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1 Algl Reserh 7 () 7 Contents lists ville t SieneDiret Algl Reserh journl homepge: Impt of nutrient stress on ntioxidnt prodution in three speies of mirolge Koen Goiris,, Willem Vn Colen, Isel Wilhes, Fián León-Tmriz, Lu De Coomn, Koenrd Muylert KU Leuven Tehnology Cmpus Ghent, Fulty of Engineering Tehnology, Deprtment of Miroil nd Moleulr Systems (M S), Cluster for Bioengineering Tehnology (CBeT), Lortory of Enzyme, Fermenttion nd Brewing Tehnology (EFBT), Geroeders De Smetstrt, 9 Ghent, Belgium KU Leuven Kulk, Reserh Unit Aquti Biology, Etienne Seln, 8 Kortrijk, Belgium Universidd de Cuen, Fulty of Chemistry, Shool of Biohemistry nd Phrmy, Av. de Aril, Cuen, Eudor rtile info strt Artile history: Reeived August Reeived in revised form Novemer Aepted Deemer Aville online xxxx Keywords: Mirolge Nutrient stress Physiologil hnges Antioxidnt formtion Mirolge re novel soure of sustinle nturl ntioxidnts with vrious pplitions, inluding food preservtion. To optimize ntioxidnt prodution in mirolge, we investigted the influene of nutrient limittion on ntioxidnt ontent in three speies, Pheodtylum triornutum, Tetrselmis suei nd Chlorell vulgris. Mirolge were ultivted in th ulture under nutrient replete, P- nd N-limited onditions. Totl ntioxidnt tivity of the iomss ws mesured using Trolox equivlent ntioxidnt pity nd squre wve voltmmetry. Additionlly, ontents of rotenoids, phenolis, toopherols nd sori id were mesured. Nutrient limittion, prtiulrly N-limittion, resulted in low ntioxidnt ontent. Both phenoli nd rotenoid ontents were signifintly redued in nutrient-limited ultures. In ontrst toopherols nd sori id levels were higher in nutrient-limited ultures, prtiulrly under P-limittion. Our results indite tht nutrient stress is not n effetive strtegy to enhne overll ntioxidnt ontent in mirolge, lthough it my e useful to enhne prodution of some vitmin ntioxidnts suh s toopherols or sori id. Elsevier B.V. All rights reserved.. Introdution Antioxidnts re widely used s dditives in the food industry to prevent lipid oxidtion []. Although the helth enefits of ntioxidnts re sometimes questioned [], ntioxidnts re nevertheless inresingly eing ommerilized s nutreutils nd food supplements []. In the food industry, there is growing tendeny to reple syntheti ntioxidnts, suh s utylted hydroxytoluene of utylted hydroxynisole, with nturl lterntives. Importnt soures of nturl ntioxidnts inlude, mongst others, rosemry, green te nd grpe seeds []. Mirolge re promising new soure of nturl ntioxidnts [ ]. At present, two speies of mirolge re lredy used ommerilly for the prodution of rotenoid ntioxidnts, i.e. Dunliell for the prodution of et-rotene nd Hemtoous for the prodution of stxnthin. In ddition to rotenoids, mirolge lso ontin other potentilly vlule ntioxidnts, suh s toopherols (vitmin E), sori id (vitmin C) or phenoli ompounds. Toopherols re widely used s dditives to prevent oxidtion of edile oils. Vitmin E ontents in mirolge (..%) n e higher thn those in virgin olive oil, ommon soure of nturl toopherols (.%; USDA Ntionl Nutrient Dtse for Stndrd Referene, ). Mirolge lso Corresponding uthor. E-mil ddress: koen.goiris@kuleuven.e (K. Goiris). ontin high onentrtions of vitmin C (..%), sometimes higher thn the vitmin C ontent found in ornges (..%; USDA Ntionl Nutrient Dtse for Stndrd Referene, ). In terrestril plnts (e.g. in te nd grpes), phenolis re n importnt group of ntioxidnts []. Severl reent studies showed tht phenoli ompounds lso ontriute signifintly to ntioxidnt tivity in mirolge [7,,]. The iohemil omposition of mirolge often responds strongly to nutrient stress. In the pst yers, numerous studies hve investigted the influene of nutrient stress on lipid umultion in mirolge (e.g. []). Nutrient stress lso results in the genertion of free rdil speies in the ell nd my therefore result in hnges in the ontent of ntioxidnts. It is well-known tht nutrient stress n indue umultion of rotenoids in severl speies of mirolge suh s Dunliell [], Hemtoous [] or other hlorophytes like Senedesmus []. Muh less is known out the influene of nutrient stress on other lsses of ntioxidnts in mirolge, lthough n inrese in oth lphtoopherol [] nd sori id [7] ontent hs een oserved in Dunliell slin upon nitrogen stress. In terrestril plnts, inresed formtion of polyphenols is oserved when exposed to vrious forms of oxidtive stress [8], inluding nutrient strvtion [9]. Likewise, n inrese in phlorotnnins, speifi lss of polyphenols, hs een oserved under limited nitrogen vilility in the rown seweed Loophor vriegt []. In mirolge, -9/ Elsevier B.V. All rights reserved.

