Energy Fuels 2009, 23, 5179–5183 Published on Web 09/02/2009
: DOI:10.1021/ef900704h
Production of Biodiesel Fuel from the Microalga Schizochytrium limacinum by Direct Transesterification of Algal Biomass Michael B. Johnson and Zhiyou Wen* Department of Biological Systems Engineering, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061 Received May 7, 2009
Producing biofuel from microalgae has gained renewed interest recently. Schizochytrium limacinum is a heterotrophic microalga that is capable of producing high levels of biomass and total fatty acid. The objective of this work is to explore the potential of producing biodiesel fuel from this alga using different biodiesel preparation methods, including oil extraction followed by transesterification (a two-stage method) or direction transesterification of algal biomass (a one-stage method). When freeze-dried biomass was used as feedstock, the two-stage method resulted in 57% of crude biodiesel yield (based on algal biomass) with a fatty acid methyl ester (FAME) content of 66.37%. The one-stage method (with chloroform, hexane, or petroleum ether used in transesterification) led to a high yield of crude biodiesel, whereas only chloroform-based transesterification led to a high FAME content. When wet biomass was used as feedstock, the one-stage method resulted in a much-lower biodiesel yield. The biodiesel prepared via the direct transesterification of dry biomass was subjected to ASTM standard tests. Parameters such as free glycerol, total glycerol, acid number, soap content, corrosiveness to copper, flash point, viscosity, and particulate matter met the ASTM standards, while the water and sediment content, as well as the sulfur content did not pass the standard. Collectively, the results indicate the alga S. limanicum is a suitable feedstock for producing biodiesel via the direct transesterification method.
conditions.5 The biodiesel made from this heterotrophically grown alga was of good quality.6 Recently, the heterotrophic alga Schizochytrium limacinum was investigated for its potential of producing omega-3 polyunsaturated fatty acid (DHA, C22:6, n-3) using biodiesel-derived crude glycerol as a lessexpensive substrate.7 In addition to DHA, S. limacinum also contains a high level of total fatty acid (∼50% of dry biomass), which is ideal for making biodiesel.7 In the algal biodiesel production processes, fatty acid methyl esters (FAME), the chemical composition of biodiesel, are commonly prepared by transesterification of algal oil using either acid or alkali as a catalyst.1,8-10 Unlike terrestrial feedstock such as soybean or canola seed, from which oil can be extracted by crushing followed with solvent extraction, releasing oil from algal cells is hindered by rigid cell walls. Mechanically crushing algal biomass (in either a mudlike wet form or a powder-like dry form) to extract oil is also difficult to be implemented using the existing crushing equipment.
1. Introduction Microalgae have long been considered as a promising alternative and renewable feedstock source for biofuels. Recently, the algae-for-fuel concept has gained renewed interest with energy prices fluctuating widely.1,2 Compared with terrestrial plants, microalgae have a high oil content and growth rate; mass algal cultivation can be performed on unexploited lands using saline water in arid regions, thus avoiding competition for limited arable lands. Currently, algal biofuel production is still limited mainly by several factors associated with algal culture. An autotrophic culture system such as an open pond typically generates a cell concentration of 0.1-1 g/L (i.e., 0.01%-0.1%, w/v);3,4 harvesting algal cells from this dilute suspension is costly. In addition, the culture system is prone to be contaminated and taken over by native species; the oil yield of the algae is relatively low. As an alternative to autotrophic culture systems, heterotrophic algal culture provides a promising way to overcome these bottlenecks. For example, the green alga Chlorella protothecoides has shown a high cell density (51.2 g/L) and oil content (>50%, dry basis) in a fed-batch heterotrophic culture
(5) Xiong, W.; Li, X. F.; Xiang, J. Y.; Wu, Q. Y. High-density fermentation of microalga Chlorella protothecoides in bioreactor for microbio-diesel production. Appl. Microbiol. Biotechnol. 2008, 78 (1), 29–36. (6) Xu, H.; Miao, X. L.; Wu, Q. Y. High quality biodiesel production from a microalga Chlorella protothecoides by heterotrophic growth in fermenters. J. Biotechnol. 2006, 126 (4), 499–507. (7) Pyle, D. J.; Garcia, R. A.; Wen, Z. Y. Producing docosahexaenoic acid (DHA)-rich algae from blodiesel-derived crude glycerol: Effects of impurities on DHA production and algal biomass composition. J. Agric. Food Chem. 2008, 56 (11), 3933–3939. (8) Demirbas, A. Production of Biodiesel from Algae Oils. Energy Sources, Part A-Recovery Util. Environ. Eff. 2009, 31 (2), 163–168. (9) Liu, B.; Zhao, Z. Biodiesel production by direct methanolysis of oleaginous microbial biomass. J. Chem. Technol. Biotechnol. 2007, 82 (8), 775–780. (10) Canakci, M.; Van Gerpen, J. Biodiesel production via acid catalysis. Trans. ASAE 1999, 42 (5), 1203–1210.
