Resea ch A icle
Recei ed: 22 June 2024 Re ised: 25 Oc obe 2024 Published online in Wiley Online Lib a y: 20 No embe 2024
(wileyonlinelib a y.com) DOI 10.1002/jc b.7785
An ibac e ial pe o mance o Co–Zn e i e
nanopa icles unde isible ligh i adia ion
Alicia G. Gubieda,a
*
Ana Abad-Díaz-de-Ce io,aAna Ga cía-P ie o,b
M. Luisa Fdez-Gubieda,cLau a Ce e a-Gabalda,d,e
Edua do O doqui-Huesa, Al onso Co nejo and C is ina Gómez-Polo
Abs ac
BACKGROUND: To add ess wa e sca ci y and p omo e sus ainable esou ce managemen , mo e e ficien and cos -e ec i e
wa e ea men solu ions a e necessa y. Pa icula ly, pa hogens in d inking wa e a e a opic o g owing conce n. One p om-
ising echnology is he use o pho oca aly ic nanopa icles ac i a ed by isible ligh as an ibac e ial agen s. This s udy ocuses
on he cha ac e iza ion and an ibac e ial p ope ies o Co–Zn e i e nanoca alys s, es ed agains Esche ichia coli.
RESULTS: The Co
x
Zn
1−x
Fe
2
O
4
(x=0, 0.1, 0.4 and 0.6) e i es we e syn hesized by he co-p ecipi a ion me hod. S uc u al, mo -
phological and op ical analyses confi med ha hese nanopa icles ha e a cubic spinel s uc u e, wi h sizes o a ound 10 nm,
and band gap ene gies sui able o isible ligh ac i a ion (1.4–1.7 eV). The an ibac e ial e ficacy o he nanopa icles agains
E. coli was es ed and compa ed wi h hei pho oca aly ic pe o mance employing phenol as o ganic pollu an model (highes
phenol deg ada ion o x=0.6). Specifically, he an ibac e ial capaci y o hese nanopa icles was e alua ed by compa ing he
abili y o bac e ia o g ow a e being incuba ed wi h he nanopa icles unde isible ligh and in he da k. I was ound ha
nanopa icles wi h lowe cobal con en (x=0 and 0.1) significan ly educed bac e ial cul u abili y unde isible ligh . T ans-
mission Elec on Mic oscopy analysis e ealed ha nanopa icles wi h cobal con en caused bac e ia o sec e e biofilm, po en-
ially o e ing some p o ec ion agains he nanopa icles.
CONCLUSION: ZnFe
2
O
4
nanopa icles show he highes an ibac e ial e ec amongs hose es ed. This is a ibu ed o he com-
bined ac ion o Zn
2+
ion elease and he pho oca aly ic e ec unde isible ligh . Fu he mo e, Zn migh inhibi p o ec i e bio-
film sec e ion, leading o highe an ibac e ial e ec s.
© 2024 The Au ho (s). Jou nal o Chemical Technology and Bio echnology published by John Wiley & Sons L d on behal o Soci-
e y o Chemical Indus y (SCI).
Keywo ds: Co–Zn e i es; pho oca aly ic nanopa icles; an ibac e ial nanopa icles
INTRODUCTION
In ec ions ela ed o con amina ed wa e esou ces by wa e -
bo ne pa hogens a e a majo global conce n o wa e quali y.
Acco ding o he 2030 Agenda o Sus ainable De elopmen 's
SDG 6, which aims o ensu e access o wa e and sani a ion o
all, 2.2 billion people s ill lacked sa ely managed d inking wa e
in 2022, including 703 million wi hou a basic wa e se ice.
1
Dia -
hea, mainly linked o limi ed access o imp o ed wa e and san-
i a ion, is he second leading cause o child mo bidi y and
mo ali y wo ldwide, and esponsible o mo e han 90% o
dea hs in child en unde 5 yea s o age in low- and middle-
income coun ies.
2
Esche ichia coli, besides being one o he majo
pa hogens associa ed wi h wa e bo ne diseases, is commonly
used as an indica o o ecal con amina ion o e i y wa e qual-
i y.
3,4
Fu he mo e, he ex ensi e use o an ibio ics in bo h e e -
ina y and human medicine has p omo ed he occu ence o
an ibio ic- esis an bac e ia, he p ima y con ibu o o an imic o-
bial esis ance and one o he mos wo ying public heal h p ob-
lems. In ac , an imic obial esis ance is expec ed o kill
*Co espondence o: AG Gubieda, Depa amen o de Inmunología, Mic obiolo-
gía y Pa asi ología, Uni e sidad del País Vasco (UPV/EHU), Leioa 48940,
Spain, E-mail: [email p o ec ed]
aDepa amen o de Inmunología, Mic obiología y Pa asi ología, Uni e sidad del
País Vasco (UPV/EHU), Leioa, Spain
bDepa amen o de Física Aplicada, Uni e sidad del País Vasco (UPV/EHU), Bil-
bao, Spain
cDepa amen o de Elec icidad y Elec ónica, Uni e sidad del País Vasco
(UPV/EHU), Leioa, Spain
dSpLine, Spanish CRG beamline a he Eu opean Synch o on Radia ion Facili y,
G enoble, F ance
eICMM-CSIC Can oblanco, Mad id, Spain
Depa amen o de Ciencias & Ins i u e o Ad anced Ma e ials and Ma hema -
ics (INAMAT²), Uni e sidad Pública de Na a a, Pamplona, Spain
© 2024 The Au ho (s). Jou nal o Chemical Technology and Bio echnology published by John Wiley & Sons L d on behal o Socie y o Chemical
Indus y (SCI).
This is an open access a icle unde he e ms o he C ea i e Commons A ibu ion-NonComme cial-NoDe i s License, which pe mi s use and
dis ibu ion in any medium, p o ided he o iginal wo k is p ope ly ci ed, he use is non-comme cial and no modifica ions o adap a ions a e made.
