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 oflipids from microalgal biomass, viz. Soxhlet extraction, the Bligh and Dyer method, and sonication. Theapproach is mechanistic in the sense that we have tried to determine the physical mechanism of extraction oflipids (cell disruption or diffusion across a cell wall) from microalgae using microscopic analysis of extractedbiomass. We have also assessed the relative influence of the solvent (or extractant) selectivity and the intensityof convection in the medium on the overall lipid yield. None of the techniques used produced completedisruption of the cells, not even sonication. Thus, the prominent mechanism of lipid extraction was diffusionacross a cell wall. Moreover, the selectivity of the solvent was found to be the most dominating factor inoverall lipid extraction by diffusion than the intensity of bulk convection in the medium.

1. Introduction

Fast depletion of fossil fuels and increasing energy demandsfrom transportation and energy sectors makes the quest foralternative fuel from renewable sources mandatory. Anotherstate of affairs that has put greater emphasis on the use of fuelsderived from renewable sources is global warming due togreenhouse gas emissions. The fuels derived from renewablesources such as biomass are carbon neutral, i.e. they do notmake any net contribution to CO2 in the atmosphere.1 The mostpopular alternate liquid fuel for petroleum derived gasoline anddiesel is biodiesel, which is essentially alkyl (methyl or ethyl)ester of fatty acids. Biodiesel is synthesized by the process oftransesterification in which the triglycerides in vegetable oilsreact with short chain alcohol such as methanol and ethanol toyield esters of fatty acids and glycerol as major byproducts inthe presence of an acidic, basic, or enzyme catalyst.2-4 Thefeedstock for biodiesel is vegetable oil derived from crops suchas 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 beenused. Despite extensive research on laboratory and pilot scale,the economy of biodiesel is not very attractive.5-9 The majorcause leading to this effect is the limiting yield of oil per hectareof plantation of crop. For most of the conventional oil cropmentioned above, the oil yield per hectare rarely exceeds 1.5tons even for genetically modified species. An alternate sourcefor oil feedstock is in the form of a lipid from microalgae.1,10-15

Conventionally microalgae have been cultivated for food andnutritional products such as beta carotene, vitamin C, Omega3, etc. However some species of microalgae contain a highquantity of lipids (approximately 50% or more for geneticallymodified species), and thus, microalgae cultivation is nowemerging as an economically viable source of oil feed stockfor biodiesel.16,17 In contrast to the conventional oil cropsmentioned above, the distinct advantages of microalgae are highgrowth rate, high biomass production, less growth time, andlow land use.11,18 In addition, microalgae cultivation has beenan effective means of utilizing (or fixing) the CO2 produced inpower plants. Typically, production of 100 tons of microalgalbiomass fixes 183 tons of CO2.

11 Thus, reduction in greenhousegas emission is a complementary benefit of microalgal route tobiodiesel.

Extraction of lipids from microalgal mass forms an importantstep in the overall process of biodiesel manufacture. There havebeen several methods or techniques reported in literature suchas Soxhlet extraction (with n-hexane as solvent), the Bligh andDyer method with a mixture of chloroform and methanol assolvents, a microwave oven technique, supercritical fluid extrac-tion, ultrasound-assisted extraction, and pressurized fluid extrac-tion. The exact mechanism of these techniques for the extractionof oil is different although most of the techniques involvedisruption of the microbial cell for release of oil droplets presentin cytoplasm.19,20 The choice of a particular technique for lipidextraction depends on several factors such as the type of species,the initial lipid content, and the amount of biomass treated perunit time. For microalgal species with low initial lipid content,the choice of extraction technique is a critical factor for theoverall process design, as small loss of lipid in extraction canhamper the overall economy. In this paper, we have addressedthis issue by comparing three techniques, viz. Soxhlet extraction,Bligh and Dyer, and ultrasonic extraction. The model speciesis Scendesmus, which is found abundantly in the river Brah-maputra and its tributaries. The typical lipid content of thesespecies is 10-12% w/w of the total biomass (dry basis). Unlikevegetable oils from various crops mentioned earlier, the lipidsfrom microalgae contains significant amount of polyunsaturatedfatty acids such as arachidonic acid, eicosapentaenoic acid,docosahexaenoic acid, γ-linolenic acid, and linolenic acid.21,22

These acids contain 3 or 4 double bonds and, thus, aresusceptible to oxidation. Moreover, the presence of severaldouble bonds also renders a slight polar character to the acidmolecule.

