Growth phase significantly decreases the DHA-to-EPA ratio in marine microalgae

Transcription

1 Aquult Int (2017) 25: DOI /s Growth phse signifintly dereses the DHA-to-EPA rtio in mrine mirolge Peter Boelen 1,3 Audrey vn Mstrigt 1 Henk H. vn de Bovenkmp 2 Hero J. Heeres 2 Anit G. J. Bum 1 Reeived: 11 Ferury 2016 / Aepted: 13 August 2016 / Pulished online: 25 August 2016 The Author(s) This rtile is pulished with open ess t Springerlink.om Astrt Mirolge re the prinipl produers of long-hin polyunsturted ftty ids (LC-PUFAs) suh s eiospentenoi id (EPA) nd dooshexenoi id (DHA) in mrine eosystems. Alge re used in quulture systems s diret or indiret feed for zooplnkton, filter-feeding mollusks nd lrvl stges of rustens nd fish. Therefore, it is neessry to selet nutrient-rih strins, with high levels of EPA nd/or DHA, preferly during the stge of rpid growth. During the ourse of lgl growth (exponentil to sttionry phse), mny mirolgl speies umulte lipids, espeilly triylglyerols. However, reltively little is known out the effet of growth phse on LC-PUFA umultion. In the present study, solute nd reltive EPA nd DHA levels of seven representtive speies of mrine mirolge were determined during different growth phses in th ulture. Four speies (Pheodtylum triornutum, Thlssiosir weissflogii, Thlssiosir pseudonn nd Rhodomons slin) umulted ftty ids during growth. In ll these speies, intrellulr EPA levels were higher during the lte sttionry growth phse thn during exponentil growth. In ontrst, n inrese in DHA ontent ws not oserved nd therefore the DHA-to-EPA rtio ws signifintly lower in lte sttionry phse ultures. These results n e used to improve the nutritionl vlue of mirolge ultivted for pplition in mrine quulture systems. Keywords Aquulture Growth phse Ftty ids LC-PUFAs Phytoplnkton & Peter Boelen p.oelen@rug.nl Deprtment of Oen Eosystems, Energy nd Sustinility Reserh Institute, University of Groningen, Nijenorgh 7, 9747 AG Groningen, The Netherlnds Green Chemil Retion Engineering, Engineering nd Tehnology Institute Groningen, University of Groningen, Nijenorgh 4, 9747 AG Groningen, The Netherlnds BioSolr Cells, P.O. Box 98, 6700 AB Wgeningen, The Netherlnds