428
10 million people by 2050 and cos he global economy $100
illion.
5,6
Mic obial pa hogens a e gene ally inac i a ed in was ewa e
ea men s h ough con en ional chemical oxida ion agen s like
chlo ine, chlo ine dioxide, chlo amines and ozone.
7
Howe e ,
hese o en esul in he gene a ion o ha m ul disin ec ion byp o-
duc s. UV echnologies o e an al e na i e disin ec ion echnique,
al hough he highe cos , ene gy consump ion and lowe e ec-
i eness o UV- esis an pa hogens limi hei wide p ac ical
applica ion. Among he cu en p oposed echnologies o he
elimina ion o mic obial pa hogens, ad anced oxida ion p o-
cesses s and ou based on he gene a ion o highly eac i e oxy-
gen species (ROS).
8
Pa icula ly, pho oca aly ic an ibac e ial
p ocedu es s and ou , whe e he gene a ion o ROS is s imula ed
by ligh i adia ion.
9
He e ogeneous pho oca alys s such as TiO
2
and ZnO ha e been ex ensi ely s udied o deg ading a b oad
ange o o ganic pollu an s and pa hogenic mic oo ganisms.
10-13
13 Howe e , mos widely employed pho oca alys s a e cha ac e -
ized by a wide band gap (ca 3.2 eV, TiO
2
), making hem mainly
ac i e unde UV ligh i adia ion. Fo p ac ical pu poses, he ac i-
a ion o a semiconduc o unde isible (sola ) ligh would acili-
a e he de elopmen o low-cos wa e disin ec ion sys ems,
whe e he elimina ion o o ganic pollu an s and mic obial pa ho-
gens would be ca ied ou syne gis ically. Acco dingly, di e en
isible-ligh -ac i e pho oca aly ic semiconduc o s ha e been p o-
posed o he simul aneous emo al o o ganic and mic obial pol-
lu an s, including semiconduc o he e os uc u es
14-16
and doped
TiO
2
(Ag,
17
C ,
18
N
19
), among o he s.
Despi e ex ensi e esea ch ac i i y in ecen decades, comme -
cial applica ions o pho oca alysis in was ewa e ea men a e
cu en ly sca ce due o inhe en p ocess challenges (e.g. low
pho ocon e sion e ficiency) and echnological limi a ions
(e.g. ene gy consump ion). The sepa a ion o he pho oca aly ic
agen om he medium, a oiding seconda y con amina ion,
and i s subsequen euse is one o he mos impo an issues
when op imizing a p ocedu e. Thus, magne ic ma e ials ha e
eme ged as po en ial al e na i es o he easy and comple e sep-
a a ion o ca alys s using ex e nal magne ic field g adien s. Mos
s a egies a e based on nanos uc u ed magne ic ma e ials, such
as co e–shell nanopa icles, whe e a magne ic co e (i.e. Fe
3
O
4
)is
usually coa ed wi h a pho oca aly ic ac i e semiconduc o .
20-22
The supe pa amagne ic egime (negligible alues o coe ci i y
and emanence) cha ac e is ic o magne ic nanopa icles is ad is-
able in hese sys ems o a oid pa icle agg ega ion and o achie e
op imal dispe sion in an aqueous medium.
Spinel e i e pho oca alys s ha e demons a ed op imal pe o -
mance o he deg ada ion o o ganic pollu an s in wa e unde
isible i adia ion.
21,23
The combina ion o hei na ow band
gap (wi hin he isible ligh ange) and hei e imagne ic na u e,
which allows euse in se e al cycles h ough magne ic sepa a ion,
makes hese pho oca alys s p omising candida es o was ewa e
ea men s. In pa icula , he pho oca aly ic esponse o Co–Zn
spinel e i es has been widely analyzed in he li e a u e.
24-30
Fu -
he mo e, hey ha e a ac ed significan in e es due o hei
high magne ic momen s (high sa u a ion magne iza ion alues),
achie ed by he inco po a ion o Co in he ZnFe
2
O
4
spinel cell
s uc u e.
31
Bulk ZnFe
2
O
4
(an i e omagne ic wi h Néel empe a-
u e T
N
=10 K) is a no mal spinel, whe e Fe
3+
ca ions an i e o-
magne ically coupled occupy oc ahed al Bsi es, and Zn
2+
ca ions (nonmagne ic) a e p e e en ially loca ed a he e ahed al
Aposi ions. Howe e , mixed s a es (i.e. Fe
3+
ca ions in bo h Band
Asi es) gi e ise o he appea ance o e imagne ism in his spinel
and he desi ed supe pa amagne ism in he nanoscale egime.
Since CoFe
2
O
4
is an in e se spinel, whe e Co
2+
ca ions end o
occupy Bsi es, he eplacemen o Co by Zn leads o a displace-
men o A-Fe
3+
ions o he oc ahed al Bsi es and o an ini ial
inc ease o he ne magne ic momen as Zn concen a ion
inc eases ollowed by a decay o highe Zn concen a ions ha
is asc ibed o he null magne ic momen o Zn
2+
ions.
29,31
Thus,
op imum magne ic esponse is ound o mixed Co–Zn e i e
nanopa icles: maximum alues o he magne ic momen o he
spinel cell (maximum sa u a ion magne iza ion) and supe pa a-
magne ic beha io .
As o hei pho oca aly ic esponse, mos s udies ocus on he
decomposi ion o dyes, eaching deg ada ion a es close o
100%,
24-27
while he deg ada ion o phenolic compounds is mo e
limi ed and hei pho oca aly ic e ficiency significan ly educed.
29
Ne e heless, i is impo an o highligh ha all s udies show
op imal pho oca aly ic beha io o hose mixed composi ions
linked o maximum alues o magne ic momen s o he Co–Zn
spinel cell. In ac , spin-pola iza ion e ec s on he imp o emen
o isible pho oca alysis is a opic o cu en in e es ,
32
mos ly
linked o he inhibi ion o cha ge ecombina ion.
