Bioca alysis and Ag icul u al Bio echnology 60 (2024) 103341
A ailable online 5 Augus 2024
1878-8181/© 2024 The Au ho s. Published by Else ie L d. This is an open access a icle unde he CC BY license
(h p://c ea i ecommons.o g/licenses/by/4.0/).
Con en s lis s a ailable a ScienceDi ec
Bioca alysis and Ag icul u al Bio echnology
jou nal homepage: www.else ie .com/loca e/bab
Sus ainable cul i a ion o Po phy idium c uen um ia ag o-
indus ial by-p oduc s: A s udy on biomass and lipid enhancemen
Gio anni Luca Russoa, An onio Luca Langello ia,*, Vi o Ve a dob,c,
Bea iz Ma in Ga ciad, Ma ia Oli ie oe, Paolo Masia,
aCAISIAL Cen e, Uni e si y o Naples Fede ico II, Via Uni e si à 133, 80055, Po ici (NA), I aly
bDepa men o Nu i ion and Food Science, Campus o Ca uja, Uni e si y o G anada, 18071, G anada, Spain
cIns i u e o Nu i ion and Food Technology ‘José Ma aix’, Biomedical Resea ch Cen e , Uni e si y o G anada, A da Del Conocimien o Sn., 18100,
A milla, G anada, Spain
dResea ch G oup “Analy ical Chemis y o Con aminan s”, Depa men o Chemis y and Physics, Resea ch Cen e o Medi e anean In ensi e
Ag osys ems and Ag i-Food Bio echnology (CIAMBITAL), Uni e si y o Alme ía, Ag i-Food Campus o In e na ional Excellence, CeiA3, 04120, Alme ía,
Spain
eDepa men o Animal Heal h, Expe imen al Zoop ophylac ic Ins i u e o Sou he n I aly, Po ici, I aly
Depa men o Ag icul u al Sciences, Uni o Food Science and Technology - Uni e si y o Naples Fede ico II, 80055, Po ici, I aly
ARTICLE INFO
Keywo ds:
Food was e
Mic oalgae
Was e alo iza ion
Response su ace me hodology
Molasses
ABSTRACT
This s udy explo ed he cul i a ion o Po phy idium c uen um using bee molasses and co n s eep
liquo (CSL) as al e na i e ag i-was e subs a es. The objec i e was o enhance he biomass and
lipid p oduc ion o P. c uen um, known o i s po en ial as an indus ial EPA p oduce . Using a
Box-Behnken Design (BBD) as pa o he esponse su ace me hodology (RSM), we in es iga ed
he e ec s o bee molasses, CSL, and F/2 nu ien s on he mic oalga's cul i a ion. The esul s
demons a ed a signi ican in luence o hese ac o s on he inal cell coun and lipid con en o e
an 8-day cul i a ion pe iod. Speci ically, op imal g ow h condi ions we e iden i ied a app oxi-
ma ely 1.78 g/L o molasses and 1.89 g/L o CSL, yielding a cell coun o 12.1 × 106cells/mL
and a lipid con en o 24.48%. Valida ion expe imen s ea i med hese indings, wi h obse ed
esul s closely aligning wi h p edic ed alues. Op imized media achie ed also a signi ican highe
con en o ca o enoids and phycobilip o eins compa ed o he s anda d media. This esea ch
highligh s he po en ial o using ag o-indus ial by-p oduc s o la ge-scale cul i a ion o P. c uen-
um, o e ing a sus ainable app oach o enhancing lipid and pigmen p oduc ion.
1. In oduc ion
Mic oalgae ep esen a pi o al ole in high added alue compounds p oduc ion due o hei capaci y o accumula e signi ican
amoun s o lipids, omega-3 oil, pigmen s o ca bohyd a es unde speci ic g ow h condi ions (Russo e al., 2021a). Apa om high
lipid yield pe uni a ea, he apid cul i a ion cycles (Adams e al., 2013), obus ness agains di e en en i onmen s such as sal wa e
o eu ophic wa e s (He e a e al., 2021), and he c ea ion o aluable by-p oduc s, e.g., p o eins and esidual biomass (Russo e al.,
2021b), a e some appealing ea u es o mic oalgae.
In he ield o omega-3 oil p oduc ion, he a ailabili y and cos o nu ien s, pa icula ly ni ogen, phospho ous and o ganic ca -
bon, play a i al ole. Omega-3 a y acids a e essen ial nu ien s enowned o hei bene icial e ec s on human heal h, no ably in e-
* Co esponding au ho .
E-mail add esses: [email protected] (A.L. Langello i), i[email p o ec ed] (V. Ve a do), bea9[email p o ec ed] (B.M. Ga cia).
h ps://doi.o g/10.1016/j.bcab.2024.103341
Recei ed 26 No embe 2023; Recei ed in e ised o m 5 July 2024; Accep ed 4 Augus 2024
Bioca alysis and Ag icul u al Bio echnology 60 (2024) 103341
2
G.L. Russo e al.
ducing he isk o hea disease, in lamma ion, and neu ological diso de s. The adi ional sou ces o omega-3, mainly ish oils, ha e
been associa ed wi h sus ainabili y challenges, such as o e ishing and con amina ion wi h hea y me als (Wang e al., 2022). How-
e e , he p oduc ion o algae oil equi es highe p oduc ion cos s i compa ed o he ish oil.
Po phy idium c uen um (also known as Po phy idium pu pu eum), a ed unicellula mic oalga, o e s a p omising al e na i e o
he sus ainable p oduc ion o omega-3 oils (Di Lena e al., 2019;Kim e al., 2021). Rich in eicosapen aenoic acid (EPA) and o he
essen ial a y acids, his mic oalga has p o en o be an e icien p oduce o lipids ha can be easily cul i a ed in a ious en i on-
men al condi ions. The g ow h o Po phy idium c uen um no only aligns wi h he cu en end o en i onmen al s ewa dship bu
also in oduces new a enues o indus ial scalabili y. Unde s anding and op imizing he g ow h o P.c uen um is, he e o e, no
me ely a scien i ic pu sui bu a global impe a i e. In ha nessing i s po en ial, esea che s and indus ies may unlock new pa hways
o add ess he in e wined challenges o heal h, sus ainabili y, and ene gy (Kim e al., 2021).
