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Antonio Ramírez Fajardo Luis Esteban Cerdán Alfonso Robles Medina Fracisco Gabriel Acién Fernández Pedro A. González Moreno Emilio Molina Grima Departamento de Ingeniería Química, Universidad de Almería, Almería, Spain Lipid extraction from the microalga Phaeodactylum tricornutum Ethanol was used for the extraction and purification of lipids from the biomass of the microalga Phaeodactylum tricornutum. This microalga is an oil-rich substrate with a high proportion of eicosapentaenoic acid (EPA). The process consisted of two steps. First, ethanol (96% vol/vol) was used to extract the lipids from the lyophilized bio- mass. Second, a biphasic system was formed by adding water and hexane to the extracted crude oil. In this way, most of the lipids were transferred to the hexanic phase while most impurities remained in the hydroalcoholic phase. The first step was carried out by two consecutive extractions at room temperature, each with 5 mL ethanol per gram of biomass, for 10 and 1.25h, respectively. Under these conditions, over 90% of the saponifiable lipids in the biomass were extracted. In the second step, the percentage of water in the hydroalcoholic phase, the hexane/hydroalcoholic phase ratio and the number of extraction steps were optimized. A water content of 40% vol/vol in the hydroalcoholic phase provided the highest lipid recovery. A recovery yield of 80% was obtained by four consecutive extractions with a hexane/ hydroalcoholic phase ratio of 0.2 (vol/vol). Equilibrium distribution data of the lipids between the hydroethanolic and the hexanic phases were also obtained in order to predict the lipid recovery yield of the extraction. This process is an alternative to the traditional methods of lipid extraction, which uses less toxic solvents and reduces the total amount of solvents used. Keywords: Extraction, lipids, purification, EPA, Phaeodactylum tricornutum, ethanol, hexane. 1 Introduction The culture of marine microalgae for the production of polyunsaturated fatty acid (PUFA)-enriched lipids has become a subject of interest in recent years, but it has not yet reached commercial scale. The only currently avail- able commercial source of n-3 PUFA is fish oil, but the amount of fish oil is continuously decreasing while the demand for PUFA is continuously on the increase. The marine diatomeaceus Phaedactylum tricornutum may possess around 30–45% of PUFA, of which eicosa- pentaenoic acid (EPA) is the major one. EPA accounts for up to 20–40% of the total fatty acids of this microalga, as a function of culture conditions [1–3]. EPA is an essential fatty acid for the human metabolism and is involved in the blood lipid equilibrium, lowers triglyceride levels in blood serum, reduces the degree of platelet aggregation, is anti- inflammatory [4] and prevents hypertriglyceridemia [5] and various carcinomas [6]. The extraction and purification of PUFA can be carried out by procedures such as solvent extraction [3, 7], urea inclusion method, HPLC [1, 2, 8, 9] or selective enzymatic reactions [10]. The method selected must avoid heat and oxidation of PUFA [11, 12]. It must also be acceptable in terms of toxicity, handling, safety and cost, especially if the PUFA are to be used in baby food or in pharmacolog- ical applications. In previous works [1–3, 7–9] free fatty acid (FFA) extracts, used to purify PUFA, were obtained by a three-step process: (a) direct saponification of bio- mass-oil, (b) extraction of unsaponifiable lipids, and (c) extraction of purified FFA. Solvents of low toxicity such as ethanol and hexane were used. In this work, the aim was to study the extraction of the saponifiable lipids from the microalga P. tricornutum, also using these low-toxicity solvents, but as lipids, i.e. without any saponification. In order to reduce the amount of sol- vent, the equilibrium and the kinetics of the oil extraction were also studied. The process developed here may be used as a first step for obtaining oils with a high content in n-3 PUFA. This oil extract can then be used, e.g., for increasing the content of a specific PUFA by selective enzymatic reactions [10]. Correspondence: Alfonso Robles Medina, Departamento de Ingeniería Química, Universidad de Almería, Almería 04120, Spain. Phone: 134–950–015065, Fax: 134–950–015484, e-mail: [email protected] 120 DOI 10.1002/ejlt.200600216 Eur. J. Lipid Sci. Technol. 109 (2007) 120–126 © 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com Research Paper