Previous authors have considered extraction of lipids frommicroalgal biomass through various methods. Mecozzi et al.23

have recommended the use of diethylether as an extractionsolvent for lipids with ultrasonication as it prevents oxidativemodifications of lipids. Pernet and Trembley24 have insisted ongrinding followed by ultrasonication for complete extraction oflipids from microalgae. Cravatto et al.25 and Virot et al.26 havereported enhancement in lipid extraction from microalgae withapplication of ultrasonication and microwaves. Recently, Leeet al.27 have compared various extraction techniques formicroalgae such as autoclaving, bead beating, sonication, and10% NaCl solution extractant. However, in this study the exactphysical mechanism of oil extraction was not explored. Second,Lee et al.27 also did not explore the relative impact of the nature

* To whom correspondence should be addressed. Phone: 91-361-258 2258. Fax: 91-361-2582291. E-mail: vmoholkar@iitg.ac.in.

Ind. Eng. Chem. Res. 2010, 49, 2979–2985 2979

10.1021/ie9016557 2010 American Chemical SocietyPublished on Web 02/04/2010

of the solvent (extractant) and method of extraction on theoverall lipid yield. In this paper, we have made an attempt toinvestigate these issues. With the dual approach of couplingexperimental results with microscopic analysis of extractedbiomass and estimation of the shear stress (for technique ofsonication) using a mathematical model, we have tried to revealthe interconnections between three factors influencing theextraction process, viz. the nature of fatty acids present in thelipids, the nature of extractant, and the technique of extraction.We have also demonstrated how the overall yield of lipids is aresult of the relative impact of these factors.

2. Aim and Approach

As noted earlier, the aim of this study is to do mechanisticassessment of the efficacy of the various techniques forextraction of lipid from microalgal biomass. The lipids aremainly located in the cytoplasm of the algal cell in the form ofdroplets of size ∼30 nm or so.28 Given this, one can postulatetwo basic mechanisms by which extraction of a lipid canpossibly occur: (1) diffusion of lipids across the cell wall, ifthe algal biomass is suspended in the solvent with higherselectivity and solubility (or large partition coefficient) for lipidsand (2) disruption of the cell wall with release of cell contentsin the solvent. The relative contribution of each of thesemechanisms depends on the extraction technique. It could beeasily perceived that diffusive mechanism will have lessefficiency (in terms of long extraction time and smaller yieldof lipid) due to the slow diffusion of lipid molecules across thecell wall. On the other hand, a disruptive mechanism is likelyto cause faster extraction of lipids with high yields, as it involvesthe direct release of the lipid droplets in cytoplasm in to thebulk liquid with rupture of cell wall. We have selected threeextraction techniques for comparative study: (1) Soxhlet extrac-tion with n-hexane as extractant, (2) the Bligh and Dyer methodwith a chloroform methanol mixture as extractant, and (3)ultrasonic extraction with both n-hexane and chloroform metha-nol solution as extractant. We try to speculate a priori as towhich of the two mechanisms is likely to contribute most tothe overall lipid extraction in each technique. Soxhlet extrac-tion29 essentially involves percolation of the solvent throughthe biomass sample which is dried and ground into smallparticles. The solvent is taken in the flask 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 severalcycles of extraction, the solvent containing the extracted oil istaken out and solvent is evaporated to recover the lipid. TheSoxhlet extraction, thus, does not involve application of anyshear stress to the biomass (provided the pregrinding of biomassis relatively mild and aimed at loosening of biomass clustersinto small particles). With this, the principal extraction mech-anism is likely to be diffusion.

The Bligh and Dyer method30 involves simultaneous extrac-tion and partitioning by mixing of the microalgal cell suspensionin water with a mixture of chloroform and methanol. Themixture forms two phases after completion of extraction. Thelower phase containing chloroform with dissolved lipid isseparated to extract the lipid. Similar to Soxhlet extraction, theBligh and Dyer method also does not involve application ofshear stress to the algal biomass, and hence, the predominantmechanism is expected to be diffusion.

