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This is an Accep ed Manusc ip o an a icle published by ELSEVIER in
MARINE POLLUTION BULLETIN, Vol. 117 , on 2017, a ailable a :
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Manusc ip D a
Manusc ip Numbe : MPB-D-16-01358R1
Ti le: Bioaugmen a ion wi h bac e ia selec ed om he mic obiome
enhances A h ocnemum mac os achyum me al accumula ion and ole ance
A icle Type: Resea ch Pape
Keywo ds: A h ocnemum mac os achyum; endophy ic bac e ia; me al
pollu ion; PGPB; phy o emedia ion; izosphe ic bac e ia
Co esponding Au ho : D . En ique Ma eos-Na anjo,
Co esponding Au ho 's Ins i u ion:
Fi s Au ho : Sal ado a Na a o-To e
O de o Au ho s: Sal ado a Na a o-To e; José M Ba cia-Pied as;
Miguel A Ca iedes; Eloisa Pajuelo; Susana Redondo-Gómez; Ignacio D
Rod íguez-Llo en e; En ique Ma eos-Na anjo
Abs ac : A glasshouse expe imen was designed o in es iga e he ole o
bac e ial conso ia isola ed om he endosphe e (CE) and izosphe e (CR)
o A h ocnemum mac os achyum on i s me al up ake capaci y and ole ance
in plan s g own in me al pollu ed sedimen s. A. mac os achyum plan s we e
andomly assigned o h ee bioaugmen a ion ea men s (CE, CR and wi hou
inocula ion) du ing 120 days. Bioaugmen a ion wi h bo h bac e ial
conso ia enhanced A. mac os achyum capaci y o accumula e ions in i s
oo s, while shoo ions concen a ion only inc eased wi h CE ea men .
Fu he mo e bioaugmen a ion amelio a ed he phy o oxici y le els, which
was e lec ed in an inc emen o plan g ow h o 59 and 113% o shoo s
and 52 and 98% o oo s wi h CE and CR ea men s, espec i ely. This
e ec was suppo ed by bac e ia bene icial e ec on pho ochemical
appa a us and he modula ion o i s oxida i e s ess machine y. These
indings indica ed ha bac e ia selec ed om he mic obiome can be
claimed o imp o e A. mac os achyum me al emedia ion e iciency.
Highligh s
 Endo- and izosphe e bac e ial ole on A. mac os achyum me al esponse was analysed
 Bac e ial bioaugmen a ion enhanced he e iciency o me al phy oex ac ion
 Bac e ial inocula ion mi iga ed he impac o pollu ion on A. mac os achyum g own
 They posi i ely a ec i s pho ochemical appa a us
 They a ou ed he modula ion o i s oxida i e s ess machine y
*Highligh s
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Bioaugmen a ion wi h bac e ia selec ed om he mic obiome enhances
A h ocnemum mac os achyum me al accumula ion and ole ance
Sal ado a Na a o-To e1, José M. Ba cia-Pied as2,3, Miguel A. Ca iedes1, Eloísa
Pajuelo1, Susana Redondo-Gómez2, Ignacio D. Rod íguez-Llo en e1, En ique Ma eos-
Na anjo2*
1Depa amen o de Mic obiología y Pa asi ología, Facul ad de Fa macia, Uni e sidad de
Se illa. c/ P o eso Ga cía González, 2, 41012-Se illa, Spain.
2Depa amen o de Biología Vege al y Ecología, Facul ad de Biología, Uni e sidad de
Se illa. 1095, 41012-Se illa, Spain.
3IFAPA, Cen o Las To es –Tomejil. C a Se illa-Cazalla, km 12.200. 41200 Alcalá
del Río, Se illa, Spain.
*Co esponding au o :
D . En ique Ma eos Na anjo
e-mail: [email p o ec ed]
Tel.: + 34 – 954557064
Fax: + 34 – 954-4615780
*Manusc ip (clean - no acked changes)
Click he e o iew linked Re e ences
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Abs ac
A glasshouse expe imen was designed o in es iga e he ole o bac e ial
conso ia isola ed om he endosphe e (CE) and izosphe e (CR) o A h ocnemum
mac os achyum on i s me al up ake capaci y and ole ance in plan s g own in me al
pollu ed sedimen s. A. mac os achyum plan s we e andomly assigned o h ee
bioaugmen a ion ea men s (CE, CR and wi hou inocula ion) du ing 120 days.
