Isolation, Identification and Biotechnological Applications of a Novel, Robust, Free-living Chlorococcum (Oophila) amblystomatis Strain Isolated from a Local Pond

applied sciences A icle Isola ion, Iden i ica ion and Bio echnological Applica ions o a No el, Robus , F ee-li ing Chlo ococcum (Oophila) amblys oma is S ain Isola ed om a Local Pond Nádia Co eia 1,2 , Hugo Pe ei a 3, Joana T. Sil a 1, Tamá a San os 3, Ma ia Soa es 1, Ca olina B. Sousa 3,4, Lisa M. Schüle 3, Ma ga ida Cos a 1, João Va ela 3,* , Leonel Pe ei a 2 and Joana Sil a 1 1Allmic oalgae Na u al P oduc s S.A., Resea ch and De elopmen Depa men —Rua 25 de Ab il, nº 19, 2445-413 Pa aias, Po ugal; [email p o ec ed] (N.C.); [email p o ec ed] (J.T.S.); ma ia.soa es@allmic oalgae.com (M.S.); [email p o ec ed] (M.C.); [email p o ec ed] (J.S.) 2MARE—Ma ine and En i onmen al Sciences Cen e, Depa men o Li e Sciences, Uni e si y o Coimb a, 3000-456 Coimb a, Po ugal; [email p o ec ed] 3CCMAR—Cen e o Ma ine Sciences, Uni e si y o Alga e, Gambelas, 8005-139 Fa o, Po ugal; [email p o ec ed] (H.P.); [email p o ec ed] (T.S.); ca olb [email p o ec ed] (C.B.S.); [email p o ec ed] (L.M.S.) 4Global Heal h and T opical Medicine (GHMT), Ins i u o de Higiene e Medicina T opical, Uni e sidade No a de Lisboa, 1349-008 Lisboa, Po ugal *Co espondence: [email p o ec ed]; Tel.: +351-289-800-900; Fax: +351-289-800-069 Recei ed: 23 Ma ch 2020; Accep ed: 23 Ap il 2020; Published: 27 Ap il 2020   Abs ac : Biop ospec ion o no el au och honous s ains is key o he success ul indus ial-scale p oduc ion o mic oalgal biomass. A no el Chlo ococcum s ain was ecen ly isola ed om a pond inside he indus ial p oduc ion acili y o Allmic oalgae (Lei ia, Po ugal). Phylogene ic analysis based on 18S ibosomal ibonucleic acid ( RNA) gene sequences sugges s ha his isola e is a no el, ee-li ing Oophila amblys oma is s ain. Howe e , as ou phylogene ic da a s ongly sugges s ha he a o emen ioned axon belongs o he genus Chlo ococcum, i is he e p oposed o ename his species as Chlo ococcum amblys oma is. In o de o cha ac e ize he bio echnological po en ial o his no el isola e, g ow h pe o mance and biochemical composi ion we e e alua ed om he pilo (2.5-m 3 ) o indus ial (10-m 3 ) scale. The highes maximum a eal p oduc i i y (36.56 g · m −2· day −1 ) was eached in a 10-m 3 ubula pho obio eac o (PBR), as compa ed o ha ob ained in a 2.5-m 3 PBR (26.75 g · m −2· day −1 ). Chlo ococcum amblys oma is displayed high p o ein con en (48%–56% d y weigh (DW)) and mode a e le els o o al lipids (18%–31% DW), ca bohyd a es (6%–18% DW) and ashes (9%–16% DW). Fu he mo e, he lipid p o ile was domina ed by polyunsa u a ed a y acids (PUFAs). The highes pigmen con en s we e ob ained in he 2.5-m 3 PBR, whe e o al chlo ophylls accoun ed o 40.24 mg · g −1 DW, ollowed by lu ein wi h 5.37 mg · g −1 DW. O e all, his ee-li ing Chlo ococcum amblys oma is s ain shows g ea po en ial o nu i ional applica ions, coupling a p omising g ow h pe o mance wi h a high p o ein con en as well as ele an amoun s o PUFAs, chlo ophyll, and ca o enoids. Keywo ds: biop ospec ion; mic oalgae; Chlo ococcum (Oophila) amblys oma is; pho obio eac o s; indus ial-scale p oduc ion; bio echnological applica ions Appl. Sci. 2020,10, 3040; doi:10.3390/app10093040 www.mdpi.com/jou nal/applsci Appl. Sci. 2020,10, 3040 2 o 14 1. In oduc ion Mic oalgal biodi e si y emains la gely unexplo ed and biop ospec ion e o s may ep esen an oppo uni y o disco e no el me aboli es and p omo e biomass p oduc ion a lowe cos s [ 1 , 2 ]. These mic oo ganisms ha e been widely ecognized as a co ne s one o bioeconomy and bio e ine y de elopmen , due o hei high applicabili y as eeds ocks o ood, eed, bioplas ics, bio e ilize s, and bio uels as well as o was ewa e ea men and CO 2 mi iga ion [ 3 – 5 ]. In Eu ope, mac o- and mic oalgae we e ecognized in he S a egic Ene gy Technological Plan (SET-PLAN) as a pa o he key- alue chain o ad anced bioma e ials p oduc ion. Po ugal, as a coas al coun y, has excellen clima ic condi ions and a g ea po en ial o a sus ainable indus ial mic oalgal cul i a ion [ 5 ]. Ne e heless, he high cos o mic oalgal p oduc ion is s ill he main es ic ion associa ed wi h he comme cializa ion o biomass o di e en commodi ies [5,6]. The wo ld’s inc easing popula ion and he p edic ions o an insu icien p o ein supply ha e led o esea ch on new al e na i e and uncon en ional p o ein sou ces [ 7 ]. Algal biomass is cu en ly conside ed as a good candida e o his pu pose since hese pho osyn he ic aqua ic mic oo ganisms a e able o syn hesize all he essen ial amino acids and hei cul i a ion does no compe e o a able land, as hey can be g own in bio eac o s [ 3 , 5 ]. P o eins a e he main cons i uen s o mic oalgae and one o he mos impo an p oduc s o mic oalgae-based bio e ine ies [ 8 ]. In e es ingly, hese p o eins, and pep ides he eo , a e e y in e es ing o unc ional ood applica ions due o hei biological ac i i ies, in pa icula hose ela ed o he p e en ion and educ ion o hype ension, oxida i e s ess, cance , diabe es, in lamma ion, and immune diso de s [ 9 ]. Mo eo e , as mic oalgae con ain la ge amoun s o pigmen s (e.g., ca o enoids), many o hem