This is a sel -a chi ed e sion o an o iginal a icle. This e sion
may di e om he o iginal in pagina ion and ypog aphic de ails.
Au ho (s):
Ti le:
Yea :
Ve sion:
Copy igh :
Righ s:
Righ s u l:
Please ci e he o iginal e sion:
CC BY 4.0
h ps://c ea i ecommons.o g/licenses/by/4.0/
Supplemen ing ai wi h CO2 s ipped om eci cula ing aquacul u e imp o es g ow h o
wo g een mic oalgae in aquacul u e was ewa e
© 2023 The Au ho s. Published by Else ie B.V.
Published e sion
Pi honen, Juhani; Koukka, Silja; Pulkkinen, Ka ja
Pi honen, J., Koukka, S., & Pulkkinen, K. (2023). Supplemen ing ai wi h CO2 s ipped om
eci cula ing aquacul u e imp o es g ow h o wo g een mic oalgae in aquacul u e was ewa e .
Aquacul u e, 567, A icle 739242. h ps://doi.o g/10.1016/j.aquacul u e.2023.739242
2023
Aquacul u e 567 (2023) 739242
A ailable online 6 Janua y 2023
0044-8486/© 2023 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/).
Sho communica ion
Supplemen ing ai wi h CO
2
s ipped om eci cula ing aquacul u e
imp o es g ow h o wo g een mic oalgae in aquacul u e was ewa e
Juhani Pi honen
*
, Silja Koukka, Ka ja Pulkkinen
Uni e si y o Jy askyla, Depa men o Biological and En i onmen al Science, P.O. Box 35, FIN-40014 Uni e si y o Jy askyla, Finland
ARTICLE INFO
Keywo ds:
Ci cula economy
Dissol ed nu ien s
Ca bon dioxide
Nu ien apping
Pho obio eac o
ABSTRACT
To imp o e sus ainabili y and o implemen he p inciples o ci cula economy in aquacul u e, we es ed he
possibili y o boos he cap u e o ni a e by wo g een mic oalgal species om eci cula ing aquacul u e sys em’s
(RAS) was ewa e by supplemen ing ai wi h ca bon dioxide s ipped om a RAS. Ca bon dioxide addi ion
inc eased cell densi ies o Mono aphidium g i i hii and Haema ococcus plu ialis in pho obio eac o s du ing 9-day
g owing pe iods. Howe e , g ow h a es and ni a e up ake a es we e only imp o ed o M. g i i hii. Addi ion o
CO
2
dec eased pH o he medium wi h M. g i i hii which likely also a ec ed posi i ely on algal g ow h and
nu ien up ake. These labo a o y scale expe imen s sugges ha mic oalgal cul i a ion o p oduce aluable
biomass could be connec ed o a RAS o dec ease ni a e and CO
2
emissions om aquacul u e.
1. In oduc ion
A eci cula ing aquacul u e sys em (RAS) is a echnology used o
suppo in ensi e aquacul u e p oduc ion on land, especially ishes, wi h
limi ed use o wa e . Al hough co e ing only a small ac ion o he o al
aquacul u e p oduc ion wi hin EU (Bos ock e al., 2016), RAS has gained
popula i y du ing he las decade (Bos ock e al., 2016; Eumo a, 2021)
due o he possibili y o s ic con ol o he sys em and con inuous
p oduc ion unde s able en i onmen al condi ions. In RAS, he wa e is
passed h ough di e en echnical ins alla ions o emo e solids, o
ans o m ammonia eleased by he ish in o less ha m ul ni a e, o s ip
excess CO
2
, and o kill pa hogens, be o e pumping he wa e back o he
ish anks. Du ing eci cula ion he concen a ion o dissol ed nu ien s
inc eases in he sys em and his inc ease is commonly con olled by
dilu ing he sys em wi h esh wa e . The RAS e luen ypically con ains
high concen a ion o dissol ed nu ien s, e.g. ni a e up o 100 mg/l
(Da idson e al., 2014) and phospha e up o 45 mg/l ( an Bussel e al.,
2013) ha e been epo ed, bu ins ead o disposing he nu ien - ich
wa e in o na u al wa e cou ses o wa e ea men plan s, i could be
u he used o g owing plan s (Goddek e al., 2019) o mic oalgae
(S e ˇ
ci´
c e al., 2019; Ramli e al., 2020).