The method of ultrasonic extraction involves sonication orultrasound irradiation of the suspension of algal biomass in asuitable solvent. Conventionally, water is used as a solvent for

ultrasound irradiation; however, in the present situation wherethe extract (i.e., lipid) is hydrophobic, an organic solvent ispreferred. Ultrasound manifests its physical and chemical effectsthrough the phenomena of cavitation which are nucleation,growth, and transient impulsive collapse of tiny bubbles in theliquid, driven by bulk pressure variation due to ultrasoundwaves. The well-known chemical effect of cavitation is thegeneration of highly reactive radicals due to dissociation of theentrapped vapor molecules in the cavitation bubble at extremeconditions reached inside the bubble at the moment of transientcollapse.31-33 This phenomena occurs in both aqueous (as inrefs 31-33) and organic liquid medium (as in the present study),provided the pressure amplitude of ultrasound is sufficientlyhigh. The principal physical effect is generation of intenseconvection in the bulk medium. However, the convectiongenerated by cavitation has contribution due to two physicaleffects, viz. microturbulence (which is intense oscillating motionof liquid with low to moderate velocities) and shock waves(which are high pressure waves emitted by the bubble, withamplitudes as high as 30-50 bar). The mathematical model forthe radial dynamics of cavitation bubbles with which themagnitudes of the microturbulence velocity and pressureamplitude of the shock waves can be estimated is provided asSupporting Information with this manuscript. As a result of theseeffects, both diffusion and disruption are likely to contribute tothe extraction of the lipid. The microturbulence causes mixingof biomass with solvent (without induction of shear stress), andhence, diffusive extraction is likely to occur. On the other hand,shock waves are likely to cause rupture of cell wall of algalbiomass due to which disruptive extraction is also likely tocontribute.34-36

We shall compare the extraction process by two means: first,the exact quantity of lipid extracted with any technique (aspercentage of dry biomass) and, second, microscopic assessmentof extracted biomass.

3. Material and Methods

3.1. Analytical Reagents. All reagents were of analyticalgrade. BG medium components (viz. NaNO3, K2HPO4 ·3H2O,MgSO4 ·7H2O, CaCl2 ·2H2O, citric acid, ferric ammoniumcitrate, EDTA dinitrium-salt, Na2CO3, and micronutrients),chloroform, methanol, n-hexane, and sodium chloride wereobtained from Merck (Mumbai, India) and used as received.For preparation of the BG culture medium solution, deionizedwater from the Milli Q Plus (Elix 3, Millipore SA) watertreatment system was used.

3.2. Maintenance of Microalgal Culture. A fresh watermicroalgal strain, Scenedesmus sp., with reported lipid contentbetween 6 and 10% was provided by Defense ResearchLaboratory (DRL) Tezpur. Algal culture was maintained in 250mL, cotton plugged Erlenmeyer flasks, containing 150 mL ofliquid BG culture medium incubated at 25 °C. Biomass wasproduced in a pond type photobioreactor (refer to Figure 1)designed by IIT Guwahati, with the following physical condi-tions maintained: illuminance ) 1200 lx, relative humidity )70%, pH ) 7.5. The concentration or number density of algalcells was assessed with a UV-visible spectrophotometer bymonitoring optical density of samples drawn from the photo-bioreactor at 686 nm. Algal cells were harvested after 28-30days of incubation, at which absorbance was found to bemaximum.

3.3. Harvesting of Biomass. Biomass was harvested in twostages, viz., first, by allowing the algal biomass to settle naturallywith removal of the upper layer of water and, second, by

2980 Ind. Eng. Chem. Res., Vol. 49, No. 6, 2010

centrifugation of the lower concentrated biomass suspensiontaken in 50 mL polyethylene centrifuge tubes (Tarson) at 6000rpm for 10 min (centrifuge: Hermle Z-300, Germany). Followingcentrifugation, the supernatant was discarded and the biomasspellets settled at the bottom were taken out and allowed to dryin warm air at 45 °C for 4-5 days in an oven. Dried microalgalpellets were finely powdered using pestle and mortar beforeextraction of lipids.