28
Howe e ,
despi e nume ous s udies on hei pho oca aly ic beha io , he
ac i a ion o he mic obial esponse by isible i adia ion in Co–
Zn e i e nanopa icles (pho oca aly ic an ibac e ial e ec )is
sca cely analyzed in he li e a u e and mos s udies a e ocused
on ZnFe
2
O
4
, using di usion-based me hods.
33-35
Acco dingly, he aim o he wo k epo ed he e was o analyze
he pho oca aly ic an ibac e ial ac i i y o Co
x
Zn
1−x
Fe
2
O
4
nano-
pa icles (x=0, 0.1, 0.4 and 0.6) employing E. coli. The selec ed
composi ions ha e been shown o p esen isible pho oca aly ic
ac i i y o he decomposi ion o phenol and oluene.
29
The cul-
u abili y o E. coli was s udied a e incuba ing he bac e ia wi h
he nanopa icles unde bo h isible ligh and in da kness, a di -
e en ime poin s and concen a ions. Resul s show an absence
o an ibac e ial e ec o Co- ich nanopa icles, despi e hei
enhanced pho oca aly ic ac i i y in phenol deg ada ion. This is
a ibu ed o bac e ia sec e ing a p o ec i e biofilm ma ix, which
is inhibi ed by Zn in he absence o Co.
EXPERIMENTAL PROCEDURE
Syn hesis o pho oca alys s
Co
x
Zn
1−x
Fe
2
O
4
nanopa icles (x=0, 0.1, 0.4 and 0.6) we e syn he-
sized by he co-p ecipi a ion me hod,
21
whe e Fe(NO
3
)
3
·9H
2
O
(Lab Kem, 98.0–101.0%, CAS: 7782-61-8), Zn(NO
3
)
2
·6H
2
O (Sigma
Ald ich, ≥99.0%, CAS: 10196-18-6) and Co(NO
2
)
3
·6H
2
O (Me ck,
≥99.0%, CAS: 10026-22-9) we e mixed s oichiome ically in an
aqueous solu ion, adding NaOH solu ion (1 mol L
−1
) d opwise
unde cons an s i ing un il he pH was 13. The p ecipi a e, col-
lec ed by cen i uga ion, was washed, d ied a 50 °C o e nigh
and calcined a 400 °C o 6 h.
Cha ac e iza ion o pho oca alys s
The s uc u e o he nanopa icles was analyzed h ough powde
X- ay di ac ion (XRD) (B uke D8 Ad ance) wi h monoch oma ed
Cu K⊍1 adia ion (⊗=1.54056 Å) and scanning ansmission elec-
on mic oscopy (STEM) wi h a high-angle annula da k field
de ec o , STEM-HAADF (Tecnai field emission gun ope a ed a
300 kV). Di use eflec ance spec oscopy (DRS) was conduc ed
wi h a UV/Vis/NIR Jasco V-670 o e alua e he band gap ene gy
calcula ed using he Kubelka–Munk unc ion. A SQUID magne-
ome e (Quan um Design MPMS XL7) was employed o
An ibac e ial pe o mance o Co–Zn e i e nanopa icles www.soci.o g
J Chem Technol Bio echnol 2025; 100: 428–437 © 2024 The Au ho (s).
Jou nal o Chemical Technology and Bio echnology published by John Wiley & Sons L d on behal o Socie y o Chemical Indus y (SCI).
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cha ac e ize he high magne ic field magne iza ion (applied mag-
ne ic field o 6 T) a 50 and 300 K, and he hys e esis loops o he
samples a oom empe a u e.
The pho oca aly ic ac i i y o wo selec ed samples (x=0and
0.6) was checked employing phenol as o ganic con aminan model
in wa e . Phenol was selec ed as a model pollu an because i is a
non- ola ile con aminan commonly ound in indus ial was ewa-
e . I s concen a ion in was ewa e can ange om <10 mg L
−1
o se e al housands o millig ams pe li e .
36
The ini ial concen a-
ion was chosen based on epo ed oxici y le els o phenolic com-
pounds in he li e a u e, which ange om 9 o 25 mg L
−1
.
37
A suspension o nanopa icles (pH =6) was p epa ed by mixing
24 cm
3
o wa e , 1 cm
3
o phenol solu ion (500 mg dm
−3
) and
20 mg o pho oca alys and s i ed in he da k which co esponds
o an ini ial phenol concen a ion o 20 mg mL
−1
. A e 60 min in
he da k, he suspension was pho oi adia ed wi h a 300 W xenon
lamp (ScienceTech) employing a pho o eac o (V=25 cm
3
)
equipped wi h a qua z window. The op ical pa h included a cu -
o fil e ha ansmi ed ligh o wa eleng h g ea e han
400 nm. Aliquo s collec ed a di e en imes (60 and 120 min)
o eac ion we e analyzed employing a high-pe o mance liquid
ch oma og aphy (HPLC) sys em (Agilen 1100) equipped wi h
e ac i e index de ec o and an UV de ec o (269 nm). The mea-
su emen s we e pe o med using a C18 column (Phenomenex
Gemini 5 μm, 110A; 250 ×4.6 mm, PN 00G-4435-E0) wo king a
45 °C unde isoc a ic flow condi ions o 0.5 cm
3
min
−1
. Du ing
HPLC measu emen s, a mobile phase composed o 70% ace oni-
ile ( / ), 29.5% wa e and 0.5% o hophospho ic acid (85%
w/w solu ion) was used a a flow a e o 0.5 cm
3
min
−1
.
Cha ac e iza ion o leaching o me al ions (Fe
2+/3+
,Zn
2+
and
Co
+3
) in aqueous media was pe o med in samples dilu ed in
Milli-Q wa e (pH 7). The pho oca alys s we e collec ed wi h cen-
i uga ion, o isola e he pho oca alys s om he ions leached
in o he supe na an . Bo h he p ecipi a ed pho oca alys s and
he supe na an we e diges ed in 300 μL o ni ic acid a 80 °C
o 24 h. The diges ed solu ions we e hen dilu ed 60 imes o
analysis o Co, Zn and Fe concen a ion using induc i ely coupled
plasma a omic emission spec ome y (ICP-AES; Agilen 5110).