Lipid extraction from the microalga Phaeodactylum tricornutum

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Page 1: Lipid extraction from the microalga Phaeodactylum tricornutum

Antonio Ramírez FajardoLuis Esteban CerdánAlfonso Robles MedinaFracisco Gabriel Acién FernándezPedro A. González MorenoEmilio Molina Grima

Departamento de IngenieríaQuímica,Universidad de Almería,Almería, Spain

Lipid extraction from the microalgaPhaeodactylum tricornutum

Ethanol was used for the extraction and purification of lipids from the biomass of themicroalga Phaeodactylum tricornutum. This microalga is an oil-rich substrate with ahigh proportion of eicosapentaenoic acid (EPA). The process consisted of two steps.First, ethanol (96% vol/vol) was used to extract the lipids from the lyophilized bio-mass. Second, a biphasic system was formed by adding water and hexane to theextracted crude oil. In this way, most of the lipids were transferred to the hexanicphase while most impurities remained in the hydroalcoholic phase. The first step wascarried out by two consecutive extractions at room temperature, each with 5 mLethanol per gram of biomass, for 10 and 1.25 h, respectively. Under these conditions,over 90% of the saponifiable lipids in the biomass were extracted. In the second step,the percentage of water in the hydroalcoholic phase, the hexane/hydroalcoholicphase ratio and the number of extraction steps were optimized. A water content of40% vol/vol in the hydroalcoholic phase provided the highest lipid recovery. Arecovery yield of 80% was obtained by four consecutive extractions with a hexane/hydroalcoholic phase ratio of 0.2 (vol/vol). Equilibrium distribution data of the lipidsbetween the hydroethanolic and the hexanic phases were also obtained in order topredict the lipid recovery yield of the extraction. This process is an alternative to thetraditional methods of lipid extraction, which uses less toxic solvents and reduces thetotal amount of solvents used.

Keywords: Extraction, lipids, purification, EPA, Phaeodactylum tricornutum, ethanol,hexane.

1 Introduction

The culture of marine microalgae for the production ofpolyunsaturated fatty acid (PUFA)-enriched lipids hasbecome a subject of interest in recent years, but it has notyet reached commercial scale. The only currently avail-able commercial source of n-3 PUFA is fish oil, but theamount of fish oil is continuously decreasing while thedemand for PUFA is continuously on the increase. Themarine diatomeaceus Phaedactylum tricornutum maypossess around 30–45% of PUFA, of which eicosa-pentaenoic acid (EPA) is the major one. EPA accounts forup to 20–40% of the total fatty acids of this microalga, asa function of culture conditions [1–3]. EPA is an essentialfatty acid for the human metabolism and is involved in theblood lipid equilibrium, lowers triglyceride levels in bloodserum, reduces the degree of platelet aggregation, is anti-inflammatory [4] and prevents hypertriglyceridemia [5]and various carcinomas [6].

The extraction and purification of PUFA can be carried outby procedures such as solvent extraction [3, 7], ureainclusion method, HPLC [1, 2, 8, 9] or selective enzymaticreactions [10]. The method selected must avoid heat andoxidation of PUFA [11, 12]. It must also be acceptable interms of toxicity, handling, safety and cost, especially ifthe PUFA are to be used in baby food or in pharmacolog-ical applications. In previous works [1–3, 7–9] free fattyacid (FFA) extracts, used to purify PUFA, were obtainedby a three-step process: (a) direct saponification of bio-mass-oil, (b) extraction of unsaponifiable lipids, and(c) extraction of purified FFA. Solvents of low toxicity suchas ethanol and hexane were used.

In this work, the aim was to study the extraction of thesaponifiable lipids from the microalga P. tricornutum, alsousing these low-toxicity solvents, but as lipids, i.e. withoutany saponification. In order to reduce the amount of sol-vent, the equilibrium and the kinetics of the oil extractionwere also studied. The process developed here may beused as a first step for obtaining oils with a high content inn-3 PUFA. This oil extract can then be used, e.g., forincreasing the content of a specific PUFA by selectiveenzymatic reactions [10].

