New BIOTECHNOLOGY 79 (2024) 50–59
A ailable online 19 Decembe 2023
1871-6784/© 2023 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-
nc-nd/4.0/).
Phy os abiliza ion o me al(loid)s by en eme gen mac ophy es ollowing a
90-day exposu e o indus ially con amina ed g oundwa e
Blanca Velasco-A oyo
a
,
b
,
*
,
1
, Sand a Cu iel-Aleg e
a
,
c
, Aqib Hassan Ali Khan
a
, Ca los Rumbo
a
,
Daniel P´
e ez-Alonso
c
, Ca los Rad
c
, He wig De Wilde
d
, Al edo P´
e ez-de-Mo a
e
,
Rocío Ba os
a
,
**
a
In e na ional Resea ch Cen e in C i ical Raw Ma e ials o Ad anced Indus ial Technologies (ICCRAM), Uni e si y o Bu gos, Cen o de I+D+I, Plaza Misael
Ba˜
nuelos s/n, 09001 Bu gos, Spain
b
Depa men o Bio echnology and Food Science, Uni e si y o Bu gos, Plaza Misael Ba˜
nuelos, s/n, 09001 Bu gos, Spain
c
Resea ch G oup in Compos ing (UBUCOMP), Uni e si y o Bu gos, Facul y o Sciences, Plaza Misael Ba˜
nuelos s/n, 09001 Bu gos, Spain
d
TAUW Belgi¨
e n , Dep . o Soil and G oundwa e , Waaslandlaan 8A3, 9160 Loke en, Belgium
e
TAUW GmbH, Dep . o Soil and G oundwa e , Landsbe ge S . 290, 80687 München, Ge many
ARTICLE INFO
Keywo ds:
Rhizos abiliza ion
Eme gen mac ophy e
G oundwa e
Me al
Me alloid
Plan g ow h p omo ing bac e ia
ABSTRACT
Be e unde s anding o mac ophy e ole ance unde long exposu e imes in eal en i onmen al ma ices is
c ucial o phy o emedia ion and phy oa enua ion s a egies o aqua ic sys ems. The me al(loid) a enua ion
abili y o 10 eme gen mac ophy e species (Ca ex ipa ia, Cype us longus, Cype us o undus, I is pseudaco us,
Juncus e usus, Ly h um salica ia, Men a aqua ica, Ph agmi es aus alis, Sci pus holoschoenus, and Typha angus i olia)
was in es iga ed using eal g oundwa e om an indus ial si e, o e a 90-day exposu e pe iod. A “phy obial”
ea men was included, wi h 3 plan g ow h-p omo ing hizobac e ial s ains. Plan s exposed o he pollu ed
wa e gene ally showed simila o educed ae ial biomass compa ed o he con ols, excep o C. ipa ia. This
species, along wi h M. aqua ica, exhibi ed imp o ed biomass a e bioaugmen a ion. Phy o emedia ion mecha-
nisms accoun ed o mo e han 60% o As, Cd, Cu, Ni, and Pb emo al, whils abio ic mechanisms con ibu ed o
~80% emo al o Fe and Zn. Concen a ions o me al(loid)s in he oo s we e gene ally be ween 10–100 imes
highe han in he ae ial pa s. The mac ophy es in his wo k can be conside ed “unde g ound a enua o s”, mo e
app op ia e o hizos abiliza ion s a egies, especially L. salica ia, M. aqua ica, S. holoschoenus, and
T. angus i olia. Fo I. pseudaco us, C. longus, and C. ipa ia; ha es ing he ae ial pa s could be a complemen a y
phy oex ac ion app oach o u he emo e Pb and Zn. O all he plan s, S. holoschoenus showed he bes balance
be ween biomass p oduc ion and up ake o mul iple me al(loid)s. Resul s also sugges ha mul iple phy os-
a egies may be possible o he same plan depending on he inal emedial aim. Phy obial app oaches need o
be u he assessed o each mac ophy e species.
In oduc ion
En i onmen al con amina ion by me al(loid)s is a global p oblem
associa ed wi h an h opogenic ac i i ies, con amina ing p is ine soil and
wa e [1]. In he las 20 yea s, legisla ion has been de eloped o help
educe me al(loid)s in p oduc s, emo e hem om was e and emedia e
Abb e ia ions: BCF, Bioconcen a ion Fac o ; BPW, Pollu ed Wa e +Plan G ow h P omo ing Rhizobac e ia (PGPR) “RBM” Mix u e; CB, Con ol Rhizobac e ia
Mix u e; CW, Con ol Wa e ; DO, Dissol ed Oxygen; DT, Decision T ee; DW, D y Weigh ; EC, Elec ical Conduc i i y; FTW, Floa ing T ea men We land; PGPR, Plan
G ow h P omo ing Rhizobac e ia; MFS, Mic ocosms Floa ing Sys em; FW, F esh Weigh ; ICP-MS/MS, Induc i ely Coupled Plasma-Mass Spec ome y iple quad-
upole; ICP-OES, Induc i ely Coupled Plasma-Op ical Emission Spec ome y; ORP, Redox Po en ial; PW, Pollu ed Wa e ; RBM, Rhizobac e ia Mix u e; TDS, To al
Dissol ed Solids; TF, T ans e ence Fac o .
* Co esponding au ho a : In e na ional Resea ch Cen e in C i ical Raw Ma e ials o Ad anced Indus ial Technologies (ICCRAM), Uni e si y o Bu gos, Cen o
de I+D+I, Plaza Misael Ba˜
nuelos s/n, 09001 Bu gos, Spain.
** Co esponding au ho .
E-mail add esses: [email p o ec ed] (B. Velasco-A oyo), [email p o ec ed] (R. Ba os).
1
P esen add ess: Depa men o Bio echnology and Food Science, Uni e si y o Bu gos, Plaza Misael Ba˜
nuelos, s/n.
Con en s lis s a ailable a ScienceDi ec
New BIOTECHNOLOGY
jou nal homepage: www.else ie .com/loca e/nb
h ps://doi.o g/10.1016/j.nb .2023.12.003
Recei ed 7 Feb ua y 2023; Recei ed in e ised o m 27 No embe 2023; Accep ed 16 Decembe 2023
New BIOTECHNOLOGY 79 (2024) 50–59
51
exis ing con amina ion [2]. Phy o emedia ion o e s a o dable and
eco- iendly solu ions o ea men o soil and wa e con amina ion. By
le e aging plan s’ na u al capabili ies, hese me hods abso b and accu-
mula e pollu an s, add essing di e se sou ces o me al(loid)s and
o ganic con aminan s [3]. Selec ed plan s mus show ole ance o
ele a ed pollu an concen a ions, as g ow h, and easonable a ail-
abili y, as hese p ope ies a y wi h e e ence o plan and he ype o
con amina ion [4,5]. Aqua ic phy o emedia ion uses eme gen mac o-
phy es o he emo al and deg ada ion o pollu an s, ha g ow in he
wa e bodies ma gins, ei he in subme ged and eme gen condi ion,
allowing bo h he con aminan emo al om he wa e column and he
oo ing subs a e [4]. Cas illo Lo ía e al. [6] assessed he oxici y o Pb
in oo s and ae ial pa s o subme ged Sal inia biloba and sugges ed he
use o his wa e e n o managemen o esidual wa e bodies
con amina ed wi h Pb. Schück and G ege [4] sc eened he capaci y o
34 we land plan species o emo e me als (Cd, Cu, Pb, and Zn) in wa e ,
and iden i ied ha Ca ex pseudocype us and Ca ex ipa ia we e he mos
e icien and e sa ile.
