambiotic Ltd., Tel Aviv 67021, Israel
a r t i c l e
i n f o
Article history: Received 7 September 2010 Received in revised form 29 November 2010 Accepted 1 December 2010 Available online 17 December 2010 Keywords: Bio-diesel Nannochloropsis Microwave irradiation Sonication Catalyst
a b s t r a c t This work offers an optimized method for the direct conversion of harvested Nannochloropsis algae into bio-diesel using two novel techniques. The first is a unique bio-technology-based environmental system utilizing flue gas from coal burning power stations for microalgae cultivation. This method reduces considerably the cost of algae production. The second technique is the direct transesterification (a one-stage method) of the Nannochloropsis biomass to bio-diesel production using microwave and ultrasound radiation with the aid of a SrO catalyst. These two techniques were tested and compared to identify the most effective bio-diesel production method. Based on our results, it is concluded that the microwave oven method appears to be the most simple and efficient method for the one-stage direct transesterification of the as-harvested Nannochloropsis algae. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Bio-diesel, which is made from renewable biological sources, has gained importance as an alternative energy source. When considering the pollution caused by the combustion of conventional petroleum-based diesel (McKendry, 2002; Reijnders, 2006), a shift to non-conventional sources such as bio-diesel is inevitable. Biodiesel, which consists of fatty acid methyl esters (FAME), has lower CO, CO2 and hydrocarbon emissions; in addition, it is non-toxic and biodegradable (Barnard et al., 2007; Valente et al., 2010). The production of bio-diesel using various materials, such as plants, microalgae, and animal fat, has been attempted as an alternative energy source (Patil et al., 2010). Microalgae have certain advantages compared to other energy crops: (1) Microalgae do not require fresh water and can grow in salt water or even contaminated water, at sea or in ponds, thus preserving fresh water that is becoming an ever more rare and valuable natural resource. (2) Furthermore, algae can be cultivated on land that is unsuitable for food production. The cultivation of maize as a biomass fuel source has caused the significant erosion of arable land, whereas microalgae cultivation does not require arable land, and thus does not displace food crops. (3) Moreover, compared to other biofuel crops, microalgae grow much faster and contain more energy per unit weight. Microalgae can generate as much as 40 times more oil ⇑ Corresponding author. Tel.: +972 3 5318315; fax: +972 3 7384053. E-mail address: [email protected] (A. Gedanken). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.12.004
per acre than other plants used for biofuels (Schenk et al., 2008). (4) In addition, microalgae can produce both biofuels and valuable co-products such as omega 3 and several metabolites of economic interest, such as carotenoids (e.g., astaxanthin, lutein), vitamin E (alpha–tocopherol), and poly unsaturated fatty acids (arachidonic and c linoleic) (Scott et al., 2010). Thus, they have a large potential for feed, food, cosmetics, and pharmaceutical industries, which makes their conversion to bio-diesel cost efficient. In the current work we explore the optimization of bio-diesel production, probing the combination of two techniques: (1) A revolutionizing ecologically-based environmental system utilizing flue gas from coal burning power stations for microalgae cultivation. Seambiotic Ltd. has developed a unique bio-technology process for reducing significantly the cost of algae production. (2) Bio-diesel production by the transesterification process directly from the crude dried solid microalgae (without the initial lipid extraction step) using microwave and sonochemical methods. Various methods have already been used for bio-diesel production from microalgae biomass. All methods consist of two separate stages: (1) Lipid extraction using different technique such as Soxhlet extraction (with n-hexane as solvent), the Bligh and Dyer method with a mixture of chloroform and methanol as solvents, a microwave oven technique, supercritical fluid extraction, ultrasound-assisted extraction, and pressurized fluid extraction (Ranjan et al., 2010; Lee et al., 2010). (2) Bio-diesel production by the transesterification of algal oil using either acid or alkali as a catalyst (Ross et al., 2010). Recently, a one-stage method has been
