enhancemento of eicosapentaenoic acid - spitulina platensis (cyanobacteria)

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Enhancement of eicosapentaenoic acid (EPA) andlinolenic acid (GLA) production by manipulating

algal density of outdoor cultures of Monodussubterraneus (Eustigmatophyta) and Spirulinaplatensis (Cyanobacteria)Hu Qiang

a, Hu Zheungu

a, Zvi Cohen

a& Amos Richond

a

aMicroalgal Biotechnology Laboratory, The Jacob Blaustein Institute for Desert

Research, Ben-Gurion University of the Negev, Sede Boker Campus 84990, Israel

Published online: 03 Jun 2010.

To cite this article:Hu Qiang , Hu Zheungu , Zvi Cohen & Amos Richond (1997) Enhancement of eicosapentaenoicacid (EPA) and linolenic acid (GLA) production by manipulating algal density of outdoor cultures of Monodus

subterraneus (Eustigmatophyta) and Spirulina platensis (Cyanobacteria), European Journal of Phycology, 32:1, 81-86, DOI:

10.1080/09541449710001719395

To link to this article: http://dx.doi.org/10.1080/09541449710001719395

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Eur..Phycol.1997),2:1-86.rinted in Great Britain 81~~~~~~~~~~~~

Enhancement of eicosapentaenoic acid EPA) and y-linolenicacid GLA) production by manipulating algal density of outdoorcultures of Monodus subterraneus Eustigmatophyta) andSpirulina platensis Cyanobacteria)

HU QIANG , HU ZHENGYUt, ZVI COHEN AND AMOS RICHMONDMicroalgal Biotechnology Laboratory, The Jacob BlausteinInstitute for Desert Research, Ben-Gurion University of the Negev, Sede Boker Campus84990, IsraelReceived 20 November 1995; accepted 10 June 1996)The effect of algal density on cell growth and composition with special reference to the production of eicosapentaenoic acid (EPA) and'y-linolenic acid GLA) was investigated in semi-continuous outdoor cultures of Monodus subterraneusand Spirulina platensis. In bothspecies, an exponential decrease in specific growth rate and a positive skewed pattern in biomass productivity were associated with anincrease in density. In M. subterraneus, the highest EPA cell content (38 of dry weight) occurred at the optimal density of c. 4 g l- ,i.e. the density that yields the highest output rate of biomass per culture volume or area. In S. platensis, high density was associated witha decrease in total fatty acid content, but the relative abundance of GLA increased. GLA content was therefore fairly stable at c. 1% ofdry weight throughout the range of algal density tested. Fatty acid desaturation seemed to be associated with an increase in culturedensity of S. platensis, resulting in an increase in the proportion of the fatty acids 16:1 and GLA and a decrease in 16:0 and 18:2. Usinga flat plate reactor with a narrow light-path and intensive stirring which facilitated high cell concentration,we obtained maximal EPAproductivity of 589 mg 1- I day-l in M. subterraneusand maximal GLA productivity of 26'4 mg 1- day-l in S. platensis. The former isthe highest EPA production rate reported to date for any microalga, and the latter is the first report on GLA productivity by microalgae.The data presented herein suggest that maintaining very high, yet optimal culture densities (i.e. 4-10 g -1) in enclosed reactorsrepresents an efficient operating mode for enhancing productivity of desired polyunsaturated fatty acids.Key words: cell concentration, eicosapentaenoic acid (EPA), y-linolenic acid GLA), Monodus subterraneus, polyunsaturated fatty acid(PUFA), productivity, Spirulina platensis

IntroductionPolyunsaturated fatty acids (PUFAs) have been recog-nized as an essential component in human nutrition aswell as for aquaculture feed (Richmond, 1990; Benemann,1992). Some PUFAs have been reported to have ther-apeutic effects, particular attention having been given to'y-linolenic acid (GLA, 18:3w6) and eicosapentaenoic acid(EPA, 20:5w3) (Belay et al., 1993; Borowitzka, 1995;Cohen et al., 1995). Marine fish oil is at present themajor source of EPA, and GLA is extracted commerciallyfrom the plants evening primrose, blackcurrant (Traitler etal., 1984) and borage (Wolf et al., 1983), as well as fromfungi (Shimizu et al., 1988). The rapidly increasing phar-maceutical interest coupled with the limited availability ofGLA and EPA prompted research aimed at the productionof these fatty acids from microalgae (Cohen et al., 1995;Borowitzka, 1995).