*Author to whom correspondence should be addressed: Tel.: 1-(540) 231 9356. Fax: 1-(540) 231 3199. E-mail:
[email protected]. (1) Chisti, Y. Biodiesel from microalgae. Biotechnol. Adv. 2007, 25 (3), 294–306. (2) Hu, Q.; Sommerfeld, M.; Jarvis, E.; Ghirardi, M.; Posewitz, M.; Seibert, M.; Darzins, A. Microalgal triacylglycerols as feedstocks for biofuel production: Perspectives and advances. Plant J. 2008, 54 (4), 621–639. (3) Tsukahara, K.; Sawayama, S. Liquid fuel production using microalgae. J. Jpn. Pet. Inst. 2005, 48 (5), 251–259. (4) Grima, E. M.; Belarbi, E. H.; Fernandez, F. G. A.; Medina, A. R.; Chisti, Y. Recovery of microalgal biomass and metabolites: process options and economics. Biotechnol. Adv. 2003, 20 (7-8), 491–515. r 2009 American Chemical Society
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2.2. Preparation of Biodiesel from Algal Biomass. Biodiesel (FAME) was prepared from algal biomass through two methods: (M1) oil extraction from algal biomass followed by transesterification and (M2) direct transesterification of algal biomass (see Figure 1). For each method, both freeze-dried biomass and wet biomass were used as feedstock (Figure 1). Extraction-Transesterification. The algal oil extraction procedure was adapted from the protocol described by Bligh and Dyer.16 Freeze-dried algal biomass (1 g) or wet algal biomass (1 g dry weight equivalent) was placed in a solvent-proof chamber of a bead beater (Bio Spec Products, Inc., Bartlesville, OK). The dry biomass was added with 5 mL of distilled water. When wet biomass was used, the water contained in the wet biomass (∼80%, w/w) served as the water used for extraction.16 The chamber was added with 6 mL of chloroform and 12 mL of methanol, along with 1.00-mm glass beads. The mixture was blended for 2 min and transferred to a new centrifuge tube. Chloroform and distilled water (6 mL each) were respectively used to rinse the bead beater chamber via blending, and then transferred to the tube, which was thoroughly mixed using a vortex. The contents in the tube were centrifuged at 7232 g for 10 min. The organic layer containing the algal oil was then transferred to a preweighed glass vial. The mass of lipids (oil) were determined after the solvent was evaporated using N2. For the transesterification of algal oil, a mixture of methanol (3.4 mL), sulfuric acid (0.6 mL), and chloroform (4.0 mL) was added to the algal oil and heated at 90 °C for 40 min. The samples were thoroughly mixed during heating. After the reaction was completed, the samples were cooled to room temperature and mixed with 2 mL distilled water, and then time was allowed for phase separation. The lower phase, which contained biodiesel (FAME), was collected and transferred to a preweighed glass tube. The solvent was evaporated using N2, and the mass of biodiesel was determined gravimetrically. Direct Transesterification. Freeze-dried algal biomass (1 g) or wet algal biomass (1 g dry weight equivalent) was placed in a glass test tube and mixed with 3.4 mL of methanol and 0.6 mL of sulfuric acid. Depending on the experimental deign (see Figure 1), 4.0 mL of solvent (chloroform, hexane, or petroleum ether) was added to the tube, or there was no solvent addition; in this case, an additional 4.0 mL of methanol was added. The reaction mixture was heated at 90 °C for 40 min and the samples were well-mixed during heating. After the reaction was completed, the tubes were allowed to cool to room temperature. Then, 2 mL distilled water was added to the tube and mixed for 45 s. The tubes were allowed to separate into two phases. If needed, the tubes were centrifuged at 7232 g for 8 min to accelerate phase separation. The solvent layer that contained biodiesel (FAME) was collected and transferred to a preweighed glass vial. The solvent was evaporated using N2, and the mass of biodiesel was determined gravimetrically. 