Correspondence: Alfonso Robles Medina, Departamento deIngeniería Química, Universidad de Almería, Almería 04120,Spain. Phone: 134–950–015065, Fax: 134–950–015484,e-mail: [email protected]

120 DOI 10.1002/ejlt.200600216 Eur. J. Lipid Sci. Technol. 109 (2007) 120–126

© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com

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Eur. J. Lipid Sci. Technol. 109 (2007) 120–126 Lipid extraction from microalgae 121

2 Materials and methods

2.1 Microalgal biomass

Lyophilized biomass of the marine microalga Phaeo-dactylum tricornutum UTEX 640 was used as an oil-richsubstrate with a high proportion of EPA. Cells were grownin an outdoor tubular photobioreactor, harvested by cen-trifugation at 18006g and then stored at 28 7C until use.This microalga contains an average saponifiable lipidcontent of 6–7%. The fatty acid profile of the biomass,determined in triplicate by gas chromatography (GC), isshown in Tab. 1. This table shows that this microalga is in-deed rich in EPA (23 wt-% of total fatty acids or 1.4 wt-%of dry biomass).

2.2 Lipid extraction from biomass

Lipid extraction was performed as shown in Fig. 1.First, crude oil was extracted from the lyophilized bio-mass by ethanol (96% vol/vol). It was then purified byforming a hydroalcoholic solution-hexane two-phasesystem, where the purified oil is transferred to thehexane phase.

Tab. 1. Fatty acid composition of biomass from Phaeo-dactylum tricornutum and of crude oil extracted from thismicroalga.

Fatty acids Biomass§ Biomass$ Crude oil$

14:0 0.47 7.4 7.216:0 1.14 17.7 16.116:1n-7 1.15 17.9 19.216:2n-4 0.25 4.0 4.516:3n-4 0.39 6.0 6.716:4n-1 0.06 1.0 0.918:0 0.05 0.7 0.618:1n-9 0.12 1.8 1.518:1n-7 0.09 1.5 1.518:2n-6 0.15 2.4 2.818:3n-3 0.04 0.7 0.718:4n-3 0.04 0.6 0.720:4n-6 0.14 2.2 2.320:4n-3 0.05 0.8 0.620:5n-3 (EPA) 1.41 23.0 23.724:0 0.05 0.8 0.522:5n-3 0.22 3.4 1.422:6n-3 (DHA) 0.17 2.7 2.5Others 0.43 5.6 6.7

§ Percentage of fatty acids with respect to the total dryweight of biomass.$ Percentage of fatty acids with respect to the total fattyacids.

2.2.1 Extraction of crude microalgal oil frombiomass

In a typical experiment (Fig. 1), 10 g lyophilized biomasswas treated with 50 mL ethanol (96% vol/vol) in a 250-mLvessel with constant magnetic agitation at 500 rpm for24 h at room temperature. The mixture obtained was thenfiltered and the biomass residue was washed with2625 mL ethanol (96% vol/vol), or this biomass residuewas again extracted with 50 mL ethanol (96% vol/vol) for1 h. Each of the lipidic extracts obtained was analyzed bygas chromatography (GC) to determine the amount ofsaponifiable lipids extracted and their fatty acid compo-sition. All extracts obtained in this step were then put to-gether to form the crude oil extract, which was later puri-fied. In this first step, we studied the ratio of the amount oflyophilized biomass to the ethanol (96% vol/vol) volume,the number of extraction steps, the use of other extractionsolvents and the extraction rate. For the latter, sampleswere taken at increasing times.