The con inuous sea ch o economical and sus ainable nu ien sou ces o mic oalgae g ow h has di ec ed a en ion owa ds ag i-
cul u al was e p oduc s. Bee molasses (BM), a by-p oduc o suga p oduc ion, con ains suga s, i amins, and a ious o ganic acids,
which could make i sui able o mic oalgae g ow h (Piasecka e al., 2017). The global p oduc ion o BM is es ima ed o ange be-
ween 65 and 70 million me ic ons annually, wi h a ma ke alue o app oxima ely 7 billion US dolla s (S&P global, 2023). The BM
ep esen s an easily ob ainable was e in Eu ope, since EU (F ance and Ge many in pa icula ) de ains he highes p oduc ion o bee
pulp wi h 13 mT bee pulp gene a ion and abou 32 mT o molasses (Diwan e al., 2018). Co n s eep liquo (CSL) is ano he ag o-
indus ial was e widely used as cheap subs a e o biop ocessing. I is a by-p oduc o co n we -milling, is ich in amino acids and i -
amins, making i a po en ial nu ien sou ce o mic oalgae (Kim e al., 2020). S udies like Jung e al. (2010) ha e shown ha hese
by-p oduc s can be used as subs a es o bac e ial e men a ion (Jung e al., 2010), indica ing hei po en ial applicabili y o mi-
c oalgae cul i a ion. The u iliza ion o nu ien s de i ed om BM and CSL ep esen s a p omising pa hway. These sou ces con ain a
weal h o ca bohyd a es, amino acids, i amins, and mine als essen ial o mic oalgae g ow h (Nakaha a e al., 1996;Piasecka e al.,
2017). P e ious wo k has shown ha BM can be an e icien ca bon sou ce o di e en mic oo ganisms (Piasecka e al., 2017,2020;
Schmid e al., 2005), while CSL has been used as an o ganic ni ogen supplemen (Maddipa i e al., 2011;Mohammad Mi zaie e al.,
2016;Russo e al., 2023). In ac , nume ous s udies ha e explo ed he use o hese ag icul u al was e ma e ials o e men a ion and
mic obial g ow h (Kim e al., 2020) bu hei applica ion in mic oalgae cul i a ion emains ela i ely an unde explo ed ield. The use
o BM and CSL ep esen s a sus ainable app oach by alo izing ag icul u al by-p oduc s, which a e o en conside ed was e. This no
only helps in was e managemen bu also signi ican ly educes he cos o cul i a ion media.
Ea lie esea ch on aqua ic p o is s has shown p omising esul s using di e en ag icul u al was e p oduc s, such as Scenedesmus
obliquus g own in b ewe y was ewa e (Na a o-López e al., 2020), Chlo ella ulga is cul i a ed using dai y e luen (Pe e e al.,
2021) and Euglena g acilis on spen b ewe y g ain and CSL (Kim e al., 2020). Ne e heless, s udies ocusing on BM and CSL o mi-
c oalgae g ow h, pa icula ly ega ding biomass and lipid p oduc ion, a e limi ed.
In his s udy, we explo e o he i s ime he po en ial o BM and CSL as al e na i e nu ien sou ces o he g ow h o P. c uen um.
We e alua e hei e ec i eness in e ms o biomass yield, lipid p oduc i i y, and a y acid composi ion, compa ing hem wi h con-
en ional g ow h media. We also analyze he sus ainabili y and economic ad an ages o hese ag icul u al by-p oduc s. To he bes o
ou knowledge, his is he i s comp ehensi e s udy assessing he use o BM and CSL o P. c uen um cul i a ion a ge ing biomass and
lipid p oduc ion. The ou comes o his s udy will p o ide an impo an con ibu ion o he ield o al e na i e omega-3 a y acids oil
p oduc ion and may lead o mo e en i onmen ally iendly and cos -e ec i e mic oalgae cul i a ion p ac ices.
2. Ma e ials and me hods
2.1. Mic oalga and cul u e condi ions
P. c uen um (RCC653) was ob ained om he Rosco Cul u e Collec ion (Rosco , F ance) and p e-cul u ed o 10 days in s e il-
ized seawa e wi h modi ied F/2 medium con aining 75 mg NaNO3, 5 mg NaH2PO4·H2O, 4.12 mg Na2EDTA, 3.11 mg FeCl3·6H2O,
0.02 mg MnCl2·4H2O, 0.02 mg ZnSO4·7H2O, 0.01 mg CoCl2·6H2O, 0.01 mg CuSO4·5H2O, 0.006 mg Na2MoO4·2H2O, 30 mg
Na2SiO3, 0.2 mg hiamine-HCl, 0.01 mg i amin B12, and 0.1 mg bio in pe li e (Kim e al., 2021). The cul u es we e main ained
wi h a wo king olume o 200 mL in 500 mL E lenmeye lasks a 21 °C and 140 pm using an o bi al shake . Con inuous illumi-
na ion o 150 μmol pho ons m−2s−1was p o ided o he lasks.
2.2. Ag i-was e u iliza ion and sc eening expe imen s
To es ablish he g ow h pe o mance o he mic oalga unde in es iga ion, wo ypes o g ow h sc eening we e conduc ed: he
i s , e alua ing he g ow h o he biomass wi h a supplemen a ion o a ious sou ces o o ganic ca bon; he second by e alua ing bio-
mass g ow h wi h a ious concen a ions o BM, CSL, and a mix o he wo.
Fo he i s sc eening, he o ganic ca bon sou ces e alua ed we e glucose (5 g/L), suc ose (4.75 g/L) and uc ose (5 g/L), supple-
men ed o he s anda d media wi h he same concen a ion o ca bon (2 g L.1 o C). These o ganic ca bon sou ces we e selec ed as
hese a e he majo suga p esen in BM and CSL. Fo he second sc eening, di e en ypes o medium we e e alua ed: A medium wi h
1.5 g/L o BM; media wi h 3 g/L BM; media wi h 6 g L −1o BM; media wi h 1 g L −1o CSL; media wi h 2 g L −1o CSL; media wi h
3 g L −1o CSL; mixed media wi h 1.5 g L −1o BM and 1 g L −1o CSL; mixed media wi h 3 g L −1o BM and 1 g L −1o CSL; media
wi h 1.5 g L −1o BM and 2 g L −1o CSL; media wi h 3 g L −1o BM and 2 g L −1o CSL. The BM and CSL used in his expe imen was
al eady used and cha ac e ized in ou p e ious wo k (Russo e al., 2023). BM is cha ac e ized by e y high suga ( o al suga s 667 g/
kg) and ash con en (81.9 g/kg) while CSL was ound o be he iches in e ms o p o eins, wi h a concen a ion o 305 g/kg, bu also
in lac ic acid (121.2 g/kg).