An ibac e ial e ec
The an ibac e ial e ec o he nanopa icles was e alua ed on
E. coli CECT 100. The s ain was g own a 37 °C and shaking
(250 pm) in Lu ia–Be ani (LB) b o h o 3 h o each exponen ial
g ow h phase, and ha es ed by cen i uga ion a 15 000 ×g
using a able op cen i uge. The cells we e washed h ee imes
in Milli-Q wa e and dilu ed o each a densi y o 10
5
bac e ia
pe millili e . The bac e ial suspensions we e incuba ed o 5–8h
in Milli-Q wa e (non-supplemen ed and supplemen ed wi h 1–
200 μgmL
−1
o nanopa icles), a 25 °C and shaking (90 pm),
bo h in da kness and unde isible ligh i adia ion. The illumina-
ion sys em used o isible ligh i adia ion was composed o Phi-
lips fluo escen ubes (TL5 HO 54W/965 and TL5 HO 54W/840),
wi h i adiance se o 125 W m
−2
. Fo each expe imen al condi-
ion, samples we e pe iodically collec ed o de e mine he num-
be o cul u able bac e ia (exp essed as CFU mL
−1
). The numbe
o cul u able bac e ia was assessed using he mic od ople
me hod, on LB aga pla es incuba ed o 24 h a 37 °C.
The in e ac ion o he nanopa icles wi h he bac e ia was
assessed wi h TEM. Wi h his aim, bac e ia we e incuba ed a
10
8
bac e ia pe millili e wi h 200 μgmL
−1
o each e i e o
4 h. A e incuba ion, bac e ia we e collec ed by pipe ing, a oid-
ing cen i uga ion o he sample o p e en agg ega ion o he
nanopa icles and changes in in e ac ion wi h he bac e ia.
The samples we e hen deposi ed on o glow discha ged ca bon-
coa ed coppe g ids. TEM images we e acqui ed in collabo a ion
wi h he Analy ic and High Resolu ion Mic oscopy in Biomedicine
Se ice (SGIke ), o he Uni e si y o he Basque Coun y
(UPV/EHU). Images we e acqui ed wi h a JEOL JEM-1400 Plus elec-
on mic oscope a an accele a ing ol age o 120 kV.
RESULTS
Physicochemical and pho oca aly ic cha ac e iza ions
As p e iously epo ed,
29,31
he syn hesis p ocedu e p o ides spi-
nel nanopa icles wi h spinel cell s uc u e and mean g ain sizes o
a ound 10 nm. As an example, Figs 1and 2show, espec i ely, he
XRD pa e ns and STEM mic og aphs o some selec ed samples:
ZnFe
2
O
4
(x=0; Figs 1(a) and 2(a)) and Co
0.6
Zn
0.4
Fe
2
O
4
(x=0.6;
Figs 1(b) and 2(c)). While CoZn nanopa icles a e cha ac e ized
by a single cubic spinel s uc u e (Fd 3mspace g oup) (Fig. 1(b)),
he occu ence o ZnO in x=0 nanopa icles as seconda y phase
(P63mc space g oup) is clea ly isible (Fig. 1(a)). Rie eld efine-
men o he XRD di ac og ams p o ides he ollowing c ys alli e
size, d, alues o he se o Co
x
Zn
1−x
Fe
2
O
4
analyzed samples:
20 40 60 80
).u.a(y isne nI
(b)
Co
0.6
Zn
0.4
Fe
2
O
4
20 40 60 80
).u.a(y isne nI
2θ (deg ees) 2θ (deg ees)
(a)
ZnFe
2
O
4
ZnO
ZnO
ZnO
Figu e 1. XRD pa e ns o he selec ed Co
x
Zn
1−x
Fe
2
O
4
nanopa icles: (a) x=0; (b) x=0.6. Expe imen al () and Rie eld fi ing (—, in ed) cu es. The
di e ence be ween he in ensi ies is shown a he bo om (in blue). The B agg eflec ions o ZnO a e shown in (a) (j, in black), oge he wi h hose o
he cubic spinel s uc u e (j, in ed).
www.soci.o g AG Gubieda e al.
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Jou nal o Chemical Technology and Bio echnology published by John Wiley & Sons L d on behal o Socie y o Chemical Indus y (SCI).
J Chem Technol Bio echnol 2025; 100: 428–437
430
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x=0.6, d=5.4 nm; x=0.4, d=7.3 nm; x=0.1, d=6.0 nm. Fo x=0,
d=5.1 nm (spinel, 88 w %) and d=29.3nm (ZnO, 12 w %).
Sligh ly la ge alues o he nanopa icle dimensions can be
deduced om he his og ams o he nanopa icle diame e s e al-
ua ed h ough he TEM mic og aphs (8.9, 8.4, 9.0 and 9.2 nm o
x=0, 0.1, 0.4 and 0.6, espec i ely). Mo eo e , he biphasic na u e
o he ZnFe
2
O
4
sample can be also deduced om he analysis o
i s his og am ( wo nanopa icle dis ibu ions; Fig. 2(b)) in compa -
ison wi h ha shown by he es o he analyzed samples (see
Fig. 2(d), as an example).
Rega ding he su ace a ea (B unaue –Emme –Telle (BET)), as
p e iously epo ed,
29
he nanopa icles display he ypical hys-
e e ic cu e ype V (wi hou ini ial knee asc ibed o he o ma ion
o adso p ion monolaye ), acco ding o IUPAC classifica ion. No
clea end wi h xwas de ec ed in ei he BET su ace a ea o po e
diame e (66.5–92.5 m
2
g
−1
, 6.7–7.8 nm, espec i ely).