2 K. Goiris et l. / Algl Reserh 7 () 7 polyphenols hve so fr reeived little ttention. Although there is no informtion ville on the impt of nutrient stress on the phenoli ontent of mirolge, other types of environmentl stress, suh s exposure to metls [] or UV stress [], resulted in inresed phenoli ontent. For mirolge to eome n eonomilly vile soure of ntioxidnts, it is importnt to mximize the ontent of ntioxidnts in the iomss. The im of this study ws to investigte to wht extent nutrient stress n e used to modulte the ontent of ntioxidnts in mirolge. Therefore, we evluted the response to nutrient stress of totl ntioxidnt tivity s well s the ontent of seprte lsses of ntioxidnts (rotenoids, phenolis, sori id nd toopherols) in mirolge. We ompred the influene of oth N nd P-stress on ntioxidnt ontent. We rried out experiments with three speies of mirolge to investigte whether the influene of nutrient stress on the ontent of ntioxidnts is similr in different mirolgl speies. We seleted three speies tht were previously shown to possess high overll ntioxidnt tivity [], i.e. the mrine mirolge Pheodtylum triornutum nd Tetrselmis suei, nd the freshwter Chlorell vulgris.. Mterils nd methods.. Mirolge ulture onditions The hlorophytes C. vulgris (SAG strin -B) nd T. suei (CCAP strin /), nd the ditom P. triornutum (UGent strin Pt8) were ultured in L orosilite glss ottles (Durn, Shott AG, Minz, Germny) using modified versions of Wright's Cryptophyte (WC) medium prepred with either deionized wter for C. vulgris or with syntheti sewter ( g L Homrsel, Zoutmn, Belgium) for T. suei nd P. triornutum. The medium ws sterilized y utolving. Vitmins were dded fter utolving through sterile. μm PTFE filters. Cultures were illuminted using : light:drk regime ( μmol photons m s Philips Cool White fluoresent tues) nd mixed with moisturized filter-sterilized ir (flow rte: L h ). The inoulum ws grown for dys in stndrd Wright's Cryptophyte medium prepred in slt wter or freshwter (depending on the speies), ontining mm N nd. mm P. From these ultures, ml inoulum ws dded to 9 ml of the ulture medium with different N nd P-onentrtions. The nutrient-replete tretments ontined mm N nd. mm P (N:P molr rtio :), the nitrogen-limited tretment. mm N nd. mm P (N:P molr rtio ) nd the P-limited tretment ontined mm N nd. mm P (N:P rtio ). After 8 dys of ultivtion, the iomss ws hrvested y entrifugtion, rinsed with deminerlized wter for C. vulgris ultures or. M mmonium formte for T. suei nd P. triornutum nd then immeditely freeze-dried. The dry iomss ws stored frozen t C under nitrogen tmosphere until further nlysis... Monitoring of mirolgl ultures Mirolgl growth ws monitored dily, h fter the strt of the light period y mesuring optil density t nm []. Quntum yield of photosystem II (F v /F m ), whih is good inditor of nutrient stress, ws lso mesured dily. To ensure tht ll smples hd similr iomss density when mesuring F v /F m, the optil density ( nm) of the ulture ws stndrdized t.. F v /F m ws mesured using n Aqupen C (Photon Systems Instruments, Drsov, Czeh Repuli) fter min of drk dpttion of the mirolge. Mirosopi oservtions were performed using n Olympus BX mirosope equipped with Olympus DP-soft imge proessing softwre. Cell undne ws determined using n improved Neuuer ounting hmer. Cell volume ws lulted ording to Hillernd et l. [] using ell mesures otined with the imge nlysis softwre. Biomss yield ws determined y weighing the totl mount of hrvested iomss fter freeze-drying. N nd P-ontents of the iomss smples were mesured on the lst dy of the