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Mechanistic Assessment of Microalgal Lipid Extraction

Amrita Ranjan, Chetna Patil, and Vijayanand S. Moholkar*
Department of Chemical Engineering, Indian Institute of Technology Guwahati, Guwahati-781 039, Assam, India

In this paper, we have attempted to make a comparative assessment of the three techniques for extraction of lipids from microalgal biomass, viz. Soxhlet extraction, the Bligh and Dyer method, and sonication. The approach is mechanistic in the sense that we have tried to determine the physical mechanism of extraction of lipids (cell disruption or diffusion across a cell wall) from microalgae using microscopic analysis of extracted biomass. We have also assessed the relative inuence of the solvent (or extractant) selectivity and the intensity of convection in the medium on the overall lipid yield. None of the techniques used produced complete disruption of the cells, not even sonication. Thus, the prominent mechanism of lipid extraction was diffusion across a cell wall. Moreover, the selectivity of the solvent was found to be the most dominating factor in overall lipid extraction by diffusion than the intensity of bulk convection in the medium.
1. Introduction Fast depletion of fossil fuels and increasing energy demands from transportation and energy sectors makes the quest for alternative fuel from renewable sources mandatory. Another state of affairs that has put greater emphasis on the use of fuels derived from renewable sources is global warming due to greenhouse gas emissions. The fuels derived from renewable sources such as biomass are carbon neutral, i.e. they do not make any net contribution to CO2 in the atmosphere.1 The most popular alternate liquid fuel for petroleum derived gasoline and diesel is biodiesel, which is essentially alkyl (methyl or ethyl) ester of fatty acids. Biodiesel is synthesized by the process of transesterication in which the triglycerides in vegetable oils react with short chain alcohol such as methanol and ethanol to yield esters of fatty acids and glycerol as major byproducts in the presence of an acidic, basic, or enzyme catalyst.2-4 The feedstock for biodiesel is vegetable oil derived from crops such as soybean, palm, and canola. In developing countries like India, the use of edible oil for biodiesel is impractical, and hence, nonedible oils such as Jatropha and Karanja have also been used. Despite extensive research on laboratory and pilot scale, the economy of biodiesel is not very attractive.5-9 The major cause leading to this effect is the limiting yield of oil per hectare of plantation of crop. For most of the conventional oil crop mentioned above, the oil yield per hectare rarely exceeds 1.5 tons even for genetically modied species. An alternate source for oil feedstock is in the form of a lipid from microalgae.1,10-15 Conventionally microalgae have been cultivated for food and nutritional products such as beta carotene, vitamin C, Omega 3, etc. However some species of microalgae contain a high quantity of lipids (approximately 50% or more for genetically modied species), and thus, microalgae cultivation is now emerging as an economically viable source of oil feed stock for biodiesel.16,17 In contrast to the conventional oil crops mentioned above, the distinct advantages of microalgae are high growth rate, high biomass production, less growth time, and low land use.11,18 In addition, microalgae cultivation has been an effective means of utilizing (or xing) the CO2 produced in power plants. Typically, production of 100 tons of microalgal biomass xes 183 tons of CO2.11 Thus, reduction in greenhouse gas emission is a complementary benet of microalgal route to biodiesel.
* To whom correspondence should be addressed. Phone: 91-361258 2258. Fax: 91-361-2582291. E-mail: vmoholkar@iitg.ac.in.

