Filtration of Nordic recirculating aquaculture system wastewater : Effects on microalgal growth, nutrient removal, and nutritional value

This is a sel -a chi ed e sion o an o iginal a icle. This e sion may di e om he o iginal in pagina ion and ypog aphic de ails. Au ho (s): Ti le: Yea : Ve sion: Copy igh : Righ s: Righ s u l: Please ci e he o iginal e sion: CC BY 4.0 h ps://c ea i ecommons.o g/licenses/by/4.0/ Fil a ion o No dic eci cula ing aquacul u e sys em was ewa e : E ec s on mic oalgal g ow h, nu ien emo al, and nu i ional alue © 2021 The Au ho (s). Published by Else ie B.V. Published e sion Calde ini, Ma co L.; S e čić, Čedomi ; Taipale, Sami; Pulkkinen, Ka ja Calde ini, M. L., S e čić, Č., Taipale, S., & Pulkkinen, K. (2021). Fil a ion o No dic eci cula ing aquacul u e sys em was ewa e : E ec s on mic oalgal g ow h, nu ien emo al, and nu i ional alue. Algal Resea ch, 60, A icle 102486. h ps://doi.o g/10.1016/j.algal.2021.102486 2021 Algal Resea ch 60 (2021) 102486 A ailable online 10 Sep embe 2021 2211-9264/© 2021 The Au ho (s). Published by Else ie B.V. This is an open access a icle unde he CC BY license (h p://c ea i ecommons.o g/licenses/by/4.0/). Fil a ion o No dic eci cula ing aquacul u e sys em was ewa e : E ec s on mic oalgal g ow h, nu ien emo al, and nu i ional alue Ma co L. Calde ini * , ˇ Cedomi S e ˇ ci´ c, Sami Taipale, Ka ja Pulkkinen Uni e si y o Jy askyla, Depa men o Biological and En i onmen al Science, P.O. Box 35, FI-40014, Uni e si y o Jy askyla, Finland ARTICLE INFO Keywo ds: Amino acids Biological con amina ion Bio emedia ion Fa y acids Reci cula ing aquacul u e sys em ABSTRACT Mic oalgal bio emedia ion o eci cula ing aquacul u e sys em (RAS) was ewa e ep esen s an al e na i e o was ewa e ea men wi h he po en ial o gene a e aluable biomass. This s udy e alua ed he e ec s o emo ing biological con amina ion and suspended solids om No dic a ea RAS was ewa e h ough il a ion wi h 0.45 μ m il e s on he pe o mance and nu i ional alue o mic oalgae. All h ee es ed g een mic oalgae (Haema ococcus plu ialis, Mono aphidium g i i hii, and Selenas um sp.) we e able o g ow in aw (un il e ed) and il e ed RAS was ewa e . Cul i a ion in aw RAS was ewa e dec eased he ω -3 and ω -6 a y acid con en o H. plu ialis as compa ed o il e ed was ewa e , while no di e ences in cell densi y, nu ien emo al, o a y acid and amino acid con ibu ion we e seen o any mic oalgae be ween he ea men s. Fil a ion o was ewa e signi ican ly educed he con en o ac inobac e ial a y acid bioma ke s in mic oalgal cul u es compa ed o aw was ewa e . The di e ence in ac inobac e ial a y acid con en be ween aw and il e ed was ewa e was species-speci ic. Ou esul s sugges ha wi h ca e ul selec ion o mic oalgal species, RAS was ewa e can be used o he p oduc ion o high-quali y mic oalgal biomass o u he applica ions, such as aquacul u e eeds, wi h no need o emo e indigenous biological con aminan s and suspended solids. 1. In oduc ion Aquacul u e is one o he wo ld's as es -g owing ood indus ies and i s u he de elopmen and in ensi ica ion a e expec ed in almos all egions o he wo ld. Eu ophica ion o aqua ic ecosys ems caused by exposu e o nu ien - ich (pa icula e and dissol ed) aquacul u e was ewa e (WW) ep esen s one o he main isks o he en i onmen al sus ainabili y o aquacul u e [1,2]. Reci cula ing aquacul u e sys em (RAS) ep esen s an en i onmen ally supe io op ion o adi ional open sys ems ( low- h ough and ne -pen) as i acili a es he on-si e ea men o wa e . Low wa e enewal a es equi ed by mode n RAS (<10% o o al olume pe day) esul in smalle olumes o WW, while he was e ou pu depends on p oduc ion o biomass and is no educed compa ed o open sys ems. This concen a ion o was e in o educed olumes o WW p o ides an oppo uni y o imp o e was e managemen and nu ien ecycling [2,3]. Despi e echnological imp o emen s in physi- cochemical wa e pu i ica ion p ocesses, aquacul u e WW ea men emains expensi e while aluable nu ien s p esen in WW a e no eco e ed [4]. Tha is why economically easible sus ainable solu ions a e equi ed o RAS WW ea men . Bio emedia ion o WWs ep esen s a sus ainable al e na i e o adi ional physicochemical ea men s, whe e li ing o ganisms a e employed o he emo al o con aminan s. Al hough bio emedia ion o WW holds p omising oppo uni ies as a sus ainable solu ion o nu ien ecycling in RAS, o da e, mos esea ch e o has been alloca ed o s udy aquaponics (aquacul u e coupled wi h hyd oponics) [5]. Among al e na i e o ganisms o bio emedia ion o RAS WW, mic oalgae ep esen a powe ul bio echnological pla o m o he p oduc ion o high alue p oduc s and quali y biomass [6]. Mos impo an ly, hei use o bio emedia ion o di e en indus ial and municipal WWs has been p o en e ec i e [6,7,8,9]. Gi en ha mic oalgae a e a na u al ood sou ce o o ganisms commonly p oduced in aquacul u e (e.g. ish la ae, c us acean, and mollusks) [10], o RAS en e p ises, mic oalgae cul i a ion could no only suppo WW ea men bu he gene a ed mic oalgal biomass could se e as eeds ock o complemen o eplace aquacul u e eeds [11,12]. In addi ion, biological ha es o mic oalgae (e.g. by zooplank on o ish la ae) could elimina e he ope a ional cos s associa ed wi h chemical o mechanical ha es ing o mic oalgal cells (up o 90% o he o al cos o mic oalgae p oduc ion) [6,13,14]. Depending on he species and cul i a ion condi ions, mic oalgae can * Co esponding au ho . E-mail add ess: [email p o ec ed] (M.L. Calde ini). Con en s lis s a ailable a ScienceDi ec Algal Resea ch jou nal homepage: www.else ie .com/loca e/algal h ps://doi.o g/10.1016/j.algal.2021.102486 Recei ed 6 Decembe 2020; Recei ed in e ised o