experiment. An mount of dry iomss ws digested using lkline persulfte digestion [] nd the N nd P- ontents in the digestte were mesured s nitrte nd phosphte using miroflow TehnionTM segmented flow nlysis system (QuAAtro Sel Anlytil, Brn+Luee, Germny) following the pplition notes of the mnufturer (Brn+Luee, pplition notes Q- - rev. nd Q-- rev., )... Anlysis of totl ntioxidnt tivity Extrts for mesuring ntioxidnt tivity were otined using n ethnol/wter (/, v/v) mixture s previously desried []. Antioxidnt tivity ws mesured y the Trolox equivlent ntioxidnt pity (TEAC) ssy [] nd y mens of squre wve voltmmetry []. Wheres TEAC mesures the rdil svenging tivity y oth hydrogen tom trnsfer nd eletron trnsfer, squre wve voltmmetry only mesures the ility of ntioxidnts to donte eletrons. For the TEAC ssy, ABTS + rdil tion ws generted y retion of ABTS with potssium persulfte. The retion mixture ws llowed to stnd in the drk for h t room temperture nd ws used within two dys. The ABTS + solution ws diluted with deionized wter to give n sorne of.7 ±. t 7 nm. μl of smple ws mixed with.9 ml of diluted ABTS + solution nd the sorne of the resulting mixture ws mesured fter min inution t room temperture. For squre wve voltmmetry (SWV), n Autol PGSTAT (Metrohm Autol B.V., Utreht, The Netherlnds) potentiostt ws used for ll mesurements. The potentiostt ws equipped with glssy ron working eletrode ( mm dimeter), titnium mesh ounter eletrode oted with RuO /IrO lloy (Mgneto speil nodes B.V., Shiedm, The Netherlnds) nd Ag AgCl in M KCl referene eletrode. Tetrutylmmonium perhlorte ws dded to the extrts to inrese the ondutivity of the smples. The working eletrode ws onditioned for s t potentil of. V. The mesuring onditions were s follows: frequeny Hz, mplitude mv, step size mv. For oth ntioxidnt ssys, ntioxidnt tivity ws lirted ginst the syntheti toopherol nlog Trolox (-hydroxy-,,7,8-tetrmethylhromn--roxyli id) (.. mm in ethnol/ wter /, v/v)... Anlysis of different lsses of ntioxidnts Phenoli ontent ws mesured in the sme extrts s those used for ssessing totl ntioxidnt tivity, nd ws determined y the Folin Ciolteu proedure s desried y Goiris et l. []. For this, μl extrt ws mixed with. ml of Folin Ciolteu regent (previously diluted tenfold with distilled wter) nd llowed to stnd t room temperture for min. Next,. ml sodium ironte solution ( g L ) ws dded to the mixture. After inution for 9 min t room temperture, the sorne ws mesured t 7 nm. Totl phenolis were lirted ginst glli id stndrds nd re expressed s mg glli id equivlent (GAE) g iomss. Crotenoids nd hlorophylls were extrted y soniting the iomss in methnol/wter (9/, v/v) mixture, ontining trns-β-po- 8 -rotenl s internl stndrd. Pigments were seprted on Wters Spherisor ODS olumn ( μm;. mm), set t C with Wters Alline 9 hromtogrph using the ternry grdient desried in Wright et l. [7]. Eluted omponents were deteted with Wters 99 PDA detetor nd quntified t nm. Asori id ws quntified following the protool of Brown et l. [8]. Freeze-dried iomss ws mixed with MPA solution (queous solution of g L met-phosphori id + 8 g L eti id), in the presene of EGTA-glutthione to prevent oxidtion of sori id during extrtion. After extrtion, smples were entrifuged nd ph of the superntnt ws djusted with sodium ette uffer (ph.). The extrted sori id ws derivtized using susequent tretment