Correspondence to: A. Richmond. Fax: +972-7-6570198. e-mail: [email protected].*Present ddress: Marine Biotechnology Institute, Kamaishi Laboratories,Heita 3-75-1, Kamaishi, Iwate 026, Japan.t Present address: Department of Phycology, Institute of Hydrobiology,Chinese Academy of Sciences, Wuhan, Hubei 340072, P.R. China.

The freshwater eustigmatophyte Monodus subterraneusis a unicellular alga with a relatively high content of EPA(Iwamoto & Sato, 1986) and is regarded as one of themost promising algal EPA producers (Iwamoto & Sato,1986; Cohen, 1994). The effects of environmental factorson the total fatty acid content as well as production rateof EPA in this species have been elucidated underlaboratory conditions (Iwamoto & Sato, 1986; Cohen,1994). The feasibility for mass cultivation of this algaoutdoors is of interest, but cellular EPA content and itsoutput rate under these circumstances have not beensufficiently studied. Spirulina platensis, a multicellularfilamentous cyanobacterium, represents the best algalsource for GLA to date (Cohen et al., 1993; Tanticharoenet al., 1994). S. platensis is commercially cultivated in openponds the world over, primarily as a health food and alsofor animal feed (Richmond, 1990).

Cell concentration is among the major factors affectingthe photosynthetic activity and biomass productivity ofmicroalgal cultures (Soeder, 1980; Vonshak et al., 1982;Tredici et al., 1991; Sukenik et al., 1991; Torzillo et al.,1994; Hu & Richmond, 1994b), but information concern-ing the effect of cell concentration on gross chemical

Eurv J.Phycol. (1997), 32: 81-86. Printed in Great Britain 81


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Hu Qiang et al.content and particularly on fatty acid composition islacking. Results in the few available reports do no tindicate a clear pattern of effects. According to Cohenet al. (1988), an increase in cell concentration resulted in adecrease in the EPA proportion of total fatty acids in thered alga Porphyridium cruentum, whereas in Monodussubterraneus EPA content increased under similar circum-stances (Cohen, 1994). Chrismadha & Borowitzka (1994)observed only a slight effect of cell concentration on thechemical composition of the diatom Phaeodactylum tricor-nutum. Most importantly, in all previous studies relativelylow cell concentrations, i.e. less than 2 g dry weight 1-1,were used. Information on the effect of much higher cellconcentrations on cell content and the production rate ofthe major PUFAs is thus not available.

The past decade has seen continued progress in theattempt to establish cultures of m icroalgae with high cellconcentrations by using new types of photobioreactors(see reviews by Lee, 1986; Chaumont, 1993). Maximalbiomass concentration sustainable in reactors with anarrow light path is higher by more than an order ofmagnitude than that used in open raceways (Torzillo et al.,1994; Lee & Palsson, 1994; Lee et al., 1995), the highestreported cell concentration being 34 g l- I (Hu et al.,1996). High-concentration cultures would not only sus-tain a much higher volumetric productivity of biomass,but should also markedly reduce operating costs. Thiswork attempted to assess quantitatively the effect of awide range of cell concentrations (from 1 to 34 gl-1 ) onthe fatty acid composition and GLA and EPA productionrates in S. platensis and M. subterraneus, respectively,grown outdoors in a flat plate photobioreactor (Hu &Richmond, 1994a).