wi h known an ioxidan p ope ies and biomedically ele an bioac i i ies, mic oalgal biomass can be used o o i y o supplemen ood, eed, and cosme ics, imp o ing hei basic ma ke alue while p o iding inc eased heal h bene i s [ 10 ]. This is in line wi h he expec ed inc ease in he wo ldwide ma ke size o na u al pigmen s ueled by a g owing demand o such p oduc s by he consume s [3,5]. Biop ospec ing o no el mic oalgal species and p omo ing indus ial biomass p oduc ion is key o imp o ing he cu en po olio o a ailable s ains o di e en bio echnological applica ions. The sc eening o molecules and ac i i ies o bio echnological in e es as well as he unde s anding o mic oalgal ecosys em unc ioning a e essen ial o de elop sus ainable and economical solu ions o oday’s ci iliza ion and en i onmen al needs [11–13]. The p esen wo k aimed o cha ac e ize and e alua e he bio echnological po en ial o a new isola e ob ained om a pond a he Allmic oalgae acili ies whose 18S ibosomal deoxy ibonucleic acid ( DNA) sequence is iden ical o ha o Oophila amblys oma is. In e es ingly, his sequence clus e s oge he wi h hose o he S ephanosphae inia mac oclade belonging o he genus Chlo ococcum, sugges ing ha O. amblys oma is should be eclassi ied as Chlo ococcum amblys oma is. As he bio echnological use o his no el isola e was he ocus o he p esen wo k, i s g ow h pe o mance in he pilo and indus ial-scale sys ems was assessed. In addi ion, i s composi ion in e ms o p o ein, lipids, ca bohyd a es, and pigmen s we e e alua ed o de e mine he nu i ional po en ial o his mic oalga. 2. Ma e ials and Me hods 2.1. En i onmen al Sampling and S ain Isola ion The mic oalga used in his wo k, Chlo ococcum amblys oma is, was ob ained om a wa e sample collec ed om a eshwa e pond in he p oduc ion acili y o Allmic oalgae, loca ed in Pa aias (Lei ia, Po ugal). The isola ion p ocess was aken acco ding o Pa in e al. [ 14 ]. B ie ly, o isola e no el mic oalgal s ains, samples we e cen i uged se e al imes in o de o sepa a e mic oo ganisms acco ding o hei densi y g adien . The cells we e hen successi ely dilu ed and s eaked on pla es con aining g ow h medium (Sameca, Po o, Po ugal) whose composi ion is based on Guilla d’s F/2. Di e en colonies we e sepa a ed and obse ed mic oscopically. S eak-pla ing was pe o med in o de o ob ain unialgal, non-axenic, colonies. These isola ed cul u es we e hen s o ed in solid and Appl. Sci. 2020,10, 3040 3 o 14 liquid media on Allmic oalgae cul u e collec ion a 17 ◦ C and 12 h o ligh i adiance pe day, unde a con inuous pho on lux densi y o 20 µmol·m−2·s−1. 2.2. Mic oscopic Obse a ion and Molecula Iden i ica ion Mic oscopic images we e acqui ed in a Zeiss AXIO Scope A1 mic oscope wi h a Zeiss AXIOCAM 503 colo came a and ZEN Blue 2.5 li e so wa e (Ca l Zeiss Mic oscopy GmbH, Jena, Ge many), using he 40-x lens. Mic oscopy was ca ied ou using di e en ial in e e ence con as (DIC). Phylogene ic analysis was pe o med h ough Maximum-likelihood and Bayesian in e ence using a pa ial sequence o C. amblys oma is 18S RNA gene, which was deposi ed in GenBank wi h he accession numbe MT026583. The DNA sample was ob ained, and i s sequence was gene a ed as desc ibed in Pe ei a e al. [ 15 ] using he 18SUni Fo and 18SUni Re p ime s. The subs i u ion models ha bes i he da a we e selec ed using M Model es 2 .2.3 [ 16 ] and PAUP* .4.0b10 [ 17 ] applying he Akaike in o ma ion c i e ia (AIC; Akaike 1974). The maximum-likelihood analysis was pe o med using RaxML .7.0.4 [ 18 ], assuming a GTR+G+I subs i u ion model, wi h 400 boo s ap eplica es. Pos e io p obabili ies we e de e mined by Ma ko Chain Mon e Ca lo (MCMC) sampling in M Bayes .3.1.2 [ 19 , 20 ]. M Bayes analysis was also conduc ed using he model GTR+G (ns =6 a es =gamma) wi h six chains o 10,000,000 MCMC gene a ions, sampling e e y 1000 h gene a ion and using he de aul o all he o he se ings. The MCMC uns con e gence and bu n-in we e de e mined h ough he analysis o he gene a ions s log p obabili y plo using he ace analysis ool TRACER 1.6 [ 21 ]. Using he bu n-in, 20% o he ees we e disca ded. ML bes and consensus ee and BI consensus ee we e gene a ed using makeConsensusT ee.py command and edi ed wi h he g aphical iewe FigT ee .1.4.2 [22]. 2.3. Scale-Up and Biomass P oduc ion All expe imen s desc ibed we e pe o med a Allmic oalgae indus ial acili ies be ween Janua y and June 2018. A concen a ed cul u e medium (Sameca, Po o, Po ugal) based on Guilla d’s F/2 and supplemen ed wi h 25 µ M o i on (Hubel Olh ã o, Po ugal) was added un il a inal concen a ion o 10 mM o ni a e was eached. Mic oalgal cul u es we e g own in he labo a o y in 5-L bubble column eac o s a 24 ± 1 ◦ C wi h con inuously injec ed comp essed 0.2- µ m il e ed ai , unde a con inuous pho on lux densi y o 100 µ mol · m −2· s −1 . The pH o he cul u e medium was kep be ween 7.5 and 8.0 by pe iodical CO2injec ion. The scale-up o he ou doo pilo and indus ial-scale eac o s was pe o med by using i e labo a o y eac o s o inocula e a 0.25-m 3 la panel (FP) (Figu e 1a). Cul u es we e ae a ed wi h 0.2- µ m il e ed ai , he pH was also main ained be ween 7.5 and 8.0 by CO 2 pulse injec ion and he empe a u e inside he FP was