3 K. Goiris et l. / Algl Reserh 7 () 7 with sori id-oxidse nd o-phenylene dimine. Chromtogrphi nlysis of the derivtized smples ws performed on Wters Alline HPLC y injeting μl liquots on n Allteh Altim C8 μm olumn (. mm) nd isorti elution with (/8, v/v) mixture of methnol nd.8 M potssium dihydrogen phosphte uffer (ph 7.8) t flow rte of ml min.thefluoresene detetor ws set t n exittion wvelength of nm nd emission wvelength of nm. Toopherol ontent ws mesured s desried y Brown et l. [9]. For the extrtion of toopherols, equl mounts of iomss nd sori id were mixed with lkline ethnol (ethnol nd 8 g L KOH / v/v) nd inuted for min t 7 C. After ooling the smple, the toopherols were extrted from the liquid phse using hexne. After evporting the hexne under strem of nitrogen, the residue ws re-dissolved in methnol nd injeted onto n Allteh Altim C8 μm olumn (. mm). Toopherols were eluted from the olumn with mixture of wter:methnol (/98, v/v) t flow rte of. ml min nd deteted y fluoresene detetor (exittion 9 nm; emission nm).. Results nd disussion.. Growth of the ultures Biomss growth s estimted from optil density ws in generl lower in nutrient-limited when ompred to the nutrient-replete ultures (Fig. ). For Chlorell nd Tetrselmis, growth ws impired y N s well s P-limittion while for Pheodtylum growth ws only impired in the P-limited ulture. The iomss yield t the end of the experiment ws signifintly lower in the nutrient-limited ultures thn in the nutrient-replete ulture for ll three speies (Fig. ). For Chlorell nd Tetrselmis, thefinl iomss yield did not differ signifintly etween the N- nd P-limited ultures. For Pheodtylum, the finl iomss yield ws signifintly lower in the N-limited thn in the P-limited ulture. Dt on ell undne nd ell volume (Fig. ) suggest tht differenes in iomss yield were used y oth hnges in ell undne nd hnges in ell size. In Chlorell ultures, the lower iomss yield in the P-limited tretment ws primrily used y lower ell undne, while it ws due to omintion of lower ell undne nd smller ells in the N-limited tretment. In the Pheodtylum ultures, N- nd P-limittion resulted in derese in ell undne ut ell volume ws unffeted. In the Tetrselmis ultures, ell volume deresed in response to N- nd P-stress ut ell undne ws not ffeted. The derese in ell size ws more pronouned in the N-limited thn in the P-limited Tetrselmis ultures. A derese in ell volume ut not ell numers upon nutrient limittion hs een reported efore in Tetrselmis []. In nutrient-stressed ultures, iomss prodution often ontinues ut the nutrient ontent of the iomss dereses. The nutrient ontent of the iomss is therefore often etter inditor of nutrient stress thn the growth rte or the iomss yield. Biomss of the N-limited ultures hd out threefold lower N-ontent thn iomss of the nutrient-replete ultures (Fig. ). The N-ontent ws lso signifintly redued in the P-limited ultures. Similrly, the P-ontent ws up to times lower in the P-limited ultures thn in the nutrient-replete ultures. Agin, the P-ontent of the iomss ws lso slightly lower in the N-limited ultures. This redued uptke of phosphorus under N-limittion hs een desried erlier for Senedesmus []. The hlorophyll ontent of the iomss ws signifintly lower in the nutrient-stressed ultures thn in the nutrient-replete ultures (Fig. ). The derese in hlorophyll ontent ws most pronouned in the N-stressed ultures. This derese in hlorophyll ontent due C. vulgris P. triornutum. T. suei 8. O.D. (A nm ) Fv/Fm ultivtion time (h) ultivtion time (h) ultivtion time (h) ontrol P-lim N-lim Fig.. Optil density (sorne t nm) nd PSII quntum yield (F v /F m ) s funtion of time in nutrient replete, phosphorus limited or nitrogen limited ultures of C. vulgris, P. triornutum nd T. suei. Error rs orrespond to stndrd devition of triplite ultures.