Fish and bac e ial espi a ions in a RAS inc ease he wa e CO
2
concen a ion and i mus be s ipped be o e pumping he wa e o ish
anks o a oid he po en ial nega i e e ec s o high CO
2
concen a ion
on ish (Mo a e al., 2019; Sko , 2019). To inc ease he sus ainabili y
and o dec ease he ca bon oo p in o RAS-p oduced ish, i would be
ideal o use no only he dissol ed nu ien s bu also he excess CO
2
o
p oduc ion o pho oau o ophic o ganisms. Mic oalgae could se e as an
op ion o his pu pose, as mic oalgal g ow h can be s imula ed by
mode a e (up o 5%) CO
2
addi ion (Chekano e al., 2017). I has also
been sugges ed ha by pumping oom ai h ough mic oalgal bio-
eac o s hey could se e as oom ai il e s (Cheng e al., 2006). As he
RAS a e p ac ically always buil indoo s, i would be bene icial o il e
CO
2
- ich ai om CO
2
s ippe s wi h mic oalgae: i s , i would dec ease
he ene gy need o en ila ion (impo an especially in colde clima e
zones in win e when ou doo ai mus i s be hea ed in he en ila o s)
and second, i could inc ease biomass p oduc ion in algal bio eac o s.
The e is a lo o in o ma ion a ailable sepa a ely on he use o
di e en ypes o was ewa e s o p oducing mic oalgae h ough
bio emedia ion (Ch is enson and Sims, 2011; Li e al., 2019; Chai e al.,
2021), as well as o he e ec s o addi ion o CO
2
o algal cul u es (Goli
e al., 2016; Ne es e al., 2019). Howe e , he e appea s o be e y li le
in o ma ion a ailable on he combina ion o nu ien and CO
2
cap u e
om was ewa e using mic oalgae (Molazadeh e al., 2019). In p e ious
expe imen s, we ound ha g een mic oalgae (Chlo ophycae) a e sui -
able candida es o be cul i a ed in RAS was ewa e a a ela i ely low
empe a u e (~17 ◦C) (S e ˇ
ci´
c e al., 2019; Calde ini e al., 2021). He e
we es ed i he g ow h and nu ien up ake o wo p e iously es ed
species, Mono aphidium g i i hii and Haema ococcus plu ialis, could be
u he enhanced when cul i a ed in RAS was ewa e supplemen ed
* Co esponding au ho .
E-mail add esses: [email p o ec ed] (J. Pi honen), [email p o ec ed] (S. Koukka), [email p o ec ed] (K. Pulkkinen).
Con en s lis s a ailable a ScienceDi ec
Aquacul u e
jou nal homepage: www.else ie .com/loca e/aquacul u e
h ps://doi.o g/10.1016/j.aquacul u e.2023.739242
Recei ed 3 June 2022; Recei ed in e ised o m 3 Janua y 2023; Accep ed 5 Janua y 2023
Aquacul u e 567 (2023) 739242
2
wi h CO
2
s ipped om a RAS. Ou hypo hesis was ha CO
2
supple-
men a ion would inc ease mic oalgal cell densi y and hei NO
3
-N
up ake.
2. Ma e ials and me hods
Two sepa a e expe imen s we e ca ied ou be ween Ap il – June
2021 in he labo a o y o he depa men o biological and en i on-
men al science, Uni e si y o Jy ¨
askyl¨
a, Finland. The algal species used
we e M. g i i hii (s ain: NIVA-CHL 8, No way) and H. plu ialis (K-0084
(NIVA), Sweden). The algae we e cul i a ed in pho obio eac o s, which
consis ed o anspa en plas ic unnels ( o al olume 1.5 l). Ten unnels
we e a ached in one ow, abou 2.5 cm apa and co e ed wi h ans-
pa en plas ic lids o a oid excess e apo a ion and o dec ease he
likelihood o con amina ion. E e y o he unnel (n =5) ecei ed oom
ai om below wi h an ai pump h ough an ai s one (on a e age 500
ml/min), and e e y o he (n =5) was connec ed o an ai pump loca ed
in an ai igh plas ic bag ecei ing CO
2
ich ai om an expe imen al
size RAS ( o al olume abou 4.5 m
3
) ickling il e (Fig. 1), which was
used o s ip CO
2
om wa e and o add O
2
. One side o he unnel was
illumina ed cons an ly wi h a LED ligh (18W, AP67 T8 ubes, Valoya
Oy, Finland) wi h ligh in ensi y o c. 100
μ
E m
−2
s
−1
. The wa e used in
he pho obio eac o o igina ed om ano he expe imen al size RAS
( o al olume abou 750 l), housing ainbow ou (Onco hynchus
mykiss), and i was passed h ough a 48
μ
m mesh be o e use. Tempe -
a u e in he pho obio eac o s a ied be ween 17 and 18 ◦C. These
ea ing condi ions we e simila in bo h expe imen s.
Algal densi y was quan i ied daily by coun ing on a haemocy ome e
(Bü ke ) unde a mic oscope wi h 100×magni ica ion. Wa e empe -
a u e and NO
3
-N concen a ion we e measu ed wi h YSI Qua o mul i-
p obe me e (Yellow Sp ing Ins umen s, USA) and pH wi h Eu ech PC
450 (The mo Scien i ic Eu ech, Singapo e). The incoming ai CO
2
con-
cen a ion was measu ed wi h Ai Con ol COACH CO2- Moni o (Dos-
mann elec onic GmbH, Ge many) om emp y unnels connec ed o ai
pumps om lab ai o RAS ickling il e ai .