3.4. Microalgal Lipid Extraction Procedure. The powderedbiomass was treated by four methods for extraction of lipids asdescribed below:

Bligh and Dryer Method.30 In this method, lipids wereextracted from microalgal biomass with a mixture of chloroformand methanol (in a ratio of chloroform:methanol ) 3:1 v/v) assolvent. The exact protocol was as follows: 2 g of dried algalbiomass was mixed with sterile sand. This mixture was crushedwith a pestle and mortar with simultaneous addition of 15 mLchloroform to make a fine biomass suspension. To this suspen-sion, 5 mL of methanol was added. Further, 6 mL of salinesolution (1% w/w aqueous NaCl solution) was also added tothe mixture (to avoid binding of some acidic lipids to denaturedlipids), which essentially yielded a chloroform:methanol:salinemixture in the ratio 3:1:1.2 v/v. This mixture was vigorouslyshaken and allowed to stand in a separating funnel for phaseseparation. The lipids preferentially partitioned in the lowerphase, i.e. chloroform, which was separated and filtered toremove suspended biomass particles. The filtrate was thentreated in a rotavapor (Buchi Labortechnik, Model: R-200/V/Basic) for removal of solvent. The lipids left in the flask aftercomplete vaporization of chloroform were weighed.

Soxhlet Extraction.29 This is a common method for extrac-tion of solutes from various kinds of solids in semicontinuousmode. Soxhlet’s procedure essentially involves washing of solidmass with a suitable solvent that has high solubility andselectivity for the solute. For most of the organic solutes, suchas lipids in the present study, an organic solvent such asn-hexane is used. The extraction solvent is vaporized in a round-bottom flask, and these vapors are condensed in a condenser

placed over the flask. The condensed (hot) solvent is allowedto percolate through the powdered biomass (5 g) kept overWhatmann filter paper in a thimble before returning or refluxinginto the round-bottom flask. The lipids are selectively extractedinto the solvent during percolation. After several runs ofevaporation/condensation/percolation of solvent through biom-ass, the round-bottom flask (containing mixture of solvent withextracted lipids) is taken out and the solvent is vaporized usinga rotavapor to recover lipids.

Ultrasound-Mediated Bligh and Dryer Method. In thismethod, a fine suspension of algal biomass in a chloroform-methanol mixture (prepared in exactly the same way asdescribed earlier) was sonicated using a microprocessor-basedand programmable processor (Sonic & Materials, model VCX500, frequency 20 kHz, power 500 W). The schematic diagramof the setup is shown in Figure 2. Sonication of the biomasssuspension was carried out in a jacketed vessel made ofborosilicate glass (volume: 100 mL). The dimensions of thisvessel are given in caption of Figure 2. The diameter of thesonicator probe (made of titanium alloy) was 25 mm. Theultrasonic processor has variable power output control, whichwas set at 20% during experiments, resulting in a net consump-

Figure 2. Schematic of the ultrasonic processor used for sonication of thealgal biomass [legend: (1) sonicator probe; (2) glass reactor with coolingjacket (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 watercirculation; (6) port for withdrawl of sample; (7) microprocessor controlunit.

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

Figure 1. Schematic of the pond type photobioreactor used for cultivationof algal mass [legend: (1) main body of the photobioreactor (MOC: SS308); (2) seatable bench for ponds; (3) roof of photobioreactor (with suitabledimensions); (4) photosynthetic lamps; (5) paddle wheel for rotation of algalsuspension; (6) spacer at 2 ft distance; (7) perforated sparger tube for CO2

sparging; (8) lux meter for measurement of light intensity; (9) indicator fortemperature, pH, and CO2 concentration in the pond; (10) uninterruptedpower supply (UPS) of sufficient capacity; (11) baffles for the direction ofwater flow].