Extraction of lipids from microalgal mass forms an important step in the overall process of biodiesel manufacture. There have been several methods or techniques reported in literature 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 uid extraction, ultrasound-assisted extraction, and pressurized uid extraction. The exact mechanism of these techniques for the extraction of oil is different although most of the techniques involve disruption of the microbial cell for release of oil droplets present in cytoplasm.19,20 The choice of a particular technique for lipid extraction depends on several factors such as the type of species, the initial lipid content, and the amount of biomass treated per unit time. For microalgal species with low initial lipid content, the choice of extraction technique is a critical factor for the overall process design, as small loss of lipid in extraction can hamper the overall economy. In this paper, we have addressed this issue by comparing three techniques, viz. Soxhlet extraction, Bligh and Dyer, and ultrasonic extraction. The model species is Scendesmus, which is found abundantly in the river Brahmaputra and its tributaries. The typical lipid content of these species is 10-12% w/w of the total biomass (dry basis). Unlike vegetable oils from various crops mentioned earlier, the lipids from microalgae contains signicant amount of polyunsaturated fatty acids such as arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, -linolenic acid, and linolenic acid.21,22 These acids contain 3 or 4 double bonds and, thus, are susceptible to oxidation. Moreover, the presence of several double bonds also renders a slight polar character to the acid molecule. Previous authors have considered extraction of lipids from microalgal biomass through various methods. Mecozzi et al.23 have recommended the use of diethylether as an extraction solvent for lipids with ultrasonication as it prevents oxidative modications of lipids. Pernet and Trembley24 have insisted on grinding followed by ultrasonication for complete extraction of lipids from microalgae. Cravatto et al.25 and Virot et al.26 have reported enhancement in lipid extraction from microalgae with application of ultrasonication and microwaves. Recently, Lee et al.27 have compared various extraction techniques for microalgae such as autoclaving, bead beating, sonication, and 10% NaCl solution extractant. However, in this study the exact physical mechanism of oil extraction was not explored. Second, Lee et al.27 also did not explore the relative impact of the nature

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of the solvent (extractant) and method of extraction on the overall lipid yield. In this paper, we have made an attempt to investigate these issues. With the dual approach of coupling experimental results with microscopic analysis of extracted biomass and estimation of the shear stress (for technique of sonication) using a mathematical model, we have tried to reveal the interconnections between three factors inuencing the extraction process, viz. the nature of fatty acids present in the lipids, the nature of extractant, and the technique of extraction. We have also demonstrated how the overall yield of lipids is a result of the relative impact of these factors. 2. Aim and Approach As noted earlier, the aim of this study is to do mechanistic assessment of the efcacy of the various techniques for extraction of lipid from microalgal biomass. The lipids are mainly located in the cytoplasm of the algal cell in the form of droplets of size 30 nm or so.28 Given this, one can postulate two basic mechanisms by which extraction of a lipid can possibly occur: (1) diffusion of lipids across the cell wall, if the algal biomass is suspended in the solvent with higher selectivity and solubility (or large partition coefcient) for lipids and (2) disruption of the cell wall with release of cell contents in the solvent. The relative contribution of each of these mechanisms depends on the extraction technique. It could be easily perceived that diffusive mechanism will have less efciency (in terms of long extraction time and smaller yield of lipid) due to the slow diffusion of lipid molecules across the cell wall. On the other hand, a disruptive mechanism is likely to cause faster extraction of lipids with high yields, as it involves the direct release of the lipid droplets in cytoplasm in to the bulk liquid with rupture of cell wall. We have selected three extraction techniques for comparative study: (1) Soxhlet extraction with n-hexane as extractant, (2) the Bligh and Dyer method with a chloroform methanol mixture as extractant, and (3) ultrasonic extraction with both n-hexane and chloroform methanol solution as extractant. We try to speculate a priori as to which of the two mechanisms is likely to contribute most to the overall lipid extraction in each technique. Soxhlet extraction29 essentially involves percolation of the solvent through the biomass sample which is dried and ground into small particles. The solvent is taken in the ask and is evaporated. The vapors are cooled in a condenser located above the sample, and the condensed solvent is trickled down through the biomass; where it extracts the lipid or oil from biomass. After several cycles of extraction, the solvent containing the extracted oil is taken out and solvent is evaporated to recover the lipid. The Soxhlet extraction, thus, does not involve application of any shear stress to the biomass (provided the pregrinding of biomass is relatively mild and aimed at loosening of biomass clusters into small particles). With this, the principal extraction mechanism is likely to be diffusion. The Bligh and Dyer method30 involves simultaneous extraction and partitioning by mixing of the microalgal cell suspension in water with a mixture of chloroform and methanol. The mixture forms two phases after completion of extraction. The lower phase containing chloroform with dissolved lipid is separated to extract the lipid. Similar to Soxhlet extraction, the Bligh and Dyer method also does not involve application of shear stress to the algal biomass, and hence, the predominant mechanism is expected to be diffusion. The method of ultrasonic extraction involves sonication or ultrasound irradiation of the suspension of algal biomass in a suitable solvent. Conventionally, water is used as a solvent for