The accumula ion o me al(loid)s in he plan is la gely in luenced by
he me al(loid) phy oa ailabili y, he a e o abso p ion by he oo s,
and he ansloca ion om oo s o ae ial issues [7]. Mos species u ilize
oo bioac i a ion mechanisms o enhance oo abso p ion ( hizosphe e
acidi ica ion, sec e ion o o ganic acids, me al chela ion, o enzyme
p oduc ion o inc ease a ailable nu ien s). The main limi a ion o
mac ophy e me al(loid) up ake is he oxici y ha he a ge pollu an
can cause. Howe e , de oxi ica ion mechanisms allow species o a oid
he nega i e e ec s o he me al(loid)s; o example, mo e han 50% o
he Ca, Cd, Co, Fe, Mg, Mn, and Zn eco e ed in he oo s o Pis ia
s a io es was a ached o he ex e nal oo su aces indica ing he abili y
o he plan o exclude me al(loid)s and, hus, main ain ole able le els
in e nally [8]. Newe e and By ne [9] also s a ed ha he ex en o he
oo sys em a ec s he abili y o mac ophy es o emo e me al pollu -
an s, wi h ib ous oo sys ems being supe io o ap oo , due o hei
la ge su ace a ea. En i onmen al ac o s ha a e also impo an o he
up ake and accumula ion o me al(loid)s include empe a u e, ligh , pH,
and salini y [10].
Plan g ow h-p omo ing hizobac e ia (PGPR) and myco hizal ungi
ob ain C-sou ces o hei me abolism om he plan du ing “phy obial”
pa ne ships; in e u n, hey p omo e plan g ow h, dec ease me al(loid)
s oxici y, and/o imp o e he biodeg ada ion o pe sis en o ganic
compounds [11]. The applica ion o PGPR can enhance he g ow h o
hype accumula o plan s by imp o ing hei me al(loid) a ailabili y,
ole ance, and accumula ing capaci y. PGPR can p oduce me aboli es
which aid in he solubiliza ion and p o ision o essen ial nu ien s o he
plan , alle ia ing s ess a he same ime [12]. Fou mac ophy es
inc eased hei emo al capaci y o i e ace me als (Fe, Mn, Ni, Pb,
and C ) in bac e ially assis ed Floa ing T ea men We lands (FTWs) o
he clean-up o a i icially spiked i e wa e [13]. In FTWs using
Ph agmi es aus alis bioaugmen ed wi h se e al bac e ial s ains, hea y
me als we e success ully emo ed [14]. P. aus alis also emo ed phenol
om wa e mo e e icien ly in combina ion wi h h ee bac e ial s ains
[15]. Some s udies sugges ha plan -associa ed endophy es may o e
mo e po en ial o phy obial emedia ion han plan -associa ed hizo-
sphe e bac e ia. The use o endophy es na i e o he hos plan educes
compe i ion be ween bac e ial s ains, a oiding needs o einocula ion
[16,17].
While mul iple s udies ha e assessed he po en ial o PGPR and
myco hizal ungi o imp o e plan pe o mance o ag onomical ap-
plica ions, s udies o phy o emedia ion app oaches, pa icula ly in
aqua ic sys ems, a e sca ce. In he p esen s udy, we hypo hesized ha
mac ophy es a e me al(loid) ole an plan s ha can be used o a en-
ua e mixed me al(loid) con amina ion in pollu ed wa e . We also hy-
po hesized ha a phy obial s a egy (bioaugmen a ion o plan s wi h
PGPR) can imp o e he me al(loid) emo al e iciency and g ow h o
we land mac ophy es. To es hese hypo heses, 10 eme gen aqua ic
species we e exposed o 90 days o g oundwa e indus ially
con amina ed by 7 di e en me al(loid)s in he p esence o absence o
PGPR ( h ee s ains). Plan incuba ions we e pe o med in mic ocosm
loa ing sys ems (MFS) in he g eenhouse. Plan g ow h was e alua ed
by means o ae ial biomass p oduc ion. Fo assessmen o me al(loid)s,
emo al concen a ions in g oundwa e as well as concen a ions in
ae ial pa s and oo s we e measu ed. The con ibu ion o con aminan
emo al by abio ic o phy o emedia ion mechanisms was es ima ed o
each me al(loid) and plan .
Ma e ials and me hods
Expe imen al se up
Plan le s om 10 di e en species o eme gen mac ophy es we e
p o ided by “Vi e os La Dehesa” (Valdeobispo, C´
ace es, Spain, h
ps://www. i e osladehesa.com/). The species we e Ca ex ipa ia,
Cype us longus, Cype us o undus, I is pseudaco us, Juncus e usus, Men ha
aqua ica, Ly h um salica ia, Ph agmi es aus alis, Sci pus holoschoenus, and
Typha angus i olia. S ic quali y con ol measu es o ensu e he gene ic
consis ency o plan s ock we e main ained o plan p opaga ion.
Mac ophy es o uni o m gene ic o igin we e bough ha we e p oduced
p edominan ly by p opaga ing ege a i e hizomes, u ions, o s olons.
Plan s we e acclima ed unde con olled g eenhouse condi ions o wo
mon hs. Ini ially 4 mo phologically simila plan s pe 4 L bucke we e
used, howe e , a e he one mon h o acclima ion, plan s ha we e
showing uni o m g ow h we e selec ed, while he o he plan s ha we e
ei he showing lowe o highe g ow h we e emo ed. The expe imen
was conduc ed using mic ocosms loa ing sys ems (MFS) consis ing o 4
L bucke s wi h a loa ing sys em o ex uded polys y ene holding plas ic
baske s, open a he bo om o allow oo de elopmen ; o he hole was
used o wa e sampling and moni o ing (Suppl. Fig. S1). The bo de s o
he po s we e co e ed wi h aluminium oil o a oid algae p oli e a ion.
Plan s we e dis ibu ed andomly and con enien ly o a ed o a oid
p e e ed posi ions o empe a u e and ligh . MFS’ wa e le el was
s eadily moni o ed and kep adequa e o a oid changes in media con-
cen a ion. (Suppl. Fig. S1).
A e he acclima iza ion pe iod, 4 ea men s we e applied in ip-
lica e MFS, o each o he 10-aqua ic species. A o al numbe o 132 MFS
we e in oduced (Suppl. Table S1):
•Con ol (CW): Tap wa e +Hoagland’s solu ion.
•Pollu ed Wa e (PW): Tap wa e +Hoagland’s solu ion + eal
pollu ed g oundwa e .
•Pollu ed Wa e +Plan G ow h P omo ing Rhizobac e ia (PGPR)
“RBM” Mix u e (BPW): Tap wa e +Hoagland’s solu ion +PGPR
mix u e + eal pollu ed wa e .
•Con ol Rhizobac e ia Mix u e (CB): Tap wa e +Hoagland’s solu-
ion +PGPR Mix u e.
The composi ion o Hoagland solu ion used was 5 mM KNO
3
, 5 mM
Ca(NO
3
)
2
.4 H
2
O, 67 mM Fe-EDTA, 2 mM MgSO
4
.7 H
2
O, 1 mM NH
4
NO
3
,
and 0.5 mM KH
2
PO
4
, o mac onu ien s; he ace elemen concen a-
ions we e: 50 µM H
3
BO
3
, 10 µM MnCl
2
.4 H
2
O, 1 µM ZnSO
4
.7 H
2
O, 0.3
µM CuSO
4
, and 0.5 µM Na
2
MoO
4
.2 H
2
O. Un ege a ed con ol bucke s,
wi h he same composi ion as hose ege a ed o each o he 4 ea -
men s, allowed o e alua e he p ocesses in ol ed in me al(loid)s
emo al by abio ic deposi ion. Each MFS, acco ding o he co espon-
den ea men , had he ollowing composi ion: 200 mL concen a ed
Hoagland´s solu ion (18x dilu ion); 200 mL PW (18x dilu ion); 10 mL
PGPR mix u e, namely “RBM” (Rhizobac e ia Mix u e, 1:1:1; see below
o de ails). In he case o ea men s wi h inocula ion o PGPR (+RBM),
hese we e inocula ed be o e spiking he eal pollu ed wa e in o he
co esponden MFS, o allow mic oo ganisms o p ope ly colonize he
hizosphe e and/o endophy e he plan along se e al weeks.