Ind. Eng. Chem. Res., Vol. 49, No. 6, 2010 2981

tion of 100 W. The actual ultrasound intensity in the mediumwas calibrated using a calorimetric technique.37 For a theoreticalintensity of 100 W, the ultrasound probe produced an acousticwave with pressure amplitude of 1-5 bar. The processor alsohad facility of automatic frequency tuning and amplitudecompensation, which ensured constant power delivery to themedium during sonication. The height of solvent for extraction(mixture of 15 mL methanol and 5 mL chloroform) in the glassvessel was 1.3 cm. The ultrasound probe tip was immersed 5mm below the liquid surface so as to achieve proper couplingof ultrasound to the extraction solvent. The sonication wascarried out for 30 min after which 6 mL of saline solution wasadded to the biomass suspension. This mixture was then allowed

to stand in a separating funnel for phase separation. Later, lipidswere recovered from the lower (chloroform) phase (afterfiltration to remove some suspended biomass particles) in exactlythe same manner as described earlier.

Ultrasound-Mediated Extraction in n-Hexane. In thismethod, 2 g of fine powdered algal biomass was added to 20mL n-hexane. Other experimental parameters were same as incase of the ultrasound-mediated Bligh and Dyer method. Thissuspension was sonicated for 30 min, with 100 W power input,as in the previous case. After completion of sonication, themixture was filtered to remove suspended biomass particles,and lipids were recovered from filtrate after removal of solventin the rotavapor.

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

2982 Ind. Eng. Chem. Res., Vol. 49, No. 6, 2010

4. Result and Discussion

Extraction of lipids from microalgal biomass is basically amass transfer operation, the ethnicity of which depends onseveral parameters such as the nature of the solute and solvent,the selectivity of the solvent and the level of convection in themedium. However, as discussed in section 2, the more importantfactor is the physical mechanism of extraction, which could beeither diffusion of lipids across the cell wall or direct releaseof lipids in the bulk with disruption of the cell. The solventproperties will affect the extent of extraction only in the formermechanism. For the latter mechanism, the extent of extractionshould, essentially, be independent of the solvent properties.With this preamble, we present the experimental and simulationsresults, followed by discussion on correlating the two.

4.1. Experimental Results. The extent of lipid extractionfrom biomass achieved with four techniques (as a percentageof dry algal biomass) is shown in Figure 3. The trend in theextraction is as follows: Soxhlet extraction ≈ sonication withn-hexane < Bligh and Dyer method , sonication with achloroform-methanol mixture. Micrographs of the algal bio-mass before and after extraction with various techniques areshown in Figure 4. Some peculiar features that could beobserved from these micrographs are as follows:

(1) Micrograph of biomass after Soxhlet extraction (Figure4B) does not reveal any disruption of the cells. However,the initial size of the cells (as seen from Figure 4A) seemsto be reduced, i.e., the microalgal cells shrink afterextraction.

(2) Micrograph of biomass after extraction with the Blighand 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 reducedsize.

(3) Micrographs of sonicated biomass (Figure 4D for soni-cation with n-hexane as the solvent and Figure 4E forsonication with a chloroform-methanol mixture assolvent) show larger clusters of distorted or pulpybiomass, which clearly indicates large disruption ofmicroalgal cells. Nonetheless, as in the case of bothFigure 4C and D, several intact, yet shrunk, cells are alsoseen.

These micrographs clearly reveal the contribution of diffusionand disruption mechanisms to lipid extraction in varioustechniques. For Soxhlet extraction, diffusion is the only mech-anism, while for the Bligh and Dyer method and sonication bothdiffusion and disruption contribute to extraction. The disruptionof cells in the Bligh and Dyer method is attributed to the frictionof the dry algal biomass with sterile sand particles while beingpowdered with mortar and pestle. On the other hand, celldisruption in sonication with either n-hexane or a chloroform-methanol mixture as a solvent is attributed to the shock wavesinduced by transient cavitation bubbles.

4.2. Simulations Results. The results of simulations of radialmotion of cavitation bubbles and the microturbulence and shockwaves generated by it are shown in Figure 5 and 6 for n-hexaneand a chloroform-methanol mixture as the liquid medium,respectively. It could be inferred from these figures thatmicroturbulence (which is an oscillatory motion of liquid inthe vicinity of the bubble) has a continuous character, whileshock or acoustic waves are rather discrete or intermittent. Themagnitudes of microturbulence velocity, obtained as the arith-metic mean of forward or positive (i.e., directed away from thebubble) and backward or negative (i.e., directed toward thebubble), are as follows: n-hexane ) 6.52 mm/s and chloroform-

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

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

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

Ind. Eng. Chem. Res., Vol. 49, No. 6, 2010 2983

liquid media, one can easily perceive that the extent ofconvection generated by cavitation bubbles in n-hexane is farmore intense than in the chloroform-methanol mixture.