Bioaugmen a ion wi h bo h bac e ial conso ia enhanced A. mac os achyum capaci y o
accumula e ions in i s oo s, while shoo ions concen a ion only inc eased wi h CE
ea men . Fu he mo e bioaugmen a ion amelio a ed he phy o oxici y le els, which
was e lec ed in an inc emen o plan g ow h o 59 and 113% o shoo s and 52 and
98% o oo s wi h CE and CR ea men s, espec i ely. This e ec was suppo ed by
bac e ia bene icial e ec on pho ochemical appa a us and he modula ion o i s
oxida i e s ess machine y. These indings indica ed ha bac e ia selec ed om he
mic obiome can be claimed o imp o e A. mac os achyum me al emedia ion e iciency.
Key wo ds: A h ocnemum mac os achyum, endophy ic bac e ia, me al pollu ion,
PGPB, phy o emedia ion, izosphe ic bac e ia.

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1. In oduc ion
Phy o emedia ion is a p omising clean-up solu ion o ac upon o he o ganic and
ino ganic pollu an s in a wide a ie y o con amina ed si es, by ex ac ing, deg ading o
immobilizing hem h ough he use o plan s (Ma ques e al., 2011; Weis and Weis,
2004). Plan s used o me al emedia ion should be chosen on he basis o hei po en ial
o up ake, accumula e and anspo la ge amoun o me als, as well as aking in o
accoun i s abili y o main ain high g ow h a es unde me al excess (Ma ques e al.,
2011). Some imes phy o emedia ion e iciency is comp omised by g ow h inhibi ion as
a consequence o he inc emen o me al phy o oxici y le els in plan issues (Khan e
al., 2015). Recen ly a numbe o scien is s ha e begun o explo e he possibili y o using
he mic obial popula ions which colonizing he hizosphe e (Mesa e al., 2015b; Pajuelo
e al. 2014) o he endosphe e (Do y, 2008; Mesa e al., 2015a; Newman and Reynolds,
2005; Rajkuma e al., 2009) in o de o make his echnology mo e e icacious. Among
hese bac e ial popula ions he in e es has been pa icula ly ocused plan g ow h
p omo ing bac e ia (PGPB), which ha e demons a ed he abili y o p omo e plan
g ow h unde s ess condi ions (Ahemad and Kib e , 2014; de Bashan e al., 2012;
Glick, 2010; Mesa e al., 2015a,b; Ma eos-Na anjo e al., 2015; Na a o-To e e al.,
2016a; Weyens e al., 2009). Howe e he e ec o mic obial inocula ion on plan me al
up ake does no ollow a clea pa e n, being highly dependen o sedimen and
con aminan cha ac e is ics, as well as o plan -mic obe pa ne ships (Phiele e al.,
2014; Sessi sch e al., 2013). The e o e, his s udy was designed o con ibu e o ill
hese gaps o knowledge.
A h ocnemum mac os achyum (Mo ic) C. Koch is a common halophy ic sh ub
in SW Ibe ian Peninsula, which is dis ibu ed in coas al a eas h ough Medi e anean
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basin o he Middle Eas and Asia (Redondo-Gómez e al., 2010b). In he ma shes o he
sou h-wes o Spain de elops popula ions dis ibu ed om he middle o high ele a ions
in he idal ange, which a e occasionally subjec o idal inunda ions and seasonal
hype salini y (Redondo-Gómez e al., 2010b). This species ep esen sui able model
plan o s udy plan -bac e ial in e ac ions and speci ically in e ms o me al
phy o emedia ion e iciency, since his species has demons a ed a g ea capaci y o
accumula ing me als in i s issues (Redondo-Gómez e al., 2010a), being conside ed a
sui able candida e o emedia ion o me al pollu ed coas al sedimen s (Conesa and
Schulin, 2010; Redondo-Gómez e al., 2010a). In addi ion, Na a o-To e e al. (2016b)
ecen ly isola ed di e en bac e ial s ains om he hizosphe e and endosphe e o A.