B. Velasco-A oyo e al.
New BIOTECHNOLOGY 79 (2024) 50–59
52
Moni o ing and sampling scheme
The phy o emedia ion expe imen las ed 90 days and consis ed o
he ollowing moni o ing p og am:
i. Wa e pa ame e s: pH and elec ical conduc i i y (EC), among
o he s, a egula o nigh ly in e als; and me al(loid)s concen a-
ions (de ails p o ided in he sec ions o Physical-chemical de-
e mina ions in he hizosphe e wa e , and Me al con en
de e mina ions ia ICP-OES and ICP-MS/MS in wa e and plan
issues);
ii. Plan pa ame e s, including plan biomass o oo s and shoo s/lea es
(ae ial), a 90 days (end-poin ); me al(loid)s concen a ions in bo h
compa men s; and bioconcen a ion and ans e ence ac o s (de-
ails p o ided in he sec ion o Me al con en de e mina ions ia ICP-
OES and ICP-MS/MS in wa e and plan issues).
Inocula ion and su i al o PGPR
The me al(loid)- ole an RBM (1:1:1) mix u e o 3 PGPR s ains (IR9,
En e obac e sp.; IR29, En e obac e sp.; and IR39, Pseudomonas sp.),
p e iously isola ed and cha ac e ized by he Ins i u e o Technology
Ca low (unpublished esul s), was inocula ed o he co esponding
ea men s (CB, BPW) be o e he onse o he phy o emedia ion expe -
imen . All he s ains we e indi idually cul u ed in modi ied nu ien
medium (0.5% pep one, 0.3% yeas ex ac , 0.3% NaCl) a 30 ◦C o e -
nigh . A e his incuba ion, cells we e ha es ed by cen i uga ion (20
min a 1800 g), washed once wi h Dulbecco’s phospha e-bu e ed saline
(DPBS) and e-suspended in Hoagland medium. The op ical densi y was
adjus ed o OD
600
=0.07 (≈10
8
CFU mL
−1
). Bac e ial suspensions we e
mixed in 1:1:1 a io and 10 mL o he esul ing conso ia was inocula ed
in each MFS (a a inal concen a ion o 2.5 mL L
−1
), whe e equi ed.
Fo he su i al analysis o he inocula ed PGPR in he hizosphe e in
he co esponding mic ocosms, hizomes and o he endophy ic issues
we e ha es ed and analysed. B ie ly, cul u e-dependen pla e-coun ing
me hod was used, bo h om di ec ly collec ed hizosphe e wa e ali-
quo s and plan issues, including ae ial and hizosphe e plan pa s.
Nega i e con ols we e included in e e y case. The wa e samples we e
di ec ly pla ed on o nu ien aga pla es, a se e al dilu ions. Fo oo s/
hizomes, wo dis inc p ocedu es we e ollowed: o de ec he p esence
o endophy ic bac e ia, plan issues we e su ace s e ilized wi h 70%
E OH, ollowed by hei g inding wi h 0.9% NaCl; o analysing he
p esence o hizosphe e bac e ia, s e iliza ion was omi ed. The lysa es
o g ounded ma e ial we e pla ed on o nu ien aga pla es. Pla es we e
incuba ed a 30 ◦C o 24–48 h. In hose samples wi h obse ed posi i e
bac e ial g ow h, 10 andomly selec ed colonies we e pla ed on o 1/10
s eng h nu ien aga pla es, con aining 6 mM NaAsO
2
, and we e
incuba ed a 30 ºC o up o 5 days, including R9, R29, and R39 s ains,
and Esche ichia coli, as posi i e and nega i e con ols, espec i ely.
Physiological pa ame e s
A weekly ollow-up o he pheno ypes and pe o mance o he plan s
was pe o med. F esh and d y biomass o ae ial pa s and oo s was
quan i ied g a ime ically a 90 days and exp essed in g±g. The samples
o esh biomass we e examined sho ly a e ha es ing. Fo ae ial
biomass plan we e dissec ed jus abo e he g ow h subs a e, while o
oo biomass, he g ow h subs a e was ca e ully emo ed, and he plan
oo s we e washed wi h dis illed wa e o elimina e any possibly su ace
ela ed pa icles. The oo s we e hen ai d ied and used o esh weigh
quan i ica ion. The d ied biomass ( oo and shoo ) was quan i ied a e
dehyd a ing he esh biomass in an o en ope a ing a 60 ◦C o 96 h,
howe e , when necessa y, he dehyd a ion was ca ied un il i eached a
consis en weigh .
Physical-chemical de e mina ions in he hizosphe e wa e
The physical-chemical p ope ies o he hizosphe e wa e we e
moni o ed o nigh ly using a mul ipa ame e p obe (model HI98194,
HANNA). This allows he simul aneous de e mina ion o pH and elec-
ical conduc i i y (EC), edox po en ial (ORP), dissol ed oxygen (DO),
and o al dissol ed solids (TDS). B ie ly, he mul ipa ame ic sys em was
i s calib a ed, e e y ime be o e he measu emen we e aken, using
he company p o ided calib a ion s anda d solu ion (HI9828, Mul ipa-
ame e Quick Calib a ion Solu ion HANNA). Elec ode was insed by
dis illed wa e be ween he measu emen o di e en pa ame e s in each
mesocosms.
Me al con en de e mina ions ia ICP-OES and ICP-MS/MS in wa e and
plan issues.
Majo and ace elemen s in all wa e and plan samples we e
de e mined by ICP-OES (Induc i ely Coupled Plasma-Op ical Emission
Spec ome y, SPECTRO GENESIS, AMETEK, Ge many), and/o ICP-
MS/MS (8900 Induc i ely Coupled Plasma-Mass Spec ome y iple
quad uple, Agilen , USA), espec i ely, depending on he me al(liod)s
le el in g oundwa e . Wa e samples we e il e ed a 0.22 µm and
acidi ied wi h 1 mL o concen a ed HNO
3
in a 1:10 dilu ion a io. When
necessa y, he ele an dilu ions we e p epa ed o he analysis, i he
concen a ion we e highe han he linea esponse limi . D ied plan
issue samples (0.25 - 0.5 g) we e weighed on a p ecision balance ( ±
0.1 mg), mixed wi h 2 mL o H
2
O
2
(33% / ) and 8 mL o concen a ed
ni ic acid (65% w/ ) and diges ed in a mic owa e o en (ETHOS ONE,
Miles one, USA). A e he diges ion p ocess (30 min a 180 ◦C), he
liquid was il e ed (CF/WASH110 il e pape , Ø 110 mm, Scha lau),
and adjus ed o 25 mL using MilliQ wa e . All ma e ial used we e p e-
iously washed wi h dilu ed HNO
3
and insed wi h MilliQ wa e . To
main ain he ack p ecision a mul i-s anda d o 21 elemen s o known
concen a ion (ICP mul i-elemen s anda d solu ion - 89166.180, VWR,
Ge many) and blanks we e also included (one o each 15 samples). In
each ba ch o diges ion samples, a ce i ied plan ma e ial s anda d was
in oduced (ERM-CD281 Rye g ass ce i ied ma e ial) o ack accu acy.
All he sample alues we e co ec ed wi h he espec i e o he alues
ound in blank. When necessa y, he ele an dilu ions we e p epa ed
o he analysis, i he concen a ion was highe han he s anda d
de ec able limi . The ce i ied alue o he me al(liod)s in s anded
e e ence ma e ial and hei eco e y is p esen ed in Supplemen a y
Table S2. The accu acy o ins umen measu emen anged be ween
highes o Zn wi h a eco e y o 99.01 ±3.28, while lowes o As
94.129 ±1.23 ela i e o he s anda d ce i ied e e ence ma e ial used
(ERM-CD281 Rye g ass ce i ied ma e ial) along wi h e o s o measu e
less han ±5. The a e age pe cen age eco e y o all me al(liod) was
iden i ied o be 95.56%, wi h a pe cen age RSD o 2.68%. This indica es
ha he analy ical me hod used o measu e he me al concen a ions is
accu a e and ep oducible.