5. Discussion

Comparative analysis of the extent of lipid extraction withfour techniques brings out interesting features or characteristicsof the extraction process. We try to identify these by correlatingthe experimental and simulation results. As noted earlier, theprincipal physical mechanism of lipid extraction in the Soxhlettechnique is diffusion, which is a slow process. In addition,n-hexane has nonpolar character, and hence, the selectivity ofmicroalgal lipids (comprising of unsaturated fatty acids) towardn-hexane is expected to be lower. In concurrence with this, theextent of lipid extraction is the least in the Soxhlet technique.Marginally higher lipid yield with the Bligh and Dyer methodcould be attributed to two factors, viz. additional contributionby disruption mechanism to lipid release and, second, higherselectivity of microalgal lipids toward chloroform, which haspolar nature.

However, which of these factors dominate the extraction oflipids is evident from the comparison of the lipid yield withsonication using chloroform-methanol and n-hexane as thesolvents. Despite the much lower magnitudes of microturbulenceand shock waves, the extent of lipid extraction in chloroform-methanol is ∼4 times higher than n-hexane. This anomaly couldbe explained as follows:

(1) Shock waves from the cavitation bubbles are capable ofdisrupting the microbial cells due to their high pressureamplitude and discrete nature. However, the extent ofdisruption depends on the probability of interaction ofthe cell with cavitation bubbles. This probability will,of course, directly vary with the density of microbial cellsin the solution. In the present case, this density was low(2 g of biomass in 20 mL solution). Therefore, theprobability of microbial cell-cavitation bubble interactionand, consequently, the extent of disruption is expectedto be marginal in the case of both extraction solvents.With this, the diffusion of lipids across the cell wallbecomes the limiting factor for lipid extraction with bothsolvents.

(2) The extent of diffusive extraction of lipids will dependon the magnitude or intensity of bulk convection in themedium, which in the case of sonication, is essentiallycontributed by microturbulence. The second factor is theselectivity of the solvent. As noted earlier, this factor isin favor of the chloroform-methanol mixture due to itspolar nature. The greater extraction of lipids in thechloroform-methanol mixture as compared to n-hexane,despite the much lower microturbulence velocity thann-hexane, clearly indicates that solvent selectivity is thedominant factor rather than the convection. The role ofultrasound in the extraction process seems to be more ofa physical nature that it creates intense local turbulencein the medium that sweeps away the extracted lipids awayfrom the surface of the microbial cells, and thus,maintains a constant concentration gradient for continuousdiffusion of lipids from the cells.

6. Conclusion

In this paper, we have attempted to make a mechanisticassessment of the lipid extraction from microalgal biomass.Using three extraction techniques, viz. Soxhlet extraction, theBligh and Dyer method, and sonication with two solvents, we

have tried to identify the relative influence of different factorssuch as cell disruption, lipid diffusion, bulk convection, andsolvent selectivity on the extent of lipid extraction. Completedisruption of microalgal cells (which would render the lipid yieldindependent of the solvent properties) was not achieved withany of the three techniques employed. Therefore, the contribu-tion by the diffusion mechanism to the extent of lipid extractionbecomes significant. Our results clearly reveal that the selectivityof the solvent is the most dominating factor in the overall lipidextraction rather than the intensity of bulk convection in themedium under these test conditions. We hope that these resultswould be useful for further research in lipid extraction frommicroalgal biomass as well as design of a large scale extractionprocess.

Acknowledgment

This project was funded by Defense Research and Develop-ment Organization (DRDO), Ministry of Defense, Govt of India(Project No. DRLT-P1-2006/Task 21). The authors gratefullyacknowledge helpful discussions with Dr. R. B. Shrivastava,Dr. H. K. Gogoi (Defense Research Laboratory, Tezpur), andDr. M. C. Kalita (Gauhati University). Moreover, the authorsthank Dr. M. K. Purkait (Department of Chemical Engineering,IIT Guwahati) for his help in project implementation. Theauthors also thank the referees for their meticulous evaluationof the manuscript and constructive criticism.