m 10 June 2021; Accep ed 29 Augus 2021 Algal Resea ch 60 (2021) 102486 2 ha e high nu i ional alue o aquacul u e since hey a e ich sou ces o ca bohyd a es, p o eins, lipids, and o he essen ial biomolecules such as i amins and pigmen s [12,15,16]. In pa icula , a y acids (FAs) and amino acids (AAs) a e among he mos impo an mic oalgal biochem- ical componen s as highly unsa u a ed FAs, such as eicosapen aenoic acid (EPA, 20:5 ω -3) and docosahexaenoic acid (DHA, 20:6 ω -3), a e essen ial o he g ow h and ep oduc ion o zooplank on, bi al es and ish [17,18,19]. AA composi ion, in pa icula essen ial AAs (EAAs), has been shown as an impo an componen o ood o aquacul u e o - ganisms [18,20]. In addi ion, die a y non-essen ial AAs (NEAAs) ha e been epo ed as necessa y o achie e maximal g ow h in zooplank on and ish [19,21]. To da e, mos s udies on mic oalgal bio emedia ion o RAS WW we e conduc ed in wa m geog aphical loca ions [22], hence, he applicabili y o his bio emedia ion sys em a ela i ely low empe a u es (below 20 ◦C) common o No dic a eas has ecei ed li le a en ion. In ou p e ious s udy [23], h ee eshwa e g een mic oalgae (Haema ococcus plu ialis, Mono aphidium g i i hii, and Selenas um sp.) showed adequa e g ow h a es in RAS WW when g own a low empe a u es (17 ◦C), making hem easible candida es o WW bio emedia ion. Ne e heless, despi e he sa is ac o y g ow h a es ob ained, he implemen a ion o mic oalgal bio emedia ion sys ems equi es a be e unde s anding o all possible a ibu es o WW ha may a ec mic oalgal pe o mance such as biological con amina ion and suspended solids. Na i e biological con amina ion o RAS WW ep esen s a challenge o mic oalgae cul i- a ion since non- a ge mic oalgae and bac e ia may compe e o nu- ien s and ligh o may be oxic o he cul u ed mic oalgae [24]. In addi ion, e en small numbe s o he bi o ous p o ozoa can apidly mul iply and des oy a mic oalgal cul u e [25]. Al hough ecen indings highligh he po en ial bene i s o symbio ic ela ionships be ween mic oalgae and bac e ia o WW bio emedia ion [5], mos o hese s udies o e look mic oalgal biomass in a o o nu ien emo al, which does no align wi h he objec i e o op imized mic oalgal p oduc ion. Mo eo e , s udies on non-axenic mic oalgal cul u es ha e shown ha bac e ia can igge adap a ions in he s a ch and lipid me abolism o mic oalgae leading o changes in he nu i ional alue o mic oalgae [26,27,28]. These esul s a e especially signi ican when mic oalgal biomass is in ended o be e-in oduced in o RAS as a complemen o eplacemen o aquacul u e eeds. Ne e heless, since main enance o axenic cul u es in RAS acili ies is no easible, he e ec o WW's bio- logical con amina ion on mic oalgal g ow h o i s nu i ional alue should be conside ed. Suspended solids in RAS WW may also limi mic oalgal pe o mance since, when p esen a high concen a ions, hey can educe ligh pene a ion causing sel -shading and educing in- si u pho osyn he ic ac i i y and biomass p oduc ion [29]. To his end, e alua ion o he e ec s o he educ ion o he na i e biological con amina ion and suspended solids in RAS WW be o e mic oalgal inocula ion can help a oid biomass losses o dec eases in he o e all nu i ional quali y o mic oalgae. In his s udy, we in es iga ed he use o il a ion as a one-s ep RAS WW p e- ea men o signi ican ly educe biological con amina ion and suspended solids. The objec i e o he p esen s udy was o e alua e he g ow h, bio emedia ion capaci y and nu i ional quali y o h ee mic oalgae species (H. plu ialis, M. g i i hii, and Selenas um sp.) cul u ed in ei he aw (un il e ed) o il e ed No dic RAS WW. We es ima ed he nu i ional quali y as he composi ion o mic oalgal FAs and AAs, based on hei essen ial ole in aquacul u e [17,19]. We es ed he hypo hesis “ il a ion o No dic RAS WW will imp o e g ow h, biomass, nu ien emo al o nu i ional quali y o mic oalgae compa ed o un il e ed RAS WW”. 2. Ma e ials and me hods 2.1. Reci cula ing aquacul u e sys em was ewa e RAS WW samples we e ob ained om he Na u al Resou ces Ins i u e Finland (LUKE) Laukaa ish a m [30]. WW samples we e collec ed om he wa e ou le o wo indi idual RAS, a e d um il a ion and ixed bed bio eac o ea men . Fa ming condi ions p io o sample collec ion in anks 1 and 2 we e 44 and 52 whi e ish (Co egonus la a e us) wi h a mean weigh o 453 and 437 g, espec i ely, ed wi h Raisio Ci cui Sil e 3.5 mm a 0.7% body weigh pe day. Wa e ci cula ion was se a 0.2 L s −1 and eplacemen wa e adjus ed a 250 L kg −1 o eed [30]. Samples om bo h anks we e mixed and s o ed a 6 ◦C un il used. Hal o he RAS WW was il e ed h ough 0.45 μ m sy inge il e s (Co ning, Sigma-Ald ich, USA). Fil a ion was no expec ed o p oduce an axenic WW [31], bu a he dec ease he o al o ganic and biological load be o e he inocula ion o mic oalgae. Dissol ed o ganic ca bon (DOC) o un il e ed and il e ed RAS WW was measu ed om HCl-acidi ied samples ( inal pH =2) wi h a high empe a u e ca aly ic oxida ion me hod using a ca bon and ni ogen analyze (TOC-L, Shimadzu, Japan). Tu bidi y measu es we e done in iplica es wi h a Tu b 430 IR (Xylem Analy ics LLC, USA). 