2.3. Biodiesel Yield Evaluation and FAME Characterization. The yield of biodiesel was evaluated by its weight relative to (i) the weight of oil contained in the biomass and (ii) the weight of algal biomass. The composition of FAME contained in the crude biodiesel was further analyzed via gas chromatography (GC). The biodiesel samples were dissolved in the solvent (the same type as that used in the transesterification) to bring the total volume to 20 mL. Then, 0.25 mL of this sample was added with 0.25 mL of internal standard (methylated heptadecanoic acid, 10 mg/mL) and 1.0 mL of solvent. This mix was ready to be analyzed by a Shimadzu 2010 GC system (Shimadzu Scientific Instruments, Columbia, MD) that was equipped with a flame-ionization detection (FID) device and a SGE SolGel-Wax capillary column (30 m 0.25 mm 0.25 μm). The GC operation procedure was the same as described previously.14 The fatty acids were identified by comparing the
Figure 1. Methods used in the preparation of biodiesel from algal biomass: (M1) extraction-transesterification and (M2) direct transesterification. The reagents used for each method are listed in the figure.
Preparing biodiesel via the direct transesterification of raw oleaginous materials may overcome the limitations described above. The direct transesterification has shown an increased recovery of fatty acids from a variety of samples such as marine tissues,11 yeast and fungi,9 bacteria,12 and microheterotrophs.13 Our previous work has also used direct transesterification to analyze the fatty acid content of the alga S. limacinum.7 However, reports on using direct transesterification as a way to prepare biodiesel fuel from microalgae have been limited. The objectives of this work are (i) to evaluate the potentials of producing biodiesel from the alga of S. limacinum through direct transesterification and (ii) to evaluate the quality of the biodiesel prepared from this method. 2. Experimental Section 2.1. Algal Culture and Biomass Preparation. The alga Schizochytrium limacinum SR21 (ATCC MYA-1381) was used. The experimental procedures for algal cell culture were described previously.7,14 In short, the algal cells were grown in a medium that contained 90 g/L crude glycerol (obtained from Virginia Biodiesel Refinery, West Point, VA) and 10 g/L corn steep solid (Sigma) dissolved in artificial seawater. The pH was adjusted to 8.0 before autoclaving the medium at a temperature of 121 °C for 15 min. Algae were either grown in 250-mL Erlenmeyer flasks, each containing 50 mL of the medium,7 or a 30-L fermenter15 to prepare enough biomass for large-batch biodiesel production. Algal biomass was harvested by centrifugation at 7232 g for 10 min. The cell pellet was washed twice with distilled water and its water content determined. Depending on the experimental designs, the cell pellets were either freeze-dried or used as wet biomass. (11) Meier, S.; Mjos, S. A.; Joensen, H.; Grahl-Nielsen, O. Validation of a one-step extraction/methylation method for determination of fatty acids and cholesterol in marine tissues. J. Chromatogr. A 2006, 1104 (1-2), 291–298. (12) Dionisi, F.; Golay, P. A.; Elli, M.; Fay, L. B. Stability of cyclopropane and conjugated linoleic acids during fatty acid quantification in lactic acid bacteria. Lipids 1999, 34 (10), 1107–1115. (13) Lewis, T.; Nichols, P. D.; McMeekin, T. A. Evaluation of extraction methods for recovery of fatty acids from lipid-producing microheterotrophs. J. Microbiol. Methods 2000, 43 (2), 107–116. (14) Chi, Z.; Pyle, D.; Wen, Z.; Frear, C.; Chen, S. A laboratory study of producing docosahexaenoic acid from biodiesel-waste glycerol by microalgal fermentation. Process Biochem. 2007, 42 (11), 1537–1545. (15) Chi, Z. Y.; Liu, Y.; Frear, C.; Chen, S. L. Study of a two-stage growth of DHA-producing marine algae Schizochytrium limacinum SR21 with shifting dissolved oxygen level. Appl. Microbiol. Biotechnol. 2009, 81 (6), 1141–1148.
(16) Bligh, E. G.; Dyer, W. J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 1959, 37, 911–917.
