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Phytoremediation of mixed-contaminated soil using thehyperaccumulator plant

Alyssum lesbiacum

: Evidenceof histidine as a measure of phytoextractable nickel

Andrew C. Singer

*, Thomas Bell

, Chloe A. Heywood

, J.A.C. Smith

, Ian P. Thompson

Centre for Ecology & Hydrology-Oxford, Mansﬁeld Road, Oxford OX1 3SR, UK

Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK

Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK

Received 30 June 2006; received in revised form 18 August 2006; accepted 20 August 2006

Alyssum lesbiacum was shown to phytoextract nickel from PAH-contaminated soils from which the pool of nickel accessed for phytoextraction is closely modelled by a histidine-soil extract.

Abstract

In this study we examine the effects of polycyclic aromatic hydrocarbons (PAHs) on the ability of the hyperaccumulator plant

Alyssumlesbiacum

to phytoextract nickel from co-contaminated soil. Planted and unplanted mesocosms containing the contaminated soils were repeat-edly amended with sorbitan trioleate, salicylic acid and histidine in various combinations to enhance the degradation of two PAHs (phenanthreneand chrysene) and increase nickel phytoextraction. Plant growth was negatively affected by PAHs; however, there was no signiﬁcant effect on thephytoextraction of Ni per unit biomass of shoot. Exogenous histidine did not increase nickel phytoextraction, but the histidine-extractable frac-tion of soil nickel showed a high correlation with phytoextractable nickel. These results indicate that

Alyssum lesbiacum

might be effective inphytoextracting nickel from marginally PAH-contaminated soils. In addition, we provide evidence for the broader applicability of histidine forquantifying and predicting Ni phytoavailability in soils.

Keywords:

Phytoextraction; Phytoremediation; Bioremediation; Nickel;

Alyssum lesbiacum

; Metal hyperaccumulator; Histidine; Co-contaminated soil

1. Introduction

Phytoextraction

the transfer of metal from an aqueous orterrestrial source into plant biomass

is one of the few bio-logical approaches for remediating metal-contaminated soils.Metal phytoextraction may be most effectively carried outby metal-hyperaccumulating plants, which specialise in se-questering high concentrations of metal in their above-groundbiomass

a trait shared by relatively few species in the world(Baker and Brooks, 1989; Robinson et al., 1997b). A nickelhyperaccumulator is deﬁned as a plant that concentrates nickelto at least 1000 mg Ni kg

in its shoot dry biomass (Brookset al., 1977).

Alyssum lesbiacum

(Candargy) Rech.f. is amongthe few Ni hyperaccumulators for which signiﬁcant progresshas been made in detailing the mechanism of metal uptake(Ingle et al., 2005; Kerkeb and Kra¨mer, 2003). In a numberof

Alyssum

species, a strong positive correlation has beenfound between Ni exposure and histidine production (Kra¨meret al., 1996).Ingle et al. (2005)demonstrated that the histidine biosynthetic pathway is constitutively more highly expressedin

A. lesbiacum

than in the congeneric non-accumulator spe-cies

A. montanum

. The high expression level of histidine bio-synthesis genes maintains a large pool of free histidine in theroots, which increases proportionately with Ni uptake (Ingleet al., 2005). Additional species of

Alyssum

are currently beinginvestigated for ﬁeld-scale metal phytoextraction (Angle et al.,

* Correspondingauthor.Tel.:

44(0)1865281630;fax:

44(0)1865281696.

E-mail address:

acsi@ceh.ac.uk (A.C. Singer).0269-7491/$ - see front matter

82www.elsevier.com/locate/envpol

2001; Kukier et al., 2004; Li et al., 2003a; Robinson et al.,2003).Considerable research has demonstrated the contribution of plants to remediation of soils contaminated with organicchemicals through the ‘rhizosphere effect’

the area aroundthe root where there is an increase in microbial biomass andactivity (Arthur et al., 2005; Shaw and Burns, 2003). Awealthof studies have also demonstrated the role of bioaugmentationand biodegradation-enhancing chemicals in facilitatingorganic pollutant degradation in soil (Singer et al., 2005; vanVeen et al., 1997). However, few studies have investigatedthe ability of metal-hyperaccumulating plants to extract metalseffectively in mixed-contaminated soils.Inthepresentstudy,weexaminetheeffectofbiodegradation-enhancingchemicalsandmetalchelatorsonthephytoextractionofNiinamixed-contaminatedsoil.Inviewoftherecentinterestintheapplicationof