2.2. Mic oalgae s ains and cul i a ion condi ions The h ee empe a e zone eshwa e g een mic oalgal s ains used (H. plu ialis, M. g i i hii, and Selenas um sp.) we e ob ained om he No wegian Cul u e Collec ion o Algae (NORCCA). Each mic oalga was main ained as a s ock monocul u e in algae medium MWC (Modi ied W igh 's C yp ophy e) based on Guilla d and Lo enzen [32] as desc ibed p e iously [23]. A ligh mic oscope (Leica DM 500) equipped wi h a came a (Leica ICC50 W) was used o ob ain images o mic oalgae g own in MWC media. The expe imen al cul i a ion o mic oalgae was di ided in o wo expe imen al se ies, each se ies con aining wo eplica es o each mic oalga cul i a ed in each cul u e media: un il e ed RAS WW and il e ed RAS WW (Table 1). Mic oalgae we e g own in 650 mL plas ic cul u e lasks con aining 400 mL o he cul u e as desc ibed in [23]. A he s a o each se ies o expe imen s (day 0), mic oalgal s ock monocul u es we e cen i uged a 2500g o 5 min a 17 ◦C (Mega uge 1.0 R, He aeus, Ge many) o ob ain high-densi y inoculums. Each expe imen al cul u e lask was hen inocula ed wi h 1–10% o he mic oalgal sa u a ing concen a ion de e mined in he p e ious expe - imen s in MWC media. Illumina ion was p o ided on one side o he lasks by wo LED g ow ligh s (AP67 spec um, see [23]) wi h 24:00 pho ope iod and ligh in ensi y o 60–90 μ mol pho on m −2 s −1 measu ed a he su ace o he lask by a high- esolu ion spec ome e (HP-350 HiPoin Inc., Taiwan). Room empe a u e was main ained a 17 ± 0.3 ◦C. Th oughou he expe imen , he lasks we e manually mixed wice a day wi h aqua ium magne s o keep cells in suspension. Cul i- a ion was e mina ed 6 days a e mic oalgal inocula ion, be o e he cul u es eached s a iona y phase. 2.3. De e mina ion o mic oalgal g ow h Th oughou he cul i a ion pe iod, cell densi y was es ima ed daily by cell coun om wo eplica e samples o each eplica e lask in a Table 1 Cha ac e is ics o un il e ed RAS was ewa e (WW) and il e ed RAS was ewa e (FWW) p io o mic oalgal inocula ion. Dissol ed o ganic ca bon is deno ed as DOC. Values a e shown as mean ±SD o bo h expe imen al se ies. Composi ion WW FWW NH 4 -N (mg L −1 ) 0.03 ±0.01 0.05 ±0.01 NO 2 -N (mg L −1 ) 0.03 ±0.00 0.05 ±0.00 NO 3 -N (mg L −1 ) 96.87 ±0.73 97.03 ±0.17 PO 4 -P (mg L −1 ) 3.83 ±0.07 3.76 ±0.04 N:P mola a io a 55.90 ±0.62 57.05 ±0.51 pH 7.49 ±0.28 7.39 ±0.04 Tu bidi y (NTU) 5.27 ±0.68 1.33 ±0.06 DOC (mg L −1 ) 12.99 ±0.79 12.13 ±0.59 a N:P mola a io was calcula ed om NO 3 -N:PO 4 -P. M.L. Calde ini e al. Algal Resea ch 60 (2021) 102486 3 haemocy ome e chambe (Bü ke ) wi h 100×magni ica ion (Lei z 184 Labo lux D, Ge many). The speci ic g ow h a e (d −1 ) was calcula ed om he change in cell densi y du ing he exponen ial g ow h phase acco ding o [33]. To de e mine he o al d y weigh (biomass), wo aliquo s o cul u e we e aken a he end o he cul i a ion pe iod (day 6) as desc ibed p e iously [23]. Chlo ophyll-a concen a ion was assessed spec opho ome ically wi h Shimadzu Spec opho ome e (UV-1800, Japan) om samples il e ed on a ibe il e (GF/A, Wha man, GE Heal hca e, USA) a he end o he cul i a ion pe iod (day 6) acco ding o [34]. 2.4. De e mina ion o nu ien emo al Ni a e‑ni ogen (NO 3 -N) and phospha e‑phospho us (PO 4 -P) con- cen a ions we e assessed om samples o cul u e media a he begin- ning and he end o he cul i a ion pe iod wi h es ing ki s LC399 and LCK349 (Hach, USA) acco ding o manu ac u e 's ins uc ions. Quan i- ica ion was ca ied ou in a mobile labo a o y spec ome e (LASA 100, D . Lange, Ge many). Be o e being analyzed, e e y sample was il e ed h ough a 0.22 μ m sy inge il e . Pe cen age o nu ien up ake and nu ien emo al a e (Ri) we e calcula ed as desc ibed p e iously [23]. 2.5. Fa y acid analysis Once mic oalgal cul i a ion ended (day 6), be ween 20 and 35 mL o each cul u e was il e ed h ough 3.0 μ m cellulose ni a e memb anes (Wha man, GE Heal hca e, USA). Fil e s con aining he mic oalgal sample we e hen eeze-d ied, weighed (Sa o ius CP2P, Ge many), and s o ed a −80 ◦C un il he analysis (no longe han a mon h). Fil e s con aining 2–5 mg o g een mic oalgae we e placed in o es ubes (10 mL). To al lipid ex ac ion was ca ied ou wi h chlo o o m:me hanol: wa e (4:2:1) mix u e and me hanolic H 2 SO 4 (1% / ) a 50 ◦C was used o anses e i ica ion o FA o o m a y acid me hyl es e s (FAME). FAMEs we e analyzed wi h a gas ch oma og aph equipped wi h mass de ec o (GC–MS) (Shimadzu Ul a, Japan) using helium as a ca ie gas and an Agilen (Cali o nia, USA) ZB-FAME column (30 m ×0.25 mm × 0.20 μ m) (Phenomenex, USA) o sepa a ion. The empe a u e p og am: 50 ◦C was main ained o 1 min, and hen he empe a u e was inc eased a 10 ◦C min −1 o 130 ◦C, hen by 7 ◦C min −1 o 180 ◦C, and 2 ◦C min −1 o 200 ◦C held o 3 min and inally hea ed a 10 ◦C min −1 o 260 ◦C. The column low was se a 1.10 mL min −1 . Quan i ica ion calib a ion cu es o indi idual FAs we e p epa ed wi h a y acid s anda d GLC e e ence s anda d 556 C (Nu-Chek P ep, Elysian, USA). Addi ionally, we used phospholipid FA C19:0 (PLFA 19:0) and ee FA C23:0 (La odan, Swe- den) as in e nal s anda ds o he calcula ion o he eco e y pe cen - ages. Reco e y pe cen ages we e >71% o all samples. FAs in sample spec ums we e iden i ied using e en ion imes oge he wi h speci ic ions. Quan i ica ion was based on de ec o esponses, he peak a eas we e in eg a ed using GCsolu ion so wa e ( e sion 2.41.00, Shimadzu, Japan), and samples FAs a eas alues we e in e pola ed in he calib a- ion cu e o de e mine hei concen a ion. FA con en ( μ g g −1 DW) was calcula ed using he ollowing equa ion: FAic =QFA ×VVial DW1×R% (1) whe e FA ic is he con en o an indi idual a y acid ( μ g mg −1 DW) in he sample, Q FA is he concen a ion o he a y acid ( μ g μ L −1 ) based on calib a ion cu es o GLC-566C, V Vial deno es he unning olume o he samples ( μ L), DW 1 is he d y weigh o he sample, and R % deno es he eco e y pe cen age based on in e nal s anda ds. FA pe cen alues (%) we e calcula ed ollowing he o mula: FAi%=FAic To −FAic ×100 (2) whe e FA i % is he pe cen age o con ibu ion o FA i , FA ic is he de e mined concen a ion o FA i and To -FA ic is he sum concen a ion o all iden i ied FAs. As desc ibed in [35], FAs we e hen so ed by hei mean % con ibu ion, and only FAs con ibu ing >0.5% (mean ac oss all eplica es) o he o al we e used o la e s a is ical analysis (wi hou no malizing he da a o 100%). In his wo k, we ocused on he con en and con ibu ion o i e ω -3 poly-unsa u a ed FAs (16:3 ω -3, 16:4 ω -3, 18:3 ω -3, 18:4 ω -3 and 20:5 ω -3) and ou ω -6 FAs (16:2 ω -6, 18:2 ω -6, 18:3 ω -6, and 20:4 ω -6). In addi ion o mic oalgal FAs, we quan i ied ac inobac e ial FA bioma ke s (i-14:0, i-15:0, a-15:0, i-16:0, i-17:0, and a-17:0) o each sample as an app oxima ion o he bac e ial biomass in he cul u e media. Ac inobac e ial FA con en s (ng mL −1 ) ep esen he sum o all quan i ied FAs pe mL o media and a e exp essed as bac e ial FA con en s in he Resul s sec ion. 2.6. Amino acid analysis A he end o he mic oalgal cul i a ion pe iod (day 6), be ween 7 and 10 mL o cul u e we e il e ed h ough 3.0 μ m nucleopo e poly- ca bona e il e s (Wha man, GE Heal hca e, USA). Fil e s con aining he sample we e hen eeze-d ied, weighed (Sa o ius CP2P, Ge many), and s o ed a −80 ◦C un il analysis (no longe han a mon h). Mic oalgae samples we e placed in o es ubes (10 mL) and HCl 6 N was added in su icien amoun s o ensu e ha he il e s con aining he sample we e comple ely co e ed in acid. Samples we e hen hea ed a 110 ◦C o 24 h. A e AA hyd olyza ion, HCl was e apo a ed a 110 ◦C o 20 h. We used L-no aline (Sigma-Ald ich, USA) as an in e nal s anda d. F ee AAs we e hen de i a ized u ilizing he comme cial ki EZ: aas o F ee Physio- logical Amino Acid Analysis by GC–MS (Phenomenex, USA) wi h he excep ion ha no pu i ica ion column was used du ing he p ocess. AA ch oma og aphic sepa a ion and hei pos e io iden i ica ion and quan i ica ion we e done ollowing he p o ocol desc ibed in [36]. Samples we e analyzed wi h GC–MS (Shimadzu, Japan) and a used silica capilla y column (10 m ×0.25 mm), coa ed wi h 0.2 μ m o an unknown s a iona y phase (ZB-AA, Phenomenex, USA). The iden i ica- ion o AAs was based on e en ion imes and speci ic ions. Indi idual AA calib a ion cu es we e gene a ed wi h he AA s anda d AAS-18 (Sigma- Ald ich, USA). Quan i ica ion and co ec ion o AA con en ( μ g mg −1 DW), oge he wi h he de e mina ion o AA (%), we e done as desc ibed wi h FAs (Eqs. (1) and (2)). In Eq. (1), no aline eco e y pe cen age was used as R % . Only he AAs p esen in he s anda d (AAS-180) we e iden i ied and quan i ied in he samples: eigh essen ial AA (EAAs: aline, leucine, isoleucine, h eonine, me hionine, phenylalanine, lysine, and his idine), and se en non-essen ial AA (NEAAs: alanine, glycine, se ine, p oline, aspa agine, glu amic acid, and y osine). The sum o all AAs was calcula ed as he sum o EAA and NEAA. In he case o me hionine, only e y low concen a ions o he amino acid we e de ec ed, his ag ees wi h p e ious li e a u e since me hionine can be deg aded o a ying deg ees du ing acid hyd olysis [37]. 2.7. S a is ical analysis Two-way mixed e ec s analysis o a iance (ANOVA) was used o es he e ec s o mic oalgae species ( h ee mic oalgae species) o g ow h media ( il e ed o un il e ed RAS WW) on g ow h, nu ien up- ake, and FA and AA ca ego ies. The signi icance o ixed e ec s was e alua ed using Sa e hwai e's me hod o app oxima e he deg ees o eedom. The non-independence o obse a ions wi hin each un was accoun ed o by including un as a andom ac o . The signi icance o he e ec o he un was e alua ed wi h he Likelihood Ra io Tes . Es ima ed Ma ginal Means pai wise compa ison wi h Tukey adjus - men s was used o pos hoc analysis o he mixed e ec s models. Ho- mogenei y o a iances was es ed wi h Le ene's es and no mali y o he collec ed da a was es ed wi h Shapi o–Wilk's es . Pe mu a ional mul i a ia e analysis o a iance (PERMANOVA) based on he B ay- Cu is dis ance ma ix was pe o med on FA and AA pe cen age (%) da a o es i species o media ( ea men ) di e ed s a is ically om M.L. Calde ini e al. Algal Resea ch 60 (2021) 102486 4 each o he . PERMANOVA analysis o ω -3, ω -6, EAA, and NEAA we e ca ied ou on no malized pe cen age da a o each ca ego y. Mul i a - ia e homogenei y o g oup dispe sion ( a iances) was es ed using Ma i Ande son's p ocedu e o he analysis o mul i a ia e homogenei y (PERMDISP) [38]. The limi o s a is ical signi icance in all es s was se o α ≤0.05. All s a is ical analyses we e conduc ed using R (RS udio e sion 3.6.3), mixed e ec s models we e conduc ed wi h lme4 package ( 1.1-21), he es o he analysis was ca ied ou wi h ei he R base o egan packages [39]. 3. Resul s and discussion 3.1. E ec o RAS was ewa e il a ion on cell densi y, biomass, and chlo ophyll-a Fil a ion o No dic RAS WW h ough 0.45 μ m il e dec eased he u bidi y o he media bu did no a ec he concen a ion o dissol ed nu ien s (Table 1). In con as wi h ou hypo hesis, no di e ences in cell densi y, speci ic g ow h a e, o d y weigh we e obse ed be ween mic oalgae cul i a ed in un il e ed and il e ed WW a e 6 days o cul i a ion (p >0.05; Fig. 1; Table S.1). Chlo ophyll-a concen a ion was almos ~50% lowe in H. plu ialis cul i a ed in un il e ed compa ed o il e ed WW (p <0.05; Fig. 1d; Table S.4), while M. g i i hii and Sele- nas um sp. did no p esen di e ences in chlo ophyll-a be ween ea - men s (p >0.05; Table S.3). Al oge he , ou esul s sugges ha il a ion o RAS WW does no p omo e (no supp ess) mic oalgal g ow h du ing sho - e m cul i a ion, showing ha nei he biological con amina ion no he concen a ion o suspended solids p esen in RAS WW signi i- can ly a ec mic oalgal g ow h. In p e ious s udies, di e ences in cell densi y and d y weigh be ween mic oalgae cul i a ed in un il e ed and il e ed (0.20–0.22 μ m) WW