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retention times with those of standard fatty acids, and quantified by comparing their peak area with that of the internal standard.14 2.4. Producing Biodiesel in a Large Batch and Evaluating Fuel Quality. A larger-batch algal biodiesel production was performed using the methods of direct transesterification of dry biomass (using chloroform as the solvent) (see Figure 1). Freezedried biomass (400 g) was mixed with 1.36 L of methanol, 240 mL of sulfuric acid, and 1.6 L of solvent. The mixture was placed in sealed bottles and heated in a rotary shaker (180 rpm) at 80 °C for 2.25 h. After the reaction, following a cool-down period, half the total volume of deionized water was added; then, the mixture was shaken vigorously and allowed to settle and reach a biphasic state. The solvent was collected and evaporated using a nitrogen stream while being maintained in a water bath at 50 °C. The crude biodiesel was purified by washing with warm deionized water (80 °C) three times until the pH of the wash water was at a neutral level. Butylated hydroxytoluene (BHT) was added at 100 ppm to the purified biodiesel to prevent oxidation of unsaturated FAMEs. The final biodiesel product was characterized by a commercial biodiesel producer (Chesapeake Custom Chemical, Ridgeway, VA), according to the method that is described in ASTM standard D6751.
3. Results 3.1. Characteristics of Algal Biomass. The alga S. limacinum has proven capable of growing on biodiesel-derived crude glycerol while producing a high level of DHA.14 The freeze-dried biomass appeared as a white powder containing 51% lipid, 14% proteins, and 24% carbohydrate, with 11% ash.7 The alga has a relatively simple fatty acid profile with myristic acid (C14:0), palmitic acid (C16:0), docosapentaenoic acid (C22:5), and docosahexaenoic acid (C22:6) being the major fatty acids.7 Previous work showed that the total fatty acids (TFA) accounted for 40%-50% of the dry biomass, depending on different culture conditions.7,14 3.2. Biodiesel Production from Dry Algal Biomass. The biodiesel yield from the freeze-dried algal biomass through extraction-transesterification and direct transesterification was compared (see Figure 2). The biodiesel yield was first expressed as its weight relative to the algal oil present in the biomass (see Figure 2A), as commonly used by most researchers.6,9 As shown in Figure 2A, the extraction-transesterification resulted in a biodiesel yield of 98.4%. The direct transesterification (with the three solvents) also resulted in a high biodiesel yield. When no solvent was used in the direct transesterification, biodiesel yield was very low (12.7%), indicating that solvent was essential for the reaction. Figure 2A also shows that, when chloroform or hexane was used to treat biomass, the biodiesel yield (based on algal oil) exceeded 100%. The reason may that the oil mass used in the biodiesel yield calculation was false low. Therefore, the biodiesel yield was further evaluated on the dry biomass basis. As shown in Figure 2B, the algae can produce 10%20% higher biodiesel yields using direct transesterification (with chloroform and hexane) than when using the extraction-transesterification method. The characteristics of the FAME obtained from different methods are presented in Table 1. Because of the significantly low biodiesel yield for the direct transesterification without solvent (see Figure 2), the FAME composition for this treatment was not presented. As shown in Table 1, the major FAMEs contained in the biodiesel were esters of myristic acid (C14:0), palmitic acid (C16:0), docosapentaenoic acid (C22:5), and docosahexaenoic acid (C22:6). The degree of FAME unsaturation was in the range of 2.04-2.26,
Figure 2. Biodiesel yield of the microalga S. limacinum using extraction-transesterification and direct transesterification with different solvents used: (A) yield based on algal oil and (B) yield based on dry biomass. Data represent the mean values of three replicates, and error bars show standard deviations.