Me al(loid)s up ake we e also enume a ed using bioconcen a ion
ac o (BCF) and ansloca ion ac o (TF) [17]. The BCF is desc ibed as
he capaci y o plan s o he elemen al accumula ion in o he oo s
compa men wi h espec o a subs a e, while he TF is calcula ed om
he concen a ion o he elemen in he ae ial pa o he plan compa ed
o i s concen a ion in he oo s [28].
BCF =Me al(loid)in oo
Me al(loid)con en in g ow h subs a e
TF =Me al(loid)in shoo
Me al(loid)con en in oo s
Decision ee p epa a ion
The basic idea behind decision ees (DTs) is o c ea e a model ha
p edic s he alue o a a ge a iable based on se e al inpu a iables.
Sciki -lea n is a Py hon module in eg a ing a wide ange o s a e-o - he-
B. Velasco-A oyo e al.
New BIOTECHNOLOGY 79 (2024) 50–59
53
a machine lea ning algo i hms such is Classi ica ion And Reg ession
T ee (CART). The CART algo i hm gene a es only bina y ees, whe e
nonlea y nodes always ha e wo b anches (i.e., ques ions only ha e yes/
no answe s). This algo i hm di ides he main da ase in o subse s using a
single ea u e and a h eshold c i e ion, and he Gini impu i y index is
used o calcula e he p obabili y o inco ec ly classi ying a ea u e. The
p ocess is epea ed un il he ins ance eaches a lea node (a node ha
does no esul in a di ision o he da ase du ing he ee’s cons uc-
ion). Gini impu i y index (G) is used o calcula e he amoun o p ob-
abili y o a speci ic ea u e ha is classi ied inco ec ly when selec ed
andomly:
G=∑
C
i=1
pi× (1−pi)
Whe e, C is he o al numbe o classes and p
i
is he p obabili y o picking
he da a poin wi h he class i.
In his wo k, a da ase composed o me al concen a ions measu ed
in oo s and ae ial pa s (14 a iables in o al), as well as he a iable
“plan species”, was used o classi ica ion. The da a se has 120 in-
s ances (N=120), ob ained as ollows: 3 biological eplica es/species, 4
ea men s and 10 species. The pa ame e s selec ed ollowed Gini as
c i e ion, maximum dep h o 5, whe eas he o he s we e le by de aul .
The isualiza ion o he da a in he classi ica ion ees was made wi h a
py hon lib a y o decision ee isualiza ion and model in e p e a ion
(h ps://gi hub.com/pa /d ee iz). The pe o mance o he model is
e alua ed by a 10- old c oss alida ion [18,19].
S a is ical analyses
Resul s a e he mean o a leas h ee independen biological
eplica es. One-way ANOVA was pe o med a e checking o no mali y
and homogenei y o a iance assump ions. Signi ican di e ences be-
ween means we e assessed by he Tukey’ (HSD, Hones ly-Signi ican -
Di e ence) pos -hoc es , wi h a signi icance o p<0.05. Fo mean
compa ison o me al(loid)s be ween PW and BPW ea men s, a - es
was applied. The s a is ical package IBM SPSS S a is ics .22 was used.
Resul s
Ae ial biomass
The esul s ega ding he plan ae ial esh biomass a e 90 days o
exposu e o he con amina ed g oundwa e e ealed di e ences among
ea men s wi hin each indi idual plan species. Fo mos o he s udied
species, exposu e o con amina ed g oundwa e (i.e., PW/BPW) was
ei he de imen al o had no e ec a he end o he incuba ion (Fig. 1a
o ae ial biomass and Fig. 1b o oo biomass). Signi ican di e ences
among he ou ea men s (CW, PW, CB, and BPW) we e obse ed o
C. ipa ia, J. e usus, M. aqua ica, L. salica ia, P. aus alis, and
T. angus i olia. In e es ingly, ae ial biomass o C. ipa ia was signi i-
can ly g ea e when exposed o he pollu ed wa e (PW) compa ed o he
con ol (CW). No ewo hy, o his species, he esh ae ial biomass was
signi ican ly la ge in he pollu ed bioaugmen ed ea men (BPW) han
he espec i e con ol (CB) and he pollu ed wa e ea men (PW). The
la ges ae ial biomass o all ea men s was measu ed o he BPW g own
C. ipa ia plan s, sugges ing a posi i e e ec o PGPR´on his aqua ic
mac ophy e. Fo J. e usus and M. aqua ica ae ial biomass was signi i-
can ly lowe o PW compa ed o CW; his was no obse ed o BPW,
sugges ing a bene icial e ec o he hizobac e ia bioaugmen a ion. In
he case o L. salica ia, plan s exposed o pollu ed wa e (PW and BPW)
Fig. 1. F esh Biomass [a) o ae ial, and b) o oo ] p oduced by he en-aqua ic species unde s udy a e 90 days o exposu e o he co esponden ea men . Da a
( e ical ba s) ep esen he mean o h ee biological eplica es ( ±s anda d de ia ion, SD). Di e en le e s indica e signi ican di e ences be ween ea men s o
each plan species (P<0.05, One-Way ANOVA ollowed by Tukey´s pos hoc es ). CW: con ol wa e ; PW: pollu ed wa e ; CB: Con ol bac e ia; and BPW: bac e ia
+pollu ed wa e ; FW. F esh Weigh in g ams (g).
B. Velasco-A oyo e al.
New BIOTECHNOLOGY 79 (2024) 50–59
54
showed lowe biomass han he con ols. A simila pa e n was obse ed
o P. aus alis and T. angus i olia. Finally, o C. longus, C. o undus,
I. pseudaco us, and S. holoschoenus, no signi ican di e ences we e ound
be ween he ou ea men s. Fo he oo biomass a ian esponse o
biomass p oduc ion was obse ed, no ably signi ican di e ences be-
ween ea men s o C. ipa ia, J. e usus, and L. salica ia, was no ed,
while no s a ically signi ican di e ences we e obse ed o o he plan .
In he case o C. ipa ia plan in PW showed signi ican ly highes le els,
while o J. e usus and L. salica ia highes oo biomass was no ed o
CW.
Physical-chemical pa ame e s
Changes in pH and EC o pollu ed wa e a e shown in Suppl. Figs. S2
and S3. The esul s displayed co espond o he da a measu ed in he
hizosphe e wa e o nigh ly a e 0, 15, 30 and 45 days o exposu e.
Moni o ing was inalized on day 45 co esponding o ime 3 (T3) since
pH and EC we e s abilized. Al hough esul s o pH showed a g ea
a iabili y, some gene al pa e ns we e obse ed (Suppl. Fig. S2). As a
ule, he ini ial pH o he pollu ed wa e ea men s (PW/BPW) was
1–1.5 uni s lowe han he espec i e con ols (CW/CB). This can be
a ibu ed o he acidic na u e o he pollu ed wa e . As ime p og essed,
he pH alues o he wa e inc eased signi ican ly o he pollu ed
ea men s (PW/BPW) eaching compa able le els o hose o he con-
ols (CW/CB). In gene al, pH alues anged be ween app ox. 5.5–6.5
o mos ea men s and moni o ing imes. The end was owa ds
neu aliza ion om ini ial acid pH alues, in mos o he es ed species,
excep o I. pseudaco us and, o a lesse ex en , o L. salica ia,
P. aus alis, and S. holoschoenus. In hose species, a s eady basi ica ion
was de ec ed in he PW ea men . In con as o he gene al neu al pH
endency in he PW, hose MFS co esponding o BPW ea men ended
owa ds basi ica ion. I. pseudaco us exhibi ed he lowes pH alues
al eady a T0 in PW and BPW, o la e inc ease wi h ime; whe eas in CW
and CB he end was a dec eased pH a T1 in compa ison wi h T0, o
la e aise again, especially a T3. Fo EC signi ican di e ences we e
obse ed be ween ea men s o he same species and be ween expo-
su e imes o he same ea men (Suppl. Fig. S3). As a ule, EC was
app ox. 2–3 imes g ea e o ea men s PW and BPW han hei
espec i e con ols o mos species, because o he high EC o he
o iginal pollu ed g oundwa e sample. Gene ally, he EC ange o he
pollu ed ea men s PW and BPW anged app ox. be ween 400–800 µS
cm
−1
, and o he con ols (CW and CB) be ween 200–600 µS cm
−1
. Fo
a ious plan species including C. longus, I. pseudaco us, L. salica ia,
P. aus alis, and T. angus i olia, he EC g adually dec eased a e an ini ial
inc ease a T1 o he PW and BPW ea men s. Likewise, he mul i-
pa ame ic p obe was used o he measu emen o o he pa ame e s
including dissol ed oxygen (DO) and edox po en ial (ORP) (da a no
shown). The DO alues anged be ween 2.4 o 5.2 mg L
−1
a T0 and
g adually dec eased o 2.15 o 3.5 mg L
−1
a T3, indica ing O
2
con-
sump ion in he hizosphe e wa e . The edox po en ial (ORP) anged
be ween 150 and 400 mV and, hus, is ep esen a i e o oxidic
condi ions.