we e obse ed a e mic oalgae en e ed s a iona y phase [40,41]. The e o e, i is possible ha wi h sho - e m cul i a ions, when nu ien s a e no limi ing, biological con amina ion o RAS WW does no limi mic oalgal g ow h. Since mic oalgal ha - es ing should co espond wi h he highes p oduc ion o he desi ed end-p oduc , cul i a ion imes should be op imized based on he a - ge ed use o mic oalgal biomass. Fo RAS WW, op imiza ion o cul i- a ion imes should p io i ize maximal mic oalgal biomass, nu ien emo al, and nu i ional quali y o he gene a ed biomass, hence, long cul i a ion imes whe e mic oalgae each s a iona y phase migh no be equi ed. Despi e la ge di e ences in cell densi ies (Fig. 1a), he h ee es ed mic oalgae eached e y simila d y weigh s by day 6 (Fig. 1c; Table S.5), possibly due o di e ences in cell size be ween mic oalgae species and pa icula ly he la ge size o H. plu ialis (Fig. S.1). I is impo an o poin ou ha he in luence o pa icula e ma e and biological con amina ion p esen in WW o he inal d y weigh canno be discoun ed, pa icula ly o un il e ed WW. The e o e, despi e no obse ing di e ences in cell numbe be ween ea men s, i is possible ha he eco ded d y weigh s we e sligh ly o e es ima ed. Independen o he media, M. g i i hii had a highe speci ic g ow h a e han he o he mic oalgae (0.61 ±0.03 d −1 ) (p <0.05; Fig. 1b; Table S.5), while no di e ence was obse ed be ween H. plu ialis and Selenas um sp. (0.47 ±0.04 and 0.49 ±0.03 d −1 , espec i ely) (Fig. 1b; Table S.5). Speci ic g ow h a e and cell densi y alues obse ed in his s udy ag ee wi h hose o li e a u e on he same mic oalgae species cul i a ed in e e ence algae media [23,42,43]. In addi ion, o he s udies looking a he same mic oalgae gene a/species cul u ed in di e en ypes o WW a empe a u es o 20 ◦C o highe epo ed simila o lowe speci ic g ow h a es han in his wo k (Haema ococcus [8]; Mono aphidium [9]; Selenas um [44]). This highligh s he esilience o he h ee es ed mic oalgae species used in his s udy and p esen s hem as good candida es o he bio emedia ion o o he indus ial WWs wi h simila p ope ies o No dic RAS WW. O e all, he simila g ow h a es ob ained in his and ou p e ious s udy wi h he same mic oalgal s ains and cul u e condi ions [23], suppo he use o RAS WW o e icien mic oalgal cul i a ion. 3.2. Nu ien emo al Nu ien emo al e iciency ( emo al pe cen age and emo al a e) was no a ec ed by WW il a ion (p >0.05; Fig. 2; Table S.4). This esul sugges s ha mic oalgal emo al o ni a e‑ni ogen (NO 3 -N) o phospha e‑phospho us (PO 4 -P) du ing sho - e m cul i a ion pe iods is no a ec ed by WW's biological con amina ion no suspended solids. These indings a e in line wi h p e ious s udies on mic oalgal bio e- media ion o aquacul u e WW, whe e comple e s e iliza ion o WW did no signi ican ly a ec he nu ien emo al e iciency o g een mic o- algae du ing exponen ial g ow h [27,41]. Howe e , as wi h cell densi y, species-speci ic e ec s o biological con amina ion on nu ien emo al ha e been obse ed when mic oalgae we e g own un il la e s a iona y phase [40]. Rega dless o he cul i a ion media, Selenas um sp. and M. g i i hii we e able o emo e almos all PO 4 -P in WW (~99% on a e age) (Fig. 2a,c), while H. plu ialis had a lowe (~75%) emo al e iciency han he o he mic oalgae (Fig. 2a,c; Table S.5). NO 3 -N emo al was less han ~40% o all mic oalgal species and cul i a ion media (Fig. 2b,d). Compa ison be ween species showed ha H. plu ialis had a highe NO 3 - N emo al a e han M. g i i hii in il e ed WW (p <0.05; Table S.5) while no o he di e ences in nu ien emo al we e obse ed (Table S.5). Compa ed o p e ious s udies looking a he WW Fig. 1. Densi y (Den) (a), speci ic g ow h a e (SGR) (b), d y weigh (DW) (c), and chlo ophyll-a con en (Chl a) (d) o h ee g een mic oalgae (Haema ococcus plu ialis, Mono aphidium g i i hii, and Selenas um sp.) g own in ei he un il- e ed (WW) o il e ed RAS was ewa e (FWW) o 6 days. Values a e p esen ed as mean ±SD o ou eplica es. S a is ically signi ican di e ences be ween ea men s a e shown wi h *. Compa ison o ea men s be ween mic oalgae is no p esen ed in his igu e. M.L. Calde ini e al. Algal Resea ch 60 (2021) 102486 5 bio emedia ion capaci ies o he es ed mic oalgae, he obse ed NO 3 -N and PO 4 -P emo al a es we e consis en wi h hose desc ibed a cul i- a ion empe a u es o 20 ◦C o abo e (H. plu ialis [8]; Mono aphidium [9]; Selenas um [44]). In addi ion, in ou p e ious s udy we obse ed simila PO 4 -P and highe NO 3 -N emo al a es compa ed o his s udy [23], con i ming ha mic oalgae bio emedia ion o No dic RAS WW a ca. 17 ◦C is a iable op ion o WW ea men . Ne e heless, gi en he NO 3 -N emo al pe cen age obse ed (less han 40% o all species), and conside ing ha mic oalgae nu ien emo al a es a e a ec ed by he N: P a io o he cul u e media, balancing WW's N:P a io nea op imal equi ed by mic oalgae could inc ease nu ien up ake [7]. In his s udy he N:P a io obse ed in WW was >55 (Table 1), indica ing phospho us limi a ion o mic oalgal g ow h and sugges ing ha he addi ion o phospho us o WW, p io o mic oalgal inocula ion, could ep esen an oppo uni y o imp o e NO 3 -N emo al. 3.3. RAS was ewa e il a ion and mic oalgal a y acid p o iles All mic oalgae con ained al a-linolenic acid (ALA, 18:3 ω -3) s ea - idonic acid (SDA, 18:4 ω -3), linolenic acid (LIN, 18:2 ω -6), and gamma- linolenic acid (GLA, 18:3 ω -6) (Fig. 3). None o he s udied species p e- sen ed de ec able amoun s o DHA, and only H. plu ialis con ained a achidonic acid (ARA, 20:4 ω -6) and low con en s o EPA (0.56 ±0.17 and 0.76 ±0.14 μ g mg −1 DW in un il e ed and il e ed WW, espec- i ely) (Fig. 3). PERMANOVA