25 the Production of Polyunsaturated Fatty Acids By

7/27/2019 25 the Production of Polyunsaturated Fatty Acids By

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ELSEVIER 0032.9592(94)00047-6

Process Biochemistry, Vol. 30, No. 8, 7 1 l-7 19, 1995p.Copyright 0 1995 Elsevier Science Ltd

Printed in Great Britain. All rights reserved0032.9592/95 $9.SO+O.O0

The Production of Polyunsaturated Fatty Acids byMicroalgae: from Strain Selection to ProductPurificationE. Molina Grima, J. A. Sknchez Pkrez, F. Garcia Camacho,* A. Robles Medina,A. Gimknez Gimknez & D. L6pez AlonsotDpto. Ingenieria Quimica, tDpto. Gektica, Facultad de Ciencias Experimentales, Universidad de Almeria, E-0407 1Almeria,Spain(Received 8 June 1994; revised manuscript received 12 September 1994; accepted 2 October 1994)

A selection progr amme to incr ease the cell ul ar eicosapentaenoic mid (EPA)content has been carr ied out with the micr oalga Isochrysis galbana. The selectionprocess invol ved two stages of single selection . EPA content continuouslyincreased f r om 2.4% dry weight (d. w.) of the parent cultur e to an average value of53% d. w. in the fi nal .stage. The proporti on of total EPA variati on attri butable tothe genetic variati on (heritabil ity in a broad sense) was 0.99 showing theimpor tance of the genome i n the determination ofthis fatty acid. The growth andfatty acid profile of an EPA-r ich isolate grown as a chemostat in a cylindr icalphotobi oreactor have been studied. A decrease in EPA con tent was observed(5.21% w/w to 28% w/w) at the lowest dilu tion rate D = O-024 h- , up close to themaximum growth rate, D = 0038 h- . At the same time, the biomassconcentration also decreasedfr om 1015 mgJli tre to 202 mgjl itr e over the above-mentioned range of dilu tion rate (0). Nonetheless, the EPA productivity in creaseswith D, with u maximum of 15.26 mgjlitr elday at D = 00208 h- . Furthermore,steady-state dilu tion rates may be related to average i nternal li ght intensity.Reverse-phase, high-pressure li quid chromatography (HPLC) on octudecylsil ylsemi -pr eparati ve columns was used to separate steari doni c acid (SA), EPA anddocosohexaenoi c acid (DH A) in polyunsaturated fatty acid concentr ate obtainedby the urea complexation methodfr om a atty acid soluti on pr eviously obtained bydirect saponif ication of biomass. I solate SA, EPA andDHA fraction purity was948, WO and 94*9%, respectively, with yields of lW0, 96 and 94.0%.

INTRODUCTION noids, antioxidants and long-chain polyunsatu-rated fatty acids (PUFAS). In recent years, severalMicroalgae are considered a potential source of a companies have been developing commercialwide spectrum of chemicals, including such highly applications of these products.-valuable products as phycobiliproteins, carote- The production of lipids from microalgae pro-duced in photobiorcactors, fermenters or by*To whom correspondence should be addressed. pond-farming, depending on the value of the

711


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712 E. Moha Grima et al.product, is expected to develop rapidly.5 Amongthese lipids, n-3 PUFAs in particular are beingstudied because of their role in human health,eicosapentaenoic acid being considered ofimmense therapeutic value.6 Yet, to achieve eco-nomic viability of EPA production with micro-algae, an increase in EPA content is necessary andfor this, genetic methods must ultimately beemployed.7T 8 Beyond species/strain choice,factors affecting the growth rate and biochemicalcomposition of the cell must be borne in mind,especially environmental and operating condi-tions.Another area of interest to microalgal culture isgrowth modelling, which is useful in the predic-tion of system behaviour under different condi-tions., Algal biomass production is generallylimited by light and therefore, for the study of itskinetics, it has been found necessary to modellight distribution inside the culture.12 One para-meter exerting a notable influence on the accuratecalculation of average light intensity is the bio-mass absorption coefficient, which originates inLambert-Beers law. Its estimation depends notonly on chlorophyll a content,,4 but also onother pigments and light-absorbing molecules5and must therefore be precisely determined.In this context, the marine microalga Zsochlysisgalbana has been the subject of a phenotypicselection programme for an EPA-rich strain, andgrowth conditions enhancing EPA content werestudied. Furthermore, the urea complexationmethod was used to concentrate PUFAs in bio-mass fatty acids followed by SA, EPA and DHApurification by HPLC. An overview of thesedifferent areas is presented here.MATERIALS AND METHODSSelection processPhenotypic selection to improve the EPA contentin Z. gulbana was carried out using a parentculture supplied by Dr J. F6bregas of the Univer-sity of Santiago de Compostela, Spain. Fifty-nineisolates, called generation I, were successfullyproduced from single cells isolated with a micro-pipette under the microscope. The isolate richestin EPA was selected to produce a new set of 42isolates called generation 11. All isolates werecultured under identical conditions in sphericallOOO-ml flasks. Cultures were harvested duringthe stationary phase of growth for subsequent