mac os achyum plan s om he Odiel es ua y, which exhibi ed se e al plan g ow h
p omo ing (PGP) p ope ies, e en in he p esence o hea y me als. Among all he
isola es, he s ains Vib io kanaloae RA1, Pseudoal e omonas dis inc RA8,
Pseudoal e omonas p ydzensis RA15 and S aphylococcus wa ne i RA18 isola ed om
he hizosphe e o A. mac os achyum and he s ains Kushne ia ma is la i EAod3,
Mic ococcus aloe e ae EAod10, Bacillus ie namensis EAR8 and Halomonas
zincidu ans EAR18 isola ed om he endosphe e, we e p oposed as he bes candida es
o design wo bac e ial conso ia aimed o inocula e A. mac os achyum plan s o
imp o e i s me al emedia ion po en ial (Na a o-To e e al., 2016b). Thus, in he
p esen s udy, a con inua ion o his p e ious one, we hypo hesized ha hese selec ed
bac e ial conso ia could play an impo an ole on me al up ake, accumula ion and
anspo , as well as on he mi iga ion o phy o oxici y e ec s a ising om me al
accumula ion in plan issues.
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The aim o his s udy was o in es iga e he ole o bac e ia isola ed om he
mic obiome o A. mac os achyum in i s me al up ake capaci y and ole ance o me al
excess.
2. Ma e ial and me hods
2.1. Plan and sedimen sou ce
Seeds o A. mac os achyum and samples o he i s 15 cm o sedimen a ound
he plan s we e collec ed in Ma ch 2015 om he Odiel ma shes (37º15´N, 6º58´W; SW
Spain) and subsequen ly anspo ed o he labo a o y. Then, seeds we e disin ec ed
wi h 10% sodium hypochlo i e o 10 min and washed six imes wi h s e ile dis illa ed
wa e . These seeds we e pla ed in 9% aga pla es and placed in a g ow h chambe
(AGP-700-HR ESP; Radibe , Ba celona, Spain) wi h a egime o 10 h o ligh (20 ºC,
50% HR and 35 μmol m-2 s-1, 400-700 nm) and 14 h da k (5 ºC and 50% HR) o 20
days. A e , ge mina ed seedlings we e ans e ed o indi idual po s (9 cm high x 11
cm diame e , n = 30) illed wi h pe li e. Seedlings we e main ained in a g eenhouse wi h
he ollowing condi ions: empe a u e be ween 21-25 ºC, 40-60% ela i e humidi y,
na u al dayligh o 250 as minimum and 1000 μmol m-2 s-1 as maximum ligh and
supplemen ed wi h 20% Hoagland solu ion (Hoagland and A non, 1938).
On he o he hand, sedimen ex u e, conduc i i y, pH, edox po en ial and o al
sedimen me al concen a ions (n = 5) we e measu ed using he me hod desc ibed by
Ma eos-Na anjo e al. (2011) o de e mine he physicochemical cha ac e is ic o he
collec ed sedimen . The physicochemical p ope ies o he sedimen s a e gi en in Table
1.
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2.2. Inocula ion and plan assays in pollu ed soils
Two bac e ial conso ia we e selec ed o his wo k on basis on hei PGP
p ope ies acco ding wi h he ecen wo k by Na a o-To e e al. (2016b). They
ep esen ed he bes -pe o ming s ains among all he isola es cha ac e ized in ha
s udy. Thus we employed a conso ium wi h bac e ia isola ed om he hizosphe e (CR)
o A. mac os achyum (in eg a ed by he s ains Vib io kanaloae RA1,
Pseudoal e omonas dis inc RA8, Pseudoal e omonas p ydzensis RA15 and
S aphylococcus wa ne i RA18) and a conso ium wi h ou endophy ic s ains (CE),
wo isola ed om plan phyllosphe e (Kushne ia ma is la i EAod3 and Mic ococcus
aloe e ae EAod10) and wo om oo s o A. mac os achyum (Bacillus ie namensis
EAR8 and Halomonas zincidu ans EAR18). Each s ain was cul i a ed sepa a ely in 50
mL o TSB (T yp ic Soy B o h) medium supplemen ed wi h 0.3 M NaCl a 28 ºC du ing
24 h in con inuous shaking o each 108 cells/mL. Then, cul u es we e cen i uged o
10 min a 8000 pm and pelle s we e washed wi h 50 mL o 0.9% saline solu ion,
cen i uging a he same condi ions. Pelle s we e e-suspended in 5 mL o 0.9% saline
solu ion and mixed in 50-mL Falcon ubes, o ming he CR and CE conso ia
espec i ely. Finally, bo h conso ia we e mixed sepa a ely wi h 250 mL o ap wa e
o plan inocula ion.