analysis showed no e ec o WW il a ion on he con ibu ion o he s udied ω -3 o ω -6 FAs (Table 2). Ne e heless, PERMANOVA es loses powe and is mo e p one o ype II e o s in he p esence o he e oscedas ici y. Gi en he obse ed di e ences in dispe sion o ω -6 FA con ibu ion da a (p <0.05; Fig. S.2a; Table 3), i is possible ha di e ences in he ω -6 FAs con ibu ion be ween ea men s did exis . The e ec o mic oalgae species was signi ican in he PER- MANOVA analysis and explained >85% o he obse ed di e ences in ω -3 o ω -6 FAs (Table 2). This esul is in line wi h p e ious wo k desc ibing phy oplank on axa as he mos explana o y a iable o di e ences in FA p o iles [45,46]. Al hough all mic oalgae displayed simila o al ω -3 FAs con ibu ions (~45%), SDA con ibu ions a ied om ~2.5% o ~7.5% in H. plu ialis and Selenas um sp., espec i ely (Fig. 3a). H. plu ialis p esen ed a ω -6 FA con ibu ion o ~17%, wi h LIN making up o ~76% o he o al ω -6 FA composi ion (Fig. 3c). Compa ed o p e ious s udies whe e H. plu ialis and M. g i i hii we e cul i a ed in a e e ence algae medium, we obse ed simila ω -3 and ω -6 FA p o iles [42,47], sugges ing ha he use o No dic RAS WW as a g ow h media, do no signi ican ly a ec he mic oalgal composi ion o nu i ionally impo an FAs. In e ms o FA con en , H. plu ialis had a lowe ω -3 and ω -6 con en when cul u ed in un il e ed compa ed o il e ed WW (p <0.05; Table S.6), while no di e ences we e seen o M. g i i hii o Selenas um sp. be ween ea men s (Fig. 3b,d; Table S.6). In e es ingly, since no di e ence in FA composi ion was obse ed, H. plu ialis p opo ionally dec eased he con en o ω -3 and ω -6 FAs, sugges ing ha he condi ions p esen in un il e ed RAS WW a ec ed he o al p oduc ion o FAs compa ed o il e ed WW. Since we did no quan i y o al lipids, di - e ences in o al lipid con en be ween ea men s migh exis . The e- o e, he e ec o biological con amina ion and suspended solids on lipid p oduc ion migh be unde es ima ed in his s udy. O e all, ou FA e- sul s sugges ha mic oalgal FA me abolism esponds in a species- speci ic manne o he na i e condi ions o No dic RAS WW. The ω -3 and ω -6 a io, which is a use ul indica o o FA nu i ional alue o zooplank on [48], was ~4.5 in Selenas um sp. and M. g i i hii and ~2.5 in H. plu ialis. This esul indica es ha Selenas um sp. and M. g i i hii ha e a highe FA nu i ional alue han H. plu ialis. Due o he simila ω -3 and ω -6 FA con en s seen in M. g i i hii and Selenas um sp., we ha e no eason o belie e ha one o hese mic oalgae has su- pe io nu i ional FA alue han he o he . S udies on biocon e sion o FAs ha e shown ha plank onic o gan- isms such as Daphnia can elonga e and desa u a e ALA and SDA o EPA and LIN o ARA [49,50], bo h o which a e essen ial FAs o ish g ow h and de elopmen [17]. The e o e, gi en ha ALA and LIN a e majo con ibu o s o he ω -3 and ω -6 FA con en o he es ed mic oalgae, il e - eeding Daphnia could be used o upg ade he FA nu i ional alue o he gene a ed algal biomass o la e use in RAS, as well as o dec ease he cos s associa ed wi h he mechanical ha es o mic oalgae. 3.4. RAS was ewa e il a ion and mic oalgal amino acid p o iles All 15 s udied AAs we e iden i ied in each mic oalgae species ega dless o he cul i a ion media (Fig. 4; Table S.1). Con ibu ions o o al EAAs and NEAAs we e e y simila be ween species and ea men s (~50% o bo h EAAs and NEAAs). Acco ding o PERMANOVA, mic oalgae species showed a s a is ically signi ican e ec on bo h EAA and NEAA con ibu ion (p <0.05; Table 2), explaining 27.8% and 21.5% o he a ia ion seen in EAA and NEAA, espec i ely. These esul s a e in ag eemen wi h p e ious li e a u e, whe e he o al con ibu ion o EAA and NEAA emained ela i ely cons an be ween mic oalgae species/ axa bu signi ican a ia ions we e seen a he indi idual AA le el [51]. No e ec o il a ion was seen on he con ibu ion o EAAs o NEAAs (Table 2) and composi ional da a showed o ha e a homogeneous dispe sion o bo h EAA and NEAA (Figs. S.2b, S.3; Table 3). Fil a ion o WW a ec ed nega i ely he AA con en o Selenas um sp. o bo h EAAs and NEAAs (p <0.05; Fig. 4b; Table S.6), while M. g i i hii showed a lowe EAA con en in il e ed compa ed o un il e ed WW (p <0.05; Fig. 4b; Table S.6). Howe e , since biological con aminan s in RAS WW Fig. 2. Pe cen age o PO 4 -P emo al (a), pe cen age o NO 3 -N emo al (b), PO 4 -P emo al a es (R P ) (c), and NO 3 -N emo al a es (R N ) (d) o h ee g een mic oalgae (Haema ococcus plu ialis, Mono aphidium g i i hii, and Selenas um sp.) g own in ei he un il e ed (WW) o il e ed was ewa e (FWW) o 6 days. Values a e p esen ed as mean ±SD o ou eplica es. M.L. Calde ini e al. Algal Resea ch 60 (2021) 102486 6 such as bac e ia can syn hesize all es ed AAs [52], i is possible ha he highe obse ed EAA and NEAA con en s in un il e ed WW could be a ibu ed o he p esence o bac e ia in he cul u e media. Conse- quen ly, i is possible ha he AA con en obse ed in H. plu ialis cul i a ed in un il e ed WW was also inc eased by he p esence o bac e ia in he cul u e medium. As a limi a ion o ou s udy, we did no quan i y o al p o ein con en , which could ha e shown i di e ences be ween ea men s in p o ein con ibu ion o d y biomass exis . O e all, H. plu ialis had he highes EAA and NEAA con en s o he s udied species in bo h un il e ed and il e ed WW (EAA: 139 ±6 and 139 ±4, NEAA: 148 ±5 and 145 ±4 μ g mg −1 DW, espec i ely) (p < 0.05; Fig. 4b; Table S.6). Compa ed o p e ious s udies, we obse ed simila o al AA con en s o hose desc ibed o eshwa e g een mic oalgae in non-axenic cul u es o e e ence algae media [53], indi- ca ing ha he use o No dic RAS WW as a g ow h medium does no a ec he