analysis. 6 7 Quantitative genetic analysis of fattyacid variation was carried out on collection strainsof I. galbana and Phaeodactyhun tricornutumfollowing the methodology of Caten & Jinksls andWood et al.Growth conditionsAn isolate (labelled 11-4) selected from the secondset of isolates was grown in a chemostat culture ina 5-litre computer-controlled fermenter (NewBrunswick scientific Bioflo III).The culture medium and sterilization processeshave been described previously.2o The cultureswere constantly illuminated with four OsramDulux EL (20 W) fluorescent lamps arrangedaround the culture vessel. Incident light intensitywas measured by a laboratory quantum scalarirradiance meter QSL-100 (Biospherical Instru-ment Inc., San Diego, CA). All growth experi-ments were carried out at a temperature of 2OC,CO, was injection-controlled at pH= 8.00 andthe air supply was sterilized by filtration through0.22 pm millipore filters at a rate of la.5 litrernin- I. Agitation was set at 150 rpm, equivalent toan agitator Reynolds number of 9750.Biomass absorption coefficient (K.)K, was determined by measuring the absorbanceof six I. galbana cultures with different pigmentcontent and 12 biomass concentrations between50 and 1100 mg litre-I. The intensity of lightcoming from all directions in the centre of a (4.9-cm diameter) cylindrical vessel was measured withQSL- 100 for each biomass concentration assayed.Absorbance was calculated by Lambert-Beerslaw. The absorption coefficients were calculatedby dividing the slopes of the straight lines ofabsorbance vs biomass concentration by thevessel radius.Analytical methodsLipids were extracted by the Kochert method.21Fatty acid methylation was done by direct trans-esterification following the method of Lepage &Roy.22 The analysis of methyl esters was carriedout by gas chromatography using a 30-m capillarycolumn of fused silica (SP2330, Supelco, Belle-fonte, PA, USA), with an internal diameter of0.25 mm, O-20 ym standard film, split ratio of100 : 1, and a flame ionization detector. SIGMALipid Standard 189-15, Supelco Rapeseed oilmixture and Supelco PUFA-1 patterns were used


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Production of polyunsaturated fatty acids by microalgae 713for the determination. Nonadecanoic acid wasused as an internal standard to quantify fatty acidcontent in biomass. Proteins were assayed asdescribed by Lowry et aZz3SA, EPA and DHA were isolated by the fol-lowing method: total fatty acid was obtained bydirect saponification of 5-g samples of lyophilizedbiomass at 60C for 1 h, in a hexane-ethanol(96%) 2:5 v/v system with 2.1% w/v KOH. Thefatty solution thus obtained was concentrated bythe urea fractionation methodz4 with 375 g litre-of methanol at a urea-fatty acid ratio of 4 : 1. Thefractionation of the PUFA solution concentratewas carried out by HPLC using a reverse phase,C18, 10 mmx 250 mm, 5 pm, 80 A column,methanol-water (acetic acid) 80:20 (1%) w/w aseluent, at a 3 ml mir - flow in the mobile phaseand loading 9.49 mg of PUFA concentrate in thecolumn.The fatty acid composition was determined byGLC at each step of the purification process.RESULTS AND DISCUSSIONThe selection processThe selection process involved two stages ofsingle selection (without artificial mutagenesis) ofI. gulbuna, as described above. The resultingisolates appear to follow a consistent patter, inwhich the fatty acids found at each stage of theselection process were 14:0, 16 : 0, 16: 1 n7,18:ln7, 18:4n3, 20:5n3 and 22:6n3. Never-theless, at the same time, the isolates differedwidely in their content of specific fatty acids,within each generation as well as between genera-tions (Table 1). Thus, for example, the EPA con-tent ranged between 1.8-6~6~~ d.w. among thegeneration I isolates and analogous variation wasobserved for the other fatty acids (Table 1).Similarly, the range of variation for EPA contentin generation II was 3~6-7.8% d.w.