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reported that provides direct transesterification of a microalgae biomass using conventional heating at 90 °C for 40 min with sulfuric acid as a catalyst (Johnson and Wen, 2009). The current work illustrates the optimization of a one-stage method whereby the microalgae of Nannochloropsis (containing a high lipid content of above 30% of dry biomass) is converted to bio-diesel using the direct transesterification by microwave or sonochemical irradiation, and SrO as a catalyst. Microwave and sonochemical radiation accelerate the disruption of the microalgae cells, and as a result the release of oil is made easier. SrO as a base catalyst can be separated from the reaction mixture and reused as a catalyst. Chronologically, our first efforts were directed towards the extraction of the lipid phase from the algae, and the transesterification of the lipid using MW radiation SrO as a catalyst. Later, we attempted to conduct the transesterification directly on the as-harvested algae. The current report follows these steps, and will first report on obtaining bio-diesel from the lipid phase extracted from the Nannochloropsis algae. 2. Methods 2.1. Methods Crude dried solid Nannochloropsis was obtained from the Seambiotic Company. Methanol, chloroform (Bio Lab), and strontium oxide (99.5%, Alfa Aesar) were used as obtained. Two methods have been used for bio-diesel production: sonication (Sonics and Materials, VC-600, 20 kHz, 0.5 in. Ti horn, at 40% amplitude) and microwave irradiation. A domestic microwave oven (DMO, SHARP R-390F) operating at 2.45 GHz was used for the transesterification reactions, in a batch mode under atmospheric pressure. The output of the domestic microwave reactor was 1100 W. Its modification to accommodate a distillation column has been described elsewhere (Groisman and Gedanken, 2008). This modification, passing a distillation column through the MW oven, is aimed at enhancing safe operation by preventing the liquid from splashing. The MW oven was also modified to stir the reaction cell using a magnetic stirrer. The modification was performed by replacing the bottom part of the oven with a rounded aluminum plate. The plate was carefully attached to the framework to allow magnetic stirring, as described elsewhere (Klan et al., 2001). 2.2. Nannochloropsis cultivation and harvest A culture of the marine Eustigmatophyte Nannochloropsis sp. was inoculated in outdoor raceway ponds at a concentration of less than 0.25 g/L, grown for 7 days to a concentration of 0.5 g/L. The cultures were supplied with N & P and C inorganic nutrients, where the C was supplied by coal burning scrubbed flue gas containing 13% CO2. The culture was harvested using a continuous centrifuge (GEA Westfalia) and the paste was dried with a spray dryer (Anhydro) to obtain a dry powder. 2.3. Lipid extraction and the transesterification process As mentioned above, this research was started by extracting the lipid phase from the microalgae, and only later was it found that this process can be avoided and a direct transesterification on the harvested microalgae can be performed. We will present the results of the two-stage process, namely, extraction followed by transesterification, and will emphasize the one step which is the direct transesterification of the a-harvested microalgae. Crude dried Nannochloropsis (1 g) was mixed with methanol– chloroform (1:2 v/v) for lipid extraction using two extraction methods as follows: (1) The first method is sonication. A sonicator (Sonics and Materials, VC-600, 20 kHz, 0.5 in. Ti horn, at 40% ampli-
tude) was employed for 5 min. (2) The second extraction technique was performed using a microwave oven which was also operated for 5 min at 70% power (cycle mode of 21 s on and 9 s off). This cycle mode is a function provided by the manufacturer of the microwave oven. It is not related to the reflux and/or to the distillation column added to the oven. After the lipid extraction the chloroform–methanol phase that contains the extracted lipids was separated from the microalgae powder by filtration using a funnel with slight suction, followed by the evaporation of the solvent. The mass of the lipid obtained from each sample was determined gravimetrically. For the transesterification of microalgae lipids, a mixture of methanol–chloroform (1:2 v/v) and SrO (0.3 g) was added to the microalgae lipids, and the reaction occurred using two methods, sonication and microwave irradiation. Each of these processes lasted for 2 min. After the reaction was completed, the samples were centrifuged and filtered to separate the methanol–chloroform phase that contained the FAME (fatty acid methyl esters) from the glycerol and the catalyst. The solution of methanol–chloroform was evaporated and the mass of bio-diesel (FAME) was determined gravimetrically. 2.4. Transesterification process directly from the crude dried solid microalgae Crude dried Nannochloropsis (1 g) was mixed with methanol– chloroform (1:2 v/v) and a SrO catalyst (0.3 g). The reaction mixture was heated using two different methods, sonication and microwave irradiation, as described above, for 5 min. After the completion of the reaction, the samples were centrifuged and filtered to separate the methanol–chloroform phase that contained the bio-diesel (FAME) from the microalgae powder, the glycerol and the catalyst. The solution of methanol–chloroform was evaporated, and the mass of bio-diesel (FAME) was determined gravimetrically. During the sonication process the temperature was measured by placing a digital thermocouple in the reaction cell. The temperature was found to be 50 °C. The temperature in the microwave reaction was measured by a pyrometer (M.R.C. Ltd.) as soon as the reaction was completed. The temperature was found to be 60 °C for the direct transesterification conducted by microwave. We have therefore conducted also the transesterification at 60 °C by regular reflux using the conventional protocol. The comparison between the results is presented below. 