Me al(loid) pa e ns in wa e , ae ial, and oo compa men s
The con amina ed g oundwa e collec ed om an indus ial si e in
Belgium used in his p esen s udy was cha ac e ized by high concen-
a ions (abo e pe missible le els) o me al(loid)s (Table 1), low pH
(~3.7), and high elec ical conduc i i y (~5320 µS/cm). Fu he , he
changes in me al(loid) concen a ions in wa e we e moni o ed o he
PW and BPW ea men s e e y 15 days (T1 =15 days, T2 =30 days,
and T3 =45 days). Fo bo h ea men s a signi ican me al(loid)
emo al (≥70%) was achie ed al eady a T1 (15 days) o mos plan
species. A T3 (45 days) me al(loid) emo al was p ac ically comple e
(95–100%). Fo ins ance, in he case o Zn and Cu o he PW ea men
(Suppl. Fig. S4, and Suppl. Table S3), he ini ial concen a ion alues
apidly dec eased by >99% o Cu and >90% o Zn in he p esence o
C. ipa ia and P. aus alis a T1. The emo al o Zn was s eadie in he
p esence o I. pseudaco us (69% a T1) and M. aqua ica (60% a T1)
compa ed o o he plan species. A simila pa e n was obse ed o Cu
emo al in he p esence o C. o undus (45% a T1) and M. aqua ica (68%
a T1). In gene al, emo al e iciency was simila be ween he bio-
augmen ed (BPW) and non-bioaugmen ed (PW) ea men s. Fo
C. o undus and M. aqua ica he BPW showed signi ican ly g ea e Cu
emo al a T1 han PW.
Me al(loid)s concen a ions in bo h oo s and ae ial compa men s
exhibi ed a g ea a iabili y a he end o he expe imen (90 days) o
bo h ea men s (PW and BPW) (Fig. 2). Ne e heless, o mos plan
species a gene al end was obse ed wi h highe accumula ion o me al
(loid)s (As, Cd, Cu, Ni, Pb, and Zn) in he oo s (10 o 100 imes) in
compa ison o he ae ial pa s. This was pa icula ly e iden o oo
concen a ions o Fe ( ange om 200–10000 µg Kg
−1
DW), Cu
(200–5000 µg Kg
−1
DW), Zn (100–1500 µg Kg
−1
DW) and Ni
(50–1000 µg Kg
−1
DW). Concen a ions o As, Cd and Pb in he oo s
we e be ween 1–20 µg kg
−1
DW. In he ae ial compa men , me al(loid)
s concen a ions anged om 10–200 µg kg
−1
DW o Cu, Fe, Ni and Zn,
and be ween 0.01–2µg kg
−1
DW o As, Cd and Pb. Me al(loid) up ake
also di e ed depending on he species conside ed. G ea e me al(loid)
concen a ions o bo h oo s and ae ial pa s we e gene ally ound o
L. salica ia ollowed by M. aqua ica, J. e usus, and S. holoschoenus
compa ed o o he species. Di e ences in me al(loid) up ake be ween
he bioaugmen ed (BPW) and non-bioaugmen ed (PW) ea men s we e
also ound o some species. An inc eased up ake in he p esence o
PGPR (BPW) was obse ed o he ollowing me al(loid)s and plan s: As
in he ae ial o L. salica ia, M. aqua ica, and P. aus alis; Cd in he ae ial o
J. e usus and S. holoschoenus; Cu, Fe and Ni in he ae ial o M. aqua ica;
Zn in he oo s o M. aqua ica and S. holoschoenus and in he ae ial o M.
aqua ica. In con as , a educed up ake in he p esence o PGPR (BPW)
was obse ed o he ollowing me al(loid)s and plan s: Cu in he oo s o
L. salica ia; Pb in he oo s o J. e usus and T. angus i olia as well as in he
ae ial compa men s o I. pseudaco us, M. aqua ica, and P. aus alis. To
u he unde s and he dis ibu ion and he p edominance o he a -
ge ed me allic elemen s in he oo s and ae ial pa s o he es ed species
in he PW ea men , a classi ica ion using a decision ee me hodology
was p epa ed (Suppl. Fig. S5). The p edic ion esul s in he o m o
p opo ion alues a he e minal nodes a e he species classi ied wi h
he highes concen a ion o Fe and Cu in he lea es, like M. aqua ica,
and also Fe in he oo s, like L. salica ia.
To assess he con ibu ion o phy o emedia ion e sus o he p o-
cesses (e.g., abio ic chemical p ecipi a ion) o he emo al o me al(loid)
s om he con amina ed wa e , un ege a ed con ols we e p epa ed.
He e, a ligh b ownish p ecipi a e was obse ed wi hin he MFS. Fo
hese non- ege a ed con ols, he di e ence be ween me al(loid) con-
cen a ions a T0 and T3 o a e 90-days indica ed he con ibu ion o
me al emo al by o he (abio ic) p ocesses. Based on hese esul s a mass
balance was pe o med o assess he espec i e con ibu ion o phy o -
emedia ion (% P), which included bo h phy oex ac ion and
Table 1
Ini ial concen a ions o he a ge elemen s in he
o iginal g oundwa e sample as de e mined by ICP-
OES / ICP-MS. Resul s a e exp essed in mg L
−1
.
Me als: Cadmium (Cd), Coppe (Cu), I on (Fe), Nickel
(Ni), Lead (Pb), Zinc (Zn); me alloids: A senic (As).
Me al(loid) mg L
−1
As 0.450 – 0.500
Cd 2.000 – 2.500
Cu 160 – 1000
Fe 250 – 400
Ni 130 – 150
Pb 0.200 – 0.350
Zn 70 – 320
B. Velasco-A oyo e al.
New BIOTECHNOLOGY 79 (2024) 50–59
55
hizos abiliza ion, and o he e en s (% O), o me al(loid)s emo al
(Supplem. Table S4). Fo As, Cd, Cu, Ni and Pb, emo al was caused
mainly by phy o emedia ion (>60%) o mos plan species. Fo Pb,
emo al by phy o emedia ion accoun ed o mo e han 80% o all
plan s conside ed. In con as , o Fe and Zn, emo al due o o he e en s
was p edominan o all plan species (<20% phy o emedia ion), excep
o C. o undus. A simila phenomenon was obse ed o Cd in
T. angus i olia (28% phy o emedia ion).
Bioconcen a ion and ans e ence ac o s
Tables 2 and 3 show he bioconcen a ion (BCF) and ans e ence
(TF) ac o s calcula ed o he 10-aqua ic species a e 90 days o
exposu e o he eal pollu ed g oundwa e (PW ea men ). Resul s o
he BCF alues we e e y a iable depending on he elemen and plan
conside ed (Table 2). Pe elemen and species, he h ee mac ophy es
wi h he g ea es BCF alues a e highligh ed in bold and numbe ed. The
highes BCF alues o Cu, Fe and Zn we e epo ed o L. salica ia,
T. angus i olia, and M. aqua ica. L. salica ia was also among he i s h ee
posi ions wi h he highes BCF o all he s udied elemen s: M. aqua ica
also o Cd, and T. angus i olia also o Ni and Zn. O he plan s wi h
ela i ely high BCF included J. e usus o Pb and P. aus alis and
S. holoschoenus o As. Rega ding he TF alues, hey we e below he uni
o mos species and elemen s analysed (Table 3). In e es ingly,
I. pseudaco us showed, o all elemen s, highe TF alues han he es o
he species, pa icula ly o Pb wi h a TF ~3, deno ing hype -
accumula ion capaci y o his elemen in he ae ial compa men .