The EPA content increased continuously fromthe parent culture to generation II, as may beobserved. EPA content improvement was evidentin the increase of average amounts from theparent culture to the second generation of iso-lates, rising from 2.4% to 5.3% d.w. This meansthat with only two rounds of phenotypic selectionit was possible to double the original EPA con-tent. Other lipid characters such as total PUFAs,essential fatty acids and unsaturated fatty acids,

Table 1. Average fatty acid content in two generations ofisolates of I sochrysis gafbana and heritability, h*, for the mainfatty acids in several collection strainsFatty acid I I I K-W h

14:o16:016:ln718:ln718:4n320:5n322:6n3SaturatedUnsaturatedPUFAsEFAsLipids

2.1 * 0.082,7 + 0.103~0f0.100.6 + 0.031.4+0.064.2 * 0.151.5 + 0.055.1 &O-1511.2 * 0.346.2+0.214.7kO.1718.3 + 3.84

2.1 f O-083.0 +_0.143.0 f 0.110.6 f 0.041.4 * 0.055.3kO.142.OiO.085.9 + 0,2 113.1+0.358.8 + 0.255.7 + 0.1621.4k3.73

0.01 n.s.1.70 n.s.0.08 n.s.0.7 1 ns.0.31 n.s.17.6130.46hS-65102241.6Bh14.41 h12.2Bh

0.910.970.980.210.920.920.92n.c.n.c.nc.n.c.n.c.

OContent is expressed as percent of dry weight; I, generation1; II, generation II; K-W, Kruskal-Wallis non-parametric testfor average comparisons; n.s., not significant; n.c., notcomputed.hP< 0.001.0~001


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714 E . M ol i r z a G r ima et al.one of the isolates containing large amounts ofEPA was grown in a chemostat in order to studythe relationship between growth rate and bio-chemical composition, with special emphasis onEPA productivity.In a first stage, growth-limiting factors wereestablished and modelled in order to understandthe kinetic behaviour of the culture. The variationof steady-state biomass concentration and pro-ductivity with dilution rate is shown in Fig. 1.These curves are characteristic of continuouslight-limited cultures,30 where steady-state growthdepends on the availability of light. Light intensity,Z, inside the culture is dependent on cell concen-tration, C. In dense cell cultures, there is a lightgradient along the radius of the culture vesel dueto light attenuation. Thus, as biomass concentra-tion increases, then average internal lightdecreases. Furthermore, as physiological adapta-tion of the cells seems to respond to an average31,32ight intensity,- it is necessary to calculate theaverage light intensity to which cells are exposed.Based on Lambert-Beers law, Evers12described the distribution of light in a cylindricalvessel illuminated evenly from all sides obtainingthe following expression for light intensity at agiven point within the culture:

)* sin2 #]] ] d # (1)

P (mg I-W)-14

1,000 12800 -10

6OC- 8-6

4004

200 - -2o ,,,,,,,,,I,,,,,,,..,,..~~l.~~I.~.~~~~~~l -00 0.5 1 1.5 2 2.5 3 3.5 4

D 102 h-)Fig. 1. influence of dilution rate, D, on steady-state bio-mass concentration, C, and biomass productivity, P. Thesolid lines represent theoretical valu& obtained- with themodel for both variables.

where C is the biomass concentration, K , is thebiomass absorption coefficient, S is the distancefrom the vessel surface to an internal point, 4 isthe angle of incidence of the light path, R is thevessel radius and 1, is the incident light intensityon the culture surface.Up to now, K , has been considered constantwithin each species, although several authors havereported that K , strongly depends on the pigmentcontent of the biomass. 13,33 In, a new approach,we have measured light attenuation in six culturesof the same strain of I sochvs i s ga lbam grownunder different lighting conditions, and thereforewith different pigment contents, in order to calcu-late the variation in the absorption coefficient.These results are shown in Fig. 2 and details aregiven in Table 2. It may be observed that as pig-ment content is lowered, K , decreases as much as100%. When K , is plotted against pigmentcontent, Xp, a linear relationship results, making itpossible to apply linear regression and renderingthe equation:

K , = Y;X, + Yh (2)where Yj, = 0.0199 m* g-l (biomass) andY:,= l-7357 m2 g- (pigments); r* = O-967.

Therefore, not only is the global absorptioncoefficient not constant, but it may also be consi-dered the result of two contributing factors, thepigment-free biomass, Yb, and the other pigmentabsorption, Yi, (absorption coefficient normalizedto total pigment content). The contribution of Ybto biomass absorption coefficient, K ,, varies from47 to 24% for X,, values of 1.29 x lo- and3.65 X 10e2, respectively.