In Oc obe 2015, a e ou mon hs o A. mac os achyum plan g ow h unde
glasshouse condi ions desc ibed abo e, he pe li e o A. mac os achyum plan s was
washed o and plan s we e ans e ed o indi idual 250 mL plas ic po s (one plan pe
po and each po was placed in an indi idual shallow ay) illed wi h a homogena e o
Odiel sedimen . Po s we e andomly assigned o h ee inocula ion ea men s (non-
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o m oo s o shoo s, which could con ibu e o imp o e i s accumula o po en ial. The
endophy ic conso ium used in his expe imen (EAod3, EAod10, EAR8 and EAR18)
showed he p esence o a a ie y o enzyma ic ac i i ies, such as p o eases, pec inases
and amylases (Na a o-To e e al., 2016b). Hence, hese endophy ic enzyma ic
ac i i ies may aid in pene a ion and coloniza ion o he hos plan , and also i is
possible ha endophy ic bac e ia could ha e ce ain me al up ake capaci y h ough non-
speci ic binding o he ca ion o cell su ace o by me abolic-dependen in acellula
up ake mechanism (Gadd and G i i hs, 1978), which could con ibu e o imp o e he
anspo o me als inside he plan s, since some o hem p esen ed also mobili y
(Na a o-To e e al., 2016b).
3.2. Impac o sedimen bioaugmen a ion on plan ole ance o me al s ess
Some imes he po en ial o plan s used as biological ools o me al emedia ion
is o en comp omised h ough g ow h inhibi ion by inc eased oxici y o me als in i s
issues, and especially when his accumula ion occu s in i s pho osyn he ic pa s which
a e mo e sensi i e o me al excess (Ma eos-Na anjo e al., 2008; Pe ez-Rome o e al.,
2016). In his conce n, Redondo-Gómez e al. (2010a) ound ha he inc emen o Cd
concen a ion in he issues o A. mac os achyum plan s was ollowed by 25% o
biomass educ ion a e a mon h in plan s g own a 1.35 mM Cd unde glasshouse
condi ions. By con as , he inc ease o me al accumula ion in inocula ed plan s
obse ed in ou wo k did no co ela e wi h plan g ow h inhibi ion, despi e ha hese
concen a ions we e g ea e han oxici y h eshold o plan s in i s issues (Kaba a-
Pendias and Pendias, 2000). In ac , he ela i e g ow h a e (RGR) o A.
mac os achyum plan s g own in pollu ed sedimen om Odiel ma shes ended o

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inc ease in simila deg ee wi h bo h bioaugmen a ion ea men s (ANOVA, P < 0.05;
Fig. 2A). Fu he mo e his posi i e end was e lec ed in bo h shoo and oo d y mass,
which showed a mean inc ease o 59% and 113% o shoo s and o 52% and 98% o
oo s in CE and CR inocula ion ea men s, espec i ely (Fig. 2B). Se e al ac o s could
con ibu e o explain his esponse, such as he p esence o PGP p ope ies in he
bac e ial conso iums used in his s udy (Na a o-To e e al., 2016b) and/o by
bac e ial educ ion o me al phy o oxici y in he hos plan (Ha e bu g and Ko he 2007).