p oduc ion o AA by mic oalgae. Because EAAs a e conside ed Fig. 3. P opo ion o all a y acids (FA) and pe biomass con en o ω -3 (a, b) and ω -6 (c, d) FA o h ee g een mic oalgae (Haema ococcus plu ialis, Mono aphidium g i i hii, and Selenas um sp.) g own in ei he un il e ed (WW) o il e ed (FWW) RAS was ewa e o 6 days. S a is ically signi ican di e ences in FA con en be ween ea men s a e shown wi h *. Compa isons o ea men s be ween mic oalgae a e no p esen ed in his igu e. Table 2 PERMANOVA esul s o mic oalgae ω -3 and ω -6 a y acids oge he wi h essen ial and non-essen ial amino acid (EAA and NEAA, espec i ely) con i- bu ion p o iles. Dissimila i ies in amino acid p o iles we e compa ed be ween species (Haema ococcus plu ialis, Mono aphidium g i i hii, and Selenas um sp.), ea men s (un il e ed and il e ed was ewa e ) and hei in e ac ion (species * ea men ). Rema ked in bold, s a is ically signi ican alues (p <0.05). Va iable Sou ce d Pseudo-F exp% p ω -3 Species 2 62.51 85.3 <0.01 T ea men 1 2.02 1.4 0.16 Species * ea men 2 0.80 8.0 0.47 ω -6 Species 2 103.60 90.5 <0.01 T ea men 1 3.15 1.4 0.08 Species * ea men 2 0.26 0.2 0.79 EAA Species 2 3.98 27.8 <0.01 T ea men 1 0.57 2.0 0.66 Species * ea men 2 1.07 7.5 0.40 NEAA Species 2 2.98 21.5 0.02 T ea men 1 1.05 3.8 0.34 Species * ea men 2 1.36 9.8 0.24 Table 3 Pe mu a ional Analysis o Mul i a ia e dispe sion (PERMDISP) o he con i- bu ion o essen ial and non-essen ial amino acids (EAA and NEAA, espec i ely) and ω -3 and ω -6 a y acids ac oss h ee g een mic oalgae (Haema ococcus plu- ialis, Mono aphidium g i i hii, and Selenas um sp.) g own in ei he un il e ed o il e ed was ewa e o 6 days. Rema ked in bold, s a is ically signi ican alues (p <0.05). Uni Sou ce d F p ω -3 Species 2 1.43 0.26 ω -6 Species 2 5.27 0.01 EAA Species 2 1.31 0.29 NEAA Species 2 1.88 0.18 M.L. Calde ini e al. Algal Resea ch 60 (2021) 102486 7 he same o zooplank on and ish as o insec s and humans [54] and NEAA a e desc ibed o be a key componen explaining zooplank on g ow h [19], ou esul s sugges ha all es ed mic oalgae species could se e as a nu i ious ood sou ce o zooplank on and ish. Al oge he , gi en ha he FA and AA con en s o M. g i i hii and Selenas um sp. we e no al e ed by il a ion, hey appea as good candida es o he bio emedia ion o un il e ed WW when biomass is in ended o u he use in aquacul u e. Ne e heless, i il a ion o RAS WW is possible, H. plu ialis could ha e an ad an age o e he o he es ed species due o i s EPA, ARA, and o e all highe AA con en . I is impo an o poin ou ha mic oalgal biochemical p o iles, as well as g ow h dynamics, a e suscep ible o changes in g ow h media composi ion, ype o pho obio eac o used, and o he ac o s [55]. Thus, i is possible ha di e en g ow h a es and a ia ions in he FA and AA con en s could be obse ed when scaling up he s udied p oduc ion sys em. In addi ion, in his s udy we did no analyze he ca o enoid con en o he es ed mic oalgae, and canno he e o e e alua e he e - ec o biological con amina ion and suspended solids in RAS on his nu i ionally impo an g oup o biomolecules o aquacul u e [56]. Among o he di icul ies o he use o RAS WW o mic oalgal cul i a ion is ha i s composi ion is no homogenous among acili ies, which adds o he p oblem o eplicabili y o esul s. Tha is why mo e and la ge - scale s udies a e needed o u he con i m he easibili y o he use o mic oalgae o bio emedia ion o RAS WW and he p oduc ion o nu i ionally aluable biomass. 3.5. Bac e ial biomass in mic oalgae cul u es o un il e ed and il e ed was ewa e Un il e ed WW showed an o e all highe bac e ial biomass based on ac inobac e ial FAs han i s il e ed coun e pa a e six days o mic oalgae cul i a ion (Fig. 5; Table S.3). Excluding Selenas um sp., bo h H. plu ialis and M. g i i hii cul u es had highe bac e ial biomasses when g own in un il e ed compa ed o il e ed WW (p <0.05; Fig. 5; Table S.3). The highes bac e ial biomass was obse ed in H. plu ialis cul u e, whe e almos 3- old highe biomass was obse ed in un il e ed compa ed o il e ed WW (p <0.05; Fig. 5; Table S.4). O e all, app oxima ed bac e ial biomasses in il e ed WW we e simila in all mic oalgal cul u es (Fig. 5. Table S.4). Since H. plu ialis exhibi ed high bac e ial biomass in un il e ed WW, i is possible ha compe i ion o nu ien s o o he e ec s o he p esence o bac e ia induced a s ess esponse in his mic oalgae. Commonly, H. plu ialis esponds o s ess by accumula ing as axan hin a he expense o chlo ophyll deg ada ion [57]. The e o e, he lowe chlo ophyll-a con en obse ed o his mic oalgae in un il e ed WW (Fig. 1d) could be explained by he an- si ion om g een cells o as axan hin ich cys s. Ne e heless, we did no see di e ences in cell densi y by day 6 (Fig. 1a) and no di e ences we e seen in he numbe o cys s be ween il e ed and un il e ed ea men , sugges ing ha as axan hin accumula ion had only s a ed by he end o he cul i a ion pe iod. Compa ed o H. plu ialis, Selenas um sp. and M. g i i hii p esen ed lowe bac e ial biomasses han in un il e ed WW, sugges ing ha hese wo species a e be e able o con ol bac e ial g ow h. None heless, as all species showed simila bac e ial biomasses in il e ed WW cul u es, i Fig. 4. P opo ion o all amino acids (AA) (a) and pe biomass con en (b) o essen ial and non-essen ial amino acids (EAA and NEAA, espec i ely) o h ee g een mic oalgae (Haema ococcus plu ialis, Mono aphidium g i i hii, and Selenas um sp.) g own in ei he un il e ed (WW) o il e ed (FWW) RAS was ewa e o 6 days. S a is ically signi ican di e ences in AA con en be ween ea men s a e shown wi h *. Bold le e s E and N a e used o deno e i s a is ical di e ence in