Bioca alysis and Ag icul u al Bio echnology 60 (2024) 103341
3
G.L. Russo e al.
All hese cul i a ion media we e ob ained by adding he co esponding amoun o BM o CSL o a i icial seawa e (ASW) wi hou
supplemen a ion o o he nu ien s. The pH was adjus ed a 8.0 o all he media wi h NaOH 5 M.
The sc eening ials we e conduc ed 500 mL ai -li eac o wi h a wo king olume o 350 mL. The mixing was p o ided h ough
an ai bubbling sys em equipped wi h a il e o 0.22 μm in o de o p e en any con amina ion and o p o ide oxygena ion o he cul-
u e. The ini ial cell densi y o each expe imen was 1 × 105cells/ml. To e alua e he g ow h pe o mance wi h he a ious media,
he op ical densi y was de e mined using a spec opho ome e (ONDA UV-30 SCAN, To ino, I aly) a a wa eleng h o 686 nm.
2.3. Box-behnken design
In his s udy, a Box-Behnken Design (BBD) was employed as pa o he esponse su ace me hodology (RSM) o in es iga e he e -
ec s o BM, CSL, and F/2 nu ien s on he cul i a ion o Po phy idium c uen um. This design was chosen o i s e iciency and obus -
ness in modeling he esponse su ace wi h h ee-le el ac o ial designs (Esua e al., 2021). The h ee independen a iables consid-
e ed we e molasses concen a ion (g/L), CSL concen a ion (g/L), and F/2 nu ien s concen a ion (%). Each o hese ac o s was a -
ied ac oss h ee le els, de e mined based on p elimina y s udies. The exac alues o hese le els will be de ailed in he esul s sec ion.
The BBD o h ee ac o s includes a o al o 15 expe imen al uns. Twel e o hese uns we e used o es ima e he i s -o de and
second-o de e ms in he eg ession model, and h ee addi ional cen e poin s we e included o es ima e he expe imen al e o .
The BBD also allows o he es ima ion o in e ac ion e ec s be ween he ac o s, p o iding a comp ehensi e unde s anding o he
impac o hese a iables on he cul i a ion p ocess. The expe imen s we e conduc ed wi hin a single block o a oid block e ec s.
The speci ic le els o he h ee independen a iables used in he Box-Behnken Design a e p esen ed in Table 1.
The le els o he ac o s a e coded as −1 o he low le el, 0 o he cen e le el, and +1 o he high le el. The ac ual alues co e-
sponding o hese coded le els a e based on he ange o easible ope a ion in he lab en i onmen and p elimina y expe imen al ob-
se a ions.
The Box-Behnken Design ma ix o his expe imen consis ed o 15 uns, including 12 ac o ial poin s and 3 cen e poin s. These
expe imen al uns we e conduc ed in a andom o de o minimize he e ec s o uncon olled ac o s.
In each un, he mic oalga P. c uen um was cul i a ed unde he speci ic condi ions o molasses, CSL, and F/2 nu ien le els, and
he cell coun and lipid con en (% w/w) we e measu ed as he esponse a iables.
2.4. Lipid analysis and cell coun p ocedu es o da a collec ion
The esponse a iables in his s udy we e cell coun and lipid con en o he P. c uen um cul u e. Cell coun was de e mined using a
hemocy ome e (Bu k chambe ). The cul u ed sample was i s dilu ed i necessa y, and hen placed on o he chambe . The cells we e
coun ed unde a mic oscope, and he cell coun was calcula ed based on he dilu ion ac o and he dimensions o he coun ing cham-
be . The cell coun was exp essed in e ms o millions o cells pe millili e (x106cells/mL). Fo he lipid e alua ion, he biomass was
i s ly lyophilized. The lipid con en o biomass was de e mined using a modi ied p ocedu e o Bligh and Dye me hod (Bligh and
Dye , 1959) (a well-es ablished p ocedu e o lipid ex ac ion) using chlo o o m/me hanol/wa e solu ion wi h a olume a io o
1:2:0.8, espec i ely. Following he ex ac ion, he lipids we e d ied and weighed, and he lipid con en was exp essed as a pe cen age
o he d y weigh o he algal biomass (% w/w).
All he measu emen s we e ca ied ou a he end o each expe imen al un co esponding o he BBD. Ca e was aken o ensu e
he accu acy and ep oducibili y o hese measu emen s. Quali y con ol p ocedu es, such as epea ing he coun s o he ex ac ion
o some samples, we e also implemen ed o ensu e he eliabili y o he da a (n= 3).
2.5. Fa y acid me hyl es e s (FAMEs) analysis
The a y acid me hyl es e s (FAMEs) we e p epa ed om he o al amoun o p e iously ob ained lipids by a ansme hyla ion e-
ac ion acco ding o a p e ious me hodology desc ibed wi h ce ain modi ica ions (Aued-Pimen el e al., 2004). Fo ha pu pose,
20 mg o lipid ex ac was mixed wi h 50 μL 2 N KOH in me hanol, 500 μL o n-hexane and 500 μL o me hylnonadecanoa e (Sigma,
S . Louis, MO, USA) as in e nal s anda d (1mg/mL). The mix u e was shaken by using a o ex o 2 min. The uppe laye supe na an
(FAME ex ac ) was collec ed and injec ed in o a Gas ch oma og aphy-mass spec ome e (GC-MS). Mic oalgal ex ac s we e analyzed
acco ding o he me hod condi ions epo ed by Conde e al. (2021). The analyses we e achie ed by using an Agilen 7890 A Gas
ch oma og aphy coupled o a Wa e s QUATTRO mic oTM mass spec ome e de ec o . The sepa a ion was done by using a capilla y
column DB-5MS (30 m × 0.25 mm; . . 0.25 μm) om Agilen Technologies (J&W Scien i ic, Folsom, CA, USA). The o en empe a-
u e was 58 °C o 2 min, 25 °C min−1 o 160 °C, 2 °C min−1 o 210 °C, 30 °C min−1 o 225 °C (held o 20 min). MS de ec o ope a es
wi h ioniza ion ene gy o 70 eV and a scanning ange o m/z 50–550 m/z. The condi ions we e helium as ca ie gas a 1.4 mL min−1,
inle empe a u e 220 °C, de ec o empe a u e 230 °C, 2 μL o injec ion olume (spli less). Da a we e p ocessed by using MassLynx
e sion 4.1 (Wa e s, San Jose, CA, USA).