M. g i i hii was cul u ed in ba ch mode (all medium added in he
beginning) wi h 150 ml o algal suspension added in o 850 ml o RAS
was ewa e ( il e ed h ough a 48
μ
m mesh), equalling o a 15% inoc-
ulum olume o o al olume a io and an ini ial concen a ion o 1.3 ×
10
5
cells/ml (day 0). The g ow h o he algae was moni o ed o 10 days.
Du ing he expe imen , a e age oom ai CO
2
concen a ion was 527
ppm (0.0527%; min 497 – max 563 ppm) and in he ai pumped om he
RAS, 985 ppm (0.0985%; 910–1026 ppm). H. plu ialis was cul u ed in
ed ba ch mode (medium added a p e-de ined in e als), wi h ini ial
addi ion o 65 ml o algal suspension in o 340 ml o RAS was ewa e in
ege a i e g een phase (see e.g. Shah e al., 2016, o de ails o
H. plu ialis li e cycle), equalling o a 16% inoculum olume o o al
olume a io and an ini ial concen a ion o 2.8 ×10
4
cells/ml (day 0).
405 ml o was ewa e was u he added on days 4 and 7 ( o al olume
1215 ml). A e age oom ai CO
2
concen a ion was 526 ppm ( ange
493–575 ppm) and in he ai om RAS i was 925 ppm (805–1166 ppm)
(Fig. 2).
G ow h a e (GR, d
−1
) was calcula ed o each cul u e as
(LnN
2
−LnN
1
) *
−1
, whe e N
1
and N
2
we e concen a ions o mic oalgae
(ml
−1
) in he beginning and end o he isually es ima ed exponen ial
g ow h pe iod , espec i ely. Fo M. g i i hii he pe iod was nine days.
Fo H. plu ialis we calcula ed GR sepa a ely o h ee pe iods ma ching
he addi ion o was ewa e : days 1–3, days 3–5, and days 5–6 (see
Fig. 1b), and hen calcula ed he a e age GR o each cul u e o be used
in he s a is ical analyses. S a is ical analyses we e done wi h SPSS
e sion 26 (IBM SPSS S a is ics). Possible di e ences be ween he
ea men s (ai s. CO
2
supplemen ) in SGR we e compa ed wi h inde-
penden samples - es as he sample a iances did no di e (Le ene
es ). Di e ences in cell densi y, ni a e concen a ion and pH- alue
we e analysed by compa ing daily means o he ea men s wi h GLM
epea ed measu es ANOVA. Day was included as a wi hin-subjec ac o
and ea men as a be ween-subjec ac o . The sphe ici y assump ion
was es ed wi h Maulchy’s es , and i he sphe ici y was iola ed,
G eenhouse–Geisse co ec ed alues we e used. No mali y o da a was
es ed wi h Kolmogo o –Smi no es , bu he assump ion was no al-
ways me . Resul s o mANOVA a e epo ed, as he isk o alse posi i e
esul is no much a ec ed by iola ion o no mali y o ANOVA (Lix
e al., 1996). Fo he pos -hoc compa isons Bon e oni co ec ed alues
we e used. A alue o p <0.05 was used as he le el o s a is ical
signi icance.
3. Resul s and discussion
Ca bon dioxide supplemen a ion inc eased cell densi ies (Fig. 3;
M. g i i hii mANOVA, F
1,8
=22.759, p =0.001 and H. plu ialis mA-
NOVA, F
1,8
=22.935, p =0.001). In addi ion, he in e ac ion be ween
day and ea men was signi ican wi h bo h algae (M. g i i hii: mA-
NOVA, F
2.3
=8.368, p =0.002, H. plu ialis: mANOVA, F
3.7
=3.164, p =
0.031), showing ha CO
2
addi ion inc eased algal densi ies du ing he
expe imen . A e age ±S.D. g ow h a e o e nine days o M. g i i hii
we e 0.43 ±0.01 day
−1
and 0.48 ±0.02 day
−1
in cul u es wi hou and
wi h CO
2
supplemen a ion, espec i ely (
8
=4.23, p =0.003) and o
H. plu ialis he espec i e alues we e 0.44 ±0.08 and 0.52 ±0.16 (
8
=
1.26, p =0.24). Fo H. plu ialis g ow h a e and densi ies we e simila
when compa ed o ea lie esul s in ou labo a o y in ba ch cul u e
(S e ˇ
ci´
c e al., 2019) o in expe imen s whe e di e en ypes o g ow h
media and en i onmen al condi ions ha e been es ed (Gong and Chen,
Fig. 1. A schema ic p esen a ion o he se up whe e he g ow h o mic oalgae
was compa ed be ween unnels ecei ing ei he oom ai o CO
2
- ich ai
s ipped om a ickling il e o a 4.5 m
3
Reci cula ing Aquacul u e Sys em
(RAS). Funnels (5 eplica es in bo h ea men s) we e illed wi h 1.2 l o RAS
was ewa e . Ai pumps o he CO
2
ea men we e in an ai igh plas ic bag.