C. longus and L. salica ia also exhibi ed a highe TF alue o Pb han
o he plan species. C. ipa ia gene ally p esen ed highe alues o TF
han he o he plan s, especially o Zn.
Discussion
In he p esen s udy he me al(loid) a enua ion abili y o 10 eme -
gen mac ophy e species, na i e o he Eu opean cen al and sou he n
egions, was in es iga ed using eal g oundwa e om an indus ial si e
o e a 90-day exposu e pe iod. P e iously epo ed wo k was gene ally
pe o med ei he wi h one o ew aqua ic species [20], o a he sho
Fig. 2. Concen a ion o he a ge me al(loid)s in he wo pool compa men s ( oo s and ae ial samples) as analysed by ICP-OES (Al, Cu, Pb, and Zn), and ICP-MS
(As, Cd, and Ni). The samples displayed co espond o 90-days o exposu e o PW (pollu ed g oundwa e ) o BPW (bac e ia +pollu ed g oundwa e ) ea men . Da a
( e ical ba s) ep esen he mean o h ee biological eplica es ( ±s anda d de ia ion, SD). Resul s a e exp essed in µg Kg
−1
DW. As e isks indica e signi ican
di e ences be ween he 2 ea men s (PW and BPW) o each me al(loid) and plan analysed (P <0.05, T-s uden es .
Table 2
Resul s o he calcula ed BCF o he me al(loid)s unde s udy in he en aqua ic species exposed o he PW ea men , a e 90 days in he g eenhouse.
Me al(liod)s Plan used in he s udy
C. ipa ia C. o undus C. longus I. pseudaco us J. e usus L. salica ia M. aqua ica P. aus alis S. holoschoenus T. angus i olia
As 74.24 29.37 40.29 12.47 61.33 89.04
3
46.07 95.69
2
112.90
1
73.90
Cd 12.64 24.45 15.63 16.27 24.26 60.33
1
26.22
3
23.91 26.82
2
11.06
Cu 4.42 34.94 39.80 2.43 34.44 49.92
2
47.97
3
24.66 17.13 54.79
1
Fe 108.58 413.11 399.79 11.54 327.16 780.27
1
538.97
3
424.31 342.78 712.43
2
Ni 7.95 29.08 46.10
3
13.60 34.72 72.51
1
23.89 20.90 16.61 50.49
2
Pb 82.98 147.75 38.89 13.80 551.83
1
150.95
3
80.57 73.36 120.55 192.57
2
Zn 3.95 18.91 14.24 3.78 14.12 50.09
1
19.38
3
14.23 12.51 24.45
2
In bold, o each elemen , he plan species wi h he highes alues o BCF (pe ow, h ee species pe con aminan a e highligh ed).
1. 2. 3
Indica e he h ee highes BCF
alues in dec easing o de o each elemen .
B. Velasco-A oyo e al.
New BIOTECHNOLOGY 79 (2024) 50–59
56
incuba ion imes [4], o o jus a ew elemen s [21]. In addi ion, mos
p e ious s udies ha e used syn he ic media ins ead o eal con amina ed
wa e [13,14]. A be e unde s anding o mac ophy e ole ance unde
longe exposu e imes and using eal en i onmen al ma ices is c ucial
o assessing he po en ial o hese plan s in phy o emedia ion and
phy oa enua ion s a egies o aqua ic sys ems.
The e ec i eness o phy o emedia ion la gely depends on he se-
lec ion o he app op ia e plan s. These should be well-adap ed o he
local clima e, ac i ely ake up o signi ican ly emo e o p ecipi a e in
he hizosphe e, one o mo e con aminan s om he a ge ma ix, and
show no mal g ow h and ep oduc ion, ha is, no signs o eco oxico-
logical e ec s [22]. Depending on he na u e o he con aminan , hese
plan s should ha e he capabili y o esis , deg ade and/o adso b pol-
lu an s [23,24]. Ideally, plan s used o phy o emedia ion pu poses
g ow apidly, p oduce high biomass and possess an ex ensi e oo
sys em.
A e 90 days, plan s exposed o he pollu ed wa e (PW, BPW)
gene ally showed simila o educed esh ae ial biomass compa ed o
he con ols (CW, CB) (Fig. 1). Mili´
c e al. [25] suspec ed ha he
pho osyn hesis o Da idia in oluc a a was inhibi ed as he concen a ion
o hea y me als in he subs a e inc eased; in ha s udy, concen a ions
o almos all me als we e signi ican ly highe in unde g ound o gans
han in any o he plan pa . This mechanism could pa ially jus i y he
dec ease in biomass o he ege a i e issues a e exposu e o he
con amina ed wa e . Pho osyn hesis is inhibi ed due o me al-d i en
s ess. The eac ion mechanism is ini ia ed in he hizosphe e; he
plan igge s signals which induce emobiliza ion o nu ien s ha
esul in wide oo sys ems in de imen o educed ege a i e ae ial
pa s. Induced senescence is behind his p o ec i e mechanism o
mul iple abio ic and bio ic ypes o s ess, and ul ima ely, ac i a es a
ne wo k o biochemical and molecula esponses o apidly adap and
ole a e he s ess [26]. This could ep esen a equen s a egy in
a ious we land plan s, including mos o he mac ophy es in his s udy
(Fig. 2). Me al(loid)s a e accumula ed and immobilized in he oo is-
sues o minimize dis ibu ion o he abo eg ound pa s, pa icula ly o
he pho osyn he ic issues, and, hus, a oid hei damage [27]. In e -
es ingly, ae ial biomass o C. ipa ia and I. pseudaco us o all ea men s
was gene ally g ea e han o all o he plan s. These species we e also
cha ac e ized by lowe me al(loid) accumula ion in he oo s compa ed
o he majo i y o he mac ophy es s udied, mo e dis inc i e o an
excluding ole ance s a egy.
Al hough o mos o he s udied species he ae ial biomass dec eased
when plan s we e exposed o pollu ed wa e , an opposi e end was
obse ed o C. ipa ia (Fig. 1). Fo ins ance, esh ae ial biomass
inc eased in C. ipa ia exposed o pollu ed wa e (PW) a e 90 days.
Ladislas e al. [21] es ed C. ipa ia wi hin hei s udy and epo ed ha
biomass was no a ec ed by me allic exposu e. A e sc eening 34
mac ophy e species, Schück and G ege [4] concluded ha C. ipa ia
was one o he bes pe o ming species o emo al o 4 me als and he
only one cha ac e ized by g ea e biomass a e me al exposu e. Ae ial
biomass o C. ipa ia was signi ican ly la ge o he bioaugmen ed
ea men s (CB and BPW) wi h espec o hei non-bioaugmen ed
coun e pa s (CW and PW), indica ing a po en ial bene icial e ec o
he PGPR. Fo M. aqua ica bioaugmen a ion imp o ed ae ial biomass
(BPW) ega ding he non-bioaugmen ed pollu ed ea men (PW).