Table 1
Le els o he independen a iables used in he Box-Behnken Design o he cul i a ion o P. c uen um.
Fac o Low Le el Cen e Le el High Le el
Molasses (g/L) −1 (0) 0 (1.5) +1 (3)
CSL (g/L) −1 (0) 0 (1) +1 (2)
F/2 (%) −1 (0) 0 (50) +1 (100)
Bioca alysis and Ag icul u al Bio echnology 60 (2024) 103341
4
G.L. Russo e al.
2.6. De e mina ion o ca o enoids and phycobilip o eins
The p ocedu e o de e mining he ca o enoid p o ile om he P. c uen um ex ac was al eady desc ibed in ou p io s udy (Russo
e al., 2021b). The mic oalgal ex ac s we e ob ained using an ul asonic ba h (Bandelin, Sono ex) se wi h a equency o 35 kHz.
B ie ly, 10 mg o mic oalgal sample was ex ac ed wi h 1.5 mL o e hanol (wi h 0.1% bu yla ed hyd oxy oluene), cen i uged, and
hen s o ed a −18 °C pos - il a ion.
Fo analysis, UPLC Acqui y coupled wi h XEVO-TQ-S T iple quad upole mass spec ome y was used. The sepa a ion o
ca o enoids was done in a YMC-C30 e e sed-phase column. Mobile phases included me hanol (wi h 5% wa e and 0.1% o mic acid)
and me hyl e -bu yl e he . Analysis was se in a posi i e-ion mode, wi h speci ic mass spec ome ic pa ame e s ou lined. Da a was
p ocessed using MassLynx e sion 4.1, and ca o enoids we e quan i ied agains known s anda ds. The calib a ion cu es ha e shown
a good linea i y wi h de e mina ion coe icien s highe han 0.9918. The analysis me hod had a limi o de ec ion be ween 0.02 and
2.06 μg L−1, whe eas he limi o quan i ica ion was 0.08–6.85 μg L−1.
The phycobilip o eins we e ex ac ed ollowing he p ocedu e om he me hod p esen ed by (Tounsi e al., 2023). B ie ly, a we
sample o P. c uen um biomass was ob ained by cen i uging 1 mL o he mic oalgal mix u e a 8000×g o 10 min. This we esidue
was cleaned wice using he g ow h medium h ough cen i uga ion and la e ein oduced in a sodium phospha e bu e solu ion
(100 mM, pH 6.0) o 1 mL. To isola e he phycobilip o eins om he mic oalgal esidues, a combina ion o eezing, hawing, and ul-
asonica ion was u ilized. This commenced wi h mul iple eeze- haw cycles un il he cellula was e no longe showed a clea ed
hue. A single cycle comp ised 3 h o eezing a −20 °C and an hou o hawing in oom empe a u e condi ions (20–25 °C). Following
his, he mix u e unde wen ul asonic exposu e wi h a sonica ion p obe, con igu ed a 60% ampli ude and a 0.5 cycle o 10 min.
The sepa a ion o liquid om solid was achie ed by ano he ound o cen i uga ion a 8000×g o 10 min. The esul ing clea liquid
was hen collec ed, and he phycobilip o ein le els we e de e mined using a spec opho ome e , e e encing he abso bance A565
(phycocyanin), A620 (allophycocyanin), and A650 (B-phycoe y h in) alues based on he o mulas p o ided by Ma ca i e al. (2014).
2.7. S a is ical analysis
The collec ed da a we e subjec ed o s a is ical analysis o es ablish he ela ionship be ween he independen a iables (molasses,
CSL, and F/2 nu ien s) and he esponse a iables (cell coun and lipid con en ). A esponse su ace eg ession model was i ed o
he da a. This ype o model is pa icula ly sui able o he analysis o ac o ial designs and allows o he es ima ion o bo h linea and
quad a ic e ec s o he independen a iables, as well as hei in e ac ions.
The model used in his s udy can be w i en as ollows:
Y= β0+ β1X1+ β2X2+ β3X3+ β12X1X2+ β13X1X3+ β23X2X3+ β11X1
2+ β22X2
2+ β33X3
2+A
Whe e Y is he esponse a iable (ei he cell coun o lipid con en ); X1, X2, and X3 a e he independen a iables (molasses, CSL, and
F/2 nu ien s, espec i ely); β0is he in e cep ; β1,β2, and β3a e he linea coe icien s o X1, X2, and X3, espec i ely; β12, β13, and
β23 a e he in e ac ion coe icien s o X1*X2, X1*X3, and X2*X3, espec i ely; β11, β22, and β33 a e he quad a ic coe icien s o
X12, X22, and X32, espec i ely; εis he e o e m.
An analysis o a iance (ANOVA) was also pe o med o de e mine he s a is ical signi icance o he independen a iables and
hei in e ac ions. The ANOVA esul s p o ide he sum o squa es, deg ees o eedom, mean squa e, F- alue, and p- alue o each
e m in he model, which a e c i ical o unde s anding he con ibu ion o each e m o he a ia ion in he esponse a iable.
The da a we e analyzed using IBM© SPSS© S a is ics so wa e Ve . 23 (SPSS, Inc., Chicago,IL, USA). RSM analysis was ca ied ou
using he S a is ica 7.0 package (S a So , Tulsa,OK, USA). The i o he model was checked by examining he esidual plo s and o he
diagnos ic ools. The inal model was chosen based on s a is ical signi icance o he e ms, goodness-o - i s a is ics, and he p inciple
o pa simony.
3. Resul s and discussion
3.1. G ow h pe o mance using di e en o ganic ca bon sou ces
The sc eening esul s ega ding he o ganic ca bon supplemen a ion o s anda d media o P. c uen um a e epo ed in Fig. 1.