0
200
400
600
800
1000
1200
0123456789
CO2cocen a ion (ppm)
Day
Fig. 2. Concen a ion o CO
2
in oom ai (open symbols) and ai s ipped om
a Reci cula ing Aquacul u e Sys em (RAS, illed symbols) in wo g ow h ex-
pe imen s wi h g een mic oalgae. The i s expe imen was done wi h Mona -
aphidium gi i hii (MG) and he second wi h Haema ococcus plu ialis (HP).
J. Pi honen e al.
Aquacul u e 567 (2023) 739242
3
1997; Kaewpin ong e al., 2007). Fo M. g i i hii g ow h a e and den-
si ies we e also compa able o he expe imen s conduc ed ea lie wi h
RAS was ewa e in ou labo a o y (S e ˇ
ci´
c e al., 2019; Calde ini e al.,
2021). The simila i y o g ow h a e in he p esen expe imen s o hose
epo ed ea lie demons a es ha he g ow h condi ions in ou expe -
imen we e sui able o es ing he e ec o CO
2
addi ion o hese algae.
Ni a e is one o he mos impo an nu ien s suppo ing he g ow h
o mic oalgae (Wang e al., 2019; Nu e al., 2021), and ni a e is
a ailable in high concen a ions, up o 100 mg/l (Da idson e al., 2014),
in RAS was ewa e . The e o e, by combining mic oalgal cul u es o RAS
a ming, he en i onmen al e ec s o ish a ming could be dec eased
along wi h he p oduc ion o algal biomass. E en i he wa e con-
sump ion in in ensi e RAS a ming is ypically educed o e 90% as
compa ed o low- h ough a ming (Mu ay e al., 2014), he olume o
he e luen can s ill be la ge. Taking in o accoun he ime equi ed o
algal biomass g ow h and hei equi emen o ligh , algal pho o-
bio eac o s ha e a limi ed capaci y o pu i ica ion o la ge e luen
olumes. Thus, me hods o inc ease algal g ow h and nu ien up ake
om RAS was ewa e should be de eloped o in ensi y he wa e pu i-
ica ion p ocess. In his expe imen cul u es ecei ing addi ional CO
2
s ipped om he RAS had a signi ican ly lowe ni a e concen a ion in
was ewa e when M. g i i hii was used (Fig. 4a; mANOVA, F
1,8
=
26.848, p =0.001) bu wi h H. plu ialis he a e age ni a e
concen a ion was no lowe in CO
2
ea men (Fig. 4b; mANOVA, F
1,8
=3.168, p =0.113). I seems ha he possible inc ease in nu ien up-
ake along wi h he inc ease o CO
2
concen a ion may depend on he
mic oalgal species. Nu ien up ake (bo h ammonium and ni a e) was
epo ed o inc ease wi h g een mic oalga Desmodesmus communis when
ae a ion was supplemen ed wi h 2% CO
2
(Pezzolesi e al., 2019). On he
o he hand, CO
2
addi ion (0–20%) did no ha e signi ican e ec on
nu ien (ammonium, ni i e, ni a e, phospha e) emo al e iciency
when Chlamydomonas acidophila was used e en i he algal p oduc i i y
inc eased a CO
2
concen a ions 5% and 10% (Ne es e al., 2019).
CO
2
supplemen a ion dec eased pH in cul u es wi h M. g i i hii
(Fig. 5a; mANOVA, F
1,8
=25.716, p <0.001) bu no in H. plu ialis
cul u es (Fig. 5b; mANOVA, F
1,8
=5.032, p =0.055). A lowe pH in CO
2
ea ed cul u es was expec ed as CO
2
addi ion is known o dec ease pH,
and CO
2
addi ion is also used o main ain pH a a desi ed le el in pho-
obio eac o s (Pede sen e al., 2018). In M. g i i hii cul u es, a signi i-
can d op in pH was obse ed on day wo (Fig. 5a). Simila d op and
subsequen inc ease in pH has been epo ed p e iously wi h Chlo ella
ulga is and Chlamydomonas einha d ii (Sche holz and Cu is, 2013),
and explained wi h he p e e ence o he algae o use ammonia o e
ni a e (Pezzolesi e al., 2019) as ammonia up ake eleases p o ons
causing a dec ease in pH. On he o he hand, he inc ease o pH is mos
likely linked o pho osyn hesis which is known o induce build-up o
0
2
4
6
8
10
12
14
16
18
20
0123456789
Cell densi y (10 cells ml
1
)
Day
CO Ai
b)
0
2
4
6
8
10
12
0123456789
Cell densi y (10 cells ml
-1
)
Day
CO Ai
a
)
Fig. 3. A e age (±SD, n =5) algal densi y in pho obio eac o s wi h a) Mono aphidium g i i hii and b) Haema ococcus plu ialis ae a ed wi h oom ai ( iangle, do ed
line) o CO
2
supplemen ed ai pumped om a ickling il e o a RAS (ci cle, solid line). M. g i i hii was cul i a ed in ba ch mode, H. plu ialis wi h ed-ba ch mode,
whe e 1/3 o he medium (RAS was ewa e ) was added on days 3 and 6, and measu emen s we e aken be o e and a e he addi ion. As e isks indica e s a is ical
di e ence (p <0.05) be ween he wo ea men s. No e he di e en scales on y-axes.