Materials and methodsOrganismsand growth conditionsMonodus subterraneus Petersen UTEX 151 was obtainedfrom the University of Texas Culture Collection (Austin,TX, USA) and cultivated outdoors on BG-II mediumwith varying concentrations of NaNO 3 (10-25 gl-l).Spirulina platensis (Norstedt) Geitler strain M2 of theCulture Collection of the Centro di Studio dei Micro-organismi Autotrofi of Florence was grown in Zarouk'smedium (Zarouk, 1966) in which the NaNO 3 concentra-tion was 50 gl-'. The pH values of 75 and 9-5 for M.subterraneus and S. platensis, respectively, were maintainedby adjusting the CO2/air ratio using gas flowmeters. Theoptimal culture temperatures of 32C and 36C forM. subterraneus and S. platensis, respectively, were main-tained during most of the daylight period, from c. 0830hours to c. 1800 hours.

The photobioreactorsystem and culture operationsThe cultivation system was a flat, inclined modularphotobioreactor (FIMP) designed for outdoor mass culti-

vation of photoautotrophs, consisting of a series of fourindividual 141 glass reactors measuring 70 cm high, 90 cmlong and 28 cm wide (Hu & Richmond, 1994a). Allreactors were placed in an east-west orientation inclinedto the north (receiving direct beam radiation from thesouth) at a 300 tilt angle as had previously been foundoptimal in summer for Sede Boker, Israel (latitude 30-9N).An air-bubble mixing system providing 251 of air perlitre algal suspension per minute created a very effectiveand systematic circulation in each reactor. Recycling ofthe algal suspension in the entire system was carried outby an air-lift connecting the end reactors in the cascade.Overheating of the culture was prevented by evaporativecooling of water sprayed on the front surface of thereactor. The error range of culture temperature betweenthe set-point (optimum value) and the measured valueswas 1'5C.

The study was conducted during the summer months,employing a semi-continuous culture mode to evaluatethe effect of cell concentration on the fatty acid composi-tion and production rates of EPA and GLA. The cultureswere monoalgal and were harvested every evening byremoval of the appropriate amount of culture suspension,adding fresh medium to restore the pre-set cell concen-tration. All experiments were run for at least five con-secutive days for each regime of cell concentration. Fordry weight and fatty acid analysis, the algal suspensionwas centrifuged or filtered then washed with sterilemedium. The algal biomass was essentially free frombacterial mass.

Analytical methodsSpecific growth rate h-1). When the culture was at steadystate, /z was calculated by measuring the dry weights ofsamples taken at different time periods, using the equation/L= (lnX 2 - InXI)/t 2 - t, where X, and X2 are themean dry weights at times t and t2, respectively.Dry weight measurement. Duplicate aliquots of the algalsuspension were filtered through pre-weighed glass

1.61.4

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.05

.04 ,z

.03 E

.02 2o

000 2 4 6 8 10 12 14

Culture density g 1)Fig. 1. Specific growth rate (filled circles) and biomassproduction (open circles) as affected by culture concentration ofM. subterraneus grown outdoors in the flat plate photobioreactor.Bars indicate SD.

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EPA and GLA production by outdooralgal culturesTable . Effect of cell density on fatty acid content and composition of Monodus subterraneus

Fatty acidFatty acid composition ( FA) content

( ry wt)Cell density 16:1 16:1 16:1 18:1 18:1 18:2 18:3 20:3 20:4(gl - ) 14:0 16:0 0ll a uw7a a 18:0 w9 w7 w6 w6 w6 w6 EPA EPA TFA

0-5 2-4 241 4-3 23-1 1-3 13 9-0 1'5 33 1 5 07 5 2 21'2 2'1 9-60'8 25 24-3 4 4 23-7 1-2 13 8'9 15 31 1-7 07 5-0 209 2'1 10-215 23 23-3 4-3 23'3 1-3 1'2 8'9 1-5 3-2 17 0'7 5 4 22-2 2-3 10 62-0 2-1 23-4 2-3 25-8 1-4 1'0 6'2 0-6 2-2 0-7 0-6 5 4 26-9 3-4 1274-0 2-3 20-2 2-0 26-9 1-6 0-6 4-5 - 2-0 1-0 0-3 4-7 32-1 3-8 11'85-0 1-9 22-9 4-2 25-9 1-5 0-7 5-1 1-0 2-6 0-5 0-3 5-0 29-4 3 4 11-66-0 5-2 20-9 4-2 24-9 1'1 0-6 7-0 1-0 3-0 3-0 0-3 5 4 27-8 3-2 11-77-0 3 9 22-5 3-1 28-0 - 0-7 7-2 1-2 3 4 3 4 0-2 5 3 25-6 2-7 10-4