Supporting Information Available: Mathematical model forthe radial motion of cavitation bubbles along with the numericalsolution scheme and relevant references. This material isavailable free of charge via the Internet at http://pubs.acs.org.

Literature Cited

(1) Sheehan, J.; Dunahay, T.; Benemann, J.; Roessler, P. A Look Backat the U.S. Department of Energy’s Aquatic Species Program - Biodieselfrom Microalgae; Rep. No. NREL/TP-580-24910. National RenewableEnergy Laboratory: Golden, 1998.

(2) Gerpen, J. V. Biodiesel Processing and Production. Fuel Process.Technol. 2005, 86, 1097.

(3) Ma, F.; Hanna, M. A. Biodiesel Production: A Review. Bioresour.Technol. 1999, 70, 1.

(4) Meher, L. C.; Dharmagadda, V. S. S.; Naik, S. N. Optimization ofAlkali Catalyzed Transesterification of Pongamia Pinnata Oil for Productionof Biodiesel. Bioresour. Technol. 2006, 97, 1392.

(5) Bender, M. Economic Feasibility Review for Community ScaleFarmer Cooperatives for Biodiesel. Bioresour. Technol. 1999, 70, 81.

(6) Kim, S.; Dale, B. E. Life Cycle Assessment of Various CroppingSystems Utilized for Producing Biodiesel: Bioethanol and Biodiesel.Biomass Bioenerg. 2005, 29, 426.

(7) Marchetti, J. M.; Miguel, V. U.; Errazu, A. F. Techno-EconomicStudy of Different Alternatives for Biodiesel Production. Fuel Process.Technol. 2008, 89, 740.

(8) Sharma, Y. C.; Singh, B. Development of Biodiesel: CurrentScenario. Renew. Sust. Energ. ReV. 2009, 13, 1646.

(9) Apostolakou, A. A.; Kookos, I. K.; Marazioti, C.; Angelopoulos,K. C. Techno-Economic Analysis of a Biodiesel Production Process fromVegetable Oils. Fuel Process. Technol. 2009, 90, 1023.

(10) Neenan, B.; Feinberg, D.; Hill, A.; McIntosh, R.; Terry K. Fuelsfrom Microalgae: Technology Status, Potential and Research Requirements;Rep. No. SERI/SP-231-2550. Solar Energy Research Institute: Golden,August 1986.

(11) Chisti, Y. Biodiesel from Microalgae. Biotechnol. AdV. 2007, 25,294.

(12) Chisti, Y. Biodiesel from Microalgae Beats Ethanol. TrendsBiotechnol. 2008, 26, 126.

(13) Mata, T. M.; Martins, A. A.; Caetano, N. S. Microalgae forBiodiesel Production and Other Applications: A Review. Renew. Sust.Energ. ReV. 2010, 14, 217–232.

2984 Ind. Eng. Chem. Res., Vol. 49, No. 6, 2010

(14) Khan, S. A.; Rashmi; Hussain, M. Z.; Prasad, S.; Banerjee, U. C.Prospects for Biodiesel Production from Microalgae in India. Renew. Sust.Energ. ReV. 2009, 13, 2361–2372.

(15) Huang, G. M.; Chen, F.; Wei, D.; Zhang, X. W.; Chen, G. BiodieselProduction from Micro-Algal Biotechnology. Appl. Energy 2010, 87, 38–46.

(16) Xu, H.; Miao, X.; Wu, Q. High Quality Biodiesel Production froma Microalga Chlorella protothecoides by Heterotrophic Growth in Fer-menters. J. Biotechnol. 2006, 126, 499.

(17) Miao, X.; Wu, Q. Biodiesel Production from Heterotrophic Mi-croalgal Oil. Bioresour. Technol. 2006, 97, 841.

(18) Milne, T. A.; Evans, R. J.; Nagle, N. Catalytic Conversion ofMicroalgae and Vegetable Oils to Premium Gasoline with Shape SelectiveZeolites. Biomass 1990, 21, 219.

(19) Engler, C. R. Disruption of Microbial Cells. In: ComprehensiVeBiotechnology, 2nd ed.; Moo-Yoong, M., Ed.; Pergamon Press: Oxford,1985; pp 305-324.