The g ow h cu es o P. c uen um unde di e en suga supplemen a ions e eal insigh ul ends in he o ganism's esponse o
a ying nu i ional condi ions (see Fig. 2). Among he suga s examined, glucose and suc ose we e ound o ma kedly enhance g ow h,
as e idenced by he highe op ical densi ies obse ed in he la e s ages o he moni o ing pe iod. In con as , uc ose supplemen a-
ion mi o ed he con ol condi ion, sugges ing ha i migh no be as e ec i e in p omo ing g ow h. These obse a ions could be in-
dica i e o he o ganism's speci ic me abolic p e e ences and u iliza ion pa hways. In ac , while P. c uen um is capable o au o ophic
nu i ion, i can also be cul i a ed using ce ain o ganic ca bon sou ces. P e ious s udies ha e p o ided e idence ha he g ow h o
Po phy idium spp.can be enhanced in he p esence o speci ic o ganic ca bon subs a es (Kim e al., 2021;Li e al., 2019;Oh e al.,
2009). This can be a ibu ed o he e icien up ake and assimila ion o glucose and suc ose h ough glycolysis and he subsequen i-
ca boxylic acid (TCA) cycle. These pa hways p o ide essen ial p ecu so s and ene gy (ATP and NADPH) o biomass p oduc ion and
lipid biosyn hesis. Suc ose esul ed he op imal o ganic ca bon sou ce o he g ow h o P. c uen um, showing a signi ican highe
alue o op ical densi y a e 8 days espec o he au o ophic con ol. Rega ding glucose supplemen a ion, ou da a a e in line wi h
hose o Oh e al. (2009) which ound he highes g ow h o P. c uen um when supplemen ing glucose (5–15 g/L) o he media (Oh e
al., 2009). The p onounced g ow h in he p esence o glucose and suc ose may be a ibu ed o hei mo e e icien assimila ion o he
ac i a ion o speci ic me abolic ou es ha a o g ow h (Jiao e al., 2018). Con e sely, he simila i y be ween he uc ose cu e and
Bioca alysis and Ag icul u al Bio echnology 60 (2024) 103341
5
G.L. Russo e al.
Fig. 1. Sc eening es o di e en ype o o ganic ca bon sou ce o P. c uen um cul i a ion. The basal medium used is Guilla d /2. Con ol e e s o Guilla d /2 media
wi hou supplemen a ion o o ganic ca bon. Da a a e epo ed as mean (n = 3) ± SD.
Fig. 2. Compa ison o he inal cell coun , lipid con en and EPA concen a ion o P. c uen um a e 8 days o cul i a ion unde a ious supplemen a ion condi ions.
The blue ba s ep esen he inal cell coun (mean ± SD). The ed line plo illus a es he lipid con en (% w/w) o he co esponding condi ions. The g een line ep-
esen s he EPA con en (% o o al a y acids). (Fo in e p e a ion o he e e ences o colo in his igu e legend, he eade is e e ed o he Web e sion o his a i-
cle.)
he con ol (wi hou supplemen a ion o o ganic ca bon) may e lec a limi ed abili y o u ilize uc ose as an ene gy sou ce o he e-
qui emen o speci ic condi ions o i s e ec i e up ake. Despi e he need o u he in es iga ion o unde s and he exac mecha-
nism, he e idence sugges s ha P. c uen um exhibi s supe io g ow h unde mixo ophic condi ions compa ed o s ic ly au o ophic
condi ions.

Bioca alysis and Ag icul u al Bio echnology 60 (2024) 103341
6
G.L. Russo e al.
3.2. Sc eening esul s wi h al e na i e ag i-was e subs a es
Since he o ganic ca bon supplemen a ion lead o a g ow h boos , we es ed he g ow h pe o mances o media composed exclu-
si ely o BM, CSL and a mix o hem.
The igu e p esen s an insigh ul compa ison o he inal cell coun and lipid con en in P. c uen um unde di e en supplemen a-
ion condi ions o e an 8-day cul i a ion pe iod. The esul s highligh he signi ican in luence o di e en ag i-was e supplemen a-
ions on bo h cell g ow h and lipid accumula ion in P. c uen um.
The con ol (wi hou addi ional supplemen a ion) esul ed in a baseline cell coun , es ablishing a e e ence o compa ison. The
cell coun was no ably enhanced wi h 1.5 g/L and 3 g/L BM supplemen a ion, eaching a peak a 3 g/L. Howe e , u he inc ease in
molasses concen a ion led o a decline in cell coun . A mode a e inc ease in cell coun was obse ed wi h 1 g/L and 2 g/L CSL, while
4 g/L CSL esul ed in a sligh decline. The combina ion o molasses and CSL showed a syne gis ic e ec on cell g ow h, especially in
he condi ions o 3 g/L o BM +1 g/L CSL and 3 g/L o BM +2 g/L CSL. In ou p e ious wo k, we obse ed ha also he dia om
Phaeodac ylum ico nu um is capable o u ilize he nu ien s p esen in BM and CSL (Russo e al., 2023). BM was al eady used succes-
ully o he g ow h o he e o ophic mic oalgae like Galdie ia sulphu a ia (Schmid e al., 2005). The s udy by Schmid e al. (2005)
poin ed ou an inhibi o y e ec o BM when used in high concen a ions (10 g/L), and his is in line wi h ou indings whe e he high-
es con en o molasses showed a signi ican lowe cell densi y espec o he con ol. In gene al, molasses se es as a ich sou ce o
ca bon, p ima ily in he o m o suga s, which is a undamen al p e equisi e o mixo ophic mic oalgal me abolism (Mohammad
Mi zaie e al., 2016). Howe e , he concen a ion o molasses in he cul u e medium equi es ca e ul calib a ion. Bo h de icien and
excessi e concen a ions can impede he cellula p ocesses, leading o subop imal g ow h and lipid accumula ion. Simila ly, CSL con-
ains an a ay o nu ien s including amino acids, i amins, and mine als, which con ibu e o he o e all nu i ional quali y o he
cul u e medium (Kim e al., 2020;Russo e al., 2023). P e ious esea ch has consis en ly shown ha hese by-p oduc s can enhance
mic obial g ow h and p oduc yield. Fo ins ance, he use o bee molasses and CSL has been associa ed wi h inc eased biomass p o-
duc ion and highe yields o aluable me aboli es, such as lipids and p o eins.
Rega ding he lipid con en , he lowes amoun s we e obse ed o he con ol (11.58%) and o he medium composed only o
CSL. The lipid con en o he con ol was highe han hose o Rebolloso Fuen es e al. (2000) bu lowe han o he ound in li e a u e
o he same s ain (Yongmani chai and Wa d, 1991). A subs an ial inc ease in lipid con en was e iden wi h molasses, peaking a
3 g/L concen a ion. CSL supplemen a ion also esul ed in an inc ease in lipid con en , bu less p onounced compa ed o molasses.