0
5
10
15
20
25
30
35
40
0123456789
Ni a e (mg l-1)
Day
CO Ai
a
)
0
2
4
6
8
10
12
14
16
18
20
0123456789
Ni a e (mg l-1)
Day
CO Ai
b)
Fig. 4. A e age (±SD, n =5) wa e ni a e (NO
3
-N) concen a ion in pho obio eac o s wi h a) Mono aphidium g i i hii and b) Haema ococcus plu ialis ae a ed wi h
oom ai ( iangle, do ed line) o CO
2
supplemen ed ai pumped om a ickling il e o a RAS (ci cle, solid line). M. g i i hii was cul i a ed in ba ch mode,
H. plu ialis wi h ed-ba ch mode, whe e 1/3 o he medium (RAS was ewa e ) was added on days 3 and 6, and measu emen s we e aken be o e and a e he addi ion.
As e isks indica e s a is ical di e ence (p <0.05) be ween he wo ea men s.
J. Pi honen e al.
Aquacul u e 567 (2023) 739242
4
OH
−
and CO
2
up ake, as well as o he ni a e assimila ion (La sdo e ,
2006; Geada e al., 2017). As he op imal pH o cul i a ion o
M. g i i hii and H. plu ialis is close o neu al (Sa ada e al., 2002; Fujii
e al., 2008), CO
2
addi ion caused a dual posi i e e ec on he g ow h o
bo h algae: i s , i made he pH mo e a ou able o he algal g ow h,
and second, i p o ided he algae wi h ino ganic ca bon o allow o
inc ease in g ow h a e.
In he CO
2
ea men , CO
2
concen a ion pumped in o he cul u es
om he RAS ickling il e was a ound 900–1000 ppm. Summe el
e al. (2000) epo ed ha ai CO
2
concen a ion in a cascade column
il e ou le a ied be ween c. 1000 and 8400 ppm depending on he
packing dep h o he s ipping column, gas- o-liquid (i.e. ai o wa e )
a io and wa e CO
2
concen a ion, esul ing in an inc ease o he ai CO
2
concen a ion be o e and a e he il e om c. 70 o 500% (Summe el
e al., 2000). In ou sys em he inc ease o CO
2
concen a ion be ween
he inle ( oom ai pumped in o he ickling il e ) and ou le ( om he
il e ) ai was on a e age om 75% ( he expe imen wi h H. plu ialis) o
86% (M. g i i hii). This ela i ely low inc ease in CO
2
concen a ion was
mos likely due o low ish densi y (less han 5 kg/m
3
) in ou RAS.
Howe e , ainbow ou could be ea ed in densi ies be ween 50 and
100 kg/m
3
in RAS (Roque d’O bcas el e al., 2009). In such in ensi e
se ings CO
2
concen a ion o he ai om he degasse could be ex-
pec ed o be much highe han in he p esen expe imen , and conse-
quen ly also he g ow h o he algae could po en ially be u he
inc eased.
Di e en ypes o was ewa e s ha e been used o p oducing
mic oalgae (La sdo e , 2006), and CO
2
in indus ial lue gases ha e
been used o boos he g ow h o mic oalgae. A he same ime mic o-
algae se e as biological cleane s o was ewa e , and hey assimila e he
g eenhouse gas CO
2
(Geada e al., 2017). In aquacul u e, especially on
land-based RAS, mic oalgae could be used o cap u e bo h he dissol ed
nu ien s and CO
2
o mi iga e hei en i onmen al e ec s and o p o-
mo e ci cula economy in aquacul u e. In con as o many o he ypes
o was ewa e s and lue gases, RAS was ewa e and CO
2
a e oid o
me als o o he de imen al pollu an s ha could limi he use o
mic oalgae also o o he pu poses han biodiesel p oduc ion (Goswami
e al., 2021). In conclusion, he cu en esul s can be ega ded as a p oo
o concep , sugges ing ha he g ow h o mic oalgae g own in RAS
was ewa e can easily be inc eased by using CO
2
s ipped om a RAS,
hus gi ing an op ion o make aquacul u e p oduc ion mo e sus ainable
and en i onmen ally iendly. O he wo es ed algal species M. g i i hii
appea ed o be mo e esponsi e han H. plu ialis in e ms o g ow h a e
and nu ien up ake o he addi ion o CO
2
. The expe imen s we e done
in labo a o y scale wi h a ela i ely low concen a ion o ni a e and CO
2
and hus, u he expe imen a ion would be needed in a se ing com-
pa able o comme cial a ming, which could be expec ed o inc ease
algal g ow h a e and nu ien up ake. This labo a o y scale expe imen
does no pe mi us o make easonable es ima es o o e all e iciency o
his ype o in eg a ed sys em in comme cial ish a ms. Pilo -scale ex-
pe imen s would be needed o make an app oxima e es ima e o com-
me cial easibili y o such a sys em, and he p o i abili y will depend
e y much on he end-use o he selec ed algae. Howe e , ou sys em
indica es ha in eg a ion o mic oalgal cul u es wi h RAS is an op ion
owa ds g eene and ecologically sus ainable aquacul u e.