2 578 Aquult Int (2017) 25: Introdution Due to their high nutritionl vlue, mirolge re importnt feed soures in quulture systems (Ptil et l. 2005). Cultivted mrine lge n e used diretly, s live feed for (lrvl stges of) ivlves nd rustens, or indiretly, s food for zooplnkton suh s rotifers whih, in turn, re used to feed rustens or smll fish lrve (Muller-Feug 2000; Chuton et l. 2014). Besides this, severl mirolgl speies re urrently eing investigted y the quulture industry s fish mel or fish oil replement in the diet of ommerilly frmed fish (Kousoulki et l. 2015; Sprgue et l. 2015; Sørensen et l. 2016). Mrine lge re key orgnisms in the prodution of essentil long-hin polyunsturted ftty ids (LC-PUFAs) suh s eiospentenoi id (EPA) nd dooshexenoi id (DHA). These n-3 PUFAs re neessry for optiml nutrition nd stress tolerne of mrine fish, espeilly t the lrvl nd juvenile stges (Khozin-Golderg et l. 2011, nd referenes therein). Most ftty ids in lgl ells re present s prt of memrne lipids (e.g., phospho- or glyosyl-glyerides) or s prt of storge lipids, minly triylglyerols (TAGs), in the ytosol (Thompson 1996). LC-PUFA ontent nd omposition my show signifint differenes etween nd within lgl lsses. Eh lgl lss hs roughly its own ftty id omposition, nd the EPA DHA ontent etween lgl lsses is highly vrile (Gushin nd Hrwood 2006; Lv et l. 2010; Boelen et l. 2013). For ost-effetive prodution of nutrient-rih mrine mirolge, with high levels of EPA nd/or DHA, it is neessry to selet highly produtive strins while optimizing ultivtion onditions. Besides txon-speifi EPA nd DHA vriility, intrellulr LC-PUFA ontent my e influened y growth onditions, suh s temperture, irrdine nd CO 2 onentrtion (e.g., Jing nd Go 2004; Pl et l. 2011; Boelen et l. 2013). During the ourse of th growth or when grown under stress, mny mirolgl speies umulte lipids, espeilly TAGs (Hu et l. 2008). For exmple, when ultured under nitrte or silite strvtion, TAGs umulted up to % of totl dry weight in the mrine ditoms Thlssiosir pseudonn nd Pheodtylum triornutum (Yu et l. 2009). Although most of the LC-PUFAs n e found in struturl memrne lipids (Roessler 1990), LC-PUFAs ould e prtitioned to or deposited in TAGs during the sttionry phse of growth (Khozin-Golderg et l. 2002; Tonon et l. 2002; Guihéneuf nd Stengel 2013). In three of the four speies investigted y Tonon et l. (2002) (Nnnohloropsis oult, T. pseudonn nd Pvlov lutheri), this resulted in higher ellulr EPA ontent. Tonon et l. (2002) lso reported umultion of DHA in T. pseudonn nd P. lutheri, lthough the inorportion into TAGs ws signifintly lower nd umultion strted lter during the ourse of th growth. In other studies, EPA or DHA ontent per lgl iomss ws redued or not signifintly ffeted y ulture ge or nutrient limittion (Klein Breteler et l. 2005; Hsio nd Blnh 2006; Gong et l. 2013; Nlder et l. 2015). The im of this study ws to investigte intrellulr umultion of the essentil n-3 LC-PUFAs EPA nd DHA during different growth phses. Therefore, solute nd reltive EPA nd DHA levels of seven representtive speies of mrine mirolge were determined during growth in th ulture. The implitions of our results for the nutritionl vlue of mrine mirolge nd therey their pplition in quulture systems will e disussed.

3 Aquult Int (2017) 25: Mterils nd methods Experimentl setup Seven speies of mrine lge were seleted, representing different txonomi groups (Tle 1). The seleted speies were known to hve high EPA nd/or DHA ontent nd were often lredy used in quulture. The ultures were otined from the Rosoff Culture Colletion (RCC, Rosoff, Frne) nd the Ntionl Center for Mrine Alge nd Miroiot (NCMA, formerly CCMP, Mine, USA). The ultures were grown in limte room t 20 C inf/2 enrihed (Guillrd nd Ryther 1962) filter-sterilized sewter djusted to slinity of 35 %. The speies were sujeted to light drk yle of 16:8 h (L:D), with photon flux density of 90 lmol photons m -2 s -1. Before the strt of the experiment, stok ultures were limted for t lest 2 weeks to the ulture onditions in semiontinuous mode. The ultures were ultured in triplite in Fernh flsks with working volume of pproximtely 0.5 L. Cell ounts nd photosystem II (PSII) fluoresene prmeters were determined regulrly to define growth phse nd mximum quntum effiieny of PSII. PSII fluoresene mesurements re widely used to reognize stress onditions of the photosyntheti pprtus. A deline in mximum quntum effiieny of PSII often ours fter the exponentil phse of growth, when nutrients eome limited. Smples for ftty id nlysis nd lgl iovolume were olleted during exponentil growth (EXP), t the end of the exponentil growth phse or the eginning of the sttionry phse (STAT) nd fter t lest 5 dys fter the eginning of the sttionry phse, usully fter 14 dys of th growth (LATE STAT). To e le to mesure true inter- nd intrspeifi differenes, tking into ount differenes in totl ftty id ontent reltive to lgl iomss, the solute mounts of EPA nd DHA were determined, using lgl iovolume s iomss unit. Algl iovolume ws lulted from ell ounts nd ell size mesurements. Cell numers were determined using Coulter XL-MCL flow ytometer (Bekmn Coulter, Mimi, FL, USA) s desried y vn de Poll et l. (2005). To lulte the ell volume of the lge, smples of out 1.5 ml of ulture were nlyzed using n inverted mirosope. The sizes of 50 ells were mesured, nd ell volume ws lulted ording to stereometri formuls s given in Hillernd et l. (1999). PAM fluorometry PSII fluoresene prmeters were mesured with pulse-mplitude-modulted hlorophyll fluorometer (Wter-PAM; Heinz Wlz GmH, Germny). Smples (5 ml) were Tle 1 Detils of the investigted speies Strin Clss Strin# Arevition Pheodtylum triornutum Billriophyee CCMP2558 PT Thlssiosir weissflogii Billriophyee CCMP1049 TW Thlssiosir pseudonn Billriophyee RCC950 TP Skeletonem osttum Billriophyee RCC70 SC Emilini huxleyi Prymnesiophyee CCMP2112 EH Isohrysis gln Prymnesiophyee RCC179 IG Rhodomons slin Cryptophyee CCMP1319 RS

