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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.
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