depending on different methods (see Table 1). The total FAME yield varied significantly with the different transesterification methods. The direct transesterification (with chloroform) resulted in the highest FAME yield and content. When hexane and petroleum ether were used in the reaction, the FAME yields were significantly reduced (see Table 1), although the gravimetric crude biodiesel yield was comparable to that of chloroform-based direct transesterification (see Figure 2). Table 1 also shows that the extraction-transesterification and direct transesterification (chloroformbased) resulted in a high FAME content (63%-66%). 3.3. Biodiesel Production from Wet Algal Biomass. To reduce the algal biodiesel production cost associated with the dewatering and drying of the biomass, the possibility of using freshly harvested wet algae (through centrifuge) as biodiesel feedstock was investigated. As shown in Table 2, when the extraction-transesterification method was used to treat the wet biomass, the oil extracted and the biodiesel yield was comparable to those obtained from the freeze-dried biomass. Both the wet and dry biomasses had similar FAME compositions; however, the total FAME yield from the wet biomass was ∼20% less than that from the dry biomass. The FAME content in the wet-biomass derived biodiesel was also lower (see Table 2). In the direct transesterification method, the wet and dry biomass resulted in a similar crude biodiesel yield. However, the wet biomass resulted in a high portion of unknown FAME. The FAME yield and FAME content from the wet biomass was also significantly lower than that from the dry biomass. 3.4. Scaled-Up Production and Characterization of Algal Biodiesel. The aforementioned results indicate that the direct 5181
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Table 1. FAME Characterization of the Biodiesel Produced from Extraction-Transesterification and Direct Transesterification Methodsa,b Direct Transesterification
FAME composition C14:0 (% FAME) C16:0 (% FAME) C22:5 (% FAME) C22:6 (% FAME) others (% FAME) degree of FAME unsaturation total FAME yield % of algal oil (w/w) % of dry biomass (w/w) FAME content in biodiesel (%)
extraction-transesterification
chloroform
hexane
petroleum ether
5.25 ( 0.15 56.51 ( 0.63 5.21 ( 0.12 29.72 ( 0.90 3.31 ( 0.25 2.04 ( 0.06
5.33 ( 0.01 56.70 ( 0.06 5.12 ( 0.02 29.70 ( 0.07 3.16 ( 0.03 2.04 ( 0.005
4.47 ( 0.05 54.72 ( 0.31 5.91 ( 0.07 32.67 ( 0.30 2.22 ( 0.67 2.26 ( 0.02
4.44 ( 0.13 53.50 ( 1.60 5.67 ( 0.14 31.57 ( 1.13 4.82 ( 0.01 2.18 ( 0.08
63.70 ( 4.41 36.80 ( 2.55 66.37
72.79 ( 3.68 42.05 ( 2.12 63.47
10.49 ( 0.22 6.06 ( 0.14 9.15
11.13 ( 0.12 6.43 ( 0.08 9.71
a Data represent the mean values of three replicates ( standard deviations. b Degree of FAME unsaturation = [1.0 (% monene) þ 2.0 (% diene) þ 3.0 (% triene) þ 4.0 (%tetraenes) þ 5.0 (% pentanes) þ 6.0 (% hexane)]/100.22
Table 2. Biodiesel Yield and Its FAME Characterization Prepared from Wet and Dry Biomass Using Extraction-Transesterification and Direct Transesterificationa,b,c Extraction-Transesterification wet biomass oil content (% dry biomass) biodiesel yield % of algal oil % of dry biomass FAME composition C14:0 (% FAME) C16:0 (% FAME) C22:5 (% FAME) C22:6 (% FAME) others (% FAME) degree of FAME unsaturation total FAME yield % of algal oil (w/w) % of dry biomass (w/w) FAME content in biodiesel (%)
dry biomass
Direct Transesterification wet biomass
dry biomass
95.98 ( 3.72 55.45 ( 2.15
108.84 ( 2.67 66.97 ( 1.70
114.68 ( 0.16 66.25 ( 0.22
4.00 ( 0.20 48.16 ( 0.72 5.61 ( 0.33 35.15 ( 2.38 7.09 ( 1.81 2.30 ( 0.10
5.25 ( 0.15 56.51 ( 0.63 5.21 ( 0.12 29.72 ( 0.90 3.31 ( 0.25 2.04 ( 0.06
38.06 ( 4.73 4.78 ( 0.42 23.51 ( 2.76 36.83 ( 10.24 1.75 ( 0.09
5.33 ( 0.01 56.70 ( 0.06 5.12 ( 0.02 29.70 ( 0.07 3.16 ( 0.03 2.04 ( 0.005
51.12 ( 1.49 31.45 ( 0.92 52.66
63.70 ( 4.41 36.80 ( 2.55 66.37
8.45 ( 2.97 5.20 ( 1.83 7.76
72.79 ( 3.68 42.05 ( 2.12 63.47
61.53 ( 0.72
57.77 ( 0.42
97.07 ( 0.70 59.73 ( 0.45
a Chloroform was used in the direct transesterification. b Data represent the mean values of three replicates ( standard deviations. c Degree of FAME unsaturation = [1.0 (% monene) þ 2.0 (% diene) þ 3.0 (% triene) þ 4.0 (%tetraenes) þ 5.0 (% pentanes) þ 6.0 (% hexane)]/100.22
Table 3. Characteristics of Biodiesel Fuel Producing from S. limacinum Properties
ASTM method
flash point (closed cup) moisture content water and sediment content acid number free glycerin total glycerin corrosiveness to copper kinematic viscosity (at 40 °C) sulfur content soap particulate matter check