4 K. Goiris et l. / Algl Reserh 7 () 7 ell undne( ells ml - ) Cell volume (µm ell - ) Biomss yield (mg) N-ontent (mg g - DW) P-ontent (mg g - DW) hlorophyll (mg g - DW) 8 8 C. vulgris P. triornutum T. suei ontrol ontrol ontrol to nutrient stress is well-known phenomenon, lso known s lehing []. The quntum yield of photosystem II or F v /F m is useful inditor of stress onditions in mirolge nd this prmeter is often redued s result of nutrient stress []. F v /F m remined stle in the nutrientreplete ultures of ll speies ut delined signifintly in nutrientlimited ultures (Fig. ). This deline strted lredy fter dys nd ws prtiulrly pronouned in the N- nd P-limited Pheodtylum ultures nd the N-limited Tetrselmis ulture. F v /F m is prmeter tht requires only few ml of living ulture nd few minutes to mesure. It is therefore useful tool for routine monitoring of nutrient stress in mirolgl ultures. Fig.. Overview of ulture prmeters (ell density, ell volume nd iomss yield), iomss omposition (nitrogen, phosphorus nd hlorophyll ontents) in three mirolgl speies, grown for 8 dys in nutrient replete, phosphorus limited or nitrogen limited medium. Sttistilly different vlues (p =., one-wy ANOVA, with Holm Sidk postho test, n = ) re indited y different letter for eh mirolgl speies. Error rs orrespond to stndrd devition of triplite ultures... Totl ntioxidnt tivity Two methods were used to quntify the overll ntioxidnt tivity of the iomss: TEAC nd SWV (Fig. ). Antioxidnt tivity of the hrvested iomss s determined y TEAC nd SWV ws times higher in the nutrient replete ultures thn in the nutrient-limited ultures. Nutrient stress resulted in lrger redution of the ntioxidnt tivity thn P-stress. In our previous study [], totl ntioxidnt tivity of ommeril smples from Chlorell, Pheodtylum nd Tetrselmis ws higher in generl. In tht study, however, mirolge were ultured in outdoor photoioretors tht were exposed to nturl sunlight while in this study rtifiil lights with muh lower intensity were used. At high TEAC (µmol TE.g - DW) SWV (µmol TE.g - DW) TPC (mg GAE.g - DW) rotenoid (mg.g - DW) vit C (mg.g - DW) vit E (mg.g - DW) 8,8,,,, C. vulgris P. triornutum T. suei 7 ontrol ontrol ontrol Fig.. Antioxidnt tivity (TEAC nd SWV ssy), totl phenoli ontent (TPC), rotenoid ontent, vitmin C ontent nd vitmin E ontent in three mirolgl speies grown for 8 dys in nutrient replete, phosphorus limited or nitrogen limited medium. Sttistilly different vlues (p =., one-wy ANOVA, with Holm Sidk postho test, n = ) re indited y different letter per ntioxidnt prmeter for eh mirolgl speies. Error rs orrespond to stndrd devition of triplite ultures.