(20) Geciova, J.; Bury, D.; Jelen, P. Methods for Disruption of MicrobialCells for Potential Use in the Dairy Industry - A Review. Int. Dairy J.2002, 12, 541.

(21) Belarbi, E.-H.; Molina Grima, E.; Chisti, Y. A Process for HighYield and Scaleable Recovery of High Purity Eicosapentaenoic Acid Estersfrom Microalgae and Fish Oil. Enzyme Microb. Technol. 2000, 26, 516.

(22) Meng, X.; Yang, J.; Xu, X.; Zhang, L.; Nie, Q.; Xian, M.Biodiesel Production from Oleaginous Microorganisms. RenewableEnergy 2009, 34, 1.

(23) Mecozzi, M.; Amici, M.; Romanelli, G.; Pietrantonio, E.; Deluca,A. Ultrasound Extraction and Thin Layer Chromatography Flame IonizationDetection Analysis of Lipid Fraction in Marine Mucilage Samples.J. Chromatogr., A 2002, 963, 363.

(24) Pernet, F.; Tremblay, R. Effect of Ultrasonication and Grindingon the Determination of Lipid Class Content of Microalgae Harvested onFilters. Lipids 2003, 38, 1191.

(25) Cravatto, G.; Boffa, L.; Mantegna, S.; Perego, P.; Avogadro, M.;Cintas, P. Improved Extraction of Vegetable Oils Under High IntensityUltrasound and/or Microwaves. Ultrason. Sonochem. 2008, 15, 898.

(26) Virot, M.; Tomao, V.; Ginies, C.; Visinoni, F.; Chemat, F.Microwave Integrated Extraction of Total Fats and Oils. J. Chromatogr., A2008, 1196-1197, 57.

(27) Lee, J.-Y.; Chan, Y.; Jun, S.-Y.; Ahn, C.-Y.; Oh, H.-M. Comparisonof Several Methods for Effective Lipid Extraction from Microalgae.Bioresour. Technol. 2010, 101, S75–S77.

(28) Silverberg, B. A.; Stokes, P. M.; Ferstenberg, L. B. IntranuclearComplexes in a Cooper-Tolerant Green Alga. J. Cell Biol. 1976, 69, 210.

(29) Soxhlet, F. The Weight Analytic Determination of Milk Fat (inGerman). Polytechnisches J. (Dingler’s) 1879, 232, 461.

(30) Bligh, E. G.; Dyer, W. M. A Rapid Method of Lipid Extractionand Purification. Can. J. Biochem. Physiol. 1959, 37, 911.

(31) Hart, E. J.; Henglein, A. Free Radical and Free Atom Reactions inthe Sonolysis of Aqueous Iodide and Formate Solutions. J. Phys. Chem.1985, 89, 4342.

(32) Hart, E. J.; Henglein, A. Sonochemistry of Aqueous Solutions:Hydrogen-Oxygen Combustion in Cavitation Bubbles. J. Phys. Chem. 1987,91, 3654.

(33) Suslick, K. S. Sonochemistry. Science 1990, 247, 1439.(34) Save, S. S.; Pandit, A. B.; Joshi, J. B. Microbial Cell Disruption:

Role of Cavitation. Chem. Eng. J. (Lausanne) 1994, 55, 678.(35) Shirgaonkar, I. Z.; Lothe, P. R.; Pandit, A. B. Comments on the

Mechanism of Microbial Cell Disruption in High Pressure and High SpeedDevices. Biotechnol. Prog. 1998, 14, 657.

(36) Balasundaram, B.; Pandit, A. B. Significance of Location ofEnzymes on Their Release During Microbial Cell Disruption. Biotechnol.Bioeng. 2001, 75, 607.

(37) Sivasankar, T.; Paunikar, A. W.; Moholkar, V. S. MechanisticApproach to Enhancement of the Yield of a Sonochemical Reaction. AIChEJ. 2007, 53, 1132.

ReceiVed for reView October 24, 2009ReVised manuscript receiVed January 9, 2010

Accepted January 18, 2010

IE9016557

Ind. Eng. Chem. Res., Vol. 49, No. 6, 2010 2985