Se e al s udies ha e sugges ed a posi i e e ec on me al emo al and
biomass p oduc ion when plan s we e bioaugmen ed wi h mic oo gan-
isms as pa o a “phy obial” ea men [28,29]. Ta a e al. [30] bio-
augmen ed P. aus alis and Typha domingensis in FTWs wi h a bac e ial
conso ium consis ing o h ee s ains o ea indus ial ex ile was e-
wa e . The au ho s obse ed imp o ed emo al e iciency ollowing
bioaugmen a ion. Fahid e al. [31] obse ed o Cype us lae iga us a
73.48% educ ion in hyd oca bon concen a ions a e phy o -
emedia ion combined wi h bioaugmen a ion. In mos cases, he plan
biomass inc eased a e bioaugmen a ion. Plan ole ance o s ess can
be imp o ed by PGPR ha p omo e bo h plan g ow h and de elopmen
h ough he elease o phy oho mones. Nawaz e al. [14] ound ha
P. aus alis in he p esence o syne gis ic bioaugmen ed bac e ia ach-
ie ed highe oo and shoo g ow h, as compa ed o plan s wi hou
inocula ion. I should be no ed ha he s udies p e iously desc ibed
epo a posi i e e ec on one ype o plan and, hus, may no apply o
o he species. In he p esen s udy, a consis en gene al phy obial e ec
o all species was no obse ed. Ye , o C. ipa ia and M. aqua ica
bioaugmen a ion also imp o ed g ow h and, by ex en , ole ance o he
plan o he me al(loid) con amina ion. These esul s sugges ha he
sui abili y o phy obial ea men needs o be add essed case by case a
an ea ly assessmen s age, bu also has po en ial o imp o e plan pe -
o mance in he p esence o mixed con amina ions.
In addi ion o me al(loid) concen a ions and ypical pa ame e s
ela ed o plan g ow h (such as, empe a u e and ligh ), soil/wa e pH
and salini y can signi ican ly in luence me al(loid)s up ake and plan
g ow h, ul ima ely impac ing he phy o emedia ion pe o mance [10].
Fo PW and BPW ea men s, whe e plan s we e exposed o pollu ed
wa e , a g adual pH inc ease and a concomi an dec ease in EC was
obse ed as ime p og essed (Supplem. Figs. S2 and S3). The gene al pH
and EC pa e ns epo ed o mos o he mac ophy es exposed o
pollu ed wa e can be a ibu ed o he enhanced up ake o nu ien s by
he plan s, including he me al(loid)s, as well as o he biological and
physicochemical binding o pollu an s o he oo s and soil pa icles [14,
17]. S ikingly, o con ol ea men s o I. pseudaco us (CB and CW), an
ini ial acidi ica ion a T1 (15 days) and a la e eco e y a T3 (45 days)
was obse ed. The ini ial pH dec ease could be associa ed wi h he
elease o some acidic compounds in he hizosphe e by I. pseudaco us as
he plan becomes exposed o he new medium. Once he in luence o
hese compounds ceased, he pH o he wa e e u ned o i s o iginal
alue. Va ious mac ophy es ha e been desc ibed o elease bioac i e
compounds om he oo s ha may ei he enhance solubili y, so p ion
and/o sedimen a ion p ocesses in FTW. Fo ins ance, se e al s udies
epo ed he posi i e e ec o ci ic acid, a ou ing he me als
complexa ion and educing hei ee mobili y in plan s, posi i ely
impac ing he biomass p oduc ion [13,32].
In addi ion o he oo /shoo compa men aliza ion, me al(loid)
concen a ions we e measu ed in he pollu ed wa e o assess
phy o emedia ion/-a enua ion pe o mance. A e 45-days, he e was
Table 3
Resul s o he calcula ed TF o he me al(loid)s unde s udy in he en aqua ic species exposed o he PW ea men , a e 90 days in he g eenhouse.
Me al(liod)s Plan used in he s udy
C. ipa ia C. o undus C. longus I. pseudaco us J. e usus L. salica ia M. aqua ica P. aus alis S. holoschoenus T. angus i olia
As 0.01 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.00
Cd 0.03 0.08 0.03 0.11 0.21 0.01 0.00 0.00 0.00 0.04
Cu 0.07 0.00 0.00 0.04 0.01 0.00 0.00 0.00 0.01 0.00
Fe 0.02 0.00 0.01 0.04 0.01 0.01 0.01 0.00 0.00 0.00
Ni 0.19 0.04 0.04 0.35 0.08 0.04 0.20 0.11 0.08 0.11
Pb 0.11 0.07 0.59 3.25 0.15 0.53 0.02 0.18 0.26 0.02
Zn 0.46 0.06 0.07 0.32 0.18 0.09 0.13 0.11 0.10 0.04
In bold, he me allic elemen s and plan species displaying he highes alues o TF.
B. Velasco-A oyo e al.
New BIOTECHNOLOGY 79 (2024) 50–59
57
p ac ically comple e emo al o all elemen s om he pollu ed wa e .
Fo mos elemen s, comple e emo al was achie ed al eady a e 15 o
30 days. Acco ding o he mass balance conduc ed, he con ibu ion o
phy o emedia ion mechanisms (such as enhanced p ecipi a ion,
adso p ion, o up ake by plan s) accoun ed o mo e han 60% o As, Cd,
Cu, Ni, and Pb emo al, whils abio ic mechanisms con ibu ed o
app oxima ely 80% emo al o Fe and Zn. I should be no ed ha a
b ownish p ecipi a e was obse ed o mos MFS. This o m o p ecipi-
a ion has been p e iously desc ibed in simila s udies and is a ibu ed
o he o ma ion o i on (III) oxides/oxyhyd oxides [33]. This ag ees
wi h he edox po en ial measu ed, which was indica i e o oxidic
condi ions. Thus, i is p obable ha Zn emo al was la gely due o
co-p ecipi a ion wi h he i on (III) oxides/oxyhyd oxides. In hei s udy
wi h he loa ing wa e hyacin h (Eicho nia c assipes), Palihakka a e al.
[33] concluded ha emo al o Cu and Cd om wa e was mainly
a ibu able o phy o emedia ion mechanisms and ha , a neu al pH,
no in e ac ions occu ed be ween Cd and Cu and he i on (III) oxi-
de/oxyhyd oxide p ecipi a e obse ed. Simila ly, an enhanced p ecipi-
a ion o Cd and Cu due o he i on p ecipi a e was no obse ed in his
s udy.
Me al(loid) up ake and compa men aliza ion by he mac ophy es
was assessed by means o BCF and TF. Concen a ions o me al(loid)s in
he oo s we e gene ally be ween 10–100 imes la ge han in he shoo s.
Thus, he species analysed in ou wo k can be conside ed “unde g ound
accumula o s”; his is in acco dance wi h p e ious s udies [14,25].
Sawidis e al. [34] s a ed ha he inc eased accumula ion o me al(loid)s
in oo s and hizomes may be he esul o he la ge in e cellula ai
spaces ha cha ac e ize hei co ex pa enchyma. The p e e ed me al
(loid) accumula ion in he oo s suppo s he gene al no ion ha
we land plan species a e e y use ul o phy os abiliza ion a he han
phy oex ac ion s a egies [25]. Newe e and By ne [9] also epo ed ha
he ex en o he oo sys em a ec s he abili y o mac ophy es o emo e
me al pollu an s, wi h ib ous oo sys ems being supe io o ap oo
sys ems due o hei la ge su ace a ea. This was he case o all en
mac ophy es in es iga ed. Gene ally, a BCF equal o g ea e han 10 is
indica i e o hype -accumula i e plan s [35]. I should be no ed ha
hype accumula o s usually hype accumula e one o wo elemen s and,
hus, a e no pe de aul sui able o deal wi h con amina ions by mul iple
elemen s. The BCF o all plan s and elemen s analysed anged gene ally
be ween 10 - <100, excep o Fe, wi h alues be ween 100–780 o he
majo i y o species. Excep ionally, J. e usus showed a BCF o 552 o Pb.