5 K. Goiris et l. / Algl Reserh 7 () 7 light intensities, sturtion of photosystems my result in formtion of retive oxygen speies. Mirolgl ells n respond to this inresed oxidtive stress y produing more ntioxidnts, whih ould explin the higher ntioxidnt tivities in the previous study. SWV provides quntittive dt on the overll ntioxidnt tivity of the iomss, sed on the re under the urve, ut n lso provide qulittive informtion out the mjor ntioxidnts present, sed on the pek potentils [].TheSWVprofiles (Fig. ) show tht the nutrient replete iomss smples hve higher totl re under the urve, ut lso ontin ntioxidnts with lower pek potentils thn the nutrient-stressed ultures, whih indites tht these smples ontin more powerful ntioxidnts. In extrts of the ontrol ultures, distint pek t potentil of. V n e distinguished in ll three speies ut it is unler with whih ntioxidnt this pek orresponds. Further, in P. triornutum, pek t.7 V is present in the voltmmogrm of the iomss from the nutrient-replete medium. This pek n most likely e ttriuted to the presene of fuoxnthin, whih hs the sme pek potentil [] nd is n importnt primry rotenoid in P. triornutum... Content of different lsses of ntioxidnts Different lsses of ntioxidnts responded in different wy to nutrient limittion. The ontent of rotenoids nd phenolis in the iomss deresed in response to N nd P-stress (Fig. ). The ontent of rotenoids ws signifintly redued in the N-limited s well s the P-limited ultures ompred to the nutrient-replete ultures in ll three speies. In Pheodtylum, the rotenoid ontent ws more strongly redued in the N-limited thn in the P-limited tretments while for the other two speies the response to N nd P-stress ws similr. For rotenoids, we lso oserved hnge in the reltive omposition of the rotenoids. Crotenoids n e divided into photoprotetive rotenoids tht protet the ell ginst high light intensities nd free rdils, nd light-hrvesting rotenoids tht trnsfer sored light energy to hlorophyll to e used in photosynthesis. Photoprotetive rotenoids inlude ditoxnthin nd didinoxnthin in Pheodtylum nd violxnthin, ntherxnthin nd zexnthin in Chlorell nd Tetrselmis. The light-hrvesting rotenoids re fuoxnthin in Pheodtylum wheres lutein nd neoxnthin t s essory light hrvesting rotenoids in Chlorell nd Tetrselmis []. The rtio of photoprotetive rotenoids over light-hrvesting rotenoids inresed in the nutrient-stressed ultures (Tle ). This inrese ws more pronouned in the N-limited when ompred to the P-limited tretments. The derese in rotenoid ontent in nutrient-limited onditions oserved in our experiment is in shrp ontrst with the umultion of rotenoids during nutrient stress in Dunliell nd Hemtoous. Dunliell or Hemtoous re reltively unique in tht they umulte seondry rotenoids under stress onditions, often up to severl % of the iomss []. Most speies of mirolge, inluding those used in this study, minly produe primry rotenoids nd these primry rotenoids typilly derese upon stress [7]. Primry rotenoids re struturl omponents of the photosystems, where they svenge retive oxygen speies tht re produed during photosynthesis or ontriute to light sorption for photosynthesis, nd these re not umulted in highonentrtionssourresultsdemonstrte. Like rotenoids, the phenoli ontent ws lso redued in nutrientlimited tretments ompred to the ontrol tretment. Phenolis were redued in the N-limiting tretments of ll three speies studied. Phenolis were lso signifintly redued in the P-limited ultures of Chlorell nd Pheodtylum ut not in Tetrselmis. To the est of our knowledge, no previous dt on the influene of nutrient stress on the phenoli ontent of mirolge hve een pulished. In terrestril plnts [9] s well s in mrolge [], the ontent of phenolis typilly inreses in response to nutrient stress. In mirolge, other stress ftors suh s UV stress [] or metl stress [] lso use n inrese in the ontent of ntioxidnts. In mirolge however, the response of phenoli ontent to nutrient stress seems to e different thn in other orgnisms. As knowledge out the role nd identity of polyphenols in mirolge is limited, there is need for further reserh on this lss of metolites. The ontents of sori id nd toopherols (Fig. ) displyed different response to nutrient stress when ompred to polyphenols nd rotenoids s they were generlly higher in nutrient-limited tretments. In ultures of Pheodtylum nd Tetrselmis, sori id ws inresed in oth N- nd P-stressed ultures ut in Chlorell only in the P-stressed ulture. Toopherols inresed signifintly only in the P-stressed ulture for the three speies. Other studies lso reported signifint inrese in sori id under N-limited onditions in D. slin [7]. Elevted toopherol prodution under N-limited onditions ws oserved in Nnnohloropsis oult [], nd D. slin [7]. On the other hnd, Crllo-Crdens [8] found higher toopherol ontent in T. suei when nutrient deprived ells were given extr phosphorus nd nitrogen... Implitions for prodution of ntioxidnts using mirolge Despite the ft tht the ontent of sori id nd toopherols inresed under nutrient stress, totl ntioxidnt tivity of the iomss deresed. This suggests tht the ontriution of these two vitmins to the overll ntioxidnt tivity ws limited. Crotenoids nd phenolis displyed similr response s totl ntioxidnt tivity, suggesting tht these two lsses of ompounds re importnt ontriutors to the totl ntioxidnt tivity of the iomss. Whether they re oth importnt nnot e onluded from this study. Previous studies, however, C.vulgris P. triornutum T. suei. urrent density (A/dm²) E (V) E (V) E (V) ontrol P-lim N-lim Fig.. Squre wve voltmmogrms of ethnol/wter extrts from three mirolgl speies grown for 8 dys in nutrient replete, phosphorus limited or nitrogen limited medium.