ultrasound irradiation; however, in the present situation where the extract (i.e., lipid) is hydrophobic, an organic solvent is preferred. Ultrasound manifests its physical and chemical effects through the phenomena of cavitation which are nucleation, growth, and transient impulsive collapse of tiny bubbles in the liquid, driven by bulk pressure variation due to ultrasound waves. The well-known chemical effect of cavitation is the generation of highly reactive radicals due to dissociation of the entrapped vapor molecules in the cavitation bubble at extreme conditions reached inside the bubble at the moment of transient collapse.31-33 This phenomena occurs in both aqueous (as in refs 31-33) and organic liquid medium (as in the present study), provided the pressure amplitude of ultrasound is sufciently high. The principal physical effect is generation of intense convection in the bulk medium. However, the convection generated by cavitation has contribution due to two physical effects, viz. microturbulence (which is intense oscillating motion of liquid with low to moderate velocities) and shock waves (which are high pressure waves emitted by the bubble, with amplitudes as high as 30-50 bar). The mathematical model for the radial dynamics of cavitation bubbles with which the magnitudes of the microturbulence velocity and pressure amplitude of the shock waves can be estimated is provided as Supporting Information with this manuscript. As a result of these effects, both diffusion and disruption are likely to contribute to the extraction of the lipid. The microturbulence causes mixing of biomass with solvent (without induction of shear stress), and hence, diffusive extraction is likely to occur. On the other hand, shock waves are likely to cause rupture of cell wall of algal biomass due to which disruptive extraction is also likely to contribute.34-36 We shall compare the extraction process by two means: rst, the exact quantity of lipid extracted with any technique (as percentage of dry biomass) and, second, microscopic assessment of extracted biomass. 3. Material and Methods 3.1. Analytical Reagents. All reagents were of analytical grade. BG medium components (viz. NaNO3, K2HPO4 3H2O, MgSO4 7H2O, CaCl2 2H2O, citric acid, ferric ammonium citrate, EDTA dinitrium-salt, Na2CO3, and micronutrients), chloroform, methanol, n-hexane, and sodium chloride were obtained from Merck (Mumbai, India) and used as received. For preparation of the BG culture medium solution, deionized water from the Milli Q Plus (Elix 3, Millipore SA) water treatment system was used. 3.2. Maintenance of Microalgal Culture. A fresh water microalgal strain, Scenedesmus sp., with reported lipid content between 6 and 10% was provided by Defense Research Laboratory (DRL) Tezpur. Algal culture was maintained in 250 mL, cotton plugged Erlenmeyer asks, containing 150 mL of liquid BG culture medium incubated at 25 C. Biomass was produced in a pond type photobioreactor (refer to Figure 1) designed by IIT Guwahati, with the following physical conditions maintained: illuminance ) 1200 lx, relative humidity ) 70%, pH ) 7.5. The concentration or number density of algal cells was assessed with a UV-visible spectrophotometer by monitoring optical density of samples drawn from the photobioreactor at 686 nm. Algal cells were harvested after 28-30 days of incubation, at which absorbance was found to be maximum. 3.3. Harvesting of Biomass. Biomass was harvested in two stages, viz., rst, by allowing the algal biomass to settle naturally with removal of the upper layer of water and, second, by

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Figure 1. Schematic of the pond type photobioreactor used for cultivation of algal mass [legend: (1) main body of the photobioreactor (MOC: SS 308); (2) seatable bench for ponds; (3) roof of photobioreactor (with suitable dimensions); (4) photosynthetic lamps; (5) paddle wheel for rotation of algal suspension; (6) spacer at 2 ft distance; (7) perforated sparger tube for CO2 sparging; (8) lux meter for measurement of light intensity; (9) indicator for temperature, pH, and CO2 concentration in the pond; (10) uninterrupted power supply (UPS) of sufcient capacity; (11) bafes for the direction of water ow].

