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.