Absorbance

-11 .~~~~,~,I,~~,I~~~~/,~~'I','~~'~'I,(,,I,,,,~,,',/,"'I0 100 200 300 400 500 600 700 800 900 1,000 ,100C (mn I-)

Fig. 2. Influence of biomass concentration with differentpigment composition on culture absorbance.


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Production of polyunsaturated fatty acids by micr oalgae 715Table 2. Effect of pigment content on absorption coefficient, K,

M ass fraction of o t a l p i gmen t s, X, X IOI 29 207 209 2.33 3.51 3-65

Chlorophyll a (mass fraction lo*) o-79 1.22 1.31 1.52 2.09 2.12Chlorophyll c (mass fraction 10z) 0.16 0.34 @35 0.39 0.58 0.56Carotenoids (mass fraction lo*) 034 0.53 0.44 0.42 O-85 0.87FX lO* (m2g- biomass) 3.755.9996 0.9999.898 0.9999.898 6.743.9995 0.9992.959 0.9986.163

To obtain the average light intensity for thewhole of the photobioreactor, it is sufficient tointegrate eqn (1) between 0 < SIR to obtain thefollowing expression:exp { K, C[( R - S)cos $

+[R2-(R-S)2sin2~]o~5]}d~dS (3)where the average light intensity is now a functionof biomass concentration and its pigment content,bearing in mind the relationship between K, andXp (eqn (2)).A hyperbolic model modified by an exponent,n, similar to that of Moser34 for nutrient-limitingcultures, and analogous to Bannisters35 shapeparameter describing the abruptness of the tran-sition from low to high light region, has been usedto determine the relationship between specificgrowth rate and light intensity:

where pm,, is the maximal specific growth rateand 1, is a constant representing the affinity ofcells to light.Assuming perfect mixing and, further, that partof the absorbed energy can be utilized for cellmaintenance, by steady-state material balanceinto a chemostat in which ,u is expressed by eqn(4), the following expression is obtained:

&!!!!ELmI + zkIn the above m=0*00385 h- is the experimen-tally determined specific maintenance rate31 whenthe light intensity was set at zero (darkness) in abatch culture. The slope of ln( C/C,,) over time,

where C, represents the initial biomass concen-tration, gives the value of m.As mean light intensity and, consequently, thespecific growth rate depend on C and Xp, onemight suppose that the same steady state (D, C)could be reached with different (C,X,) pairs.Nonetheless, in our experimental device a uni-vocal relationship between D and C has beenfound as the same steady-state biomass concen-tration is reached when the same dilution rate isanalysed at different times and different states ofgrowth. Thus, an incident light intensity and vesselgeometry remain constant, there must be a rela-tionship between steady-state biomass concentra-tion and pigment content which subtracts thedegree of freedom in eqn (3) and makes the pairs(D, C) unique. For the system and environmentalconditions assayed here, this relationship can beexpressed by the following polynomial:

Xp= 1.12 x 1O-2-8.6x lO-jC+2.6 x 1WHC2r2 = 0.992 (6)Pairs (D, I) were obtained by applying eqns (4)and (6) with experimental values of C, X,, and D.After adjustment by non-linear regression to eqn(5), the values of the parameters and regressioncoefficient are: pmax= O-046 h-, Ik= 9.67 x 1OJ5quanta cme2 s-l, It= 1.7, r2 = 0.9975.Figure 3 represents the real and simulated data(solid lines). The saturation intensity was 40

quanta cme2 s-l as can be seen in Fig. 3. Theminimum light intensity necessary to maintain thebasal metabolism of the biomass is obtained at thelimit D=O, 1.12 X 1015 quanta cme2 s-l. This iscalled the compensation intensity.Biochemical compositionIt has been well known since the first publicationson applied algology3h that growth conditionsdetermine the biochemical composition of the


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716 E. Molina Gri rna et al.p lo2h-)

SF

1i4~~,~,.~~,,,~~,~~~,,Illll~~/~~I~ ,~~~~l.,,lll,,it, 5 10 15 20 25 30 35 40 45 50 55 60I (quanta cm-*s-l) IO-

Fig. 3. Variation of specific growth rate, ,u, with averagelight intensity, I.