main ained below 28 ◦ C by a sp inkle -like i iga ion sys em. The ea e , he 0.25-m 3 FP was used o inocula e a ubula pho obio eac o (PBR) o 2.5-m 3 (Figu e 1b) and abou 80% o his was used as inoculum o a 10-m 3 PBR (Figu e 1c). The pH was main ained unde 8.0 h ough an au oma ed CO 2 injec ion sys em and he empe a u e was kep unde 28 ◦ C by a sp inkle -like i iga ion sys em. Appl. Sci. 2019, 9, x FOR PEER REVIEW 3 o 14 2.2. Mic oscopic Obse a ion and Molecula Iden i ica ion Mic oscopic images we e acqui ed in a Zeiss AXIO Scope A1 mic oscope wi h a Zeiss AXIOCAM 503 colo came a and ZEN Blue 2.5 li e so wa e (Ca l Zeiss Mic oscopy GmbH, Jena, Ge many), using he 40-x lens. Mic oscopy was ca ied ou using di e en ial in e e ence con as (DIC). Phylogene ic analysis was pe o med h ough Maximum-likelihood and Bayesian in e ence using a pa ial sequence o C. amblys oma is 18S RNA gene, which was deposi ed in GenBank wi h he accession numbe MT026583. The DNA sample was ob ained, and i s sequence was gene a ed as desc ibed in Pe ei a e al. [15] using he 18SUni Fo and 18SUni Re p ime s. The subs i u ion models ha bes i he da a we e selec ed using M Model es 2 .2.3 [16] and PAUP* .4.0b10 [17] applying he Akaike in o ma ion c i e ia (AIC; Akaike 1974). The maximum-likelihood analysis was pe o med using RaxML .7.0.4 [18], assuming a GTR+G+I subs i u ion model, wi h 400 boo s ap eplica es. Pos e io p obabili ies we e de e mined by Ma ko Chain Mon e Ca lo (MCMC) sampling in M Bayes .3.1.2 [19,20]. M Bayes analysis was also conduc ed using he model GTR+G (ns =6 a es=gamma) wi h six chains o 10,000,000 MCMC gene a ions, sampling e e y 1000 h gene a ion and using he de aul o all he o he se ings. The MCMC uns con e gence and bu n-in we e de e mined h ough he analysis o he gene a ions s log p obabili y plo using he ace analysis ool TRACER 1.6 [21]. Using he bu n-in, 20% o he ees we e disca ded. ML bes and consensus ee and BI consensus ee we e gene a ed using makeConsensusT ee.py command and edi ed wi h he g aphical iewe FigT ee .1.4.2 [22]. 2.3. Scale-Up and Biomass P oduc ion All expe imen s desc ibed we e pe o med a Allmic oalgae indus ial acili ies be ween Janua y and June 2018. A concen a ed cul u e medium (Sameca, Po o, Po ugal) based on Guilla d’s F/2 and supplemen ed wi h 25 µM o i on (Hubel Olhão, Po ugal) was added un il a inal concen a ion o 10 mM o ni a e was eached. Mic oalgal cul u es we e g own in he labo a o y in 5-L bubble column eac o s a 24 ± 1 °C wi h con inuously injec ed comp essed 0.2-µm il e ed ai , unde a con inuous pho on lux densi y o 100 µmol.m−2.s−1. The pH o he cul u e medium was kep be ween 7.5 and 8.0 by pe iodical CO2 injec ion. The scale-up o he ou doo pilo and indus ial-scale eac o s was pe o med by using i e labo a o y eac o s o inocula e a 0.25-m3 la panel (FP) (Figu e 1a). Cul u es we e ae a ed wi h 0.2-µm il e ed ai , he pH was also main ained be ween 7.5 and 8.0 by CO2 pulse injec ion and he empe a u e inside he FP was main ained below 28 °C by a sp inkle -like i iga ion sys em. The ea e , he 0.25-m3 FP was used o inocula e a ubula pho obio eac o (PBR) o 2.5-m3 (Figu e 1b) and abou 80% o his was used as inoculum o a 10-m3 PBR (Figu e 1c). The pH was main ained unde 8.0 h ough an au oma ed CO2 injec ion sys em and he empe a u e was kep unde 28 °C by a sp inkle -like i iga ion sys em. Figu e 1. Di e en scale sys ems used o ou doo scale-up: (a) 0.25-m3 la panel pho obio eac o (FP); (b) 2.5-m3 pilo -scale ubula pho obio eac o (PBR); (c) 10-m3 indus ial PBR. Pic u es we e p o ided by Allmic oalgae, Resea ch and De elopmen Depa men , Pa aias, Po ugal. Fo he assessmen o g ow h pe o mance and biomass p oduc ion, h ee 0.25-m3 FP we e cul i a ed be ween Janua y and Feb ua y a an a e age empe a u e o 13.6 °C and ligh i adiance o 14.06 MJ.m−2.day−1. The 2.5-m3 PBR iplica es we e g own om Ma ch o May a an a e age Figu e 1. Di e en scale sys ems used o ou doo scale-up: ( a ) 0.25-m 3 la panel pho obio eac o (FP); ( b ) 2.5-m 3 pilo -scale ubula pho obio eac o (PBR); ( c ) 10-m 3 indus ial PBR. Pic u es we e p o ided by Allmic oalgae, Resea ch and De elopmen Depa men , Pa aias, Po ugal. Appl. Sci. 2020,10, 3040 4 o 14 Fo he assessmen o g ow h pe o mance and biomass p oduc ion, h ee 0.25-m 3 FP we e cul i a ed be ween Janua y and Feb ua y a an a e age empe a u e o 13.6 ◦ C and ligh i adiance o 14.06 MJ · m −2· day −1 . The 2.5-m 3 PBR iplica es we e g own om Ma ch o May a an a e age empe a u e o 17.7 ◦C and ligh i adiance o 19.04 MJ·m−2·day−1, while he indus ial PBR o 10-m3 iplica es we e g own om May o June a an a e age empe a u e o 15.5 ◦ C and ligh i adiance o abou 20.10 MJ·m−2·day−1. 