As o he op ical cha ac e iza ion h ough DRS, he analyzed
nanopa icles display band gap ene gies, E
g
, co esponding o
he isible ange. In ac , he Tauc plo , es ima ed h ough he
Kubelka–Munk unc ion FRðÞ=1−RðÞ
2=2R(Ris di use eflec-
ance), FR
ðÞ
hν
ðÞ
1=2∝hν−Eg
(hνis inciden ene gy), enables he
es ima ion o E
g
(linea ex apola ion in Fig. 3(a);
1.4 eV <E
g
<1.7 eV o he analyzed samples). Thus, op ical
abso p ion in he isible ange is confi med, which suppo s
he isible pho oca aly ic pe o mance o he analyzed
nanopa icles.
Rega ding he magne ic esponse (Fig. 3(b)), he samples a e
cha ac e ized a oom empe a u e by he ypical anhys e e ic
beha io o supe pa amagne ic nanopa icles (negligible ema-
nence and coe ci i y) leading o educed nanopa icle agg ega-
ion due o negligible in e pa icle magne ic in e ac ions.
Fu he mo e, as discussed in he in oduc ion, sa u a ion magne-
iza ion inc eases wi h he Co con en (x) as a consequence o he
pa icula ca ion dis ibu ion (Zn
2+
,Co
2+
and Fe
2+/3+
) be ween
he oc ahed al and e ahed al si es
31
(inse o Fig. 3(b), high field
magne iza ion a 6 T e sus x). The ma ked inc ease in he ini ial
magne ic suscep ibili y and sa u a ion magne iza ion o x=0.6
p o ides his sample wi h op imal cha ac e is ics o magne ic
sepa a ion (concen a ion unde ex e nal magne ic fields) while
main aining supe pa amagne ic cha ac e is ics.
Fu he mo e, he op imal pho oca aly ic pe o mance o
x=0.6 is confi med. Figu e 3(c) shows he e olu ion o he phenol
concen a ion a io a e 2 h o isible i adia ion compa ed o he
pho oca aly ic esponse o ZnFe
2
O
4
(x=0) nanopa icles, whe e
in his la e case negligible phenol deg ada ion is achie ed unde
he same expe imen al condi ions.
P e ious s udies clea ly indica e he dominan ole o Zn
2+
ca -
ions in he an ibac e ial ac i i y o ino ganic Zn compounds
(i.e. ZnO).
32
Acco dingly, he concen a ion o me al ca ions lea-
ched om he nanopa icles was s udied by means o ICP-AES,
by cen i uging he nanopa icles and analyzing bo h he p ecip-
i a ed nanopa icles and he supe na an . Table 1p esen s he
4 8 12 16
0
5
10
15
Coun s
Diame e (nm)
(b)
ZnFe
2
O
4
4 8 12 16
0
10
20
30
40
Coun s
Diame e
(
nm
)
(d)
Co0.4Zn0.6Fe2O4
(a) ZnFe2O4
(c) Co0.4Zn0.6Fe2O4
Figu e 2. (a, c) STEM images o selec ed nanopa icles and (b, d) hei nanopa icle size dis ibu ions.
An ibac e ial pe o mance o Co–Zn e i e nanopa icles www.soci.o g
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ca ion concen a ion in he supe na an and in he nanopa icles
in compa ison wi h nominal concen a ion. The ob ained concen-
a ion a ios o Co/Fe and Zn/Fe p esen in he supe na an o
he Co
x
Zn
1−x
Fe
2
O
4
e i es (x=0.1, 0.4 and 0.6) a e equi alen
o he nominal concen a ion o hese me al species in he nano-
pa icles. Thus, he de ec ed ca ions would come om he
absence o a p ope cen i uga ion o he nanopa icles and no
significan leaching can be concluded. Howe e , o x=0, Zn ca -
ions a e de ec ed in he supe na an in a highe a io han ha
p esen in he nanopa icles, indica ing ha Zn leaching is aking
place. The o al mass o Zn de ec ed in he supe na an
(0.652 mg L
−1
) co esponds o 0.12% o he o al mass o Zn in
he sample (540 mg L
−1
), indica ing ha his amoun o Zn was
leached om he ZnFe
2
O
4
pa icles.
An ibac e ial e ec o e i es
To de e mine he pho oca aly ic an ibac e ial esponse o he
selec ed Co–Zn nanopa icles, he g ow h o E. coli, he cul u abil-
i y, was analyzed in a 5 h ime pe iod unde he ollowing condi-
ions (Fig. 4): (i) unde isible ligh i adia ion as a con ol; (ii) in
he p esence o 200 μgmL
−1
o Co
x
Zn
1−x
Fe
2
O
4
nanopa icles
(x=0, 0.1, 0.4 and 0.6) in he da k; and (iii) in he p esence o
he a o emen ioned nanopa icles unde isible ligh . Cul u abil-
i y e e s o he capaci y o he bac e ia o unde go cell di ision
a e exposu e o he p e iously men ioned condi ions, and is
ep esen ed as colony o ming uni s pe millili e (CFU mL
−1
).
ZnFe
2
O
4
nanopa icles significan ly educed E. coli cul u abili y,
bo h in he da k and unde isible ligh i adia ion, wi h clea di -
e ences obse ed be ween bo h condi ions: while in da kness
Figu e 3. (a) Tauc plo (indi ec band gap) o he analyzed Co
x
Zn
1−x
Fe
2
O
4
nanopa icles. (b) Room empe a u e hys e esis loops (M: magne iza ion; ⊘
0
H:
applied magne ic field). Inse : magne iza ion a ⊘
0
H=6T,(•) a 300 K and () 50 K. (c) Phenol concen a ion a io C/C
0
(C
0
: ini ial concen a ion, 20 mg L
−1
)
a e 60 min in he da k (le ) and 60 and 120 min o i adia ion unde xenon lamp ( igh ).