cells. In a general sense, fast-growing cells syn-thesize a greater amount of proteins than lipids tomaintain the cellular division rate, as the duplica-tion period is short. At low dilution rates, that is tosay, slow-growing cells, a high proportion of lipidsand low protein content is obtained, making thelipid to protein ratio around 2. This variation isrelated to the different functions of biomoleculesin cell metabolism, during which storage products,mainly neutral lipids, are synthesized when theneed for the structural compound (i.e. proteins) isreduced (see Fig. 4).Furthermore, this change in the biochemicalcomposition has an effect on the fatty acid pro-file of the lipid fraction. Palmitic (16 :0) andpahnitoleic (16 : 1) acids constitute over 7% d.w.,with less EPA, which is around 5% d.w., in cellsgrown at low dilution rates (see Fig. 5). At anintermediate D, EPA remains, proportionally,approximately the same, although 16 : 0 and 16 :1content fall to 3*.5% and 4%, respectively. Finally,at high dilution rates, all fatty acid contentdecreases, with EPA the major fatty acid (see Fig.5). Thus, a high proportion of storage lipids, con-sisting mainly of 16 :0 and 16 : 1, are found inslow-growing cells. When light limits growth, cellshave to synthesize chloroplast membranes inorder to collect scarce light efficiently. Thesemembranes are composed of polar lipids whichare highly unsaturated. 37,38 In I . galbana, around30% of total fatty acids found in glycolipids isEPA.39 This may explain why the EPA contentremains around 5% d.w. in the range ofD= 0.0024 h-l to 0.021 h- , as in this interval

% Dry weightSOT

0'0.1 0.6 1.1 1.6 2.1 2.6 3.1 3.6 4.1D 10 (h-l)

1 Lipids +ProteinsFig. 4. Influence of dilution rate, D, on lipids and proteincontent.

% Dry weight8 i-

0.7 1.2 1.7 2.2 2.7 3.2D 10 (h-l)

* 140 * 16:O U 16~1 -=- 18~4 X 20~5 4 22~6Fig. 5. Fatty acid profiles at different dilution rates, D.

growth is strongly limited by light and the propor-tion of glycolipids is high.EPA and DHA separationThe focus of microalgal biotechnology on highlyvaluable fine chemicals requires not only masscultivation techniques and processes, but alsodevelopment of later processing methods. At thispoint, the commercialization of purified biomole-cules (SA, EPA and DHA) or the enriched lipidfractions (glycolipids) improves the added valueof the productivity system. Table 3 illustrates this,showing the different stages of fatty acid purifica-tion. The fatty acid content of I. galbana biomassand composition of an extract obtained by extrac-tion and direct saponification are given incolumns 1 and 2. By urea complexation, a PUFA


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Producti on of polyunsaturated fatty acids by microalgae 717Table 3. Fatty acid profiles (%) of initial biomass, lipidextract, urea concentrate (methanol, 4C, urea-fatty acid4 : 1) and HPLC fractions (methanol-water 1% acetic acid80:20; 3 ml min-I; 9.49 mg)

Fattyacid Biomass Extractb Urea SAd EPAd DHA14:o 10.1 10.7 0.3 - - -16:O 20.3 18.9 0.2 I.1 - -16:7n7 21.4 23.2 4.3 - - -18:O 0.7 0.5 1.5 1.1 - -18:ln9 1.4 1.7 @2- - -18:ln7 36 3.2 0.8 _ _ _18:2n6 0.9 0.9 0.2 - - 1.418:3n6 o-2 0.2 0.4 - -18:3n3 1.2 1.3 0,8 - ;::, _20:o 0.1 @2- - -20:ln9 0.2 @3 0.3 - - -18:4n3 6.4 7.3 22.6 94.8 - -20:3n6 0.4 0.2 0.2 - - -22:o - 0.1 0.1 - -20:4n6 o-7 o-7 1.1 - - 3.722:lnll 0.1 ;:: @I- - -22:1n9 - @- - -20:5n3 22.6 22.4 39.4 - 96.0 -24:0 - 0.1 0.1 - - -22:4n6 1.3 1.3 3.6 _ _ _22:5n3 0.2 0.1 0.1 - - _22:6n3 8.4 6.8 23.4 - - 94.9Total 100.0 99.9 100.0 97.0 100.0 100.0Lyophilized I . galbana biomass.Extract obtained by direct saponification at 60C during 1 hwith hexane-ethanol96% 2 : 5 v/v, 8 g KOH/380 ml.Urea concentrate obtained at a MeOH-fatty acid ratio of4 : 1 and successive crystallization of inclusion complexes at4C.dSA, EPA and DHA fractions obtained by semi-preparativeHPLC.

concentrate is obtained from this extract (column3). Finally, three fractions in which the major fattyacids are SA, EPA and DHA, respectively, areobtained by semi-preparative HPLC.