2.4. G ow h Assessmen Mic oalgal g ow h was moni o ed h ough biomass d y weigh (DW). DW was de e mined by il e ing a de ined sample olume on mic o-glass il e s (0.7 µ m, VWR), hen washed wi h an equal olume o deionized wa e and d ied using a mois u e analyze (KERN DBS, Balingen-F omme n, Ge many) a 120 ◦C. Volume ic biomass p oduc i i y (P) was calcula ed as he di e ence in biomass concen a ion, in g ams pe li e (X) du ing a speci ic pe iod o ime ( ), as shown by Equa ion (1) in which he indices 1 and 2deno e di e en ime poin s: Pg·L−1·day−1=X2−X1 2− 1 (1) The a eal biomass p oduc i i y (Pa) was calcula ed as he p oduc o he olume ic biomass p oduc i i y (P) and he olume o he PBR, in L (V) di ided by he o al g ound a ea (including non-pho ic a eas) o PBR, in squa e me e s (A) as shown in Equa ion (2): Pag·m−2·day−1=P×V/A(2) The speci ic g ow h a e ( µ ) was de e mined by he quo ien be ween he g ow h a e and he biomass concen a ion wi hin a pe iod o ime, as seen in Equa ion (3): µday−1=ln (X2/X1) 2− 1 (3) whe e X2and X1 e e o biomass concen a ion (g·L−1) a di e en ime poin s. The pho osyn he ic e iciency was de e mined as he a io be ween he highe hea ing alue (HHV) and sun i adia ion. Tempe a u e and sola adia ion we e measu ed using a me eo ological s a ion (R.M. Young, Michigan, USA) and a py anome e (Apogee UT SP-110, Logan, USA). The HHV was calcula ed as indica ed in Equa ion (4) and as p e iously ecommended by Callejón-Fe e e al. [23]: HHVkJ·g−1=−3.393 +0.507.C−0.341.H+0.067.N(4) Elemen al analysis o C, H and N we e pe o med as desc ibed in Sec ion 2.5. 2.5. Biochemical Composi ion The biochemical composi ion was pe o med wi h biomass g own un il he la es exponen ial phase, which was eeze d ied p io o analysis. Elemen al analysis o C, H and N we e pe o med using a Va io EL III (Va io EL, Elemen a Analyse Sys em, GmbH, Hanau, Ge many) acco ding o he manu ac u e ’s p ocedu e. To al p o eins we e calcula ed by mul iplying he Ncon en gi en by elemen al analysis wi h he s anda d con e sion ac o o 6.25 [24]. To al lipid con en was de e mined ollowing he Bligh & Dye [ 25 ] me hod wi h some modi ica ions [ 15 ]. B ie ly, biomass was ex ac ed wi h a mix u e o chlo o o m, me hanol, and wa e (1:2:0.8, : : ) and homogenized wi h an IKA Ul a-Tu ax dispe se (IKA-We ke GmbH, S au en, Ge many) on ice o 60 s. Cen i uga ion allowed phase sepa a ion and he o ganic phase was ans e ed o new essels. A e wa ds, a known olume (0.7 mL) was pipe ed o p e-weighed ubes Appl. Sci. 2020,10, 3040 5 o 14 and e apo a ed a 60 ◦ C. The esul ing d ied esidue was weighed and compa ed wi h he DW o p o ide an accu a e de e mina ion o he lipid ac ion. The ash con en was de e mined by he weigh di e ence be o e and a e he combus ion o he biomass. Biomass was hea ed a 550 ◦ C du ing 8 h using a u nace (J. P. Selec a, Sell ho n R9-L, Ba celona, Spain). Ca bohyd a es we e calcula ed by he di e ence o he o he mac onu ien s analyzed. Fa y acids we e con e ed in o he co esponding a y acid me hyl es e s (FAME) acco ding o Lepage & Roy [ 26 ] p o ocol, modi ied by Pe ei a e al. [ 15 ]. FAME we e analyzed in a gas ch oma og aphy–mass spec ome y (GC-MS) analyze (B uke SCION 456/GC, SCION TQ MS) equipped wi h a ZB-5MS capilla y column (30 m × 0.25 mm o in e nal diame e , 0.25 µ m o ilm hickens, Phenomenex), using helium as he ca ie gas. The empe a u e p og am was 60 ◦ C o 1 min, 30 ◦ C · min −1 o 120 ◦ C, 5 ◦ C · min −1 o 250 ◦ C and inal inc ease o 20 ◦ C · min −1 o 300 ◦ C. Injec ion empe a u e was 300 ◦ C. Fo iden i ica ion and quan i ica ion o FAME, i e di e en concen a ions o he s anda ds Supelco ® 37 componen FAME Mix 41 (Sigma-Ald ich, Sin a, Po ugal) we e analyzed in o de o es ablish calib a ion cu es o each o he s anda d. Fo pigmen s ex ac ion, 10 mg o esh biomass was ex ac ed wi h glass beads in 6 mL o ace one o 10 min. The sample was cen i uged (HERMLE Z300, New Yo k, NY, USA) a 3500 pm o 10 min un il comple e loss o he pelle colo . The supe na an was analyzed using a spec opho ome e (Genesys 10S UV-VIS, MA, USA) in he scanning spec um (380 o 700 nm) and he da a we e analyzed by he Excel Add-In Sol e on Windows 2013. Ca o enoid p o iles we e de e mined acco ding o Couso e al. [ 27 ]. B ie ly, he ex ac was comple ely d ied unde ni ogen low, esuspended in high-pe o mance liquid ch oma og aphy (HPLC)-g ade me hanol and il e ed (0.22 µ m) in o an ambe HPLC ial. The sepa a ion and ch oma og aphic analysis o pigmen s we e pe o med by Me ck Hi achi LaC om Eli e HPLC (Da ms ad , Ge many) equipped wi h a diode-a ay de ec o , using a LiCh oCART RP-18 (5 µ m, 250 ×4 mm , LiCh osphe ) column and a low a e o 1 mL · min −1 . The mobile phase consis ed o e hyl ace a e as sol en A and 9:1 ( : ) ace oni ile:wa e as sol en B. The g adien p og am applied was: 0–16 min, 0%–60% A; 16–30 min, 60% A; 30–32 min, 100% A and 30–35 min 100% B. The injec ion olume was 100 µ L. Quan i ica ion o pigmen s was ca ied ou a 450 nm using calib a ion cu es o he pigmen s anda ds neoxan hin, iolaxan hin, lu ein and β -ca o ene. The HPLC ch oma og am wi h ca o enoids e en ion imes (Figu e S1) and espec i e calib a ion cu es o pigmen s anda ds (Figu es S2–S5) is gi en in Supplemen a y Ma e ials. 2.6. S a is ical Analysis S a is ical analyses we e pe o med using R so wa e ( e sion 3.6.1) h ough RS udio IDE ( e sion 1.2.1335 ). When h ee o mo e condi ions we e analyzed, ANOVA was pe o med wi h he mul iple compa isons o Tukey-HSD. Fo he compa ison o g oups o independen esul s, a -s uden es was used. A con idence le el ≥ 95% was se . Fo each es , he mean and s anda d de ia ion we e de e mined among biological iplica es. 3. Resul s and