Figure 2. Schematic of the ultrasonic processor used for sonication of the algal biomass [legend: (1) sonicator probe; (2) glass reactor with cooling jacket (height 7 cm, diam 4.5 cm, thickness 2 mm, jacket thickness 5 mm, volume 100 mL); (4 and 5) inlet and outlet ports for cooling water circulation; (6) port for withdrawl of sample; (7) microprocessor control unit.

centrifugation of the lower concentrated biomass suspension taken in 50 mL polyethylene centrifuge tubes (Tarson) at 6000 rpm for 10 min (centrifuge: Hermle Z-300, Germany). Following centrifugation, the supernatant was discarded and the biomass pellets settled at the bottom were taken out and allowed to dry in warm air at 45 C for 4-5 days in an oven. Dried microalgal pellets were nely powdered using pestle and mortar before extraction of lipids. 3.4. Microalgal Lipid Extraction Procedure. The powdered biomass was treated by four methods for extraction of lipids as described below: Bligh and Dryer Method.30 In this method, lipids were extracted from microalgal biomass with a mixture of chloroform and methanol (in a ratio of chloroform:methanol ) 3:1 v/v) as solvent. The exact protocol was as follows: 2 g of dried algal biomass was mixed with sterile sand. This mixture was crushed with a pestle and mortar with simultaneous addition of 15 mL chloroform to make a ne biomass suspension. To this suspension, 5 mL of methanol was added. Further, 6 mL of saline solution (1% w/w aqueous NaCl solution) was also added to the mixture (to avoid binding of some acidic lipids to denatured lipids), which essentially yielded a chloroform:methanol:saline mixture in the ratio 3:1:1.2 v/v. This mixture was vigorously shaken and allowed to stand in a separating funnel for phase separation. The lipids preferentially partitioned in the lower phase, i.e. chloroform, which was separated and ltered to remove suspended biomass particles. The ltrate was then treated in a rotavapor (Buchi Labortechnik, Model: R-200/V/ Basic) for removal of solvent. The lipids left in the ask after complete vaporization of chloroform were weighed. Soxhlet Extraction.29 This is a common method for extraction of solutes from various kinds of solids in semicontinuous mode. Soxhlets procedure essentially involves washing of solid mass with a suitable solvent that has high solubility and selectivity for the solute. For most of the organic solutes, such as lipids in the present study, an organic solvent such as n-hexane is used. The extraction solvent is vaporized in a roundbottom ask, and these vapors are condensed in a condenser

Figure 3. Extent of lipid extraction (as a percentage of dry algal biomass) with different techniques.

placed over the ask. The condensed (hot) solvent is allowed to percolate through the powdered biomass (5 g) kept over Whatmann lter paper in a thimble before returning or reuxing into the round-bottom ask. The lipids are selectively extracted into the solvent during percolation. After several runs of evaporation/condensation/percolation of solvent through biomass, the round-bottom ask (containing mixture of solvent with extracted lipids) is taken out and the solvent is vaporized using a rotavapor to recover lipids. Ultrasound-Mediated Bligh and Dryer Method. In this method, a ne suspension of algal biomass in a chloroformmethanol mixture (prepared in exactly the same way as described earlier) was sonicated using a microprocessor-based and programmable processor (Sonic & Materials, model VCX 500, frequency 20 kHz, power 500 W). The schematic diagram of the setup is shown in Figure 2. Sonication of the biomass suspension was carried out in a jacketed vessel made of borosilicate glass (volume: 100 mL). The dimensions of this vessel are given in caption of Figure 2. The diameter of the sonicator probe (made of titanium alloy) was 25 mm. The ultrasonic processor has variable power output control, which was set at 20% during experiments, resulting in a net consump-

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Figure 4. Representative micrographs of the algal biomass before and after extraction of lipids with various techniques: (A) original algal cells of Scendesmus sp.; (B) biomass after extraction with Soxhlet apparatus; (C) biomass after extraction with the Bligh and Dyer method; (D) biomass after extraction using sonication with n-hexane as solvent; (E) biomass after extraction using sonication with a chloroform-methane mixture as solvent.

tion of 100 W. The actual ultrasound intensity in the medium was calibrated using a calorimetric technique.37 For a theoretical intensity of 100 W, the ultrasound probe produced an acoustic wave with pressure amplitude of 1-5 bar. The processor also had facility of automatic frequency tuning and amplitude compensation, which ensured constant power delivery to the medium during sonication. The height of solvent for extraction (mixture of 15 mL methanol and 5 mL chloroform) in the glass vessel was 1.3 cm. The ultrasound probe tip was immersed 5 mm below the liquid surface so as to achieve proper coupling of ultrasound to the extraction solvent. The sonication was carried out for 30 min after which 6 mL of saline solution was added to the biomass suspension. This mixture was then allowed

to stand in a separating funnel for phase separation. Later, lipids were recovered from the lower (chloroform) phase (after ltration to remove some suspended biomass particles) in exactly the same manner as described earlier. Ultrasound-Mediated Extraction in n-Hexane. In this method, 2 g of ne powdered algal biomass was added to 20 mL n-hexane. Other experimental parameters were same as in case of the ultrasound-mediated Bligh and Dyer method. This suspension was sonicated for 30 min, with 100 W power input, as in the previous case. After completion of sonication, the mixture was ltered to remove suspended biomass particles, and lipids were recovered from ltrate after removal of solvent in the rotavapor.