As might be expected, the same fatty acidprofile is observed in biomass and in the lipidextract. After that, saturated and monosaturatedfatty acids were well eliminated by the ureamethod. So, as an example, while the palmitic acidpercentage diminishes from 10.7 to 0*3%, EPAand DHA percentages increase from 22.4 to39.4% and from 7-O to 23.4%, respectively. Table4 shows a comparison of data on fish oils from theliterature and those reported here for I. gulbanabiomass. The inclusion complex methodology wassimilar in the above-mentioned studies, except forAckman et a1.,40 who used ethanol as the ureasolvent instead of methanol. The values of theconcentration factor, R, reached for polyunsatu-rated fatty acids by Ackman et aL40 are the

Table 4. Comparison of the fatty acid composition ofseveral fish oils and lipids in biomass in the present study,before and after urea fractionationFatty acid Lipid Urea Rb

Saturated 9-o 1.1 0.12Monounsat. 67.8 6.9 0.10SA 0.8 3:.: 6.38EPA 5.4 6.02DHA 3.9 29.2 7.49Saturated 30.8 - -Monounsat. 27.7 0.3 0.01SA 1.7 7.2 4.20EPA 13.9 36.8 2.60DHA 13.6 40.9 3.01Saturated 18-3 1.0 0.05Monounsat. 50.8 3.3 0.06SA 2.4 10.0 4.20EPA 12.1 27.6 2.30DHA 11.7 44.6 3.80Saturated 31.8 2.3 0.07Monounsat. 27.4 5.8 0.21SA 7.0 22.6 3.22EPA 22.4 39.4 1.76DHA 7.0 23.4 3.34Wrea concentrate.Wrea concentrate-lipid ratio.

Reference40

41

24

Present study

Table 5. Comparison of the purity of SA, EPA and DHAobtained by HPLC separation from I. galbana biomass(present study) and fish oilsReferences

~--Present study414243445

18:4n394.893.1

-

Purity (%)20:5n3

96.085.691.0

91-969097.3

22 6n394.983.185.5

GY-

highest, although the differences may be due tothe initially high proportion of monounsaturatedfatty acids, which gives rise to greater richness inPUFAs.In Table 3, the purities in SA, EPA and DHAof the three fractions obtained by HPLC are alsoshown. Major fatty acid yields in each of thesefractions of SA, EPA and DHA were 100, 99.6and 94-O%, respectively. Table 5 shows that puri-ties reached in this study are slightly higher thanthose reported in the literature.

It must be kept in mind that purity is suscep-tible to improvement only at the expense of a


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718 E. M olina Grima et al.decrease in yield, which depends on the collectioncut-off point of fractions. However, only Tokiwaet aZ.42 and Cohen & Cohen45 report values forthe yields obtained. Tokiwa et a1.42 reported66.7% for EPA and 53.3% for DHA and Cohen& Cohen45 reported 85% for EPA, much lowerthan the 99.6% and 94.0% for EPA and DHArespectively, found in this study.

ACKNOWLEDGEMENTThis research has been supported by a grant fromthe Con&ion Interministerial de Ciencia y Tecno-logia (CICYT), (BIO 91-0652).

REFERENCES1.

2.

3.

4.

5.6.

7.

8.

9.

Borowitzka, L. J. & Borowitzka: M., B-carotene fromDunaliella salina. Paper presented at 6th InternationalConference on Applied Algology. Process in Bio-technology of Photoautotrophic Microorganisms, C&skeBudijovice, Czech Republic, 6-l 1 September 1993.Radmer, R. J., Products from algae grown in closed reac-tor. Paoer oresented at 6th International Conference onApplierd Algology. Process in Biotechnology of Photo-autotrophic Microorganisms, C&ske Budijovice, CzechRepublic, 6-l 1 September 1993.Tantichavoen. M.. Bunnap. B.. Siangdung. W. & Chana-wongse, L., &ltivation ;f & in&a u&g secondarytrated starch wastewater. Paper presented at 6th Inter-national Conference on Applied Algology. Process inBiotechnology of Photoautotrophic Microorganisms,C&ske Budijovice, Czech Republic, 6-l 1 September1993.Viera, V., Break even point analysis for microalgae bio-technology products. Paper presented at 6th Interna-tional CGnfkrence on Applied Algology. Process inBiotechnology of Photoautotrophic Microorganisms,Ciske Budijovice, Czech Republic, 6-l 1 September1993.Kyle, D., Production and use of lipids of microalgae.Lipid Technology, May-June (1992) 59-64.Yongmanitchai, W. & Ward, 0. P., Omega-3 fatty acids:alternative sources of production. Proc. Bi ochem., 24(1989) 117-25. .Cohen, Z. & Shiran, D., Biosynthese overproduction ofEPA in microalgae. Paper presented at 6th InternationalConference on Applied Algology. Process in Biotech-noloev of Photoautotroohic Microorganisms, C&skeBud&vice, Czech Repubhc, 6-11 Septekber 1993.L&X Alonso. D.. SBnchez P&ez. J. A., Garcia Sgnchez,J. i., Garcia &macho, F. & Molina Grima, E., Improve-ment of the EPA content in isolates of I sochrysisgalbana. J. Mar . Biotech., l(l 993) 147-9.Kennedy, M. J., Reader, S. L. & Davies, R. J., Fatty acidproduction characteristics of fungi with particularemphasis on gamma linolenic acid production. Bio-technol. Bi oeng., 42 (1993) 625-34.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26