Ou hizosphe e bac e ial conso ium was in eg a ed by he s ains RA1, RA8, RA15
and RA18, while he endophy ic conso ium was o med by he s ains EAod3,
EAod10, EAR8 and EAR18. Bo h bac e ial conso ia ha e a mix o bac e ia isola ed
om he izosphe e and om he endosphe e o A. mac os achym wi h he bes PGP
p ope ies, such as indolace ic acid (IAA) and side opho es p oduc ion, phospha e
solubilisa ion, bio ilm o ma ion and ni ogen ixa ion in p esence o hea y me als
(Na a o-To e e al., 2016b). In pa icula IAA is a key compound in cell di ision,
elonga ion and plan issues di e en ia ion (Pa en and Glick 2002) and auxins ha e a
majo ole in oo g ow h and la e al oo o ma ion (Ljung, 2013; Remans e al., 2008;
Zhao, 2010), which could con ibu e o explain he be e g ow h o A. mac os achyum
plan s wi h he bioaugmen a ion ea men s. Ou esul s a e consis en wi h o he s whe e
an enhanced in plan g ow h was eco ded a e inocula ion wi h izosphe ic and
endophy ic bac e ial s ains (Abou-Shanab e al. 2003; Mesa e al., 2015a; Na a o-
To e e al. 2016a).
Mo ing o bac e ial educ ion o me al phy o oxici y in plan s, he e a e
inc easing epo s indica ing ha me al esis an bac e ia can diminish he me al s ess
o plan s (Ahemad and Kib e , 2014; Mesa e al., 2015a,b; Rakjuma e al., 2012; Ullah
e al., 2015; Yang e al., 2009). In his pa icula , he Mic ococcus aloe e ae EAod10,
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Halomonas zincidu ans EAR18 and Pseudoal e omonas dis inc RA8 s ains used in
his wo k ha e shown he abili y o o m bio ilms (Na a o-To e e al., 2016b), which
could con ibu e o dec ease phy o oxici y h ough o seques e o me als and o
minimize he ansloca ion o excessi e me als in o shoo s. Fu he mo e, i has been
desc ibed ha oxic pollu an s aken up by he plan may be deg aded in o plan by
endophy es (Khan and Do y, 2011), al hough he mechanism a e no well unde s ood.
On he op o ha , o he bac e ial bene icial e ec s on plan me al ole ance ha e been
ela ed o osmo ic adjus men , an ioxidan sys em modula ion and enhanced up ake o
mine als (Compan e al., 2005; Khan e al., 2015; Rajkuma e al., 2009), as well as
wi h he p o ec ion o he pho osyn he ic unc ions (Mesa e al., 2015a; Rincon e al.,
2008). Rega ding o bioaugmen a ion impac on pho osyn he ic appa a us o A.
mac os achyum, ou esul s e ealed ha ne pho osyn he ic a e (AN) alues we e
g ea e in plan s g own in sedimen bioaugmen ed wi h he bac e ial conso iums,
showing in bo h cases (CE and CR ea men s) a inc emen c. 65% espec o non-
inocula ed ea men a e 120 days o ea men (one-way ANOVA, P < 0.01; Fig. 3A).
S oma al conduc an e (gs) and in e cellula CO2 concen a ion (Ci) showed ends ha
we e highly simila o ha o AN, wi h inc emen s a ound 125% and 170% o gs and
33% and 42% o Ci, espec i ely (one-way ANOVA, P < 0.01; Fig. 3B and C). In
addi ion, maximum quan um e iciency o PSII pho ochemis y (F /Fm) and quan um
e iciency o PSII (

PSII) alues a midday did no a y wi h he bioaugmen a ion
ea men s espec o he con ol a e 120 days o expe imen , wi h alues a ound 0.82
and 0.57 o F /Fm and

PSII, espec i ely (Table 1. Supplemen a y ma e ial). Ob ained
esul s sugges ed ha he inc ease in g ow h can also be a ibu ed o he imp o emen
in he pho osyn he ic ca bon assimila ion o A.mac os achyum plan s g own in
bioaugmen ed sedimen s.