The lipid con en in mixed mediums was gene ally highe , indica ing a a o able e ec on lipid accumula ion. These ag o-was e sub-
s a es showed also a lipid concen a ion boos o P. ico nu um, which is in line o ou p e ious s udy (Russo e al., 2023). Mo eo e ,
ano he s udy epo ed ha supplemen a ion o BM o g ow h media inc eased he lipid con en o Chlo ella ulga is (Kendi lioğlu
Şimşek and Ce in, 2022). Mixo ophic mode has been widely epo ed o inc ease he lipid yield o mic oalgae cul i a ion espec he
classic au o ophy. The ecen s udy o Piasecka e al. (2020) in es iga ed he ole o BM in he g ow h o Te adesmus obliquus unde
di e en cul u e condi ions. The au ho s epo ed a lipid p oduc i i y doubled espec he au o ophic con ol which could be due o
a p o ec i e mechanism in algal cells induced by exposu e o nu ien s ess (Piasecka e al., 2020). P. c uen um is ecognized o be an
excellen candida e o he p oduc ion o long-chain polyunsa u a ed a y acids, especially EPA (Hu e al., 2018;Russo e al., 2021a;
Yongmani chai and Wa d, 1991)
The esul s demons a e a complex ela ionship be ween suga supplemen a ion and bo h cell g ow h and lipid accumula ion in P.
c uen um. Speci ic concen a ions o molasses and CSL, indi idually o in combina ion, could op imize he cell coun and lipid con-
en .
3.3. BBD esul s and eg ession analysis
The cul i a ion o P. c uen um is a complex p ocess in luenced by a ious en i onmen al and nu i ional ac o s. Among hese, ou
s udy ocused on he impac s o molasses, CSL, and F/2 nu ien concen a ions, which we e iden i ied as c i ical pa ame e s in he
cul i a ion p ocess. The esponse su ace me hodology (RSM), g ounded in eg ession analysis, was employed in ou s udy o sc u i-
nize he impac o h ee key ac o s: BM concen a ion; CSL concen a ion; and F/2 nu ien concen a ion, on wo esponse a iables:
cell coun and lipid con en o P. c uen um. This ad anced s a is ical echnique acili a ed a comp ehensi e unde s anding no only o
he main e ec s bu also he in e ac ion e ec s o he ac o s on he esponse a iables.
The expe imen al uns and esul s o BBD a e epo ed in Table 2.
The highes cell coun was obse ed in Run 15 (12.171 × 106cells/mL), whe e 1.5 g/L o BM, 2 g/L o CSL, and no /2 nu ien s
we e used. In con as , he lowes cell coun was seen in Run 7 (2.31 × 106cells/mL) wi h no BM, 0 g/L o CSL, and a 50% concen a-
ion o /2 nu ien s. The highes lipid concen a ion was obse ed in Run 10 wi h 24.75% w/w, showcasing he same composi ion
ha ga e ise o he maximum cell coun . On he o he hand, he minimum lipid concen a ion was ound in Run 5 wi h 12.34% w/w,
which had 3.0 g/L o BM, no CSL, and a 50% concen a ion o /2 nu ien s.
The signi icance o BBD was es ed by analysis o a iance (ANOVA) and epo ed in Table 3. P- alue lowe han 0.05 was consid-
e ed signi ican in he analysis.
The eg ession equa ion o cell coun ob ained om he model has been shown in eq. (1)
Bioca alysis and Ag icul u al Bio echnology 60 (2024) 103341
7
G.L. Russo e al.
Table 2
Box-behnken design and esul s o biomass g ow h and lipid con en wi h supplemen a ion o bee molasses, co n s eep liquo and s anda d nu ien s ( /2).
Run Fac o Assignmen Responses (Y)
X1(g/L) X2(g/L) X3 (%) Cell coun (x106/mL) Lipid (% w/w)
1 1.5 1 50 11.59 17.23
2 3.0 1 100 8.92 22.14
3 1.5 1 50 11.80 18.34
4 1.5 0 100 8.74 13.84
5 3.0 0 50 5.21 12.34
6 3.0 1 0 8.26 24.32
7 0.0 0 50 2.31 11.34
8 0.0 2 50 7.99 19.75
9 1.5 1 50 10.34 18.45
10 3.0 2 50 7.16 24.75
11 1.5 0 0 3.65 17.45
12 0.0 1 0 9.53 16.88
13 0.0 1 100 9.23 19.33
14 1.5 2 100 10.95 23.32
15 1.5 2 0 12.17 22.85
Coded alues; X1Bee molasses, X2Co n s eep liquo ; X3 /2 nu ien s.
Table 3
Analysis o a iance o cell coun and lipid p oduc i i y om Box-Behnken Design used o P. c uen um.
Sou ce DFaAdj SSbAdj MScP-Value
Cell coun Lipids Cell coun Lipids Cell coun Lipids
Model 9 114.33 245.669 12.704 27.297 0.008 0.004
Linea 3 44.44 203.241 14.815 67.747 0.008 0.001
Molasses (g/L) 1 0.030 33.008 0.030 33.008 0.877 0.007
CSL (g/L) 1 42.17 168.361 42.177 168.361 0.002 0.001
F/2 (%) 1 2.239 1.872 2.238 1.872 0.219 0.346
Squa e 3 56.23 30.697 18.745 10.232 0.005 0.042
Molasses 21 27.64 0.008 27.647 0.008 0.004 0.950
CSL 21 29.81 3.745 29.811 3.745 0.004 0.201
F/2 21 0.84 25.257 0.849 25.257 0.426 0.012
2-Way In e ac ion 3 13.65 11.731 4.551 3.910 0.084 0.199
Molasses x CSL 1 3.49 4.000 3.495 4.000 0.139 0.189
Molasses x F/2 1 0.23 5.359 0.230 5.359 0.671 0.139
CSL x F/2 1 9.92 2.372 9.928 2.372 0.061 0.294
E o 5 5.66 8.650 1.131 1.730
Lack-o -Fi 3 4.41 7.739 1.471 2.580 0.311 0.154
Pu e E o 2 1.24 0.911 0.623 0.455
To al 14 119.99 254.320
R2= 96.28, P, p obabili y; CSL, co n s eep liquo .
aDF, deg ee o eedom.
bSS, sum o squa es.
cMS, mean squa es.