6 K. Goiris et l. / Algl Reserh 7 () 7 Tle Overview of pigment omposition in three mirolgl speies grown for 8 dys under nutrient replete, phosphorus limited or nitrogen limited medium. Sttistilly different vlues (p =., one-wy ANOVA, with Holm Sidk postho test, n = ) re indited y different letter for eh mirolgl speies. Chlorell vulgris Pheodtylum triornutum Tetrselmis suei Control P-lim N-lim Control P-lim N-lim Control P-lim N-lim Crotenoids (mg g DW) LP (mg g DW).8 ±..9 ±.. ±. 7.8 ±..7 ±.. ±.. ±.. ±.. ±..9 ±..8 ±.8. ±..78 ±.. ±.. ±..99 ±..9 ±.. ±. LP/LH rtio. ±..8 ±..9 ±..7 ±.. ±..8 ±.. ±..7 ±.. ±. De-epoxidtion stte. ±.. ±.. ±.. ±.. ±.. ±..7 ±..7 ±..8 ±. Chlorophyll (mg g DW).8 ±..7 ±.. ±..8 ±.. ±.. ±.. ±..9 ±..9 ±. Light proteting pigments ditoxnthin + didinoxnthin (DT + DD) or violxnthin + ntherxnthin + zexnthin (VAZ). Light proteting pigments / light hrvesting pigments; (DT + DD )/ (Chl + fuoxnthin) or VAZ / (Chl +Chl). De-epoxidtion stte: DT / (DD + DT) or (zexnthin + ntherxnthin) / VAZ []. hve indited tht rotenoids nd phenolis n oth hve omprle ontriution to totl ntioxidnt tivity []. The response of totl ntioxidnt tivity s well s the ontent of different lsses of ntioxidnts ws omprle in the three speies studied. This suggests tht our oservtions n e generlized to other speies of mirolge. Mirolge suh s Hemtoous nd Dunliell tht mssively umulte seondry ntioxidnts, however, re likely to disply different response, ut these speies re extremophiles tht re proly the exeption rther thn the rule. Although the overll response of ntioxidnts ws similr, there were nevertheless ovious differenes etween the three speies with respet to the ontent of speifi lsses of ntioxidnts: Pheodtylum hd higher rotenoid ontent thn the other speies, Tetrselmis hd higher ontent of toopherols nd Chlorell hd high ontent of sori id. If mirolge re to e used for ommeril prodution of ntioxidnts, the overll ntioxidnt yield of the ulture is more relevnt prmeter thn the ontent of ntioxidnts in the iomss. The ntioxidnt yield n e lulted y multiplying iomss yield nd ntioxidnt tivity per unit of iomss. The totl ntioxidnt yield is muh higher in nutrient-replete onditions thn in nutrient-limited onditions euse oth iomss prodution nd ntioxidnt ontent of the iomss re mximum when nutrients re non-limiting. The ntioxidnt yield ws times lower in P-limited onditions thn in nutrient-replete onditions. For N-limittion, the ntioxidnt yield ws even 7 times lower. Cultivtion under nutrient-replete onditions will minly result in high yield of rotenoids nd phenoli ntioxidnts. For prodution of toopherols or sori id, nutrient-limited ulture onditions re more fvorle. Beuse nutrient limittion results in n inrese in toopherol nd/or sori id ontent ut derese in iomss produtivity, the degree of nutrient stress will need to e optimized in order to mximize the yield of these ntioxidnts. When mirolge re nutrient-limited, the flow of eletrons from the photosystems to the eletron trnsport hin is impired nd retive oxygen speies re formed [9]. The impirment of the photosystems is illustrted y the derese in F v /F m upon nutrient limittion. The