Discussion 3.1. Mo phological Cha ac e iza ion The mic oalgal specimen collec ed in he eshwa e pond a he acili ies o Allmic oalgae was iden i ied based on bo anical, mic o-mo phological ea u es and axonomy as Chlo ococcum Meneghini. A ouche was deposi ed in he Allmic oalgae cul u e collec ion wi h numbe 0030CN. Vege a i e cells o he symbio ic algae appea ed g ass-g een, li e as soli a y cells o in empo a y g oups o inde ini e o m wi hou a mucilaginous en elope, a e ellipsoidal o sphe ical and a y in size om 8 o 25 µ m. Rep oduc ion wi hin he genus Chlo ococcum is mainly asexual, by bi lagella e zoospo es (o aplanospo es in case o wa e s ess) ha do no become sphe ical upon cessa ion Appl. Sci. 2020,10, 3040 6 o 14 o mo ili y bu e ain an o oid, ellipsoid shape o some days [ 28 , 29 ]. By ligh mic oscopy, in h ee-week-old liquid cul u es, g een ege a i e cells wi h a hollow, pa ie al chlo oplas wi h o wi hou an open su ace we e obse ed. In he basal pa o he chlo oplas , a sphe ical py enoid was p esen and he nucleus was in a cen al posi ion in he cell (Figu e 2a). Tempo a y g oups o cells o inde ini e o m and wi hou a mucilaginous en elope (Figu e 2c) we e seen. In h ee-day-old liquid cul u es, nume ous zoospo angia (Figu e 2d– ), zoospo es (Figu e 2b) and young g een ege a i e cells became e iden . Zoospo es had wo lagella, an an e io s igma, one sphe ical py enoid in he cen al egion and a nucleus pos e io o py enoid. Zoospo es had an ellipsoidal o m (3.59 ± 0.43 µ m wide and 8.56 ± 0.82 µ m long), young g een cells had an ellipsoidal o o oid o m (6.70 ± 1.23 µ m wide and 9.66 ± 1.23 µ m long) and ma u e g een cells we e sphe ical (15.14 ± 2.34 µ m). P olonged obse a ions o cul u es show ha his Chlo ococcum mul iplica ion seems o be simila o Chlo ococcum in usionum Meneghini, mainly h ough he endogenous di ision o he con en o he ege a i e cell by p og essi e clea age o a mo he p o oplas in o uninuclea e segmen s [ 30 ]. The numbe o zoospo es migh a y acco ding o he size o he mo he cell. Once comple ely o med, zoospo es ee hemsel es by bu s ing he zoospo angium memb ane (Figu e 2e) due o excess p essu e inside. Appl. Sci. 2019, 9, x FOR PEER REVIEW 6 o 14 cen al egion and a nucleus pos e io o py enoid. Zoospo es had an ellipsoidal o m (3.59 ± 0.43 µm wide and 8.56 ± 0.82 µm long), young g een cells had an ellipsoidal o o oid o m (6.70 ± 1.23 µm wide and 9.66 ± 1.23 µm long) and ma u e g een cells we e sphe ical (15.14 ± 2.34 µm). P olonged obse a ions o cul u es show ha his Chlo ococcum mul iplica ion seems o be simila o Chlo ococcum in usionum Meneghini, mainly h ough he endogenous di ision o he con en o he ege a i e cell by p og essi e clea age o a mo he p o oplas in o uninuclea e segmen s [30]. The numbe o zoospo es migh a y acco ding o he size o he mo he cell. Once comple ely o med, zoospo es ee hemsel es by bu s ing he zoospo angium memb ane (Figu e 2e) due o excess p essu e inside. Figu e 2. Mic oscopic images o Chlo ococcum amblys oma is li e s ages using he di e en ial in e e ence con as (DIC): (a) ege a i e cell; (b) zoospo e; (c) g oup o cells wi h inde ini e o m; (d) zoospo angium; (e) and ( ) bu s o zoospo angium memb ane and ealizing o zoospo es. n = nucleus, p = py enoid, s = s igma, z = zoospo e, zp = zoospo angium. Scale ba (a) and (b) = 2 µm, (c) o ( ) = 5 µm. 3.2. Molecula Iden i ica ion and Phylogene ic Analysis Fo he de e mina ion o he axonomic classi ica ion o he new isola e wi hin he Chlo ococcaceae, he amily o which he genus Chlo ococcum belongs, molecula phylogene ic analysis was ca ied ou . Sequences o 18S RNA genes om se e al Chlo ococcaceae mic oalgae we e analyzed using Bayesian (BI) and Maximum Likelihood (ML) in e ence. Chlo ella ulga is 18S RNA gene was used as an ou g oup. BI and ML ees opology (Figu e 3) indica es ha he s ain isola ed om a pond loca ed a Allmic oalgae indus ial acili ies (accession numbe : MT026583) belongs o a axonomic uni sis e o C. minu um/C. aqua icum, wi h an almos maximum pos e io p obabili y (0.99) and a 93% ML boo s ap alue. In e es ingly, C. ellipsoideum appea s o be an ou g oup, b anching o hese axa wi h maximum suppo . Al hough his isola e does p esen simila i ies o C. in usionum, he holo ype o he genus, conce ning i s ep oduc ion, phylogene ic analysis s ongly sugges s ha belongs o he S ephanosphae inia mac oclade, a he han o he Moewusinia, he mac oclade o C. in usionum (Figu e 3) [31]. Mo eo e , upon submi ing ou sequence o GenBank, he au ho s ound ha his sequence is 100% iden ical o an Oophila amblys oma is (Kings on isola e, Oophila clade A) sequence wi h he accession numbe KY091671 [32] and o o he sequences ha ha e no been classi ied down o he species le el. This inding is qui e in e es ing, as he phylogene ic da a he e p esen ed s ongly indica es ha O. amblys oma is does belong o he genus Chlo ococcum. This species is o en isola ed as a symbion in eggs o se e al amphibian species, bu i s p ope axonomy has emained unce ain [32]. Recen ly, i has been epo ed ha his species can also occu as ee-li ing mic oalgae [33]. Taken oge he , i is he e Figu e 2. Mic oscopic images o Chlo ococcum amblys oma is li e s ages using he di