10'0 7-1 23-0 - 30-5 - 0-8 8-9 - 2-4 2-4 - 4 7 20-9 2-4 10-512-0 2-6 28-4 - 29-4 - 0-9 7-8 1'1 2-7 2-7 - 5-2 20-5 1-9 9-3

Cultures were semi-continuous, harvested daily in the late afternoon. The data shown are mean values of at least two independent samples, each analysedin duplicate. Maximal difference between duplicates was less than 10 .TFA, total fatty adds; EPA, eicosapentaenoic acid.aTentative assignment.

microfibre filters (Whatman, GF/C), washed with 05 NHC, dried in an oven at 105C for 4 h and then weighed.

Fatty acidanalysis. Freeze-dried cells were transmethylatedwith methanol-acetyl chloride according to Cohen et al.(1993), heptadecanoic acid being added as an internalstandard. Fatty acid methyl esters were identified by co-chromatography with authentic standards (Sigma, StLouis, MO) and by calculation of the equivalent chainlength. Fatty acid content was determined by comparingeach peak area with that of the internal standard andcorrected accordingly. Gas chromatographic analysis wasperformed on a Supelcowax 10 (Supelco, Bellefonte, PA)fused silica capillary column (30 m x 032 mm) at 200C(FID, injector and flame ionization detector temperature2300C, split ratio 1: 100).

Results and discussion

Growth rate and biomass productionThere was a clear relationship between algal density andbiomass production in M. subterraneus. An increase inalgal density resulted in an exponential reduction in thespecific growth rate, whereas productivity of biomassfollowed a positive skewed pattern in response to increas-ing algal densities (Richmond, 1988). In M. subterraneus,increasing algal densities resulted in increased productivityup to the optimal density; a further increase in algal densityabove optimal led to a gradual decline in productivity(Fig. 1). Growth and biomass production of S. platensis(not shown) followed essentially the same pattern as thatof M. subterraneus, except that the optimal algal densitywas 82 1-I7 g - and maximal productivity was

Table 2. Effect of culture density on fatty acid content and composition of Spirulinaplatensis

Fatty acid contentFatty acid composition ( f TFA) (%)dry wt)Mean cell concentrationin the morning (g 1) 16:0 16:1 18:0 18:1 18:2 GLA GLA TFA

2-2 46-7 4-1 1-2 4-8 22-4 20-6 1I0 5-24-1 45-1 4-0 0-9 5 5 21-5 20-2 1-0 5-06-5 43-9 5'1 1-3 5-8 18-9 23-5 1I0 4 48-3 40-0 4 7 11 4 7 16-4 25-7 11 4-8

10-2 44-2 5-8 1-3 5 5 18-4 26-1 1'1 4-015-5 39-0 6-1 11 5-0 16-6 25-6 09 3 520-7 36-1 7-1 1-0 5 4 16-0 26-4 09 3625-1 36-6 7 3 1-4 5-0 15-8 275 10 3530-8 38-9 6-4 1I0 4-1 15-7 25-4 09 3-834-2 40-0 6-3 1-0 4-1 15-0 24-8 0-9 38

Cultures were semi-continuous, harvested daily in the late afternoon. The data shown are mean values of at least two independent samples, each analysedin duplicate. Maximal difference between duplicates was less than 10 .GLA, y-linolenic acid; TFA, total fatty acids.

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Hu Qiang et al.

0 2 4 6 8 10 12 1.

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Fig. 2. The proportion of 18:2 (open circles) and 18:3 (filledcircles) in the total fatty acids as affected by algal density inS. platensis.