CRediT au ho ship con ibu ion s a emen
Juhani Pi honen: Concep ualiza ion, Me hodology, Fo mal anal-
ysis, In es iga ion, Da a cu a ion, W i ing – o iginal d a , Supe ision,
P ojec adminis a ion. Silja Koukka: Fo mal analysis, In es iga ion,
Da a cu a ion, W i ing – e iew & edi ing, Visualiza ion. Ka ja Pulk-
kinen: Concep ualiza ion, Me hodology, Fo mal analysis, In es iga ion,
Resou ces, Da a cu a ion, W i ing – e iew & edi ing, Supe ision.
Decla a ion o Compe ing In e es
The au ho s decla e ha hey ha e no known compe ing inancial
in e es s o pe sonal ela ionships ha could ha e appea ed o in luence
he wo k epo ed in his pape .
Da a a ailabili y
Da a will be made a ailable on eques .
Acknowledgemen s
We wan o hank Ta u Koponen o he help in unning he expe i-
men wi h M. g i i hii and Juha Ahonen o main aining he RAS.
Re e ences
Bos ock, J., Lane, A., Hough, C., Yamamo o, K., 2016. An assessmen o he economic
con ibu ion o EU aquacul u e p oduc ion and he in luence o policies o i s
sus ainable de elopmen . Aquac. In . 24, 699–733. h ps://doi.o g/10.1007/
s10499-016-9992-1.
an Bussel, C.G.J., Mahlmann, L., K oeckel, S., Sch oede , J.P., Schulz, C., 2013. The
e ec o high o ho-phospha e wa e le els on g ow h, eed in ake, nu ien
u iliza ion and heal h s a us o ju enile u bo (Pse a maxima) ea ed in in ensi e
eci cula ing aquacul u e sys ems (RAS). Aquac. Eng. 57, 63–70. h ps://doi.o g/
10.1016/J.AQUAENG.2013.08.003.
Calde ini, M.L., S e ˇ
ci´
c, ˇ
C., 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 Res. 60, 102486 h ps://doi.o g/10.1016/j.
algal.2021.102486.
Chai, W.S., Tan, W.G., Halima ul Munawa oh, H.S., Gup a, V.K., Ho, S.H., Show, P.L.,
2021. Mul i ace ed oles o mic oalgae in he applica ion o was ewa e
bio ea men : a e iew. En i on. Pollu . 269, 116236 h ps://doi.o g/10.1016/J.
ENVPOL.2020.116236.
Chekano , K., Schas naya, E., Solo chenko, A., Lobako a, E., 2017. E ec s o CO
2
en ichmen on p ima y pho ochemis y, g ow h and as axan hin accumula ion in he
chlo ophy e Haema ococcus plu ialis. J. Pho ochem. Pho obiol. B 171, 58–66.
h ps://doi.o g/10.1016/j.jpho obiol.2017.04.028.
Cheng, L., Zhang, L., Chen, H., Gao, C., 2006. Ca bon dioxide emo al om ai by
mic oalgae cul u ed in a memb ane-pho obio eac o . Sep. Pu i . Technol. 50,
324–329. h ps://doi.o g/10.1016/j.seppu .2005.12.006.
Ch is enson, L., Sims, R., 2011. P oduc ion and ha es ing o mic oalgae o was ewa e
ea men , bio uels, and biop oduc s. Bio echnol. Ad . 29, 686–702. h ps://doi.o g/
10.1016/J.BIOTECHADV.2011.05.015.
Da idson, J., Good, C., Welsh, C., Summe el , S.T., 2014. Compa ing he e ec s o high
s. low ni a e on he heal h, pe o mance, and wel a e o ju enile ainbow ou
5
6
7
8
9
10
0123456789
p
H
Day
CO Ai
a
)
5
6
7
8
9
10
0123456789
pH
Day
CO Ai
b)
Fig. 5. A e age (±SD, n =5) wa e pH in
pho obio eac o s wi h a) Mono aphidium
g i i hii and b) Haema ococcus plu ialis
ae a ed wi h oom ai ( iangle, do ed line)
o CO
2
supplemen ed ai pumped om a
ickling il e o a RAS (ci cle, solid line).