Guterman, H., Vonshak, A. ALBen-Yaakov, S., A macro-model for ouidoor algal mass production. Biotechnol.BioenR., 35 (1990) 809-19.Mark< H., Modelling of algal production system. InAl gal Bi omass: Producti on and Use, ed. G. Shelef & C. J.Soeder. Elsevier North Holland Biomedical Press,Amsterdam, 1980, pp. 361-83.Evers, E. G., A model for light-limited continuouscultures: growth, shading and maintenance. Biocechnol.Bioeng., 38 (199 1) 254-9.Falkowski, P. G., Dubinsky, Z. & Wyman, K., Growth-irradiance relationshio in Dhvtoolankton. Limnol.Oceanogr., 30 (1985) 3ii-21. a + LSukcnik, A., Falkowski, P. G. & Bennett, J., Potentialenhancement of photosynthetic energy conversion inaleal mass culture. Biotechnol . Bioenn., 30 (1987)970-7. I ,Lanrrdon. C.. On the causes of intersoecific differencesin thv g&v&-irradiance relationship ior phytoplankton.II. A general review. J. Plank. Res.. 10 (1988)1291-372.L6pez Alonso, D., Molina Grima, E., Sgnchez P&ez, J.A., Garcia Stichez, J. L. & Garcia Camacho, F., Isola-tion of clones of Zsochrysis galbana rich in eicosapentae-noic acid. Aquacul tur e, 102 (1992a) 363-7 1.Lbpez Alonso, D., Molina Grima, E., SBnchez P&ez, J.A., Garcia Stichez, J. L. & Garcia Camacho, F., Fattyacid variation among different isolates of a single strainof isochtysis galbana. Phytorhemistry, 31 (19926)3901-4.Caten, C. E. & Jinks, J. L., Quantitative genetics. InSecond Intemuti onal Svmoosium on the Genetics ofIndustrial Microorganiims, ed. K. D. McDonald.Academic Press. New York. 1976. DD. 93-l 11.Wood, A. M., iande, R. & Fryxeli,G. A., Quantitativegenetic analysis of morphological variation in an antarc-tic diatom grown at two light intensities. J. Phycol., 23(1987) 42-54.Molina, E., Martinez, M. E., S?inchez, S., Garcia, F. &Contreras, A., Growth and biochemical compositionwith emphasis on the fatty acids of Tetruselmis sb_ Appl.Microbial. Biotechnol., 36 (1991) 21-5.Kochert, G., Quantitation of the macromolecular com-ponents of microalgae. In Handbook of Phycological*Methods. Physiologizal and Bi ochemical tiethbds, eh. J.Hellebust & S. Cragie. Cambridge University Press,Cambridge, 1978, pp. 189-95.Lepage, G. & Roy, C., Improved recovery of fatty acidthrough direct transesterification without prior extrac-tion or purification. J. L ipi d Res., 25 (1984) 1391-6.Lowry, O., Rosebrough, N., Farr, A. & Randall, R.,Protein measurement with the folin phenol reagent. J.Biol. Chem.. 193 (1951) 265-75.Haagsma, G., van Gent; C. M., Luten, J. B., de Jong, R.W. & van Doom, E., Preparation of an w3 fatty acidconcentrate from cod liver oil. J. Am. Oil Chem. Sot., 3(1982) 117-18.keg& de1 Castillo, C. I., L6pez Alonso, D., FertindezSevilla, J. M., Garcia Stichez, J. L. & Acitn FernBndez,F. G., .Heridability of fatty acid content in Zsochrysisnalbana and Phaeodactylum tricornutum. Paperpresented at 6th International Conference on AppliedAlaologv. Process in Biotechnology of PhotoautotrophicM&oorganisms, C&ske Budijo&e, Czech Republic,6-l 1 September 1993.De la Noue, J. & De Pauw, N., The potential of micro-algal biotechnology: a review of production and uses ofmicroalgae. Biotech. Adv., 6 (1988) 725-70.