Table 1. Nominal me al ionconcen a ion o Co and Zn ela i e o Fe in Co
x
Zn
1−x
Fe
2
O
4
pa icles, and ICP-AES analysis o me al ion concen a ions in
nanopa icles and supe na an , ela i e o i on concen a ion
xNominal [Co]/[Fe] Nominal [Zn]/[Fe] Nanopa icle [Co]/[Fe] Nanopa icle [Zn]/[Fe] Supe na an [Co]/[Fe] Supe na an [Zn]/[Fe]
0—0.580 0.000 (1) 0.584 (4) 0.00 (2) 0.8 (1)
0.1 0.053 0.522 0.055 (1) 0.508 (2) 0.000 (3) 0.53 (6)
0.4 0.211 0.348 0.215 (3) 0.322 (1) 0.216 (1) 0.331 (2)
0.6 0.316 0.232 0.320 (1) 0.223 (1) 0.320 (4) 0.226 (1)
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hese nanopa icles caused comple e lack o cul u abili y by he
4 h ma k, hei e ec was significan ly s onge unde isible ligh
i adia ion, and comple e lack o cul u abili y was achie ed by he
2 h ma k. These esul s sugges ha he abili y o E. coli o su i e
and di ide is s ongly a ec ed by he p esence o hese e i e
nanopa icles, and ha he e ec is syne gis ically enhanced by
he combina ion o isible ligh i adia ion and he nanopa icles.
Howe e , he e ec on cul u abili y was less p onounced o
x=0.1 nanopa icles, being almos negligible o highe Co con-
en s (x=0.4 and 0.6) ei he in da k condi ions o unde isible
ligh i adia ion. As Fig. 4(b) shows, o x=0.1 in da kness, only
a 10- old dec ease in cul u abili y was obse ed du ing he 5 h
pe iod, bu unde isible ligh i adia ion he p esence o nanopa -
icles led o a 100- old dec ease in cul u abili y (10
5
CFU mL
−1
in
he isible ligh con ol e sus 10
3
CFU mL
−1
in he p esence o
nanopa icles and ligh ). These findings sugges ha he bac e ial
inhibi ion e ec o he nanopa icles dec eases as he Co/Zn a io
(x) inc eases.
Focusing on he nanopa icles wi h enhanced an ibac e ial
esponse (x=0), he e ec o he nanopa icle concen a ion on
he an ibac e ial e ec was analyzed unde simila expe imen al
condi ions (wi h and wi hou isible ligh , a e 5 h o incuba ion;
Fig. 5).
As can be seen, no significan changes in cul u abili y we e
ound o concen a ions lowe han 12.5 μgmL
−1
unde da k
condi ions, al hough a highe concen a ions, x=0 nanopa i-
cles showed oxici y in he da k (Fig. 5(a)). Howe e , unde ligh
condi ions a 10- old dec ease in cul u abili y was obse ed a
nanopa icle concen a ions as low as 12.5 μgmL
−1
. This e ec
is compa able o ha obse ed wi h x=0.1 nanopa icles using
a 16- old highe concen a ion (200 μgmL
−1
) (Fig. 4(b)). Fu he -
mo e, when inc easing he concen a ion o x=0 nanopa icles
abo e 12.5 μgmL
−1
unde ligh condi ions, a syne gic e ec
was e iden and he cul u abili y sha ply dec eased eaching neg-
ligible alues o concen a ions abo e 50 μgmL
−1
.
Finally, cul u abili y was analyzed a he selec ed concen a ion
o 20 μgmL
−1
o x=0 as a unc ion o he incuba ion ime (Fig. 5
(b)). While his concen a ion o nanopa icles caused a small
e ec on cul u abili y in da kness (10- old dec ease), cul u abili y
dec eased by ou o de s o magni ude a e 8 h o incuba ion
unde isible ligh i adia ion. These esul s indica e ha concen-
a ion could be adjus ed depending on he ime o ea men
desi ed.
In e ac ion o e i es and E. coli
To in es iga e i he changes in bac e ial cul u abili y we e due o
changes in in e ac ion be ween he nanopa icles and E. coli, he
mo phology o he bac e ia a e incuba ion wi h he e i es
was analyzed using TEM. Fo his expe imen , bac e ia we e incu-
ba ed in dis illed wa e con aining 200 μgmL
−1
o nanopa icles
unde isible ligh i adia ion o a 4 h pe iod. As shown in Fig. 6,
Zn e i e nanopa icles (x=0) accumula ed on he su ace o
he bac e ia, sugges ing in e ac ion be ween he nanopa icles
and he bac e ial wall, al hough he bac e ial s uc u e emained
seemingly in ac . In con as , Co–Zn e i e nanopa icles induced
changes in bac e ial mo phology, wi h he bac e ia p esen ing
sec e ion-like changes in hei ex e io . In bac e ia incuba ed wi h
x=0.1 nanopa icles, hese sec e ions we e isible mos ly in he
bac e ial wall, and in some cases along he bac e ial pili, in he
a ea closes o he bac e ial wall. Incuba ion wi h he x=0.4
Ligh Ligh +Nanopa icles (200 µg/mL)
Nanopa icles (200 µ
g
/mL)
012345
10
0
10
1
10
2
10
3
10
4
10
5
10
6
ime (h)
CFU/mL
012345
10
0
10
1
10
2
10
3
10
4
10
5
10
6
ime (h)
CFU/mL
012345
10
0
10
1
10
2
10
3
10
4
10
5
10
6
ime (h)
CFU/mL
x = 0
012345
10
0
10
1
10
2
10
3
10
4
10
5
10
6
ime (h)
CFU/mL
(a) (b)
(c) (d)
x = 0.1
x = 0.4 x = 0.6
Figu e 4. (a–d) Cul u abili y o bac e ia incuba ed in wa e unde isible ligh i adia ion, in he p esence o 200 μgmL
−1
o Co
x
Zn
1−x
Fe
2
O
4
e i es in
da kness, and in he p esence o 200 μgmL
−1
o doped e i es unde isible ligh i adia ion. G aphs indica e colony o ming uni s a six ime poin s, om
0 o5h.