2.5. Characterization The yield of bio-diesel was evaluated by its weight relative to the weight of the microalgae biomass. A bio-diesel product was analyzed by 1H NMR spectroscopy (Bruker) and recorded on a 200 or 300 MHz spectrometer. The chemical shifts were referenced to CDCl3. The conversion percentage was calculated directly from the integrated areas of the triglyceride and FAME signals. The composition of FAME contained in the bio-diesel was further analyzed via gas chromatography (Shimadzu GC 2010) with a flame ionization detector (FID) and a DB-23 Agilent column (length: 60 m, ID: 0.25 mm, Film: 0.15 mm). A FAME Mix C4–C24 was used as an external standard. The bio-diesel samples were dissolved in hexane. Carrier gas: helium, flow rate 109 mL/min. Column oven temperature program: initial temp: 50 °C, hold time 1 min, increasing the temperature to 175 °C at a heating rate of 25 °C/min. When the temperature was further increased to 230 °C the heating rate was changed to 4 °C/min, hold time 5 min, total time: 24.75 min. The microalgae biomass was analyzed and characterized before and after the lipid extraction and the transesterification reaction by light microscopy (Apo-Tome AxioImager.z1 microscope), and Scanning Electron Microscopy (SEM, JSM-840, JEOL).
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3. Results and discussion 3.1. Nannochloropsis cultivation by Seambiotic Under the limitations of current technology, algae can theoretically convert up to 15% of the photosynthetically available solar irradiation (PAR), or roughly 6% of the total incident radiation, into new cell mass. From the technology for gas transfer and cleaning, command control of its concentration in cultivation ponds, and its absorption in algae for energy-rich products, high-yield, oil-rich algae strains that grow in open-pond systems have been developed. Carbon dioxide feed is the biggest cost item in the long run of algal cultivation. Nutrients are other key requirements for microalgal growth. Seambiotic maintains a 1000-m2 site with eight open raceway ponds that can produce approximately 20 g/m2/ day of algae. The algae ponds are situated several 100 m from the power plant smokestacks. For carbon, Seambiotic uses flue gas from a power plant that combusts coal and contains 13% of free of charge CO2. The flue gas is undergoing a cleaning process and is then mobilized to the ponds. The carbon dioxide and nitrogen from the flue gas are consumed by the microalgae. Carbon is a key requirement, as the composition of microalgae is about 45% carbon. This is generally supplied as CO2. Theoretically, for each gram of microalgae about 2 g of CO2 are required, based on a mass balance. This provides both a source of carbon for enhancing algal growth and a means for capturing CO2 before it is released to the atmosphere, thus leading to net greenhouse gas (GHG) emission reduction. Seambiotic uses seawater that the power station utilizes for cooling the turbines. Scheme 1 outlines the stages in the cultivation process of Seambiotic.
3.2. Comparison of bio-diesel production methods Our main goal was to explore the possibility of conducting a transesterification of the harvested microalgae without separating the lipid phase. After accomplishing these goals, i.e., the transesterification of the harvested microalgae by sonication and MW radiation, we started the process of the optimization of bio-diesel production from the microalgae. In order to obtain the optimum conditions, various experiments have been conducted (Scheme 2, and Fig. 1). Since this is the first report in which sonochemistry and MW are being used for the transesterification of harvested microalgae, we carried out a comparison among six different reactions. (1) The first reaction was carried out sonochemically and in two steps, namely, extraction and transesterification. (2) These two steps were performed using microwave radiation. (3) A one-step direct transesterification using sonication. (4) A one-step transesterification by microwave. (5) Direct transesterification without the initial extraction step by a regular reflux technique. (6) In addition, a one-step transesterification reaction with identical and same amounts of precursors was conducted for 5 min as a control reaction at room temperature without using microwave or sonication. Fig. 1 shows the bio-diesel yields of the various reactions conducted on the microalgae biomass. The yield of bio-diesel was estimated by its weight relative to the weight of the microalgae biomass. As shown in Fig. 1, the reaction performed by the two separate steps, extraction and transesterification, using sonication, yielded 18.9% bio-diesel, whereas using microwave irradiation resulted in a higher bio-diesel yield of 32.8%. Moreover, a direct