M. g i i hii was cul i a ed in ba ch mode,
H. plu ialis wi h ed-ba ch mode, whe e 1/3
o he medium (RAS was ewa e ) was added
on days 3 and 6, and measu emen s we e
aken be o e and a e he addi ion. As e isks
indica e s a is ical di e ence (p <0.05) be-
ween he wo ea men s.
J. Pi honen e al.

Aquacul u e 567 (2023) 739242
5
Onco hynchus mykiss wi hin wa e eci cula ing aquacul u e sys ems. Aquac. Eng.
59, 30–40. h ps://doi.o g/10.1016/J.AQUAENG.2014.01.003.
Eumo a, 2021. F eshwa e aquacul u e in he EU. In: Luxembou g. h ps://doi.o g/
10.2771/594002.
Fujii, K., Nakajima, H., Anno, Y., 2008. Po en ial o Mono aphidium sp. GK12 o ene gy-
sa ing as axan hin p oduc ion. J. Chem. Technol. Bio echnol. 83, 1578–1584.
h ps://doi.o g/10.1002/jc b.1978.
Geada, P., Vasconcelos, V., Vicen e, A., Fe nandes, B., 2017. Mic olagal biomass
cul i a ion. In: Ras ogi, R.P., Madamwa , D., Pandey, A. (Eds.), Algal G een
Chemis y. Else ie , Ams e dam, pp. 257–284.
Goddek, S., Joyce, A., Ko zen, B., Bu nell, G.M., 2019. Aquaponics Food P oduc ion
Sys ems. Sp inge , Na u e Swi ze land. h ps://doi.o g/10.1007/978-3-030-15943-
6.
Goli, A., Shami i, A., Talaiekhozani, A., Esh iaghi, N., Aghamohammadi, N., A oua, M.K.,
2016. An o e iew o biological p ocesses and hei po en ial o CO
2
cap u e.
J. En i on. Manag. 183, 41–58. h ps://doi.o g/10.1016/J.JENVMAN.2016.08.054.
Gong, X., Chen, F., 1997. Op imiza ion o cul u e medium o g ow h o Haema ococcus
plu ialis. J. Appl. Phycol. 9, 437–444. h ps://doi.o g/10.1023/A:1007944922264.
Goswami, R.K., Ag awal, K., Shah, M.P., Ve ma, P., 2021. Bio emedia ion o hea y
me als om was ewa e : a cu en pe spec i e on mic oalgae-based u u e. Le .
Appl. Mic obiol. 75, 701–717. h ps://doi.o g/10.1111/lam.13564.
Kaewpin ong, K., Sho ip uk, A., Pow ongsook, S., Pa asan , P., 2007. Pho oau o ophic
high-densi y cul i a ion o ege a i e cells o Haema ococcus plu ialis in ai li
bio eac o . Bio esou . Technol. 98, 288–295. h ps://doi.o g/10.1016/J.
BIORTECH.2006.01.011.
La sdo e , K., 2006. Was ewa e ea men wi h mic oalgae - a li e a u e e iew.
VATTEN 62, 31–38.
Li, K., Liu, Q., Fang, F., Luo, R., Lu, Q., Zhou, W., Huo, S., Cheng, P., Liu, J., Addy, M.,
Chen, P., Chen, D., Ruan, R., 2019. Mic oalgae-based was ewa e ea men o
nu ien s eco e y: a e iew. Bio esou . Technol. 291, 121934 h ps://doi.o g/
10.1016/J.BIORTECH.2019.121934.
Lix, L.M., Keselman, J.C., Keselman, H.J., 1996. Consequences o assump ion iola ions
e isi ed: a quan i a i e e iew o al e na i es o he one-way analysis o a iance F
es . Re . Educ. Res. 66, 579–619. h ps://doi.o g/10.3102/00346543066004579.
Molazadeh, M., Ahmadzadeh, H., Pou ian a , H.R., Lyon, S., Rampelo o, P.H., 2019. The
use o mic oalgae o coupling was ewa e ea men wi h CO
2
bio ixa ion. F on .
Bioeng. Bio echnol | www. on ie sin.o g 7, 42. h ps://doi.o g/10.3389/
bioe.2019.00042.
Mo a, V.C., Nilsen, T.O., Ge wins, J., Gallo, M., Y ebo g, E., Bae e jo d, G.,
Kola e ic, J., Summe el , S.T., Te jesen, B.F., 2019. The e ec s o ca bon dioxide on
g ow h pe o mance, wel a e, and heal h o A lan ic salmon pos -smol (Salmo sala )
in eci cula ing aquacul u e sys ems. Aquacul u e 498, 578–586. h ps://doi.o g/
10.1016/J.AQUACULTURE.2018.08.075.
Mu ay, F., Bos ock, J., Fle che , D., 2014. Re iew o Reci cula ion Aquacul u e Sys em
Technologies and hei Comme cial Applica ion. Uni es i y o S i ling Aquacul u e,
S i ling, U.K.