2 1 i 03 g 1-l day-I-distinctly higher than the valuesfor M. subterraneus.

The lower specific growth rates associated withincreasing algal densities in M. subterraneus (Fig. 1) aswell as with S. platensis (not shown) probably reflectedincreased light limitation, and the marked differences inoptimal cell concentration between M. subterraneus andS. platensis may represent differences in the maximal netphotosynthetic efficiency attainable by the species.

Fatty acid compositionThe effect of cell concentration on fatty acid compositionof M. subterraneus is shown in Table I. The major fattyacids were EPA, 16:0, 16:1w7 and 18:1w9. When algaldensity was maintained at its optimum (c. 4gl-'), theproportion of EPA as a percentage of total fatty acids washighest, reaching 321 , whereas the proportions of allother fatty acids except for I6:Iw 7 were at their lowest.Lower than optimal cell concentrations resulted in asignificant decrease in both total fatty acid content andthe proportion of EPA; this finding agreed well with that

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Culture density g 1-1)Fig. 4. EPA content (open circles) and production rate (filledcircles) as affected by algal density in M. subterraneus cultures inmid-summer. Bars indicate SD.

of Cohen (1994) that EPA content increased proportion-ally with increasing cell concentrations, from 02 to 34 g1 '. When cell concentrations rose above the optimallevel, however, EPA content and its share in the total fattyacid profile became smaller.

The major fatty acids in S. platensis (strain M2) areGLA, 16:0, 16:1, 18:0, 18:1 and 18:2 (Table 2). As celldensity increased, the profile of fatty acids changedsignificantly, resulting in an increase in 18:3 from c. 20(of total fatty acid) at a cell density of 2 g I- l to c. 28 at25 g l- I. This was associated with a decrease in 18:2 fromc. 22 to c. 15 . The proportion of 18:2 did not changeand that of 18:3 was even slightly reduced at a furtherincrease in cell density (Fig. 2). Evidently, fatty acids tendto be more desaturated at higher cell densities: in additionto the increase in GLA associated with a decrease in 18:2,the percentage of 16:0 decreased while that of 16:1increased (Table 2). Cohen et al. (1993) reported that 18:0and 18:1 occur mainly in neutral lipids, perhaps function-ing as reserve material. The concentrations of these twofatty acids were fairly stable over a wide range of algaldensities, the percentages of 18:0 and 18:1 being 1I1(SE = 0-06 , n = 10) and 51 (SE = 0-24 , n = 10)of total fatty acids, respectively (Table 2). Evidently,

300E2>4:3aSCD

30 35Culture density g 1-1)

Fig. 3. Chlorophyll content as affected by culture density. Opencircles, M. subterraneus; filled circles, S. platensis. Bars indicateSD.

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Culture density g 1-1)

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Fig. 5. GLA content (open circles) and production rate (filledcircles) as affected by algal density in S. platensis cultures inmid-summer. Bars indicate +SD.

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EPA and GLA production by outdoor algal culturesTable 3. Comparison of production rate and cell content of eicosapentaenoic acid EPA) in various species of microalgae

Cultivation Growth EPA content EPA productivityAlgal species Cultivation vessel mode conditions ( ry wt) (mg l- day - ') ReferencesNannochloropsissp. Glass column, 5 cm ID B CL a 36 Sukenik (1991)Phaeodactylum tricornutum Glass tank, 561 C LD 33 251 Yongmanitchai &Ward (1992)P. tricornutum Tubular reactor, 501 C Outdoors 19 47'8 Molina Grima et al. (1994)Monodus subterraneus Erlenmeyer, 0 11 SC CL 34 257 Cohen (1994)M. subterraneus Flat plate reactor, 141 SC Outdoors 38 589 This work

ID, internal diameter; B,batch; C, continuous; SC, semi-continuous; CL, continuous illumination; LD, light-dark cycle; outdoors, natural condition.aEPA content, 022 pg per cell.