Howe e , he species L. salica ia, T. angus i olia and M. aqua ica p e-
sen ed he bes BCF balance when all elemen s we e conside ed.
The TF de e mines he a io o me al(loid)s dis ibu ion be ween
shoo s and oo s and, in u n, he po en ial use o a plan o phy oex-
ac ion (≥1) o phy os abiliza ion (<1) s a egies [35]. In gene al, he
TF was below 0.2 o mos elemen s and plan species. Hence, he
mac ophy es s udied a e in gene al mo e app op ia e o phy os abili-
za ion s a egies. We land species end o accumula e he bulk o ace
elemen s in hei unde g ound o gans, suppo ing hizos abiliza ion
mechanisms [36]. An excep ion o his was I. pseudaco us wi h a TF o
Pb o 3.25. Han e al. [37] s udied he po en ial capaci y o wo I is
species, I. lac ea and I. ec o um, in ela ion o Pb ole ance and phy-
o emedia ion mechanisms. I is a e widely dis ibu ed pe ennial species
and a e common o namen al plan s; his is an addi ional aluable
ea u e o phy o es o a ion s a egies [37,38]. O he species wi h
ela i ely la ge TF, ye below 1, included C. longus (0.59) and L. salica ia
(0.53) o Pb, as well as C. ipa ia o Zn (0.46). Fo hese species, ha -
es ing he ae ial pa could be a sui able app oach o u he emo e Pb
and Zn om he a ge ma ix, espec i ely.
Al oge he , ou esul s sugges ha a ious o he species s udied
ul il a leas one o he c i e ia o phy o emedia ion and could be used
o a enua e me al(loid) con amina ions in aqua ic sys ems o es o a-
ion o i e ma gins. Fo ins ance, C. ipa ia and I. pseudaco us ae ial
biomass was no a ec ed when exposed o he pollu ed wa e , whe eas a
signi ican dec ease was obse ed o he o he species. Hence, hese
plan s a e highly ole an o mixed me al(loid)s con amina ions and sui
o he phy o es o a ion pu poses. The po en ial o C. ipa ia o phy-
o emedia ion app oaches was p e iously epo ed in o he s udies [4,
21]. The high TF o Pb o I. pseudaco us and me al(loid) ole ance, as
shown by simila ae ial biomass o con ols, makes his plan also
in e es ing o phy oex ac ion. In con as , L. salica ia, M. aqua ica, S.
holoschoenus, and T. angus i olia showed ela i ely high BCF o a ious
me al(loid)s, hus, ha ing po en ial o phy os abiliza ion s a egies.
S. holoschoenus also showed a good balance be ween me al(loid) ole -
ance and up ake, biomass s abili y wi h highe BCF o mul iple me al
(loid)s. The po en ial o his mac ophy e o phy o emedia ion has been
explo ed ba ely in he pas [25]. I should be no ed ha o phy oex-
ac ion i can be mo e e ec i e o ha es plan s wi h g ea e
abo e-g ound biomass and mode a e issue concen a ions o he
pollu an o in e es , a he han plan s wi h lowe biomass bu highe
issue concen a ions [39,40]. Thus, mul iple phy os a egies may be
possible o he same plan depending on he inal aim o he p ojec (e.
g., es o a ion, s abiliza ion, and ex ac ion) [41].
Conclusion
The abili y o he en eme gen mac ophy es o ole a e and up ake
me al(loid)s exposed o a eal indus ially pollu ed g oundwa e o 90-
days di e ed signi ican ly depending on he species conside ed. Six ou
o he en candida es ul illed a leas one c i e ion o phy o -
emedia ion, including C. ipa ia, I. pseudaco us, L. salica ia, M. aqua ica,
S. holoschoenus, and T. angus i olia. In gene al, me al(loid)s accumula-
ion occu ed in he oo s wi h li le ans e o he shoo s, sugges ing
ha he in es iga ed mac ophy es a e be e sui ed o phy os abiliza-
ion a he han phy o emedia ion s a egies. Among all, C. ipa ia and
I. pseudaco us showed highe ole ance o he mixed con amina ion han
he es o mac ophy es as ae ial biomass o hese species was no
a ec ed by he exposu e o he pollu ed wa e . These species ended o
accumula e less me al(loid)s in he oo s, being pa icula ly p omising
o phy o es o a ion o i e ma gins. The phy obial ea men did no
p oduce a consis en gene al enhancemen o me al(loid) up ake and
g ow h bu did show imp o ed ole ance o C. ipa ia and M. aqua ica
when exposed o he pollu ed wa e . Thus, bioaugmen a ion app oaches
may imp o e plan pe o mance in he p esence o mixed con amina-
ions, bu his needs o be u he assessed o each combina ion o
PGPR, plan and con amina ion.
Funding
This wo k has been unded by he GREENER p ojec o he Eu opean
Union’s Ho izon 2020 esea ch and inno a ion p og am (G an Ag ee-
men No. 826312). S. Cu iel-Aleg e p e-doc o al con ac was unded by
Jun a de Cas illa y Le´
on (ORDEN EDU/1508/2020, de 15 de diciemb e).
Au ho s a emen
B. Velasco-A oyo: o iginal d a p epa a ion, lab wo k and da a
analyses; S. Cu iel-Aleg e and A.H.A. Khan: Me hodology, lab wo k,
and da a analyses; C. Rumbo: mic obial analysis; D. P´
e ez-Alonso: lab
wo k, and s a is ical e ision; C. Rad, and R. Ba os: concep ualiza ion,
ex e ision, and supe ision. H. de Wilde and A. P´
e ez-de-Mo a: si e
cha ac e iza ion and g oundwa e sampling, manusc ip e ision.
CRediT au ho ship con ibu ion s a emen
Rad Ca los: P ojec adminis a ion, Resou ces, So wa e, Supe i-
sion, W i ing – o iginal d a , W i ing – e iew & edi ing. De Wilde
He wig: Concep ualiza ion, Da a cu a ion, Fo mal analysis, Me hodol-
ogy. Rumbo Ca los: Fo mal analysis, In es iga ion, Me hodology.
Daniel P´
e ez-Alonso: In es iga ion, Me hodology, Resou ces. Ba os
Rocío: In es iga ion, Supe ision, Funding acquisi ion, W i ing –
B. Velasco-A oyo e al.
New BIOTECHNOLOGY 79 (2024) 50–59
58
o iginal d a , P ojec adminis a ion, W i ing – e iew & edi ing. de-
Mo a Al edo P´
e ez: Da a cu a ion, Fo mal analysis, Me hodology.
Velasco-A oyo Blanca: Da a cu a ion, Fo mal analysis, W i ing –
o iginal d a , Concep ualiza ion, In es iga ion, P ojec adminis a ion,
Valida ion, Visualiza ion. Cu iel-Aleg e Sand a: Da a cu a ion, Fo mal
analysis, In es iga ion, Me hodology, Resou ces, So wa e. Khan Aqib
Hassan Ali: In es iga ion, So wa e, W i ing – o iginal d a , W i ing –
e iew & edi ing, Fo mal analysis, Visualiza ion.
Decla a ion o Compe ing In e es
The au ho s decla e he ollowing inancial in e es s/pe sonal e-
la ionships which may be conside ed as po en ial compe ing in e es s:
Rocio Ba os epo s inancial suppo was p o ided by Eu opean
Union’s Ho izon 2020 esea ch and inno a ion p og am. Sand a Cu iel-
Aleg e epo s inancial suppo was p o ided by Jun a de Cas illa y
Le´
on.
Acknowledgemen s
The au ho s a e g a e ul o And ea Ma ínez o he echnical help.
Au ho s also acknowledge P o . Kie an Ge maine o p o iding PGPR
s ains, and C´
esa Ma ín (Vi e os La Dehesa) o his help in choosing
plan species.
Appendix A. Suppo ing in o ma ion
Supplemen a y da a associa ed wi h his a icle can be ound in he
online e sion a doi:10.1016/j.nb .2023.12.003.
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