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4. Result and Discussion Extraction of lipids from microalgal biomass is basically a mass transfer operation, the ethnicity of which depends on several parameters such as the nature of the solute and solvent, the selectivity of the solvent and the level of convection in the medium. However, as discussed in section 2, the more important factor is the physical mechanism of extraction, which could be either diffusion of lipids across the cell wall or direct release of lipids in the bulk with disruption of the cell. The solvent properties will affect the extent of extraction only in the former mechanism. For the latter mechanism, the extent of extraction should, essentially, be independent of the solvent properties. With this preamble, we present the experimental and simulations results, followed by discussion on correlating the two. 4.1. Experimental Results. The extent of lipid extraction from biomass achieved with four techniques (as a percentage of dry algal biomass) is shown in Figure 3. The trend in the extraction is as follows: Soxhlet extraction sonication with n-hexane < Bligh and Dyer method , sonication with a chloroform-methanol mixture. Micrographs of the algal biomass before and after extraction with various techniques are shown in Figure 4. Some peculiar features that could be observed from these micrographs are as follows: (1) Micrograph of biomass after Soxhlet extraction (Figure 4B) does not reveal any disruption of the cells. However, the initial size of the cells (as seen from Figure 4A) seems to be reduced, i.e., the microalgal cells shrink after extraction. (2) Micrograph of biomass after extraction with the Bligh and Dyer method shows few distorted clusters of biomass, which are the disrupted cells. However, as in Figure 4C, several intact cells are also seen although with reduced size. (3) Micrographs of sonicated biomass (Figure 4D for sonication with n-hexane as the solvent and Figure 4E for sonication with a chloroform-methanol mixture as solvent) show larger clusters of distorted or pulpy biomass, which clearly indicates large disruption of microalgal cells. Nonetheless, as in the case of both Figure 4C and D, several intact, yet shrunk, cells are also seen. These micrographs clearly reveal the contribution of diffusion and disruption mechanisms to lipid extraction in various techniques. For Soxhlet extraction, diffusion is the only mechanism, while for the Bligh and Dyer method and sonication both diffusion and disruption contribute to extraction. The disruption of cells in the Bligh and Dyer method is attributed to the friction of the dry algal biomass with sterile sand particles while being powdered with mortar and pestle. On the other hand, cell disruption in sonication with either n-hexane or a chloroformmethanol mixture as a solvent is attributed to the shock waves induced by transient cavitation bubbles. 4.2. Simulations Results. The results of simulations of radial motion of cavitation bubbles and the microturbulence and shock waves generated by it are shown in Figure 5 and 6 for n-hexane and a chloroform-methanol mixture as the liquid medium, respectively. It could be inferred from these gures that microturbulence (which is an oscillatory motion of liquid in the vicinity of the bubble) has a continuous character, while shock or acoustic waves are rather discrete or intermittent. The magnitudes of microturbulence velocity, obtained as the arithmetic mean of forward or positive (i.e., directed away from the bubble) and backward or negative (i.e., directed toward the bubble), are as follows: n-hexane ) 6.52 mm/s and chloroform-

Figure 5. Simulations of the radial bubble motion and its physical effects in n-hexane: time history of (A) normalized bubble radius (R/Ro); (B) acoustic waves emitted by the bubble; (C) velocity of microturbulence generated by the bubble.