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Fu he mo e o he physiological e ec eco ded in his s udy was he di e en ial
modula ion o he an ioxidan esponses o A. mac o achyum plan s g own in
bioaugmen a ed sedimen s. Me al oxici y in plan s is known o induce ROS gene a ion
and as consequence oxida i e damage, which inju y plan g own and de elopmen
(Redondo-Gómez e al., 2011). Howe e plan s ha e he abili y o module an ioxida i e
enzymes ac i i ies, such as asco ba e pe oxidase (APx), ca alase (CAT), guaiacol
pe oxidise (GPx) and supe oxide dismu ase (SOD), which con ibu e o main ain he
homeos asis again he high le els o ROS (Dazy e al., 2009; Qiu e al., 2008; Redondo-
Gómez e al., 2011; Mesnoua e al., 2016). In ou expe imen he an ioxida i e enzymes
ac i i ies in shoo s and oo s we e signi ican ly a ec ed by sedimen bioaugmen a ion
ea men s a e 120 days o ea men (one-way ANOVA, P < 0.01; Fig. 4). Thus in
shoo s, APx ac i i y was signi ican ly enhanced in bo h inocula ion ea men s, and GPx
and SOD ac i i ies inc eased only in plan s g own in sedimen inocula ed wi h CE
ea men , while CAT ac i i y did no signi ican ly a y wi h he bioaugmen a ion
ea men s (Fig. 4A-D). The inc emen o SOD and GPx ac i i ies in CE ea men ,
sugges ed ha he inocula ion wi h endophy ic bac e ia could con ibu e o educe he
oxida i e s ess de i ed o he g ea e amoun o me al eco ded in his plan s. Thus,
SOD can con e supe oxide adicals o H2O2, while GPx can ca alyze decomposi ion o
H2O2 in o H2O (Dazy e al., 2009; Qiu e al., 2008), which could con ibu e o main ain
he pho osyn he ic unc ion (Redondo-Gómez e al., 2011), despi e he high le el o
me als eco ded in A. mac os achyum plan s. On he o he hand, ou esul s showed ha
in oo s APx, CAT, GPx and SOD ac i i ies we e signi ican ly lowe in plan s g own in
CE ea men , compa ed wi h he con ol and CR ea men s (one-way ANOVA, P <
0.01; Fig. 4). This esponse sugges s ha endophy ic could con ibu ed in he
modula ion o me al s ess a oo s le els, as was p e iously indica ed in Solanum
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nig um plan s inocula ed wi h he endophy ic s ain Se a ia sp. RSC-14 (Khan e al.,
2015). So i is possible, as we p e iously indica ed, ha in ce ain deg ee he me als
inside o oo s could be seques e ed by endophy ic bac e ia a oiding i s in e ac ion wi h
essen ial me abolic p ocesses o A. mac os achyum plan s; howe e his a ea dese es
u he esea ch.
Conclusion
We can conclude ha sedimen bioaugmen a ion wi h app op ia e PGPB, isola ed om
he mic obiome o A. mac os achyum plan s, con ibu e o imp o e i s phy o emedia ion
e iciency. Inocula ion wi h bo h bac e ial conso ia imp o ed me al up ake and
accumula ion in oo s. This enhancemen could be asc ibed o bac e ial e ec s on me al
mobili y and bioa ailabili y. Also ou esul s showed ha CE conso ium inc eased he
capaci y o A. mac os achyum o accumula e me als in i s shoo s, which could be linked
wi h he pene a ion and coloniza ion capaci ies, as well as wi h me al up ake o
endophy ic bac e ia. Conce ning bioaugmen a ion e ec s on plan ole ance, bo h
bac e ial conso ia con ibu ed o mi iga e phy o oxici y le el ela ed wi h me al
accumula ion in plan s issues. This e ec was e iden in e ms o g ow h and CO2
assimila ion capaci y, as indica ed he g ea e RGR and pho osyn he ic assimila ion
alues compa ed wi h i s non-inocula ed coun e pa s. Likewise, una ec ed e iciency
o PSII pho ochemis y appa a us migh indica e ha bioaugmen a ion ea men s
mi iga ed me al impac on he unc ionali y and in eg i y o he pho osyn he ic appa a us
o A. mac os achyum. Finally ou esul s indica ed ha bioaugmen a ion e ec on A.
mac os achyum me al ole ance was also asc ibed o he modula ion o an ioxida i e
enzyme machine y in i s oo s and shoo s. Consequen ly, he inocula ion wi h selec ed
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hizosphe ic and endosphe ic bac e ia is a sui able me hod which would allow o
enhance he e iciency o A. mac os achyum du ing es o a ion p og ams o pollu ed
coas al sedimen s.