D-93 D-1796 D-2709 D-664 D-6584 D-6584 D-130 D-445 D-7039 Cc-17-79 C-100
limits
actual value
130 °C min report 0.05 vol % max 0.50 mg KOH/g max 0.02 mass % max 0.24 mass % max 3 max 1.9-6.0 mm2/s 15 ppm max report yes
204 °C 0.111 mass % 0.1 vol % 0.11 mg KOH/g 0.003 mass % 0.097 mass % 1a 3.87 mm2/s 69 ppm not detected yes
comment
ASTM standard given in units of ppm visual appearance
transesterification (with chloroform) had the best performance for producing biodiesel from S. limacinum, in terms of biodiesel yield, FAME yield, and FAME content (see Figure 1 and Table 2). Therefore, this method was used to prepare a larger batch of biodiesel for ASTM standard tests. The scaled-up biodiesel production resulted in ∼200 mL of liquid fuel from 400 g of algal biomass. The ASTM standard tests of this liquid fuel indicate that the free glycerol, total glycerol, acid number, corrosiveness to copper, flash point, particulate matter check, viscosity, and soap check meet the standards. However, the water and sediment and sulfur content did not meet the ASTM standards (see Table 3).
lipid content achieved in this culture system.5,6,17 The microalga S. limacinum is capable of using crude glycerol, which is a major byproduct in the biodiesel manufacturing process, as a carbon source for its heterotrophic growth.7,14 Recently, the disposal of crude glycerol has been a major challenge, with the rapid expansion of the biodiesel industry. Previous research on the heterotrophic growth of S. limacinum on crude glycerol focused on producing docosahexaenoic acid (DHA),7,14 an omega-3 polyunsaturated fatty acid that has various applications as nutraceuticals for human health.18 A high biomass density culture (37 g/L within 40 h) has been reported for this
4. Discussion
(17) Miao, X. L.; Wu, Q. Y. Biodiesel production from heterotrophic microalgal oil. Bioresour. Technol. 2006, 97 (6), 841–846. (18) Nettleton, J. A. Omega-3 Fatty Acids and Health; Chapman & Hall: New York, 1995.
Heterotrophic algal culture is a promising method of algal biofuel production, because of the high cell density and high 5182
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algal species using a dissolved oxygen shifting strategy. The total fatty acids (TFA) content of the algal biomass was ∼50% (dry basis); 30% of the TFA were DHA. However, the remaining 70% of the TFA (mainly, C14:0, C16:0, and C22:5) have not been fully utilized. This leads us to explore the possibility of producing biodiesel fuel from this alga, so the entire fatty acids contained in the algae can be utilized. Algal biodiesel production is typically performed by oil extraction followed by the transesterification of algal oil.17 Direct transesterification of the raw biomass has also been reported in some algal13 and fungal species.9 Because of the inherent nature of a single-stage reaction, direct transesterification was much less time-consuming than extractiontransesterification. It also avoided the potential lipid loss during the extraction stage; as a result, the direct transesterification led to a higher yield of crude biodiesel and FAME (see Figure 2). However, extra care must be taken in the design of a direct transesterification method. For example, a direct transesterification without solvent had a poor conversion yield (see Figure 2). Among the three solvents tested, chloroform resulted in a much higher FAME yield and the content in biodiesel than hexane and petroleum ether (see Table 1). Previous reports evaluated biodiesel production performance using gravimetric biodiesel yield based on algal oil.9,17 While this definition was still used in this work, the biodiesel yield was also evaluated based on algal biomass. In addition, we used GC to quantify the FAME composition, FAME yield, and total FAME content in the biodiesel. These parameters provide deep insight into the quality and purity of the biodiesel fuel. For example, the three solvents in the direct transesterification resulted in a similar gravimetric biodiesel yields (see Figure 2), whereas the FAME composition obtained from the three solvents indicate that chloroform performed better (see Table 1). Biodiesel yield and the FAME composition are significantly influenced by the transesterification conditions, such as the ratio of oil/biomass to methanol, the catalyst loading, reaction time, and temperature. Complete mixing of