Cell coun (x /mL)= 0.99 + 4.152 𝑀𝑜𝑙𝑎𝑠𝑠𝑒𝑠 ( )+ 10.49 𝐶𝑆𝐿 ( )+ 0.0181 𝑓/2 (%)
10
6
𝑔
𝐿
𝑔
𝐿
− 1.216 𝑀𝑜𝑙𝑎𝑠𝑠𝑒𝑠 ( )𝑥 𝑀𝑜𝑙𝑎𝑠𝑠𝑒𝑠 ( )− 2.841 𝐶𝑆𝐿 ( )
𝑔
𝐿
𝑔
𝐿
𝑔
𝐿
×𝑥 𝐶𝑆𝐿 ( )+ 0.000192 /2 (%) 𝑥 /2 (%) − 0.623 𝑀𝑜𝑙𝑎𝑠𝑠𝑒𝑠 ( )
𝑔
𝐿
𝑔
𝐿
×𝑥 𝐶𝑆𝐿 ( )+ 0.00320 𝑀𝑜𝑙𝑎𝑠𝑠𝑒𝑠 ( )𝑥 𝑓/2 (%) − 0.0315 𝐶𝑆𝐿 (𝑔/𝐿)
𝑔
𝐿
𝑔
𝐿
*𝑓/2 (%)
(1)
Fo cell coun , he es ima ed coe icien s o he ac o s and hei in e ac ions we e ob ained om he i ed model. The model was
s a is ically signi ican o cell coun (P = 0.008). The P- alue o molasses was ound o be no signi ican (P = 0.877), indica ing
ha a ia ions in molasses concen a ion don' ha e a subs an ial impac on cell coun . Simila ly, /2 nu ien s pe cen age also didn'
show any signi ican e ec s. CSL ins ead was signi ican o cell coun (P = 0.002) e ealing i s c ucial ole in in luencing cell coun .
The squa e e ms we e also s a is ically signi ican , indica ing ha he e a e likely non-linea ela ionships be ween he ac o s and
he esponses. This is suppo ed by he s ong in luence o he squa ed e ms o molasses (P = 0.004) on cell coun and /2
(P = 0.012) on lipids. Quad a ic e ec s e e o he impac o he squa e o he ac o on he esponse a iables. These e ec s a e c u-
Bioca alysis and Ag icul u al Bio echnology 60 (2024) 103341
8
G.L. Russo e al.
cial o cap u e he po en ial non-linea ela ionships be ween he ac o s and he esponse a iables (Ha ke e al., 1995). Howe e ,
wo-way in e ac ions, al hough p esen ing a ious le els o impac , did no gene ally each a signi ican le el o 0.05. The ole o /2
nu ien s in in luencing cell coun is mul i ace ed. In ac , i is supposed ha an inc eased nu ien concen a ion would consis en ly
esul in heigh ened cell coun s. Howe e , ou da a con adic s his assump ion. A case in poin is he compa ison be ween Run 4,
wi h a 100% /2 nu ien concen a ion yielding a cell coun o 8.747 × 106, and Run 6, whe e he absence o /2 nu ien s esul ed
in a sligh ly ele a ed cell coun o 8.263 × 106. The de i ed eg ession equa ion o cell coun (1) in eg a es all hese ac o in lu-
ences and can be ins umen al in p edic ing he cell coun gi en speci ic concen a ions o molasses, CSL, and /2 nu ien s.
The same app oach was employed o he lipid con en . In Table 2 is epo ed he s a is ical esul s o he BBD used on lipid p o-
duc i i y, while in equa ion (2) is epo ed he eg ession equa ion.
𝐿𝑖𝑝𝑖𝑑 (% 𝑤/𝑤) = 14.14 + 1.4 𝑀𝑜𝑙𝑎𝑠𝑠𝑒𝑠 ( )+ 4.83 𝐶𝑆𝐿 ( )+ 0.1065 𝑓/2 (%)
𝑔
𝐿
𝑔
𝐿
+ 0.02 𝑀𝑜𝑙𝑎𝑠𝑠𝑒𝑠 ( )𝑥 𝑀𝑜𝑙𝑎𝑠𝑠𝑒𝑠 ( )− 1.007 𝐶𝑆𝐿 ( )𝑥 𝐶𝑆𝐿 ( )
𝑔
𝐿
𝑔
𝐿
𝑔
𝐿
𝑔
𝐿
+ 0.001046 𝑓/2 (%) 𝑥 /2 (%) + 0.667 𝑀𝑜𝑙𝑎𝑠𝑠𝑒𝑠 ( )𝑥 𝐶𝑆𝐿 ( )
𝑔
𝐿
𝑔
𝐿
− 0.01543 𝑀𝑜𝑙𝑎𝑠𝑠𝑒𝑠 ( )𝑥(%) + 0.0154 𝐶𝑆𝐿 (𝑔/𝐿) * 𝑓/2 (%)
𝑔
𝐿
𝑓
2
(2)
The esul s om he BBD on P. c uen um lipid p oduc i i y illumina e he impac s o molasses, CSL, and /2 nu ien concen a-
ions on he mic oalgal esponse. The model was s a is ically signi ican o lipid p oduc i i y (P = 0.004). The linea e ec s we e
p onounced, especially o CSL, wi h a P- alue o 0.001, sugges ing a ema kable in luence on lipid p oduc i i y. The linea e ec o
molasses was also signi ican (P = 0.007), indica ing i s impo ance in he lipid yield. Addi ionally, non-linea ela ionships be ween
ac o s and lipid con en a e e iden , conside ing he signi icance o squa ed e ms. Speci ically, /2 squa ed (P = 0.012) poin s o a
no able non-linea ela ionship be ween /2 concen a ion and lipid con en . This lack o linea signi icance sugges s a delica e bal-
ance o his nu ien concen a ion, which is in any case he lowes signi ican ac o o he inc ease o cell coun and lipid p oduc-
i i y. In o he wo ds, he supplemen a ion o s anda d nu ien s o he combina ion o BM and CSL was no use ul in any o he ana-
lyzed esponses. A simila esul s was ob ained also on P. ico nu um cul i a ed using cheese whey and CSL (Russo e al., 2023). In
ou s udy he s anda d /2 nu ien s we e no needed o suppo he biomass p oduc i i y, since hose p esen in CSL and BM we e su -
icien o sus ain biomass g ow h and lipid p oduc i i y.
The lack-o - i alues o bo h cell coun and lipids indica e ha he model i s he da a easonably well and he a ia ions no ex-
plained by he model a en' signi ican ly la ge. This suppo s he eliabili y o he BBD in s udying he e ec s o hese ac o s on P. c u-
en um g ow h and lipid p oduc i i y.