4 580 Aquult Int (2017) 25: drk-dpted for 15 min nd pled in ustom-mde uvette (2 9 2 m). Bsl fluoresene (F 0 ) ws mesured under wek mesuring light, nd mximl fluoresene (F m ) ws determined fter sturting light pulse (0.8 s, 4000 lmol photons m -2 s -1 ). The mximum quntum effiieny of PSII ws lulted s F v /F m = (F m - F 0 )/F m (Mxwell nd Johnson 2000). Ftty id nlysis Lipids were extrted using modified Bligh nd Dyer extrtion s desried y Gukert et l. (1988). Susequently, the lipids were sujeted to mild lkline methnoli trnsesterifition (White et l. 1979) to produe ftty id methyl esters (FAMEs). Smples of 100 ml of lgl ulture were entrifuged, nd the remining pellets were freeze-dried for 48 h. The pellets were extrted for 18 h in single-phse solvent system of 3.5 ml hloroform, 7.5 ml methnol nd 2.8 ml 50 mm phosphte uffer (ph 7.4), nd known mount of nondenoi methyl ester (Sigm) ws dded s n internl stndrd. The smples were trnsferred to 50-mL entrifuge tues, nd 3.5 ml of hloroform nd 3.5 ml of ultrpure Milli-Q wter (Millipore) were dded. After entrifugtion for 5 min, the lower hloroform phse ws dried under N 2 nd the remining lipids were resuspended in 1 ml of methnol/toluene mixture (1:1, v/v). One milliliter of 0.2 N MeOH KOH ws dded, nd the mixture ws inuted for 15 min t 37 C. After neutrlizing to ph 6 with 1 M eti id, 2 ml of hloroform nd 2 ml of Milli-Q wter were dded. After entrifugtion, the hloroform phse with the FAMEs ws reovered nd the nonpolr FAMEs were seprted from the polr ompounds over smll Al 2 O 3 olumn using dihloromethne (DCM) s eluent. The FAMEs were nlyzed on Hewlett-Pkrd 5890 gs hromtogrph equipped with oth flme ioniztion detetor (FID) nd HP 5972 mss spetrometer detetor (MSD). Smples of 5 ll were injeted using n HP 6890 utosmpler t 60 C on Restek RTX 1701 (60 m mm, film thikness 0.25 lm) olumn. The temperture ws held t 60 C for 1 min nd then inresed t rte of 10 C min -1 until 180 C nd then to finl temperture of 250 C t5 C min -1, whih ws mintined for 15 min. The injetion temperture ws 250 C, nd the detetor tempertures were oth 280 C. Long-hin ftty ids (numer of rons C14) were identified from mss spetr nd retention times y omprison with those of PUFA No. 1 stndrd mixture (Mtrey LLC, USA). Quntifition of ftty ids ws done y integrtion of pproprite pek res lirted with the known onentrtion of the dded internl stndrd. Sttistil nlysis Differenes in F v /F m, ftty id ontent or DHA-to-EPA rtio etween growth phses nd speies were nlyzed y two-wy nlysis of vrine (ANOVA) followed y simple min effets nlysis when there ws signifint intertion. Signifint (p \ 0.05) differenes mong growth phses were further nlyzed y pirwise omprisons using the Šidk djustment for multiple omprisons. Vlues of reltive EPA nd DHA ontent (% of totl ftty ids) were squre-root-trnsformed prior to nlysis. All sttistil nlyses were performed using IBM SPSS Sttistis (version 22) softwre (IBM Corportion, Armonk, NY, USA).