9/9

Production ofpolyunsatur atedfatty acids by micr oalgae 719

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

Rowlands. R. T.. Industrial strain imnrovement: muta-genesis Ad r&dom screening proiedures. EnzymeMicrob. Technol.. 6 (1984) 3-10.Craig, R., Reich&It, B. 2. & Reichelt, J. L., Geneticengineering of micro-algae. In Micro-algal Biotechno-lo& ed. h;i. A. Borowigka & L. J. Bor&itzka. Cam-bridge Univ. Press, Cambridge, 1988, DU. 415-55.Sha&h, A., Ben-Amotz, A. &&ron, h%, Production andselection of high B-carotene mutants of Dunaliellubardawil (Chlorophyta). J. Phycol., 27 (1991) 652-6.Richmond, A., A prerequisite for industrial micro-algalculture: efficient utilization of solar irradiance. InA& al BiorechnoloKv, ed. T. Stadler, .I. Mollion, M. C.Veydus, Y. Karam&os, H. Morva & D. Chritiaen. Else-vier Academic Press, Amsterdam, 1988, DD. 237-44.Molina Grima, E., SBnchez P&ez, 3: A., GarciaCamacho, F., Garcia Sgnchez, J. L. & L6pez Alonso, D.,n-3 PUFA Productivity in chemostat cultures of micro-algae. Appl. Microbial. Biotechnol., 38 (1993) 599-605.Raven, J. A., Limits to growth. In Micro-algal Biotechno-logy, ed. M. A. Borowitzka & L. J. Borowitzka.Cambridge University Press, Cambridge, 1988, pp.331-56.Sukenik. A.. Levv. R. S.. Levv. Y.. Falkowski. P. G. &Dubinsky, Z:, Op&nizingalgalb;io&ss prod&on in anoutdoor pond: a simulation model. J. Appt. Phycol., 3(1991) 191-201.Moser, A., Kinetics of batch fermentation. In Biotech-noZogy Vol. 2, ed. H. J. Rehm & G. Reed. VCH Ver-lagspesellschaft, Weinheim, 1985, up. 243-83.B&&ister, T. T.; Quantitative desbxption of steady state,nutrient-saturated algal growth, indicating adaptation.Li mnol. Oceanogr., 24 (1979) 76-96.Burlew, J. S., Atgal Culture fr om Laboratory to PilotPZant.Washington DC, Carnegie Institution, 1953.

37.

38.39.

40.

41.

42.

43.

44.

45.

Forder, J. & Steer, M. W., The use of quantitativeelectron microscopy in the study of lipid composition ofmembranes. J. Exp. Bot., 27 (1976) 1137-41.Kates, M. & Volcani, B. E., Lipid components of dia-toms. Biochem. Biophys. Acta, 116 (1966) 264-78.Molina Grima, E., Garcia Camacho, F., FerntidezSevilla, J. M., AciBn Ferntidez, F., Stichez P&ez, J. A.& Segura de1 Castillo, C. I., EPA productivity fromcontinuous culture of I sochrysis galbana. Paperpresented at 6th International Conference on AppliedAlgology. Process in Biotechnology of PhotoautotrophicMicroorganisms, Cbske Budijovice, Czech Republic,6-11 September 1993.Ackman, R. G., Ratnayake, W. M. N. & Olson, B., Thebasic fattv acid composition of Atlantic fish oils: poten-tial similahties usefuifor enrichment of polyunsa&ratedfattv acids bv urea comvlexation. J. Am. Oil Chem. Sot.,65 (1988) 136-8. -Wille, H. J., Traitler, H. & Kelly, M., Production of poly-enoic fish oil fatty acids by combined urea fractionationand industrial scale preparative HPLC. Rev. Frans.Corps. Gras., 34 (1987) 69-74.Tokiwa, S. & Kanazawa, A., Preparation of eicosapen-taenoic and docosahexaenoic acids bv reversed chasehigh performance liquid chromatograpjly. Bull. Jai. Sot.Sci. F ish., 47 (1981) 675-80.Perrut, M., Purification of polyunsaturated fatty acid(EPA and DHA) ethyl esters by preparative high per-formance liquid chromatography. LC-CC I ntern., 106(1988) 60-2.Grant, J., Multigram isolation of omega-3 fatty acidsfrom fish oil using preparative HPLC. AmericanLaboratoq May (1988) 80-5.Cohen, Z. & Cohen, S., Preparation of eicosapentaenoicacid concentrate from I orphyridium cruentum. J. Am.Oil Chem. Sac., 68 (1991) 16-19.

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