The cul i a ion o P. c uen um, as depic ed by ou esea ch, is ma kedly in luenced by BM, CSL. The obus ness o ou model, as de-
pic ed by he high R2 alue, unde sco es he c i icali y o hese ac o s in de e mining bo h cell coun and lipid p oduc i i y o he mi-
c oalga. Among he indi idual ac o s, CSL showcased he mos signi ican in luence on bo h cell coun and lipid p oduc i i y. This
migh sugges he impo ance o p o eins, o ganic acids, and mine als con ained in la ge amoun s in he CSL o he g ow h o bio-
mass and p o is s (Tan e al., 2016). The nu ien s ess on P. c uen um can indeed change i s biochemical composi ion, as epo ed in
o he s udies (Hu e al., 2018). On he o he hand, while molasses plays a mo e subs an ial ole in lipid p oduc i i y, i s impac on cell
coun is ela i ely mu ed. This could poin owa ds he ene gy- ich suga s in molasses being p ima ily channeled in o lipid syn hesis.
In ac BM, which a e ich in suga s, p esen ed a s ong in luence on lipid p oduc i i y. This is cong uen wi h he idea ha ca bohy-
d a e- ich subs a es can o en p omo e lipid accumula ion in many mic oalgae, gi en ha suga s se e as ca bon sou ces, which a e
hen ans o med in o lipids in he cells (Gao e al., 2019;Oh e al., 2009). This is also in line wi h he s udy o Gao e al. (2019) whe e
he o ganic ma e in he cul i a ion media p omo ed he mixo ophic g ow h and lipid p oduc i i y o Chlo ella sp. G-9. In algal
bio e ine ies, achie ing a high lipid con en wi hin cells is i al o ensu ing he economic iabili y high added alue compound p o-
duc ion (Russo e al., 2022). Ne e heless, cul i a ing hese algal cells unde condi ions ha p omo e lipid accumula ion, such as ni-
ogen s a a ion, coole empe a u es, ele a ed pH le els, and inc eased salini y, o en coincides wi h a educ ion in biomass p oduc-
i i y (Ab eu e al., 2012). This comp omises he o e all yield o lipid p oduc ion. Emb acing mixo ophic cul i a ion me hods
eme ges as a p omising s a egy o add ess his challenge in he con ex o P. c uen um bio e ine ies. Recen esea ch has indica ed
ha using hese cul i a ion modes can concu en ly boos bo h biomass p oduc ion and lipid accumula ion in speci ic algae s ains
(Gao e al., 2019;Russo e al., 2023). Howe e , u he esea ch is essen ial o ine- une he g ow h condi ions ailo ed o each spe-
ci ic s ain, emphasizing he desi ed a ge compounds o p oduc ion.
3.4. Op imiza ion and alida ion o cell concen a ion and lipid p oduc i i y
The op imiza ion s a egy employed in his s udy was unde pinned by he BBD. To be e isualize he esul s o BBD and o p o-
ide insigh s o he op imiza ion p ocess, a 3-D su ace plo combined wi h con ou plo was epo ed o bo h cell concen a ion and
lipid con en (Fig. 3).
The g aphical in e p e a ion o he esul s allows o he easy iden i ica ion o ends and op imal egions. The colo o he su ace
and he con ou plo bo h e lec he cell coun (Fig. 3a) and lipids (Fig. 3b), wi h da ke colo s co esponding o lowe alues and
ligh e colo s co esponding o highe alues. The con ou plo a he bo om se es as a ' oo p in ' o he su ace plo , p o iding an
addi ional pe spec i e on he esponse su ace. This can aid in iden i ying op imal condi ions o maximizing he cell coun . In ac ,
om he igu e, i can be obse ed ha he cell coun eaches a zone o maximum concen a ion, a e which he biomass dec eases a
Bioca alysis and Ag icul u al Bio echnology 60 (2024) 103341
9
G.L. Russo e al.
Fig. 3. Su ace and con ou plo o cell coun (A) and o al lipid con en (B) o Po phy idium c uen um cul i a ed wi h bee molasses and co n s eep liquo h ough box-
behnken design.
a ce ain a e. In con as , he lipid con en doesn' appea o each a maximum poin ; ins ead, i shows an inc easing end as he mo-
lasses concen a ion inc eases.
To unde s and he op imal egion, in Fig. 4 is epo ed he o e laid con ou plo be ween cell coun and lipid con en .
This o e laid plo ep esen s he o al yield (p oduc o cell coun and lipid con en ) as a unc ion o he CSL and molasses concen-
a ions, wi h he /2 nu ien concen a ion held cons an a low alue (0%). The con ou lines ep esen poin s o equal o al yield.
In Fig. 4 has been highligh ed he op imal a ea whe e he cell coun and lipid concen a ion is a hei maximum. The s a iona y poin
whe e he maximum cell coun mee s he maximum lipid con en is app oxima ely a 1.78 g/L o molasses and 1.89 g/L o CSL. These
a e he mos e ec i e concen a ions o maximizing bo h cell coun and lipid con en simul aneously in he cul i a ion o P. c uen um
ollowing he BBD. In hese condi ions a cell coun o 12.1 × 106cell/mL and a lipid con en o 24.48% should be eached. To ali-
da e he model he expe imen was epea ed wi h hese g ow h media condi ion. The esul s we e he ollowing:
11.88 ± 0.6 × 106cell/mL and 23.75 ± 0.69% o lipid concen a ion a e 8 days o cul i a ion in he same condi ions as he BBD
i s expe imen . The new da a ob ained wi h he op imal nu ien concen a ions we e no signi ican ly di e en om he p edic ed
alue, alida ing he BBD esul s.
Fig. 4. O e laid con ou plo o cell concen a ion and lipid con en in he cul i a ion o mic oalga Po phy idium c uen um. The blue lines depic he minimum and
maximum p edic ed cell concen a ions (in × 106cells/mL) ac oss a ying le els o bee molasses (g/L) and co n s eep liquo (CSL, g/L), wi h /2 nu ien s ixed a
0%. The o e laid ed lines ep esen speci ic le els o lipid con en (in % w/w). Whi e colo ed egion is he op imal egion o maximize he esponses. (Fo in e p e a-
ion o he e e ences o colo in his igu e legend, he eade is e e ed o he Web e sion o his a icle.)