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transesterification reaction using microwave resulted in an even higher bio-diesel yield of 37.1%. The direct transesterification by sonication resulted in a bio-diesel yield of 20.9%. The direct transesterification by reflux yielded 6.95%, and the control reaction marked above as number (6) yielded only a 2.9% conversion to bio-diesel in the one-step direct transesterification. These results reveal the importance of microwave irradiation or sonochemistry in accelerating the transesterification reaction. In addition, Fig. 1 indicates that using direct transesterification is not only a simpler and time saving process, as compared to the two-step process, but also yields higher amounts of bio-diesel. Fig. 1 illustrates the biodiesel conversion of microalgae lipids. The conversion of microalgae lipids to FAME (bio-diesel) was calculated by the integration of 1H NMR signals, as described elsewhere (Meher et al., 2006). The relevant signals chosen for integration were those of methoxy groups in the FAME (3.66 ppm, singlet) and those of the a-methylene protons present in all the triglyceride derivatives (2.3 ppm, triplet) of the microalgae lipids. According to Fig. 1, when using direct transesterification by microwave irradiation, we reach the highest conversion (99.9% conversion) of the triglyceride to biodiesel. The control reaction at room temperature demonstrated only a 10.7% conversion. 3.3. Fatty acid composition The nature of the fatty acid composition in the Nannochloropsis was determined by GC analysis (Table 1). The analysis was conducted on the products of the four (1)–(4) reactions mentioned above. According to Table 1, the major composition of bio-diesel, produced using the illustrated techniques, consists of methyl esters of palmitic (C16:0) and palmitoleic (C16:1) acids. The presence of a high percentage of saturated fatty acids is known to impart good oxidation resistance to the bio-diesel (Kondamudi et al., 2009). Table 1 shows a low percentage of methyl esters having a carbon chain of >18 carbons. This guarantees a low viscosity for the bio-diesel. 3.4. Characteristics of Nannochloropsis as a crude dried solid microalgae Light microscope micrographs of the microalgae biomass before and after bio-diesel production were taken (not shown). The original Nannochloropsis cells are arranged in large clusters. The micrograph of the biomass after the direct transesterification reaction by sonication shows a few broken clusters of biomass. We also observe cells that were disrupted by the sonication. Micrographs of a Nannochloropsis biomass after microwave irradiation show a more severe damage to the cell caused by the MW (not shown). Several cells appear to be unaffected by the MW, but a more careful examination reveals that the number of the cells that remain intact are reduced. The crude dried solid microalgae of Nannochloropsis were also characterized by Scanning Electron Microscopy (SEM). SEM micrograph of a Nannochloropsis biomass before bio-diesel production with various particles with an average size of 13 lm (not shown). Each particle is related to a cluster that is composed of many smaller cells. After the direct transesterification reaction using sonication and microwave, a part of the cell clusters was broken into smaller particles with an average size of 8 lm (related to sonicated biomass), and some with an average size of 6 lm (related to biomass after the microwave irradiation). The figures
Scheme 1. Seambiotic microalgae cultivation process.
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(a) Nannochloropsis biomass
MW OR Sono
MW OR Sono
Algal lipids
Methanol, Chloroform
Methanol, Chloroform, SrO
Bio-diesel (FAME)
(b) MW OR Sono OR Reflux OR None
Nannochloropsis biomass
Methanol, Chloroform, SrO
Bio-diesel (FAME)
Scheme 2. Bio-diesel production process using the following methods: (a) A two-step reaction, namely, extraction and transesterification, was performed using microwave radiation or sonochemistry. (b) A one-step direct transesterification using microwave or sonochemistry or reflux or control reaction without heating and stirring.
120
120
100
100
80
80
60
60
40
40
20
20
0
Bio-diesel yield (% of dry biomass)
Bio-diesel conversion (% of microalgae lipids)
Bio-diesel conversion (% of microalgae lipids) Bio-diesel yield (% of dry biomass)
0 Ex-trans Sono
Ex-trans MW
Direct Direct MW Reflux Sono
None
Fig. 1. Bio-diesel yield of the Nannochloropsis microalgae and bio-diesel conversion of the microalgae lipids using various techniques. Five techniques used in bio-diesel production: (Ex-trans Sono) extraction and transesterification reaction steps occurred separately using sonication, (Ex-trans MW) extraction and transesterification reaction steps occurred separately using microwave, (Direct Sono) direct transesterification without the initial extraction step using sonication, (Direct MW) direct transesterification without the initial extraction step using microwave, (Reflux) direct transesterification without the initial extraction step using regular reflux technique, and (None) extraction and transesterification reaction occurred without heating and stirring.