higher de-epoxidtion stte of the xnthophylls lso points to higher prodution of retive oxygen speies []. Therefore, n inrese in ntioxidnt ontent is expeted under nutrient limited onditions. The ft tht totl ntioxidnt ontent of the iomss deresed when nutrients were limiting my suggest tht ells re less proteted ginst these retive oxygen speies. However, this is not neessrily the se, s under nutrient-limiting onditions, the numer of photosystems is gretly redued, illustrted y the redution in hlorophyll ontent of the iomss in our experiment. While eh photosystem my produe more retive oxygen speies under nutrient-limiting onditions, the totl prodution of retive oxygen speies in the ell is not neessrily higher euse the numer of photosystems is gretly redued. In our experiments, the hlorophyll ontent of the iomss deresed muh more strongly thn the ntioxidnt ontent. This resulted in lrger numer of ntioxidnts per unit of hlorophyll under nutrient-limited onditions, espeilly in the hlorophytes (Fig. ). In other studies, the ontent of rotenoid ntioxidnt is often expressed per unit of hlorophyll rther thn per unit of iomss []. Like in our study, these studies generlly report n inrese in rotenoids per unit hlorophyll under nutrient-limited onditions. We lso oserved n inrese in the de-epoxidtion stte of xnthophylls in nutrient-stressed onditions, whih ws explined efore y n inresed rte of xnthophyll yling under these onditions []. normlised ontent per mg hl C. vulgris P. triornutum T. suei TEAC TPC CAR VIT C VIT E TEAC TPC CAR VIT C VIT E TEAC TPC CAR VIT C VIT E ontrol P-lim N-lim Fig.. Antioxidnt tivity (TEAC; μmol TE mg hlorophyll ), totl phenoli ontent (TPC; mg GAE mg hlorophyll ), rotenoid ontent (CAR; mg mg hlorophyll ), vitmin C ontent (VIT C; mg mg hlorophyll ), nd vitmin E ontent (VIT E; mg mg hlorophyll ) in three mirolgl speies grown in nutrient replete, phosphorus limited or nitrogen limited medium. Sttistilly different vlues (p =., one-wy ANOVA, with Holm Sidk postho test, n = ) re indited y different letter for eh mirolgl speies. Error rs orrespond to stndrd devition of triplite ultures.

7 K. Goiris et l. / Algl Reserh 7 () 7 7. Conlusions For the three speies of mirolge studied, nutrient limittion (N s well s P) resulted in derese in totl ntioxidnt tivity of the iomss nd derese in the ontent of rotenoids nd phenoli ompounds. The ontent of sori id nd toopherols, however, ws higher in nutrient-limited onditions thn when nutrients were non-limiting. These results indite tht ultivtion under nutrientreplete onditions should e pplied when mirolge re used for prodution of ntioxidnts for nutritionl or hemil pplitions. Aknowledgments The uthors wish to thnk the Reserh Foundtion Flnders (FWO), projets GA9N nd..9.n, for the finnil support. Referenes [] J. Pokorny, Nturl ntioxidnts for food use, Trends Food Si. Tehnol. (99) 7. [] A. Bst, G.R.M.M. Henen, Ten misoneptions out ntioxidnts, Trends Phrmol. Si. (), [] N. Jin, K.G. Rmwt, Nutreutils nd ntioxidnts in prevention of diseses, in: K.G. Rmwt, J.-M. Mérillon (Eds.), Nt. Prod. 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