2.2.2 Purification of crude microalgal oil

In a typical experiment, 60 mL water was added to 100mLof the crude oil solution to obtain a hydroalcoholic solutionwith 40% water (vol/vol). Then, 32 mL hexane was added(hexane/hydroalcoholic solution ratio of 0.2 mL/mL), form-ing a biphasic system. In this way the majority of the oil wastransferred to the hexanic phase. Extraction was per-formed at room temperature (between 20 and 25 7C), in dimlight, under an argon atmosphere and with agitation. Oncethe two phases were separated, an aliquot of the hexanephase was taken for fatty acid determination by GC. Thelipid concentration in the hydroalcoholic phase was deter-mined by a lipid balance, taking into account the initialamount of lipids in the crude oil. As shown in Fig. 1,extraction of lipids was also carried out in several steps;e.g. five extractions were carried out with 32 mL hexaneeach (total hexane/hydroalcoholic solution ratio of 1 mL/mL). The lipid extraction yield was expressed as theamount of fatty acids present in the hexane phase dividedby the amount of fatty acids present in the crude lipidextract. In this step, the influence of the following variableswas studied: the percentage of water in the hydroalcoholicsolution, the hexane/hydroalcoholic solution ratio and thenumber of extraction steps. The equilibrium distribution oflipids between the hydroalcoholic (40% water vol/vol) andhexane phases was determined by adding differentvolumes of hexane to 1 mL hydroalcoholic solution of lipidconcentration 3.6 g/mL (the hexane/hydroalcoholic solu-tion ratio varied from 0.17 to 6 mL/mL). Once equilibriumwas attained, the lipid concentration in the hexane phasewas determined by GC, while the lipid concentration in thehydroalcoholic phase was determined as detailed above.

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122 A. R. Fajardo et al. Eur. J. Lipid Sci. Technol. 109 (2007) 120–126

Fig. 1. Process for the extractionand purification of lipids from themicroalga Phaeodactylum tri-cornutum.

2.3 Calculation of lipid extraction yields andfatty acid determination

In this study, the interest is mainly focused on saponifiablelipids instead of total lipids, and the former are expected tobe in a direct relationship with the fatty acid content. Con-sequently, the yield of saponifiable lipid extraction in thecrude extract might be obtained as the ratio of fatty acids inthe crude extract to fatty acids in the initial biomass, whichwas 6–7% of the dry weight. The yield of saponifiable lipidextraction in the purified extract might be obtained as theratio of fatty acids in the purified extract (hexane phase) tofatty acids in the initial crude extract (initial hydroalcoholicsolution). The fatty acids in the biomass and in the hexaneand alcoholic phases were analyzed by capillary GC. Fattyacids in the initial biomass were determined by directtransesterification of the biomass following the method ofLepage and Roy [13]. To determine the fatty acids in thehexane and alcoholic phases, a known volume was driedunder an N2 stream, and methylation was also carried outby direct transesterification following the method ofLepage and Roy [13]. Methylation and methyl ester analy-sis by GC have been described elsewhere [7].

3 Results and discussion

3.1 Extraction of crude microalgal oil frombiomass

The fatty acid profile of a crude oil extracted from P. tri-cornutum with ethanol (96% vol/vol) is shown in Tab. 1.Obviously, this fatty acid profile is quite similar to that ofthe biomass.

Fig. 2 shows that the lipid recovery yield increases withthe extraction time, being practically constant after 20 hof extraction. The extraction rates were similar at thethree ethanol (96% vol/vol)/biomass ratios tested. Thelipid recovery yield obtained with 10 and 15 mL ethanol(96% vol/vol) per gram of biomass was around 80%,while with 5 mL ethanol (96% vol/vol) per gram of biomassit was approximately 90%. This higher yield may be dueto the fact that 5 mL ethanol (96% vol/vol) per gram ofbiomass is enough to dissolve the oil from the biomass,and to the higher agitation intensity per volume unit at thisratio. Therefore the lowest ratio tested can be used; in thisway, the consumption of solvent decreases and the crudeoil solution obtained is more concentrated. Concentra-tions below 5 mL/g led to difficulties in handling, homog-enization and agitation of the mixture.

Due to the long time necessary to attain extraction recov-ery yields around 90% (20 h), the possibility of carrying outthe extraction in several steps was studied. The firstextraction step was stopped when the increase in extrac-tion yield was lower than 1%/h, i.e. at 10 h. Tab. 2 showsthat the extraction yield attained in this first extraction stepwas 77.4%. In a second extraction of the lipids containedin the biomass residue, the recovery yield increased up to96.1% (Tab. 2). This second extraction was also carriedout with acetone, n-hexane and ethanol/hexane 3 : 1 vol/vol, producing similar results (data not shown).

The total extraction time for these two extraction stepswas 11.25 h, which is significantly lower than the timenecessary to attain a 90% recovery yield with only oneextraction step (20 h; Fig. 1).