e en ial in e e ence con as (DIC): ( a ) ege a i e cell; ( b ) zoospo e; ( c ) g oup o cells wi h inde ini e o m; ( d ) zoospo angium; ( e ) and ( ) bu s o zoospo angium memb ane and ealizing o zoospo es. n =nucleus, p =py enoid, s=s igma, z =zoospo e, zp =zoospo angium. Scale ba (a) and (b)=2µm, (c) o ( )=5µm. 3.2. Molecula Iden i ica ion and Phylogene ic Analysis Fo he de e mina ion o he axonomic classi ica ion o he new isola e wi hin he Chlo ococcaceae, he amily o which he genus Chlo ococcum belongs, molecula phylogene ic analysis was ca ied ou . Sequences o 18S RNA genes om se e al Chlo ococcaceae mic oalgae we e analyzed using Bayesian (BI) and Maximum Likelihood (ML) in e ence. Chlo ella ulga is 18S RNA gene was used as an ou g oup. BI and ML ees opology (Figu e 3) indica es ha he s ain isola ed om a pond loca ed a Allmic oalgae indus ial acili ies (accession numbe : MT026583) belongs o a axonomic uni sis e o C. minu um/C. aqua icum, wi h an almos maximum pos e io p obabili y (0.99) and a 93% ML boo s ap alue. In e es ingly, C. ellipsoideum appea s o be an ou g oup, b anching o hese axa wi h maximum suppo . Al hough his isola e does p esen simila i ies o C. in usionum, he holo ype o he genus, conce ning i s ep oduc ion, phylogene ic analysis s ongly sugges s ha belongs o he S ephanosphae inia mac oclade, a he han o he Moewusinia, he mac oclade o C. in usionum (Figu e 3) [ 31 ]. Mo eo e , upon submi ing ou sequence o GenBank, he au ho s ound ha his Appl. Sci. 2020,10, 3040 7 o 14 sequence is 100% iden ical o an Oophila amblys oma is (Kings on isola e, Oophila clade A) sequence wi h he accession numbe KY091671 [ 32 ] and o o he sequences ha ha e no been classi ied down o he species le el. This inding is qui e in e es ing, as he phylogene ic da a he e p esen ed s ongly indica es ha O. amblys oma is does belong o he genus Chlo ococcum. This species is o en isola ed as a symbion in eggs o se e al amphibian species, bu i s p ope axonomy has emained unce ain [ 32 ]. Recen ly, i has been epo ed ha his species can also occu as ee-li ing mic oalgae [ 33 ]. Taken oge he , i is he e p oposed o ename O. amblys oma is as Chlo ococcum amblys oma is. This p oposal is suppo ed no only by he esul s o Figu e 3, bu also by he la es a emp a de ining he axonomy o his species [ 32 ]. Indeed, hese au ho s hin ha ha sequences o he Oophila clade A symbion s clus e wi h hose o Chlo ococcum, unlike “Oophila” clade B isola es, which appea o be closely ela ed o axa classi ied as Chlamydomonas [32]. Appl. Sci. 2019, 9, x FOR PEER REVIEW 7 o 14 p oposed o ename O. amblys oma is as Chlo ococcum amblys oma is. This p oposal is suppo ed no only by he esul s o Figu e 3, bu also by he la es a emp a de ining he axonomy o his species [32]. Indeed, hese au ho s hin ha ha sequences o he Oophila clade A symbion s clus e wi h hose o Chlo ococcum, unlike “Oophila” clade B isola es, which appea o be closely ela ed o axa classi ied as Chlamydomonas [32]. Figu e 3. Bayesian in e ence ees o he amily Chlo ococcaceae using 18S ibosomal ibonucleic acid ( RNA) sequences show ha he no el isola e (acc. numbe : MT026583) o ms a clade sis e o C. minu um/C. aqua icum wi h C. ellipsoideum as an ou g oup. In e es ingly, he MT026583 sequence is iden ical o ha o Oophila amblys oma is, a species ha can be ei he ee-li ing [33] o a salamande egg symbion [32]. As hese esul s s ongly sugges ha O. amblys oma is belongs o he genus Chlo ococcum, i is he e p oposed ha his species should be enamed as Chlo ococcum amblys oma is as gi en in he ee. Mo eo e , hese esul s con i m p e ious da a sugges ing ha he genus Chlo ococcum can be clea ly di ided in o wo mac oclades: S ephanosphae inia and Moewusinia [31]. ML boo s ap alues (>50) and BI pos e io p obabili ies (>0.70) a e indica ed a he espec i e b anches. The accession numbe o each sequence used is gi en a e he axonomic classi ica ion o each lea node. 3.3. Scale-up and Biomass P oduc ion G ow h assays we e pe o med in PBRs o di e en olumes and geome y, namely, pilo -scale 0.25-m3 FP, 2.5-m3 ubula PBR and indus ial-scale 10-m3 ubula PBR. The biomass p oduc i i ies, speci ic g ow h a es and pho osyn he ic e iciencies o C. amblys oma is a e p esen ed in Table 1. The highe olume ic p oduc i i ies we e achie ed in he pilo -scale sys ems, eaching simila global and maximum p oduc i i ies (p ≥ 0.05) in he 0.25-m3 FP (0.07 and 0.27 g.L−1.day−1) and 2.5-m3 PBR (0.09 and 0.26 g.L−1.day−1). On he o he hand, compa ed o pilo -scale sys ems, he indus ial scale 10-m3 PBR egis e ed signi ican ly lowe global and maximum olume ic p oduc i i ies o 0.05 and 0.09 g.L−1.day−1, espec i ely (p < 0.05). The ob ained olume ic p oduc i i ies a e lowe han hose p e iously epo ed by Cabanelas e al. [34] o Alki ia (Chlo ococcum) li o ale g own in a ho izon al ubula PBR o 0.09-m3 (0.74 g.L−1.day−1 in Oslo, 0.7 g.L−1.day−1 in Wageningen, 0.58 g.L−1.day−1 in Rio de Janei o and 0.6 g.L−1.day−1 in Cadiz). Table 1. Maximum olume ic p oduc i i y, o e all olume ic p oduc i i y, maximum a eal p oduc i i y and o e all a eal p oduc i i y, speci ic g ow h a e (μ) and pho osyn he ic e iciency (PE) o pilo - and indus ial-scale ubula