Figure 6. Simulations of the radial bubble motion and its physical effects in chloroform-methanol: time history of (A) normalized bubble radius (R/ Ro); (B) acoustic waves emitted by the bubble; (C) velocity of microturbulence generated by the bubble.

methanol mixture ) 2.62 mm/s. On the other hand, the highest amplitude of the shock waves in chloroform-methanol is 100 bar, while that in n-hexane is 238 bar. Comparing the magnitudes of microturbulence velocity and shock waves in the two

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liquid media, one can easily perceive that the extent of convection generated by cavitation bubbles in n-hexane is far more intense than in the chloroform-methanol mixture. 5. Discussion Comparative analysis of the extent of lipid extraction with four techniques brings out interesting features or characteristics of the extraction process. We try to identify these by correlating the experimental and simulation results. As noted earlier, the principal physical mechanism of lipid extraction in the Soxhlet technique is diffusion, which is a slow process. In addition, n-hexane has nonpolar character, and hence, the selectivity of microalgal lipids (comprising of unsaturated fatty acids) toward n-hexane is expected to be lower. In concurrence with this, the extent of lipid extraction is the least in the Soxhlet technique. Marginally higher lipid yield with the Bligh and Dyer method could be attributed to two factors, viz. additional contribution by disruption mechanism to lipid release and, second, higher selectivity of microalgal lipids toward chloroform, which has polar nature. However, which of these factors dominate the extraction of lipids is evident from the comparison of the lipid yield with sonication using chloroform-methanol and n-hexane as the solvents. Despite the much lower magnitudes of microturbulence and shock waves, the extent of lipid extraction in chloroformmethanol is 4 times higher than n-hexane. This anomaly could be explained as follows: (1) Shock waves from the cavitation bubbles are capable of disrupting the microbial cells due to their high pressure amplitude and discrete nature. However, the extent of disruption depends on the probability of interaction of the cell with cavitation bubbles. This probability will, of course, directly vary with the density of microbial cells in the solution. In the present case, this density was low (2 g of biomass in 20 mL solution). Therefore, the probability of microbial cell-cavitation bubble interaction and, consequently, the extent of disruption is expected to be marginal in the case of both extraction solvents. With this, the diffusion of lipids across the cell wall becomes the limiting factor for lipid extraction with both solvents. (2) The extent of diffusive extraction of lipids will depend on the magnitude or intensity of bulk convection in the medium, which in the case of sonication, is essentially contributed by microturbulence. The second factor is the selectivity of the solvent. As noted earlier, this factor is in favor of the chloroform-methanol mixture due to its polar nature. The greater extraction of lipids in the chloroform-methanol mixture as compared to n-hexane, despite the much lower microturbulence velocity than n-hexane, clearly indicates that solvent selectivity is the dominant factor rather than the convection. The role of ultrasound in the extraction process seems to be more of a physical nature that it creates intense local turbulence in the medium that sweeps away the extracted lipids away from the surface of the microbial cells, and thus, maintains a constant concentration gradient for continuous diffusion of lipids from the cells. 6. Conclusion In this paper, we have attempted to make a mechanistic assessment of the lipid extraction from microalgal biomass. Using three extraction techniques, viz. Soxhlet extraction, the Bligh and Dyer method, and sonication with two solvents, we

have tried to identify the relative inuence of different factors such as cell disruption, lipid diffusion, bulk convection, and solvent selectivity on the extent of lipid extraction. Complete disruption of microalgal cells (which would render the lipid yield independent of the solvent properties) was not achieved with any of the three techniques employed. Therefore, the contribution by the diffusion mechanism to the extent of lipid extraction becomes signicant. Our results clearly reveal that the selectivity of the solvent is the most dominating factor in the overall lipid extraction rather than the intensity of bulk convection in the medium under these test conditions. We hope that these results would be useful for further research in lipid extraction from microalgal biomass as well as design of a large scale extraction process. Acknowledgment This project was funded by Defense Research and Development Organization (DRDO), Ministry of Defense, Govt of India (Project No. DRLT-P1-2006/Task 21). The authors gratefully acknowledge helpful discussions with Dr. R. B. Shrivastava, Dr. H. K. Gogoi (Defense Research Laboratory, Tezpur), and Dr. M. C. Kalita (Gauhati University). Moreover, the authors thank Dr. M. K. Purkait (Department of Chemical Engineering, IIT Guwahati) for his help in project implementation. The authors also thank the referees for their meticulous evaluation of the manuscript and constructive criticism. Supporting Information Available: Mathematical model for the radial motion of cavitation bubbles along with the numerical solution scheme and relevant references. This material is available free of charge via the Internet at http://pubs.acs.org. Literature Cited
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ReceiVed for reView October 24, 2009 ReVised manuscript receiVed January 9, 2010 Accepted January 18, 2010 IE9016557