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Eur. J. Lipid Sci. Technol. 109 (2007) 120–126 Lipid extraction from microalgae 123

Fig. 2. Extraction of lipids from P. tri-cornutum biomass: Influence of extractiontime and ethanol (96% vol/vol)/biomassratio on the lipid recovery yield. Ethanol(96% vol/vol)/biomass ratios used:(n) 15 mL/g, (D) 10 mL/g, (6) 5 mL/g.

Tab. 2. Extraction of oil from P. tricornutum biomass. Oilrecovery yields obtained after three extraction steps: Firstextraction: 10 g of biomass extracted with an ethanol(96% vol/vol)/biomass ratio of 5 mL/g; second and thirdextraction steps: the biomass residue was treated with50 mL ethanol (96% vol/vol) per step, sampling atincreasing times.

Time[min]

Lipid recoveryyield [%]§

First extraction 600 77.4Second extraction 15 13.7 (91.1)

30 15.0 (92.4)45 16.0 (93.4)60 16.9 (94.3)75 18.7 (96.1)90 18.7 (96.1)

Third extraction 20 1.6 (97.7)40 2.0 (98.1)60 2.4 (98.5)80 2.5 (98.6)

§ Total recovery yield at each sampling appears in brack-ets.

A third extraction step was carried out with the residualbiomass (Tab. 2). After 80 min an additional recovery yieldof 2.5% may be obtained, which gives a total oil extrac-tion yield of 98.6%. However, this marginal increase in theyield does not justify a third extraction step.

Therefore the lipid extraction may be realized in twoextraction steps of 10 and 1.25 h duration, respectively,and by using an ethanol (96% vol/vol)/biomass ratio of5 mL/g in every step. Under these conditions, we

obtained a total oil recovery yield of 96.1% (77.4% in thefirst extraction step and 18.7% in the second step;Tab. 2).

3.2 Purification of crude oil

In addition to saponifiable lipids (acylglycerols, phospho-lipids and glucolipids), the crude oil obtained with ethanol(96% vol/vol) contains unsaponifiable lipids such as pig-ments (carotenoids, chlorophylls, etc.), proteins, aminoacids and other lipid and non-lipid contaminants. Thepurification of the lipids may be carried out by addingwater and hexane to form a biphasic system. In this waythe lipids will remain predominantly in the hexanic phaseand the more polar compounds will remain in the hydro-alcoholic phase [7, 14]. The optimization of this purifica-tion was carried out in two steps. First, the water contentof the hydroalcoholic phase was optimized, and then thelipids were extracted with hexane.

3.2.1 Optimization of the water content of thehydroalcoholic solution

The alcoholic solution contained only the water addedwith the ethanol (96% vol/vol). An increase in the watercontent increased the solution polarity, decreased themiscibility of the alcoholic solution and hexane andincreased the lipid recovery yield when hexane wasadded, i.e. the higher the water content, the more theequilibrium distribution of the lipids between the hydro-alcoholic and the hexanic phases is displaced to thehexane phase [7]. Water was added to the crude hydro-alcoholic oil solution to attain a water content rangingbetween 20 and 80% vol/vol. Hexane was then added at

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124 A. R. Fajardo et al. Eur. J. Lipid Sci. Technol. 109 (2007) 120–126

Fig. 3. Purification of lipids contained in a hydro-alcoholic solution by extraction with hexane: Influ-ence of the water content of the hydroalcoholic so-lution on the lipid recovery yields (RH) in the hexanephase. Extractions were carried out with a hexane/hydroalcoholic solution ratio of 1 : 1 (vol/vol).

a hexane/hydroalcoholic solution ratio of 1 : 1 vol/vol.After extraction, the hexanic phase was separated andthe lipid recovery yield was evaluated.

Fig. 3 shows the lipid extraction yield obtained with hex-ane at increasing water content in the hydroalcoholic so-lution. This figure shows that the lipid extraction yieldincreases with the water content, reaching a maximum at40%. At higher water contents, the lipid extraction yielddecreased due to the formation of emulsions, the stabilityof which increased with the water content. Emulsionsmake oil extraction difficult in the hexanic phase and de-crease recovery. This result is not new, since a previouswork on extraction of fatty acids from P. tricornutum withethanol (96% vol/vol) [3] proved that the crude oil solutioncan contain up to 15 g/L of proteins, which facilitated andstabilized the hydroalcoholic/hexane emulsions. An opti-mal water content of 40% was therefore chosen to pre-vent or minimize emulsion formation.