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Acknowledgemen s
This wo k was unded by Jun a de Andalucía (P11-RNM-7274MO p ojec ), INIA (RTA
2012-0006-C03-03 p ojec ) and Minis e io de Economía y Compe i i idad (CGL2016-
75550-R p ojec ). S. Na a o-To e hanks Jun a de Andalucía and J.M. Ba cia-Pied as
hanks INIA o hei pe sonal inancial suppo s. Au ho s a e g a e ul o Uni e si y o
Se ille G eenhouse Gene al Se ices (CITIUS) o i s collabo a ion. We also hank he
use ul commen s o e iewe s who helped o imp o e he inal e sion o he
manusc ip .
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Legends o igu es
Fig. 1. E ec o bioaugmen a ion ea men s (con ol, wi hou inocula ion; CE,
inocula ion wi h a bac e ial conso ium om he endosphe e and CR, inocula ion wi h a
bac e ial conso ium om he hizosphe e; de ails in ma e ial and me hods) on o al
a senic, As (A), ch omium, C (B), coppe , Cu (C), lead, Pb (D), nickel, Ni (E) and zinc
(F) o shoo s and oo s o A h ocnemum mac os achyum plan s g own in na u al
sedimen s om Odiel ma sh o 120 days. Values a e means ± S.E. (n = 7). Di e en
le e s indica e means ha a e signi ican ly di e en om each o he (P < 0.05).
Fig. 2. E ec o bioaugmen a ion ea men s (con ol, wi hou inocula ion; CE,
inocula ion wi h a bac e ial conso ium om he endosphe e and CR, inocula ion wi h a
bac e ial conso ium om he hizosphe e; de ails in ma e ial and me hods) on ela i e
g ow h a e, RGR (A) and o al d y mass (B) in A h ocnemum mac os achyum plan s
g own in na u al sedimen s om Odiel ma sh o 120 days. Values a e means ± S.E. (n
= 7). Di e en le e s indica e means ha a e signi ican ly di e en om each o he (P <
0.05).
Fig. 3. E ec o bioaugmen a ion ea men s (con ol, wi hou inocula ion; CE,
inocula ion wi h a bac e ial conso ium om he endosphe e and CR, inocula ion wi h a
bac e ial conso ium om he hizosphe e; de ails in ma e ial and me hods) on ne
pho osyn he ic a e, AN (A), s oma al conduc ance, gs (B) and in e cellula CO2
concen a ion, Ci (C) in andom p ima y b anches o A h ocnemum mac os achyum
plan s g own in na u al sedimen s om Odiel ma sh o 120 days. Values a e means ±
S.E. (n = 7). Di e en le e s indica e means ha a e signi ican ly di e en om each
o he (P < 0.05).

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Fig. 4. E ec o bioaugmen a ion ea men s (con ol, wi hou inocula ion; CE,
inocula ion wi h a bac e ial conso ium om he endosphe e and CR, inocula ion wi h a
bac e ial conso ium om he hizosphe e; de ails in ma e ial and me hods) on
Asco ba o pe oxidase, APx (A), ca alase, CAT (B), guaiacol pe oxidase, GPx (C) and
supe oxide dismu ase, SOD (D) in shoo s and oo s o A h ocnemum mac os achyum
plan s g own in na u al sedimen s om Odiel ma sh o 120 days. Values a e means ±
S.E. (n = 3). Di e en le e s indica e means ha a e signi ican ly di e en om each
o he (P < 0.05).
Figu e(1)
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Figu e(2)
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Figu e(3)
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Figu e(4)
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Supplemen a y Da a
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