5 Aquult Int (2017) 25: Results Reltive (% of totl ftty id) nd solute (normlized to iovolume) EPA nd DHA ontent showed lrge vriility etween the tested speies (Tle 2; Fig. 1). The ditoms (Billriophyee) showed high EPA nd reltively low DHA perentges. In ontrst, the two prymnesiophytes were hrterized y high DHA nd low EPA ontent. Rhodomons slin, representtive ryptophyte speies, showed oth high EPA nd DHA perentges ompred to the other speies. Although EPA perentges in S. osttum were omprle with the other ditoms (9 % of totl ftty ids on verge), solute levels of EPA normlized to iovolume were low (\1 fglm -3 ). DHA perentges were highest in E. huxleyi (up to 20 % of totl ftty ids), while solute levels were Tle 2 Sttistil results of two-wy ANOVA for effets of growth phse nd lgl speies on reltive (% of totl ftty ids), solute (normlized to iovolume) EPA nd DHA ontent, totl ftty id ontent (fg lm -3 ), mximum quntum effiieny of PSII (F v /F m ) nd DHA-to-EPA rtio Effet on Soure of vrition df MS F p EPA % Speies <0.001 Growth phse Speies 9 growth phse <0.001 Residul DHA % Speies <0.001 Growth phse Speies 9 growth phse Residul EPA Speies <0.001 Growth phse <0.001 Speies 9 growth phse <0.001 Residul DHA Speies <0.001 Growth phse Speies 9 growth phse Residul Totl ftty ids Speies <0.001 Growth phse <0.001 Speies 9 growth phse <0.001 Residul F v /F m Speies <0.001 Growth phse <0.001 Speies 9 growth phse <0.001 Residul DHA-to-EPA rtio Speies <0.001 Growth phse <0.001 Speies 9 growth phse Residul Sttistilly signifint p vlues (p \ 0.05) re printed in old

6 582 Aquult Int (2017) 25: A EPA (%) EXP STAT LATE STAT B EPA (fg µm -3 ) EXP STAT LATE STAT C DHA (%) EXP STAT LATE STAT D DHA (fg µm -3 ) EXP STAT LATE STAT 0 0 Fig. 1 Reltive (% of totl ftty ids) (A, C) nd solute (normlized to iovolume) (B, D) EPA nd DHA ontent in seven speies of mrine lge during different growth phses. Vlues represent verges (±SD) of three replite ultures. Different letters (per speies) represent signifint differenes t p \ 0.05 mong growth phses ording to the nlysis of simple min effets. EXP = exponentil phse, STAT = sttionry phse, LATE STAT = lte sttionry phse. PT = Pheodtylum triornutum. TW = Thlssiosir weissflogii. TP = Thlssiosir pseudonn. SC = Skeletonem osttum. EH = Emilini huxleyi. IG = Isohrysis gln. RS = Rhodomons slin omprle with I. gln nd R. slin (etween 3 nd 6 fg lm -3 ). Also, the totl ftty id ontent vried signifintly etween speies (Tle 2; Fig. 2). The lowest mounts (etween 1.6 nd 8.2 fg lm -3 ) were found in S. osttum, while the highest totl ftty id ontent (122.6 ± 29.8 fg lm -3 ) ws mesured in T. pseudonn during the lte sttionry phse of growth. For ll speies, the mximum quntum effiieny of PSII (F v /F m ) deresed during growth (Fig. 2), inditing tht the ultures were sujeted to stress during the lte sttionry phse. The intertive effets of speies 9 growth phse were highly signifint (Tle 2), inditing tht the effet of growth phse on totl ftty id ontent ws not the sme for ll speies. Out of the seven speies tested, four speies (P. triornutum, T. weissflogii, T. pseudonn nd R. slin) showed signifintly enhned totl ftty id ontent in the lte sttionry phse s ompred to the exponentil growth phse (Fig. 2). Also, the solute mount of EPA in these speies ws higher during the lte sttionry growth phse thn during exponentil growth, lthough this effet ws not sttistilly signifint for P. triornutum (Fig. 1). In P. triornutum, solute EPA nd totl ftty id ontent initilly deresed with growth, ut oth levels were signifintly higher t the lte sttionry growth phse s ompred to the sttionry growth phse. Highest solute EPA levels were found in T. pseudonn nd R. slin (8.9 nd 7.5 fg lm -3, respetively). The highest inrese in EPA during th growth ws found in T. weissflogii. In this speies, the solute EPA ontent ws 3.4 times higher during the lte