3.2.2 Optimization of oil purificationwith hexane

To optimize the hexane/hydroalcoholic solution ratio andthe number of extraction steps, we determined the equi-librium distribution of the lipids between the hexanic andhydroalcoholic (40% vol/vol water) phases and the rela-tionship between the recovery yield and the hexane/hydroalcoholic solution ratio (Tab. 3, Fig. 4). Tab. 3 showsthat the recovery of purified lipids (RH) increased with thehexane/hydroalcoholic phase ratio. The partitioning equi-librium constant of oil between both phases (KL)increased with the decrease in the hexane/hydroalcoholicsolution ratio, the equilibrium constant being greatly dis-placed to the hexanic phase. This is also observed inFig. 4 which shows the equilibrium data of the distributionof lipids between the hydroalcoholic and the hexanic

Tab. 3. Purification of the lipids contained in the hydro-alcoholic solution by extraction with hexane: Influence ofthe hexane/hydroalcoholic solution (40% water vol/vol)ratio (r) on the lipid concentration in the hexane and thehydroalcoholic solution (CH and CHA, respectively), thelipid distribution coefficient between both phases(KL = CH/CHA) and the lipid recovery yield in hexane (RH).Initial lipid concentration in the hydroalcoholic extract:3.6 g/L.

Hexane/hydro-alcoholic solutionratio (vol/vol), r

CH

[g/L]CHA

[g/L]KH RH

[%]

0.17 13.67 1.32 10.37 63.40.25 9.30 1.27 7.32 64.70.33 7.19 1.20 5.99 66.60.5 4.91 1.14 4.30 68.31 2.57 1.03 2.49 71.32 1.37 0.85 1.62 76.43 0.94 0.77 1.23 78.64 0.72 0.71 1.01 80.26 0.49 0.68 0.71 81.0

phases. These data enable prediction of the lipid yieldwhen extraction is carried out in one or several steps withdifferent volumes of hexane.

To make this estimation, the miscibility degree of bothphases must be known. Taking into account the equilibri-um data for the ternary system ethanol/water/hexanegiven by Bonner [15], the hydroalcoholic (40% water) andhexane phases can be considered as totally immiscible[3, 7]. On the other hand, the equilibrium data (Fig. 4) werefitted to the equation:

CH ¼ 0:0112e5:19CHA r2 = 0.9941 (1)

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Eur. J. Lipid Sci. Technol. 109 (2007) 120–126 Lipid extraction from microalgae 125

Fig. 4. Equilibrium distribution of saponifiable lipids be-tween the hydroalcoholic (40% water vol/vol) and thehexanic phases. CH, lipid concentration in the hexanicphase; CHA, lipid concentration in the hydroalcoholicphase.

The continuous line in Fig. 4 corresponds to this equation.If the lipid extraction is carried out in one or several equi-librium extraction steps, the extraction recovery yieldscan be calculated from Eq. (1) (equilibrium data) and fromthe lipid balances at each equilibrium extraction step. Inthis way, for a specific i-equilibrium step of a series of nequilibrium extraction steps, the lipid concentrations ofthe hexanic and hydroalcohlolic phases at the end of theextraction (CHi and CHAi, respectively) will be calculated bysolving Eq. (1) and the lipid balance

CHi ¼1rðCHAi�1 � CHAiÞ (2)

where r is the hexane/hydroalcoholic solution ratio (vol/vol) and CHAi–1 is the lipid concentration in equilibriumstep i – 1. This equation was obtained taking into accountthat hexane without lipids is fed at each extraction stepand that the two phases are totally immiscible, as pre-viously commented. On knowing the lipid concentrationof the hydroalcoholic solution fed in the first step(CHAO = CHAi–1 for i = 1) and the hexane/hydroalcoholicsolution ratio, r, all the intermediate and final concentra-tions can be calculated step by step. From these con-centrations, the lipid recovery yield can also be calculatedby the equation:

RH ¼

Pn

i¼1CHiVH

CHA0VHA0

100 (3)

where VH and VHAO are the hexane volumes added at eachequilibrium extraction step and the initial volume of thehydroalcoholic phase, respectively, CHAO is the initial lipidconcentration in the hydroalcoholic solution, and n is thenumber of equilibrium extraction steps. Tab. 4 shows the

Tab. 4. Comparison between the lipid yields obtainedexperimentally by hexane extraction and predicted by theequilibrium data. Extraction carried out in five and fourequilibrium steps with a hexane/hydroalcoholic solution(40% water) ratio of 0.2 vol/vol, starting from two hydro-ethanolic oil solutions with lipid concentrations of 1.44and 3.83 g/L.

Extractionstep§

RHi [%]§ RHi experimental[%]

CHAO = 1.44 g/L1 29.72 28.32 12.06 14.43 7.10 7.94 4.91 2.85 3.72 2.6

RH = 57.51 RH = 56.0

CHAO = 3.83 g/L1 64.74 58.92 9.60 15.03 4.18 6.54 2.53 1.4

RH = 81.05 RH = 81.8

§ Yields calculated by Eqs. (1), (2) and (3).

lipid extraction yields obtained in two extractions of fiveand four equilibrium steps, starting from two hydro-alcoholic lipid solutions of concentrations 1.44 and3.83 g/L, respectively, and using a hexane/hydroalco-holic solution ratio of 0.2 vol/vol. This table comparesthe extraction yields obtained experimentally (by usingfive or four extraction steps) to those based on asequence of five or four batch extraction steps, assum-ing that equilibrium is reached in each step and that thetwo phases are totally immiscible. Good correlation be-tween the two yields can be observed (less than 6%variation), which indicates that each experimentalextraction step is almost equivalent to an equilibriumstep. This table shows that the yield is greater at higherinitial lipid concentration, which is a consequence of theequilibrium curve form (Fig. 4), since at low concentra-tions the distribution coefficient KL is also lower (Tab. 3).Therefore it is important to purify lipids starting fromconcentrated crude oil solutions; e.g. with concentra-tions of the order of those obtained when the biomassis treated with an ethanol/biomass ratio of 10 mL/g (twoconsecutive extractions with 5 mL/g each). In this case,the lipid concentration in the hydroalcoholic solution ishigher than 3 g/L, and therefore the lipid recovery yieldwould be 80% when the extraction is carried out in foursteps using 0.2 mL hexane/mL hydroalcoholic solution(Tab. 4).

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4 Conclusions

A procedure of extraction and purification of lipids fromP. tricornutum biomass has been optimized. This proce-dure consists of two steps: (i) obtaining a crude lipidextract by two-step extraction of the microalgal biomasswith ethanol (96% vol/vol) and (ii) purification of this crudelipid extract by extraction of the oil with hexane. Betweenboth steps, the water content of the hydroalcoholic lipidsolution has to be adjusted to 40% vol/vol in order tomaximize the saponifiable lipid recovery yield. This overallextraction procedure improves that of Kates [16] becauseit reduces the amount of solvents and uses more bio-compatible ones. In this way, while the procedure ofKates uses 250 mL chloroform/methanol per gram ofbiomass, this procedure only uses 10 mL ethanol and16 mL hexane per gram of biomass, achieving lipidrecovery yields of about 80%, similar to those obtainedby the former procedure.

For the purification of lipids by extraction with hexane, theequilibrium concentrations of saponifiable lipids in thehydroalcoholic (40% water vol/vol) and hexane phaseswere obtained. These data allow the prediction of the lipidrecovery yield that can be obtained with different hexane/hydroalcoholic solution ratios and with one or severalextraction steps. In this way, it has been demonstratedthat using five or four extraction steps and a hexane/hydroalcoholic solution (40% water) ratio of 0.2 vol/vol,experimental lipid yields and those predicted from theequilibrium data are quite similar.

Acknowledgments

This research was supported by grants from the Minis-terio de Educación y Cultura and Ministerio de Ciencia yTecnología (Spain), Projects PPQ2000–1220 andAGL2003–03335.

References

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[Received: October 3, 2006; accepted: December 11, 2006]

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