Ne es, F. de F., Hoinaski, L., R¨
o ig, L.R., Bianchini De ne , R., De, H., Lisboa, M., de
Fa ias Ne es, F., De ne , R.B., 2019. Ca bon bio ixa ion and lipid composi ion o an
acidophilic mic oalga cul i a ed on ea ed was ewa e supplied wi h di e en CO
2
le els. En i on. Technol. 40, 3308–3317. h ps://doi.o g/10.1080/
09593330.2018.1471103.
Nu , S., Oslan, H., Shopa we, N.F., Yuso , A.H., Rahim, A.A., Chang, C.S., Tan, J.S.,
Oslan, S.N., A umugam, K., Bin A i , A., Sulaiman, A.Z., Mohamed, M.S., My, A.B.
A., 2021. A e iew on Haema ococcus plu ialis biop ocess op imiza ion o g een and
ed s age cul u e condi ions o he p oduc ion o na u al as axan hin. Biomolecules
11, 256. h ps://doi.o g/10.3390/biom11020256.
Pede sen, T.C., Ga dne , R.D., Ge lach, R., Pey on, B.M., 2018. Assessmen o
Nannochlo opsis gadi ana g ow h and lipid accumula ion wi h inc eased ino ganic
ca bon deli e y. J. Appl. Phycol. 30, 2155–2166. h ps://doi.o g/10.1007/S10811-
018-1470-X.
Pezzolesi, L., Mazzo i, M., Vanucci, S., Pis occhi, R., 2019. Assimila ion o ino ganic
ni ogen o scaling up Desmodesmus communis (Scenedesmaceae) biomass
p oduc ion. J. Appl. Phycol. 31, 2833–2844. h ps://doi.o g/10.1007/s10811-019-
01814-9.
Ramli, N.M., Ve e h, J.A.J., Yuso , F.M., Nu ulhuda, K., Nagao, N., Ve degem, M.C.J.,
2020. In eg a ion o algae o imp o e ni ogenous was e managemen in
eci cula ing aquacul u e sys ems: a e iew. Re iew. F on . Bioeng. Bio echnol 8,
1004. h ps://doi.o g/10.3389/ bioe.2020.01004.
Roque d’O bcas el, E., Pe son-Le Ruye , J., Le Bayon, N., Blanche on, J.P., 2009.
Compa a i e g ow h and wel a e in ainbow ou ea ed in eci cula ing and low
h ough ea ing sys ems. Aquac. Eng. 40, 79–86. h ps://doi.o g/10.1016/J.
AQUAENG.2008.11.005.
Sa ada, R., T ipa hi, U., Ra ishanka , G.A., 2002. In luence o s ess on as axan hin
p oduc ion in Haema ococcus plu ialis g own unde di e en cul u e condi ions.
P ocess Biochem. 37, 623–627. h ps://doi.o g/10.1016/S0032-9592(01)00246-1.
Sche holz, M.L., Cu is, W.R., 2013. Achie ing pH con ol in mic oalgal cul u es h ough
ed-ba ch addi ion o s oichiome ically-balanced g ow h media. BMC Bio echnol.
13.
Shah, M.M.R., Liang, Y., Cheng, J.J., Da och, M., 2016. As axan hin-p oducing g een
mic oalga Haema ococcus plu ialis: om single cell o high alue comme cial
p oduc s. F on . Plan Sci. | www. on ie sin.o g 7, 531. h ps://doi.o g/10.3389/
pls.2016.00531.
Sko , P.V., 2019. CO
2
in aquacul u e. In: G osell, M., Munday, P.L., Fa ell, A.P.,
B aue , C.J. (Eds.), Ca bon Dioxide. Academic P ess, pp. 287–321. h ps://doi.o g/
10.1016/BS.FP.2019.07.004.
S e ˇ
ci´
c, ˇ
C., Pulkkinen, K., Pi honen, J., 2019. Sc eening o mic oalgae and LED g ow
ligh spec a o e ec i e emo al o dissol ed nu ien s om cold-wa e
eci cula ing aquacul u e sys em (RAS) was ewa e . Algal Res. 44, 101681 h ps://
doi.o g/10.1016/j.algal.2019.101681.
Summe el , S.T., Vinci, B.J., Pied ahi a, R.H., 2000. Oxygena ion and ca bon dioxide
con ol in wa e euse sys ems. Aquac. Eng. 22, 87–108. h ps://doi.o g/10.1016/
S0144-8609(00)00034-0.
Wang, F., Gao, B., Wu, M., Huang, L., Zhang, C., 2019. A no el s a egy o he hype -
p oduc ion o as axan hin om he newly isola ed mic oalga Haema ococcus plu ialis
JNU35. Algal Res. 39, 101466 h ps://doi.o g/10.1016/J.ALGAL.2019.101466.
J. Pi honen e al.