the reactants is another important parameter that influences the fuel quality. In this work, the transesterification conditions were adapted from a previous report in which fish oil and cod liver oil were the raw materials.19 A thorough optimization of the reaction conditions for this specific algal biomass can further increase the biodiesel yield and reduce production cost. Previous literature on direct transesterification using dried biomass has been reported.9,13,17 Our study shows that the biodiesel yield and FAME content of the wet biomass in the direct transesterification was significantly lower than those obtained from dry biomass (see Table 2), suggesting that drying the algae is necessary for direct transesterification. However, the extraction-transesterification process resulted in a similar biodiesel yield and FAME composition between the wet biomass and dry biomass (see Table 2). Because freeze-drying biomass is usually an expensive operation, a further economic analysis
is needed to choose either drying the algae for direct transesterification or using wet biomass for extractiontransesterification. The ASTM standard tests of the algae-derived biodiesel show that the fuel met most of the ASTM standards but failed the water and sediment test. Because the liquid fuel was free of particulate matter but had 0.111% (by mass) of moisture content, water could contribute significantly (or fully) to the water and sediment content (see Table 3). In the future, the water and sediment content can be improved using a water removal step. In previous studies, excess water was removed via heating at 70 °C under vacuum20 or by drying the biodiesel over anhydrous sodium sulfate.21 Another option is to heat the biodiesel under atmospheric pressure to above the boiling point of water, so that it evaporates from the system. This should be safe for the fuel prepared in this work, because the flash point is high (see Table 3). A final option that is often used in industry is to perform a dry wash using chemicals (such as magnesium silicate) so that water will not be added to the biodiesel from the wash step. The other ASTM test that the liquid fuel failed was the sulfur content. This is probably due to the use of MgSO4 in the algal culture media, and the resulting high sulfur content of the algal biomass.7 To meet the ASTM standard, the medium for this alga must be modified to use a low sulfur/sulfate composition. Another important parameter in the ASTM test is the cloud point, which indicates the cold flow properties of the biodiesel fuel. Although this parameter was not tested, it is believed the algal biodiesel will have a low cloud point, i.e., a superior cold flow property, because of the high unsaturation level of the FAME. On the other hand, if too high unsaturation results in an oxidation problem, a stabilizer (antioxidant) can be added to extend the storage life of the algal biodiesel. 5. Conclusion In summary, this work shows potential of producing biodiesel fuel from the heterotrophic microalga S. limacinum. Direct transesterification of the oleaginous biomass resulted in a higher biodiesel yield and fatty acid methyl ester (FAME) content than the extraction-transesterification method. The biodiesel produced from direct transesterification meets most of the ASTM specifications; future work must be performed to optimize the biodiesel yield, and control the fuel quality, to meet the water and sediment content, as well as the sulfur content. Acknowledgment. The authors gratefully acknowledge Virginia Cooperative Extension and USDA CSREES (No. 2006-3890903484) for their financial support of this project. Allen French at Chesapeake Customer Chemicals performed the ASTM standard tests. (20) Ma, F. R.; Hanna, M. A. Biodiesel production: A review. Bioresour. Technol. 1999, 70 (1), 1–15. (21) Veljkovic, V. B.; Lakicevic, S. H.; Stamenkovic, O. S.; Todorovic, Z. B.; Lazic, M. L. Biodiesel production from tobacco (Nicotiana tabacum L.) seed oil with a high content of free fatty acids. Fuel 2006, 85 (17-18), 2671–2675. (22) Chen, F.; Johns, M. R. Effect of C/N ratio and aeration on the fatty acid composition of heterotrophic Chlorella sorokiniana. J. Appl. Phycol. 1991, 3, 203–209.
(19) Indarti, E.; Majid, M. I. A.; Hashim, R.; Chong, A. Direct FAME synthesis for rapid total lipid analysis from fish oil and cod liver oil. J. Food Compos. Anal. 2005, 18 (2-3), 161–170.
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