accumulation of neutral lipids does not take place inresponse to an increase in cell concentration.GLA and EPA are primarily concentrated in polar lipidsand especially in galactolipids as membrane components

(Nichols &Wood, 1968; Cohen et al., 1987; 1988; Sukeniket al., 1989). The high proportion of GLA and EPA ingalactolipids suggests that these acids may be largelyconfined to photosynthesizing lamellae typical of bothcyanobacteria and eukaryotic algae (Nichols & Wood,1968; Sukenik et al., 1989; Hudson & Karis, 1974). Thereis evidence that shade adaptation takes place by increas-ing the cellular content of the photosynthetic membranecomplex as well as its surface area (Sukenik et al., 1987,1989; Berner et al., 1989). Increasing the GLA and EPAshare of the total fatty acids (Tables 1, 2) as well as thephotosynthetic pigments (Fig. 3) in response to anincrease in cell concentration and thus, presumably, tolight limitation may reflect such a photoadaptive process.Nevertheless, an increase in cell concentrations above theoptimum (above c. 7gl -1) in M. subterraneus did notaffect cell chlorophyll, which remained stable (Fig. 3),while the proportion of EPA gradually declined (Table 1).Increasing culture density above 25 gl-l in S. platensisresulted in a slight decline in the proportion of GLA(Table 2) and a gradual decrease in the photosyntheticpigments (Fig. 3). This may reflect yet another phase ofthe photoadaptative process: when light limitation stem-ming from the ultra-high cell concentrations becomesvery extreme, additional pigment accumulation and/orgreater surface area of galactolipid-associated thylakoidmembranes would lead to an even more severe attenua-tion of incident light. Thus, decreasing the relativeabundance of pigments as well as the GLA- and EPA-enriched galactolipids during very extreme self-shadingmay represent an efficient adaptation to extreme lightlimitation.

GLA and EPA productivityEPA and GLA production rates as affected by algaldensity are shown in Figs 4 and 5. In mid-summer,maximal EPA productivity of 589 mg l day- 1 (155 gdry wtl-l day - l x 38 dry weight) was achieved inM. subterraneus cultures maintained at the optimal algal

density. Likewise, maximal GLA productivity of 264 mgI-l day-l (2'2 g dry wet 1 day- l x 1 2 dry weight)was achieved in the S. platensis cultures at optimal algaldensity. The former value represents the highest reportedfor the production of EPA from microalgae either out-doors or in the laboratory (Table 3); the latter is the firstreport on production rate of GLA by S. platensis.Maintaining optimal cell concentration for maximizingproductivity of desired PUFAs, however, does no tnecessarily meet commercial considerations. The optimalstrategy regarding cell concentration will depend on thespecific practical goal. Feeding rates for animals in mar-iculture operations, for example, may be based on cellcounts per volume, rendering it desirable to minimize thenumber of microalgal cells needed to achieve the samePUFA ration per animal. Maximum PUFA content per celland not per culture volume is at a premium for mariculture(Dunstan et al., 1993), and this consideration may alsoapply to production of health food tablets. For pharma-ceutical products based on extraction of PUFAs, however,maximizing productivity of PUFAs per culture volumeseems to represent the best economic course.Previous studies aiming to achieve maximal cell con-tents and overall productivities of PUFAs have focusedprimarily on nutrient stress (e.g. Suen et al., 1987; Reitanet al., 1994). Unfavourable culture conditions, however,would reduce the overall biomass productivity, as well asintroducing instability in continuous cultures, raising thehazard for contamination and culture loss (Richmond,1990). This study demonstrated that in order to modifycell chemical content and productivity, manipulating thecell density is preferable to imposing stress, since itpermits all factors affecting growth to function at theiroptimum. Indeed, in algal mass cultures the light regimefor the single cell as affected mainly by cell density shouldrepresent the sole environmental limitation for photo-autotrophic production (Goldman, 1979; Richmond,1988).

AcknowledgementsThe technical assistance of Ben Freihoff and Li Zhen isgratefully acknowledged.

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