Table 1 Fatty acid composition of Nannochloropsis obtained using different methods. Fatty acid
C12:0 C14:0 C15:0 C16:0 C16:1 C17:0 C18:0 C18:1 C18:2 C18:3 n3 C20:3 n6 C20:3 n3 C20:5
Fatty acid composition (wt.%) Ex-trans Sono
Ex-trans MW
Direct Sono
Direct MW
ND 6.6 0.6 42.8 27.3 0.4 1.0 9.1 1.3 0.4 0.3 1.7 4.9
ND 6.2 0.6 40.2 28.3 0.4 1.0 9.6 1.4 0.3 0.3 2 6.5
1.7 7.2 0.6 46.5 27.2 0.4 1.1 9 0.7 ND ND ND 0.4
ND 6.1 0.5 38.4 27.7 0.4 1 8.8 1.2 0.7 0.5 2.1 6.5
ND: not detected.
(not shown) show a greater damage to the clusters occurring upon MW treatment. From the illustrated micrographs, the explanation for the higher yield of bio-diesel obtained by MW is made clear. Obtaining larger amounts of bio-diesel is related to the exposure of more cells to the MW by breaking it into smaller clusters, enabling a more efficient transesterification. The reason that the MW radiation is more effective in the destruction of the cells can be just a temperature effect, i.e., higher temperatures obtained in the MW oven than in the sonochemical reaction. However, this can be the reason only if we assume that the sonochemical reaction is conducted in the bulk and not in-
side the collapsing bubble, and not in the 200 nm ring around the collapsing bubble. Since the temperatures in these regions are 5000 and 2000 K, respectively (Suslick, 1989), if we rule out this possibility and assume that a higher temperature is obtained in the sonochemical reaction, we will attribute the MW effect to the ionic nature of the transition state of the transesterification reaction. This helps to couple the MW to the reacting molecules and cause the acceleration of the chemical reaction. We have compared the yield of the transesterification reaction of the direct reaction under microwave radiation with the regular reflux technique. Both reactions were conducted at 60 °C for 5 min. Using microwave we have obtained a bio-diesel yield of 37.1%, while only 6.9% were obtained using the regular reflux technique. In addition, when using direct transesterification by microwave irradiation, we reach the highest conversion (99.9%) of the triglyceride to bio-diesel. The regular reflux technique demonstrated only a 74.6% conversion. This difference is due to the heating up the reaction mixture in MW, faster than in the conventional reflux. MW radiation is more effective in the destruction of the cells and accelerates better the transesterification reaction. Although the temperature of the sonochemical process is lower than of conventional heating by reflux, the bio-diesel yield and conversion of the triglyceride to bio-diesel of the sonochemical process is much higher than regular reflux. The efficiency of the sonochemical process is due to effect of the collapsing bubbles that causes a local very high temperature leading to the breaking of more cells into smaller clusters, to oil release, and to a fast transesterifcation reaction.
4. Conclusions In summary, the current work reports on the capability of producing bio-diesel fuel from Nannochloropsis microalgae. The microalgae are prepared by a unique bio-technology-based environmental system utilizing flue gas from coal burning power stations for microalgae cultivation using seawater. This technique reduces the cost of algae production significantly. Direct transesterification (a onestage process) of the as-harvested Nannochloropsis biomass resulted in a higher bio-diesel yield content than that in a two-stage process. The paper shows that if the one step is done by the MW irradiation technique, very large amounts of bio-diesel can be obtained in 5 min. References Barnard, T.M., Leadbeater, N.E., Boucher, M.B., Stencel, L.M., Wilhite, B.A., 2007. Continuous-flow preparation of biodiesel using microwave heating. Energy Fuels 21, 1777–1781. Groisman, Y., Gedanken, A., 2008. Continuous flow, circulating microwave system and its application in nanoparticle fabrication and biodiesel synthesis. J. Phys. Chem. C 112, 8802–8808. Johnson, M.B., Wen, Z., 2009. Production of biodiesel fuel from the microalga schizochytrium limacinum by direct transesterification of algal biomass. Energy Fuels 23, 5179–5183. Klan, P., Hajek, M., Cirkva, V., 2001. The electrodeless discharge lamp: a prospective tool for photochemistry Part 3. The microwave photochemistry reactor. J. Photochem. Photobiol. A: Chem. 140, 185–189.
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