pho obio eac o s (PBR). Di e en le e s indica e signi ican di e ences be ween eac o s. Values a e gi en as means ± s anda d de ia ion (n = 3). Reac o Volume ic P oduc i i y (g.L−1.d−1) A eal P oduc i i y (g.m−2.d−1) µ (d−1) PE (%) Maximum Global Maximum Global Figu e 3. Bayesian in e ence ees o he amily Chlo ococcaceae using 18S ibosomal ibonucleic acid ( RNA) sequences show ha he no el isola e (acc. numbe : MT026583) o ms a clade sis e o C. minu um/C. aqua icum wi h C. ellipsoideum as an ou g oup. In e es ingly, he MT026583 sequence is iden ical o ha o Oophila amblys oma is, a species ha can be ei he ee-li ing [ 33 ] o a salamande egg symbion [ 32 ]. As hese esul s s ongly sugges ha O. amblys oma is belongs o he genus Chlo ococcum, i is he e p oposed ha his species should be enamed as Chlo ococcum amblys oma is as gi en in he ee. Mo eo e , hese esul s con i m p e ious da a sugges ing ha he genus Chlo ococcum can be clea ly di ided in o wo mac oclades: S ephanosphae inia and Moewusinia [ 31 ]. ML boo s ap alues (>50) and BI pos e io p obabili ies (>0.70) a e indica ed a he espec i e b anches. The accession numbe o each sequence used is gi en a e he axonomic classi ica ion o each lea node. 3.3. Scale-Up and Biomass P oduc ion G ow h assays we e pe o med in PBRs o di e en olumes and geome y, namely, pilo -scale 0.25-m 3 FP, 2.5-m 3 ubula PBR and indus ial-scale 10-m 3 ubula PBR. The biomass p oduc i i ies, speci ic g ow h a es and pho osyn he ic e iciencies o C. amblys oma is a e p esen ed in Table 1. The highe olume ic p oduc i i ies we e achie ed in he pilo -scale sys ems, eaching simila global and maximum p oduc i i ies (p≥0.05) in he 0.25-m3FP (0.07 and 0.27 g·L−1·day−1) and 2.5-m3PBR (0.09 and 0.26 g · L −1· day −1 ). On he o he hand, compa ed o pilo -scale sys ems, he indus ial scale 10-m 3 PBR egis e ed signi ican ly lowe global and maximum olume ic p oduc i i ies o 0.05 and 0.09 g · L −1· day −1 , espec i ely (p<0.05). The ob ained olume ic p oduc i i ies a e lowe han hose p e iously epo ed by Cabanelas e al. [ 34 ] o Alki ia (Chlo ococcum) li o ale g own in a ho izon al ubula PBR o 0.09-m 3 (0.74 g · L −1· day −1 in Oslo, 0.7 g · L −1· day −1 in Wageningen, 0.58 g · L −1· day −1 in Rio de Janei o and 0.6 g·L−1·day−1in Cadiz). Appl. Sci. 2020,10, 3040 8 o 14 Table 1. Maximum olume ic p oduc i i y, o e all olume ic p oduc i i y, maximum a eal p oduc i i y and o e all a eal p oduc i i y, speci ic g ow h a e ( µ ) and pho osyn he ic e iciency (PE) o pilo - and indus ial-scale ubula pho obio eac o s (PBR). Di e en le e s indica e signi ican di e ences be ween eac o s. Values a e gi en as means ±s anda d de ia ion (n=3). Reac o Volume ic P oduc i i y (g·L−1·d−1) A eal P oduc i i y (g·m−2·d−1) µ (d−1) PE (%) Maximum Global Maximum Global 0.25-m3FP 0.27 ±0.02 a0.07 ±0.00 a,b 22.31 ±1.50 a5.94 ±0.41 a0.16 ±0.00 a0.73 ±0.02 a 2.5-m3PBR 0.26 ±0.02 a0.09 ±0.02 a26.75 ±2.47 a8.65 ±1.62 a,b 0.13 ±0.03 a1.33 ±0.12 b 10-m3PBR 0.09 ±0.03 b0.05 ±0.01 b36.56 ±1.52 b14.84 ±3.98 b0.16 ±0.02 a1.84 ±0.18 c Howe e , an opposi e end was ound o he a eal p oduc i i y, whe e he highes maximum p oduc i i y was achie ed in he 10-m3PBR, eaching 36.56 g·m−2·day−1(p<0.05). The o e all a eal p oduc i i y was also highe in he 10-m 3 PBR, when compa ed o he FP (p<0.05), bu wi hou signi ican di e ences when compa ed o he 2.5-m 3 PBR (p ≥ 0.05). C. amblys oma is has shown highe a eal p oduc i i ies in compa ison wi h he same wo k o Cabanelas e al. [ 34 ] (16.6 g · m −2· day −1 in Oslo, 15.7 g · m −2· day −1 in Wageningen, 13.05 g · m −2· day −1 in Rio de Janei o and 13.5 g · m −2· day −1 in Cadiz). The explana ion o he di e ence be ween olume ic and a eal p oduc i i ies among sys ems is due o he geome y o he eac o s. As indus ial PBRs ha e a high densi y o ho izon al ubes, he lowe ubes ecei e a lowe amoun o ligh i adiance, caused by shadowing, which educes he olume ic p oduc ion. Despi e his, as he olume o biomass p oduced in he same a ea is subs an ially highe , i esul s in highe a eal p oduc i i ies. This end was p e iously epo ed o he same PBRs geome y upon he cul i a ion o Te aselmis sp. (Chlo ophy a) CTP4 [ 12 ] and Phaeodac ylum ico nu um (Bacilla iophy a) [35]. Speci ic g ow h a es we e simila among he di e en eac o s (p ≥ 0.05) and conside ably lowe compa ed o ha o A. li o ale g own in a 0.09-m 3 PBR ( om 0.46 day −1 o 0.62 day −1 ) [ 34 ]. In addi ion, he pilo -scale FP egis e ed he lowes pho osyn he ic e iciency, 0.73 % (p<0.05), while he indus ial PBR achie ed he highes alue, 1.84 % (p<0.05). Simila PEs we e epo ed o P. ico nu um using he same 2.5-m 3 PBR (1.11%) and 10-m 3 PBR (2.08%) [ 35 ]. Fa o able climac e ic condi ions and sunligh exposu e du ing he g ow h pe iod in he indus ial PBR we e bene icial o he cul u e g ow h. Highe pho osyn he ic e iciency was al eady expec ed in he indus ial PBR, since he olume o cul u e pe uni o a ea o he pho ic a ea is subs an ially highe . Fu he mo e, he FP ae a ion sys em is less e icien in compa ison o ha o he indus ial PBR, hampe ing ligh abso p ion due o bio ilm o ma ion on he walls o he eac o . 