7 Aquult Int (2017) 25: A EXP STAT LATE STAT Totl Ftty Aids (fg µm -3 ) B F v / F m Fig. 2 Totl ftty id ontent (fg lm -3 )(A) nd mximum quntum effiieny of PSII (F v /F m )(B) of seven speies of mrine mirolge during different growth phses. Vlues represent verges (±SD) of three replite ultures. Different letters (per speies) represent signifint differenes t p \ 0.05 mong growth phses ording to the nlysis of simple min effets. EXP = exponentil phse, STAT = sttionry phse, LATE STAT = lte sttionry phse. PT = Pheodtylum triornutum. TW = Thlssiosir weissflogii.tp= Thlssiosir pseudonn.sc= Skeletonem osttum.eh= Emilini huxleyi. IG = Isohrysis gln. RS = Rhodomons slin sttionry growth phse thn during exponentil growth. On the other hnd, solute levels of DHA during the lte sttionry phse were not signifintly higher thn levels in exponentilly growing ultures (Tle 2; Fig. 1d). DHA-to-EPA rtios were lulted for speies with initil EPA nd DHA levels oth higher thn 1 % (Tle 3). In ll these speies, the DHA-to-EPA rtio deresed signifintly during growth. The highest DHA-

8 584 Aquult Int (2017) 25: to-epa rtio ws mesured in R. slin during exponentil growth (0.72 ± 0.02). The highest derese ws oserved in P. triornutum. In this speies, the DHA-to-EPA rtio ws lmost four times lower during the lte sttionry growth phse thn during exponentil growth. Disussion In this study, we foused on the umultion of the essentil n-3 LC-PUFAs EPA nd DHA in th ultures of mrine lge. Although mny studies hve demonstrted umultion of (neutrl) lipids in strved ultures of mirolge, reltively little ws known out the effet of growth phse on EPA nd DHA umultion. Our study onfirms previous results tht the solute mounts of EPA nd DHA, ut lso the totl ftty id ontent vries onsiderly etween speies (Volkmn et l. 1989; Viso nd Mrty 1993; Tonon et l. 2002; Mnsour et l. 2005; Ptil et l. 2007; Boelen et l. 2013). Consequently, high interspeifi vriility in their nutritionl vlue exists. Four out of the seven investigted mirolgl speies umulted ftty ids during th growth. For E. huxleyi, I. gln nd S. osttum, no signifint umultion of lipid ompounds ould e demonstrted. Possily, other photosyntheti produts (e.g., rohydrtes) umulted in these speies when entering the nutrient-depleted stge (Hrrison et l. 1990; Grnum et l. 2002). In ll lipid-umulting speies, solute ellulr EPA levels were higher during the lte sttionry growth phse thn during exponentil growth. The reltive undnes (% of totl ftty ids) of EPA in these lge remined reltively onstnt during the stge of growth, while totl ellulr ftty id ontent ws signifintly inresed. This ould e n indition tht t lest prt of the EPA is trnsferred into or present in TAGs, sine in most lge stress-indued lipids re predominntly in the form of TAGs (Hu et l. 2008). It is hypothesized tht mirolgl TAG n serve s depot for LC-PUFAs, whih ould e moilized for the onstrution of hloroplsti memrnes under sudden hnges in environmentl onditions (Cohen et l. 2000; Khozin-Golderg et l. 2005). High proportions of LC-PUFAs present in TAGs ould e n dvntge when ulturing mirolge to produe n lterntive for fish oil (Tonon et l. 2002; Leu nd Boussi 2014). The solute mount of DHA, however, ws not ffeted, nd therefore, the DHA-to- EPA rtio in speies ontining DHA s well s EPA deresed signifintly during growth. This implies tht in these speies, the umultion of EPA nd DHA seems to e regulted y two different mehnisms. This effet ould hve onsequenes for the nutritionl qulity, sine the nutritionl vlue of lge is dependent not only on the totl Tle 3 Effet of growth phse on DHA-to-EPA rtio in three speies of mrine mirolge. DHA-to-EPA rtios were lulted for speies with initil EPA nd DHA levels oth higher thn 1 % (reltive to totl ftty ids) Strin EXP STAT LATE STAT Pheodtylum triornutum 0.43 ± ± ± 0.01 Thlssiosir weissflogii 0.35 ± ± ± 0.01 Rhodomons slin 0.72 ± ± ± 0.02 Vlues represent verges (±SD) of three replite ultures. Different letters per row (speies) represent signifint differenes t p \ 0.05 mong growth phses ording to the nlysis of simple min effets. EXP = exponentil phse, STAT = sttionry phse, LATE STAT = lte sttionry phse