EAA and/o NEAA ( espec i ely) was obse ed. Compa isons o ea men s be ween mic oalgae a e no p esen ed in his igu e. Fig. 5. Bac e ial a y acids (FAs) con en in mic oalgae cul u e media o h ee g een mic oalgae (Haema ococcus plu ialis, Mono aphidium g i i hii, and Sele- nas um sp.) g own in ei he un il e ed (WW) o il e ed WW (FWW) o 6 days. Values a e gi en as he sum o all iden i ied bac e ial FAs. S a is ically signi - ican di e ences be ween ea men s a e shown wi h *. Compa isons o ea - men s be ween mic oalgae a e no p esen ed in his igu e. M.L. Calde ini e al. Algal Resea ch 60 (2021) 102486 8 is possible ha when lowe bac e ial loads a e ini ially p esen in he cul u e medium, bac e ial g ow h dynamics a e equally a ec ed by he di e en mic oalgae species. Since we only quan i ied ac inobac e ial FAs om mic oalgae il- a es (3.0 μ m il e po e size), ou esul s mos likely unde es ima e he o al bac e ial loads o he cul u es due o bac e ia passing h ough he il e s. In addi ion, we did no ollow bac e ial g ow h du ing mic oalgal cul i a ion, which could ha e exposed aluable in o ma ion o he unde s anding o mic oalgae-bac e ia g ow h dynamics in WWs. E en hough we did no see di e ences in mic oalgal cell numbe s be ween ea men s by he end o he cul i a ion pe iod (Fig. 1a), s udies by Bolch e al. [58] and Rhee [59] showed ha a apid onse o bac e ial g ow h on he cul u e media can sho en he exponen ial g ow h phase o mic oalgae, leading o lowe cell densi ies. This is an impo an aspec o conside i he objec i e o mic oalgal p oduc ion is he accumula ion o an end-p oduc ha equi es longe cul i a ion imes han he one used in his s udy. Since biological con amina ion encompasses mo e han jus bac e ia, he e a e likely o be o he signi ican di e ences in he biological composi ion o un il e ed and il e ed RAS WW cul u es. Daily mic o- scope examina ion o mic oalgal cul u es showed negligible amoun s o non- a ge ed o ganisms and suspended solids in il e ed WW while small numbe s o p o ozoa we e obse ed in un il e ed RAS WW. Al hough we did no quan i y any obse ed mic oo ganisms p esen in he cul u es besides he a ge ed mic oalgae, we did no obse e conside able in- c eases in biological con amina ion h oughou he cul i a ion pe iod. This sugges s ha he na i e biological con amina ion o he RAS WW used o ou expe imen did no p esen a h ea o mic oalgal p oduc ion. In summa y, ou esul s show ha mic oalgal bio emedia ion o No dic RAS WW a low empe a u es (17 ◦C) ep esen s a easible and sus ainable al e na i e o con en ional physicochemical wa e ea - men s wi h he ad an age o nu ien ecycling and aluable biomass gene a ion. In addi ion, besides changes in he FA con en o H. plu ialis, we did no obse e signi ican dec eases in mic oalgal pe o mance due o he p esence o biological con amina ion and suspended solids, sug- ges ing ha il a ion o RAS WW p io o mic oalgal inocula ion migh be unnecessa y when sho - e m cul i a ions a e used. 4. Conclusions In his s udy, we showed ha mic oalgal bio emedia ion o No dic RAS WW is a easible al e na i e o con en ional WW ea men s. Bio- logical con amina ion did no a ec mic oalgal g ow h, biomass p o- duc ion, nu ien emo al, o AA and FA composi ion o mic oalgae, bu had a nega i e e ec on he ω -3 and ω -6 FA con en o one o he s udied species, H. plu ialis. O e all, ou esul s sugges ha o he gene a ion o aluable biomass, he emo al o indigenous biological con amina ion and suspended solids o RAS WW does no p oduce majo changes in he bio emedia ion capaci y o he nu i ional quali y o mic oalgae. E-supplemen a y da a o his wo d can be ound in he online e sion o he pape . CRediT au ho ship con ibu ion s a emen Ma co Calde ini: Concep ualiza ion, Me hodology, Valida ion, Fo mal analysis, In es iga ion, W i ing - O iginal D a , W i ing - Re- iew & Edi ing, Visualiza ion. ˇ Cedomi S e ˇ ci´ c: Concep ualiza ion, Me hodology, Valida ion, In es iga ion, W i ing - O iginal D a , W i ing - Re iew & Edi ing, Visualiza ion, Supe ision. Sami Taipale: Concep ualiza ion, Me hodology, Valida ion, Re- sou ces, W i ing - Re iew & Edi ing, Visualiza ion, Supe ision, P ojec adminis a ion, Funding acquisi ion. Ka ja Pulkkinen: Concep ualiza ion, Me hodology, Valida ion, Re- sou ces, W i ing - Re iew & Edi ing, Visualiza ion, Supe ision, P ojec adminis a ion, Funding acquisi ion. In o med consen , human/animal igh s No con lic s, in o med consen , o human o animal igh s a e applicable o his s udy. Da a a ailabili y Da ase s ela ed o his a icle can be ound a doi:10.17011/jyx/da ase /72716. Decla a ion o compe ing in e es None. Acknowledgmen The au ho s would like o hank labo a o y echnicians Me i Kois- inen, Emma Pajunen, and Juha Ahonen o hei help du ing he expe imen al wo k, Ossi Ke a o his help in GC–MS analysis, Juhani Pi honen and Minna Hil unen o hei commen s ha imp o ed he manusc ip , Lucas Pe uchena o his help wi h he g aphical abs ac , and Na u al Resou ces Ins i u e Finland (LUKE) Laukaa ish a m o p o iding was ewa e o he expe imen . Financial suppo o his wo k was p o ided by he Uni e si y o Jy ¨ askyl¨ a and he Eu opean Ma i ime and Fishe ies Fund (EMFF). Appendix A. Supplemen a y da a Supplemen a y da a o his a icle can be ound online a h ps://doi. o g/10.1016/j.algal.2021.102486. Re e ences [1] J.P. Blanche on, R. Pied ahi a, E.H. Eding, D.E.R. D’O bcas el, G. Lema i´ e, A. Be gheim, S. Fi els ad, In ensi ica ion o landbased aquacul u e p oduc ion in single pass and euse sys ems, in: A. Be gheim (Ed.), Aquacul u al Enginee ing and En i onmen , Resea ch Signpos , 2007, pp. 21–47. [2] R.H. 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