3.4. Biochemical Composi ion In o de o u he cha ac e ize C. amblys oma is, he biochemical composi ion o he biomass was analyzed. Fo his pu pose, only biomass p oduced in he 2.5- and 10-m 3 PBR was analyzed, since hese a e he cul u e sys ems used o p oduce biomass by Allmic oalgae in he la ge-scale indus ial acili y. 3.4.1. P oxima e Composi ion The con en o p o eins, lipids, ca bohyd a es, and ashes de ec ed in he biomass p oduced in bo h PBRs is p esen ed in Table 2. The esul s ob ained e ealed ha p o eins a e he main mac onu ien p esen in C. amblys oma is biomass. The highes p o ein con en was achie ed in mic oalgal cells g own in he 2.5-m 3 PBR, eaching mo e han 55% o hei DW (p<0.05), while he biomass p oduced in he 10-m 3 PBR egis e ed a p o ein con en o 48% DW. Mic oalgal p o eins a e app ecia ed as an addi i e o eed o a wide a ie y o animals anging om ish (aquacul u e) o pe s and a m animals and a e e y in e es ing o unc ional ood applica ions due o hei demons a ed biological ac i i ies impac ing di e en Appl. Sci. 2020,10, 3040 9 o 14 human heal h condi ions [ 3 , 9 ]. The lowe alue o p o ein a io ob ained in he biomass g own in he 10-m 3 PBR was p obably due o exposu e o he cul u es o s ess ul condi ions. E en hough he accumula ion o ca bohyd a es in mic oalgae is mainly due o he ne balance be ween pho osyn hesis and espi a ion [ 36 ], i would be expec ed ha he biomass g own in he 10-m 3 PBR showed he highes alue o ca bohyd a es, since i was exposed o highe ligh i adiance. Howe e , i was in he biomass g own in he pilo -scale PBR ha he highes pe cen age o ca bohyd a es was de ec ed, achie ing 17.43% o biomass DW (p<0.05), wi hin he ange o epo ed alues o he majo i y o mic oalgae [ 4 , 9 ]. Indeed, unde un a o able condi ions (e.g., high i adiance and ni ogen limi a ion), he excess o ca bon ixed du ing pho osyn hesis may ha e been di e ed in o s o age compounds such as lipids o ca bohyd a es [ 4 , 9 , 37 , 38 ]. This could also explain he highe lipid con en obse ed in he biomass g own in he 10-m 3 PBR (p<0.05), 31.44% DW, in compa ison o ha o mic oalgal cells g own in he 2.5-m3PBR (18.33%). Table 2. Biochemical composi ion o Chlo ococcum amblys oma is biomass g own in he 2.5- and 10-m 3 ubula pho obio eac o s (PBR). P o eins, lipids, and ca bohyd a es a e p esen ed as he pe cen age o biomass d y weigh (DW). Di e en le e s indica e signi ican di e ences be ween eac o s. Values a e gi en as means ±s anda d de ia ion (n=3). Reac o P o eins (%) Lipids (%) Ca bohyd a es (%) Ashes (%) 2.5-m3PBR 55.72 ±2.85 a18.33 ±0.97 b17.43 ±3.58 a9.88 ±5.87 a 10-m3PBR 48.22 ±0.43 b31.44 ±4.10 a5.78 ±3.99 b15.85 ±5.00 a 3.4.2. Fa y Acid P o ile In o de o assess he lipid composi ion o he biomass, he a y acid p o ile was analyzed (Table 3). The FAME p o ile o C. amblys oma is is domina ed by C16:4n-3 (21.94%–23.65% o o al a y acids; TFA), C16:0 (15.24%–19.29% o TFA), C18:4n-3 (8.61%–8.83% o TFA), C18:3n-3 (27.58%–31.40% o TFA) and C18:1 (5.34%–6.27% o TFA). In e es ingly, his mic oalga con ains a high con en o polyunsa u a ed a y acids (PUFAs), eaching 74.70% o TFA in he biomass p oduced in he 2.5-m 3 PBR, which is signi ican ly highe han he 67.10% o TFA de ec ed in he biomass ob ained om he 10-m 3 PBR (p<0.05) . Sa u a ed a y acids (SFAs) and monounsa u a ed a y acids (MUFAs) a e simila be ween he eac o s. O e all, he FAME p o ile does no ma ch wi h p e ious alues epo ed o o he Chlo ococcum mic oalgae, whe ein he FAME p o ile combines highe pe cen ages o SFAs and MUFAs, and lowe con en s o PUFAs [ 39 , 40 ], ein o cing he conclusion ha C. amblys oma is is a species dis inc om hose whose a y acid p o iles ha e been desc ibed p e iously. PUFAs a e classi ied as n-3 and n-6 and play an impo an ole in human and animal nu i ion [ 41 , 42 ]. I is epo ed ha n-3 a y acids play an impo an ole in he p e en ion o co ona y hea disease and in lamma o y disease, b ain diso de , such as Alzheime ’s disease and help in cance he apy [ 43 – 45 ], while n-6 a y acids a e p o-in lamma o y [ 41 , 46 ]. The unc ional sou ces o n-3 in mic oalgae a e no mally eicosapen aenoic (EPA, C20:5n-3) and docosahexaenoic (DHA, C22:6n-3) acids; howe e , chlo ophy es and pa icula ly eshwa e mic oalgae a e, in gene al, de icien in bo h C20 and C22 PUFAs [ 4 , 45 ]. Ne e heless, he e a e o he impo an PUFAs such as linoleic (LA, C18:2n-6), α -linolenic (ALA, C18:3n-3) and γ -linolenic (GLA, C18:3n-6) acids wi h p o en biomedical and nu aceu ical applica ions [ 42 ]. Fu he mo e, once consumed, hese a y acids a e me abolized wi hin mammalian cells. The eby, ALA is con e ed o s ea idonic (C18:4n-3) and eicosa e aenoic (C20:4n-3) acids o o m EPA, which is u he me abolized o DHA (C22:6n-3) [ 41 , 47 ]. Al hough he n-3 and n-6 PUFAs biosyn he ic pa hways sha e he same se ies o enzymes, he heal h e ec s a e dependen on he Σ n-6/ Σ n-3 a io [ 41 , 46 , 47 ], which is ecommended o be lowe han 10 by he Wo ld Heal h O ganiza ion (WHO) in o de o p e en in lamma o y, ca dio ascula and ne ous sys em diso de s [ 46 , 47 ]. The e o e, since C. amblys oma is, p esen s a PUFA/SFA a io o 4.30–2.81 and a Σ n-6/ Σ n-3 a io o 0.10–0.13, his s ain can be conside ed as a good al e na i e sou ce o die a y PUFA.