An ibac e ial pe o mance o Co–Zn e i e nanopa icles www.soci.o g
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nanopa icles on he o he hand esul ed in sec e ions bo h in he
bac e ial wall and in some cases along he ull leng h o he bac-
e ial pili. Las ly, incuba ion wi h x=0.6 nanopa icles esul ed
in a c own-like pheno ype, wi h sec e ions o a g ea e size accu-
mula ing along he bac e ial wall. In all h ee cases, he nanopa -
icles seemed o in e ac wi h he obse ed sec e ion. These
Ligh + Nanopa icles
Nanopa icles
02468
100
101
102
103
104
105
106
ime (h)
CFU/mL
Ligh
Ligh +Nanopa icles
Nanopa icles
x = 0 (20 µg/mL)
(a) (b)
x = 0
10 100
10–1
100
101
102
103
104
105
106
Concen a ion ( g/mL)
CFU/mL
Figu e 5. (a) E ec o di e en concen a ions o ZnFe
2
O
4
(x=0) nanopa icles on E. coli cul u abili y, a e 5 h o incuba ion wi h and wi hou isible
ligh i adia ion. (b) Cul u abili y o bac e ia incuba ed in he p esence o he nanopa icles in da kness and unde isible ligh i adia ion a a concen a-
ion o 20 μgmL
−1
. G aph indica es colony o ming uni s a fi e ime poin s, om 0 o 8 h.
Figu e 6. TEM images o E. coli, a e 4 h o isible ligh i adia ion and incuba ion wi h Co
x
Zn
1−x
Fe
2
O
4
nanopa icles.
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findings, in combina ion wi h he bac e ial cul u abili y assay
esul s, sugges ha he sec e ion obse ed in he TEM images
could ha e a p o ec i e e ec agains he nanopa icles and hei
pho oca aly ic e ec .
DISCUSSION
Zn-based nanopa icles (ZnO, ZnFe
2
O
4
) ha e been ex ensi ely
s udied as an ibac e ial agen s.
33-35,38
Two main mechanisms
ha e been p oposed o hei an ibac e ial ac i i y: (i) ROS gene a-
ion, ein o ced by he pho oca aly ic e ec unde ligh i adia-
ion, wi h ROS gene a ed by he pho ocha ges being able o
p oduce damage o he cell wall and o he o ganelles o he bac-
e ia, es ic ing hei g ow h and leading o bac e ial cell dea h;
and (ii) zinc ion (Zn
2+
) elease, whe e he p o eins in he bac e ial
cell wall (nega i ely cha ged a biological pH alue) cap u e he
cha ged Zn
2+
ions, leading o he o ma ion o insoluble me al
p o eina es, he inhibi ion o espi a o y chain enzymes and e en-
ually bac e ial dea h.
33
O he mechanisms, like memb ane dis-
up ion o p o ein leakage, ha e also been p oposed o he
an ibac e ial e ec o Zn e i e nanopa icles.
33
Howe e ,
he ac ual o igin o he an ibac e ial esponse o nanopa icles is
no en i ely clea in he li e a u e, and he con ibu ion o each
mechanism in he o e all esponse o bac e ia in he p esence
o nanopa icles is cu en ly a opic o deba e.
39
In his s udy he esul s poin owa ds he elease o Zn
2+
being
a key ac o o he an ibac e ial p ope ies o he e i es, as
elease o Zn
2+
was negligible in he nanopa icles wi h no an i-
bac e ial esponse (x=0.4 and 0.6), while he nanopa icles wi h
he highes an ibac e ial esponse (x=0) p esen ed Zn
2+
leach-
ing. O he esea che s ha e also ound ha Zn e i es ha e be e
an ibac e ial e ec han Co e i es
40
; howe e , he nanopa icle
syn hesis and es s pe o med a e di e en , making compa ison
di ficul . Fu he mo e, he enhanced an ibac e ial esponse unde
isible ligh i adia ion sugges s he e is a syne gic e ec be ween
Zn
2+
elease and ROS gene a ion, despi e he educed pho oca a-
ly ic pe o mance o Zn-based e i e (x=0) obse ed in phenol
deg ada ion. Howe e , and su p isingly, he mos ac i e isible
pho oca aly ic nanopa icles (highe Co concen a ions) do no
p omo e any an ibac e ial e ec .
A po en ial explana ion o his unexpec ed esul is he bac e-
ial sec e ion obse ed in bac e ia incuba ed wi h Co–Zn nano-
pa icles, which migh ha e a p o ec i e e ec . Bac e ia a e
known o p oduce an ex acellula polyme ic subs ance (EPS) o
p o ec hemsel es om ex e nal h ea s and an imic obial
agen s. This EPS ma ix could limi he pene a ion o nanopa i-
cles, hus educing he an ibac e ial e ec
13,41
and p e en ing
bonding o he nanopa icles o he bac e ial su ace, a key mech-
anism o an ibac e ial ac ion ha inc eases memb ane pe me-
abili y and acili a es ROS ans e .
29,42
In addi ion, he EPS
ma ix could educe he pho oca aly ic e ficiency o nanopa icles
h ough se e al mechanisms. Fo example, he EPS ma ix could
shield he nanopa icles om ligh , educing hei pho oca aly ic
ac i i y, o con ain o ganic compounds ha ac as sca enge s o
ROS, p e en ing hese molecules om eaching he bac e ia.
43
Addi ionally, he EPS could educe he su ace a ea o he nano-
pa icles by adso p ion, hus hinde ing pho oca aly ic eac ions.