9 Aquult Int (2017) 25: mount of EPA nd/or DHA ut lso on the reltive proportion of these LC-PUFAs (Brown 2002; Spolore et l. 2006). For fish lrvl nutrition, DHA-to-EPA rtios of etween 1 nd 2 re dvised (Rodríguez et l. 1998). For R. slin, whih ontins signifint perentges of oth DHA nd EPA nd is used s quulture feed (e.g., Ggné et l. 2010), this would imply tht lthough the totl ellulr mount of ftty ids is higher, the nutritionl vlue of this speies is lower t the end of the growth phse. Currently, severl mirolgl speies re eing investigted y the quulture industry s fish mel or fish oil replement in the diet of ommerilly frmed fish (Kousoulki et l. 2015; Sprgue et l. 2015; Sørensen et l. 2016). Repling the trditionl EPA- nd DHA-rih, ut finite, mrine ingredients, fishmel nd fish oil present signifint hllenge for the quulture industry (Sprgue et l. 2016). Promising fish oil lterntives re lipids from mirolge whih n potentilly grow t high growth rtes nd whih n reh high mximum ell densities with high levels of EPA nd/or DHA. Muh reserh hs een foused on heterotrophi DHA-produing speies suh s Shizohytrium (Kousoulki et l. 2015; Sprgue et l. 2015). However, Sprgue et l. (2015) showed tht the sene of EPA in Shizohytrium-sed fish diet signifintly impirs the overll nutritionl vlue. Sørensen et l. (2016) investigted the potentil use of the phototrophi EPA-rih P. triornutum s fish mel replement in diet for Atlnti slmon. They showed tht P. triornutum n reple up to 6 % of the fish mel without dverse effets on nutrient digestiility, utiliztion of the feed nd growth performne. In future studies, it would e useful to test mixtures of DHA-rih Shizohytrium nd EPA-rih phototrophi lgl speies s lterntive soures of EPA nd DHA in qufeeds. In our study, we showed tht intrellulr EPA levels in P. triornutum nd three other mrine lgl speies n e further enhned y optimizing the hrvest time. We onlude tht in some mrine lgl speies EPA umultes during th growth. The highest inrese in EPA ws mesured in T. weissflogii, while highest levels were mesured in T. pseudonn nd R. slin. Asolute levels of DHA were not ffeted, whih n hve onsequenes for the nutritionl vlue of these lge. This effet hs to e tken into ount when th ulturing mirolge s food soure in quulture systems. Aknowledgments We thnk Willem vn de Poll, Ptrik Rozem nd Ronld Visser for onstrutive suggestions nd tehnil support. This projet ws rried out within the reserh progrm of BioSolr Cells, o-finned y the Duth Ministry of Eonomi Affirs. Open Aess This rtile is distriuted under the terms of the Cretive Commons Attriution 4.0 Interntionl Liense ( whih permits unrestrited use, distriution, nd reprodution in ny medium, provided you give pproprite redit to the originl uthor(s) nd the soure, provide link to the Cretive Commons liense, nd indite if hnges were mde. Referenes Boelen P, vn Dijk R, Sinninghe Dmsté JS, Rijpstr WIC, Bum AGJ (2013) On the potentil pplition of polr nd temperte mrine mirolge for EPA nd DHA prodution. AMB Express 3:26 Brown MR (2002) Nutritionl vlue nd use of mirolge in quulture. In: Cruz-Suárez LE, Rique- Mrie D, Tpi-Slzr M, Gxiol-Cortés MG, Simoes N (eds) Avnes en Nutriión Auíol VI. Memoris del VI Simposium Internionl de Nutriión Auíol. 3 l 6 de Septiemre del Cnún, Quintn Roo, Méxio

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