41
Nanopa icles wi h highe Zn concen a ion may ha e a g ea e
abili y o inhibi EPS p oduc ion in E. coli han nanopa icles wi h a
highe concen a ion o Co. In e es ingly, zinc oxide nanopa icles
ha e ecen ly been obse ed o inhibi biofilm o ma ion bo h in
E. coli
44
and in Pseudomonas ae uginosa, ano he G am-nega i e
bac e ia.
45,46
This wo k highligh s he need no only o ensu e
accu a e pho oca aly ic cha ac e iza ion o he nanopa icles bu
also o conduc a comp ehensi e analysis o hei in e ac ion wi h
bac e ia, as his in e ac ion can ha e a p o ound impac on he
o e all an ibac e ial pe o mance.
Finally, he an ibac e ial esponse o ZnFe
2
O
4
, boos ed by isi-
ble ligh , should be specially ou lined. Concen a ions as low as
20 μgmL
−1
show a ma ked e ec on he cell cul u abili y a e
8 h unde isible ligh i adia ion, wi h a no iceable educ ion in
he an ibac e ial capaci y when he nanopa icles in e ac wi h
he bac e ia in he da k. This concen a ion is e y low compa ed
o o he epo ed esul s, which indica e he use o e i e concen-
a ions abo e 100 μgmL
−1
o ob ain significan an ibac e ial
esponse.
35
The highes epo ed dose o ZnFe
2
O
4
o exhibi no
oxici y when es ed in human cells is o 125 μgmL
−1
,
33
and con-
cen a ions as high as 500 μgmL
−1
ha e been shown o pose li -
le isk o aqua ic o ganisms when incuba ed o up o 100 h.
47
The e o e, he e ficiency o a 20 μgmL
−1
dose in his a icle is o
special significance. Fu he mo e, he an ibac e ial e ec is
obse ed wi hin 8 h, which is a ela i ely sho pe iod compa ed
o o he epo ed an ibac e ial nanopa icles.
42,47-49
CONCLUSIONS
This s udy desc ibes he pho oca aly ic an ibac e ial e ec o Co–
Zn e i e (Co
x
Zn
1−x
Fe
2
O
4
,x=0, 0.1, 0.4 and 0.6) nanopa icles.
The esul s show ha he Co/Zn a io mainly de e mines he
pho oca aly ic ac i i y o he nanopa icles and hei an ibac e ial
ac i i y unde isible ligh and da k condi ions. S uc u al and
mo phological cha ac e iza ion (XRD and TEM) confi ms he cha -
ac e is ic spinel s uc u e wi hou significan changes in he main
nanopa icle sizes (a ound 10 nm). Band gap ene gy es ima ion
and magne ic cha ac e iza ion suppo he op imal cha ac e is ics
o he nanopa icles o be employed as pho oca aly ic agen s o
was ewa e ea men s: op ical abso p ion in he isible ange,
high magne ic momen and supe pa amagne ic esponse a
oom empe a u e.
The enhanced pho oca aly ic pe o mance (phenol deg ada-
ion) o he e i e wi h he highes Co con en (Co
0.6
Zn
0.4
Fe
2
O
4
,
x=0.6) is confi med. Howe e , a ligh -induced dec ease in cul u -
abili y was only de ec ed o nanopa icles wi h low Co con en
(ZnFe
2
O
4
and Co
0.1
Zn
0.9
Fe
2
O
4
,x=0 and x=0.1). The op imum
an ibac e ial e ec o ZnFe
2
O
4
e en a low doses (20 μgmL
−1
)is
explained as a syne gic e ec o Zn
2+
ca ion leaching, gene a ion
o ROS enhanced by isible ligh i adia ion and inhibi ion o he
sec e ion o a p o ec i e biofilm ma ix.
Finally, his wo k highligh s he need no only o ensu e accu-
a e pho oca aly ic cha ac e iza ion o he nanopa icles, bu also
o conduc a comp ehensi e analysis o hei in e ac ion wi h bac-
e ia. This in e ac ion can ha e a p o ound impac on he o e all
an ibac e ial pe o mance.
ACKNOWLEDGEMENTS
The wo k has been pe o med unde g an s IT1479-22 unded by
he Basque Go e nmen and PID2020-116321RB-C21 unded
by MCIN/AEI/10.13039/501100011033. The au ho s acknowledge
he use o Se icio Gene al de Apoyo a la In es igación-SAI, Uni-
e sidad de Za agoza o he mic oscopy. The au ho s hank SGI-
ke (UPV/EHU/ERDF, EU) se ice o hei echnical and human
suppo , especially he Phy o on; Analy ic and High Resolu ion
Mic oscopy in Biomedicine; and IBERCRON Se ices.
An ibac e ial pe o mance o Co–Zn e i e nanopa icles www.soci.o g
J Chem Technol Bio echnol 2025; 100: 428–437 © 2024 The Au ho (s).
Jou nal o Chemical Technology and Bio echnology published by John Wiley & Sons L d on behal o Socie y o Chemical Indus y (SCI).
wileyonlinelib a y.com/jc b
435
10974660, 2025, 2, Downloaded om h ps://scijou nals.onlinelib a y.wiley.com/doi/10.1002/jc b.7785 by Uni e sidad Del Pais Vasco, Wiley Online Lib a y on [17/01/2025]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
DATA AVAILABILITY STATEMENT
The da a gene a ed du ing he cu en s udy a e a ailable om
he co esponding au ho on easonable eques .
AUTHOR CONTRIBUTIONS
CGP w o e he main manusc ip ex . CGP, LCG, EOH and AC gen-
e a ed and analyzed da a o Figs 1–3. AGG and AADC gene a ed
and analyzed da a o Figs 4–6and Table 1. AGP, MFGR, CGP and
AADC con ibu ed o he concep ion o he p ojec . All au ho s
con ibu ed o in e p e a ion o he da a and e ision o he
manusc ip .
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Jou nal o Chemical Technology and Bio echnology published by John Wiley & Sons L d on behal o Socie y o Chemical Indus y (SCI).
J Chem Technol Bio echnol 2025; 100: 428–437
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