Alyssum

formetalremediation(Angleetal.,2001; Kukier et al., 2004; Li et al., 2003a,b),

A. lesbiacum

wasassessed for Ni phytoextraction in the presence of two polycy-clic aromatic hydrocarbons (PAHs), phenanthrene and chrys-ene. These PAHs are among the most abundant constituents of organic polluted sites (e.g., coal-gas manufacturing plants:ATSDR, 1995). Among the three amendments investigated,two

sorbitan trioleate (surfactant) and salicylic acid (PAH bi-odegradativeinducer)

wereselectedfortheirpotentialcontri-bution towards PAH biodegradation. Sorbitan trioleate,a surfactant containing three C

aliphatic groups on a sugarbackbone, was applied to enhance the bioavailability of thePAHs, as had been previously demonstrated with polychlori-natedbiphenyls(Singeretal.,2000)andpentachlorophenol(un-published data). Salicylic acid, a metabolite of and inducer forthe naphthalene operon, was investigated as a soil amendmentto stimulate indigenous microbial degradation of PAHs (Chenand Aitken, 1999; Singer et al., 2000). Interestingly, salicylicacid is known to be involved in pathogen-induced defence re-sponses in plants and is present at elevated concentrations inmetal-hyperaccumulator plants in the genus

Thlaspi

, where itis implicated in the high degree of cellular tolerance towardsnickel (Freeman et al., 2005).The third amendment, histidine, was selected primarily forits Ni-chelating properties. In lieu of other natural organic-acidchelates such as citrate, oxalate, or acetate (Khodadoust et al.,2004; McGrath and Zhao, 2003), histidine was chosen for itsbiological compatibility with the presumed mechanism of Nisequestration in

A. lesbiacum

(Kra¨mer et al., 1996). Its selec-tion also lessens concerns associated with the use of EDTA,a toxic and persistent synthetic chelate employed in previousphytoextraction studies (Meers et al., 2004; Vassil et al.,1998). All soils were evaluated from two depths (0 to 5 cmand 5 to 10 cm) and fractionated into a coarse (

m)and ﬁne fraction (

m) to further examine the distributionand dynamics of Ni within the treatments. Each soil fractionwas assayed for the water-, histidine- and acid-extractableNi pools, representing pools of nickel from which a decreasingpercentage of nickel is presumed to be phytoaccessible.A histidine-utilizing, Ni-tolerant bacterium, which was pre-viously isolated from the experimental soil, was repeatedlyamended to a multi-component treatment to facilitate the dis-ruption of the Ni-histidine complex

a potentially rate-limit-ing step for the phytoextraction of Ni in

A. lesbiacum

.In this study, our aims were: (1) to assess the individual andadditive effects of salicylic acid, sorbitan trioleate and histi-dine amendment on the phytoextraction of Ni and biodegrada-tion of PAHs; (2) to examine the relationship between thewater-, histidine- and acid-extractable Ni pools and the phy-toextractable Ni pool; and (3) to examine the performanceof

A. lesbiacum

in co-contaminated (non-serpentine) soil.

2. Materials and methods

2.1. Soil contamination

Thirty kg of uncontaminated agricultural sandy-loam soil (Table 1: Wick series, sandy loam soil from the Wellesbourne National Vegetable ResearchStation, Warwickshire, UK (Whitﬁeld, 1974)) was air-dried and sieved(2 mm, Mesh No. 10) prior to spiking with Ni and PAHs. Two 10% (w/w) sub-samples of the sandy-loam soil were then spiked with either an acetone solu-tion (1:1, v/w) of phenanthrene and chrysene or an acetone-aqueous solution(1:1, v/w) of NiSO

. The spiked soil subsamples were mixed with Ni, PAH,or pristine soil (1:1) to a ﬁnal concentration of 100 mg phenanthrene andchrysene kg

soil and 50 mg Ni kg

soil as described in Section2.4. Thesoil Ni concentration of 50 mg kg

was chosen: (1) to enable detection of small shifts in the soil Ni pools owing to phytoextraction, since a larger initialsoil concentration would have masked these subtle shifts; (2) to assess the po-tential for using

A. lesbiacum

as a tool to phytoextract metals from marginallycontaminated soil; and (3) to evaluate the application of

A. lesbiacum

at theUK government target for residential sites, 50 mg Ni kg

soil (EnvironmentAgency, 2002). The soil PAH concentration of 100 mg kg

was chosen toprovide sufﬁciently realistic levels of PAH to lend insight into the efﬁcacyof nickel phytoextraction in co-contaminated soils, as well as the sub-lethallytoxic response of

A. lesbiacum

to PAHs.Table 1Physical and chemical parameters of the experimental soilMean

SD (

)Soil chemical parametersCEC (cmol kg

) 7.71

0.31 (4)pH 5.74

0.08 (3)Soil texture

% Soil particles

m 77

3 (6)% Soil particles

m 23

3 (6)% Sand 73

% Silt 12

% Clay 14

Clay% Illite 27.0

1.7 (3)% Expandable

61.7

2.5 (3)% Kaolinite 11.3

1.2 (3)Organic matterLoss on ignition (%) 2.24

0.01 (3)% Carbon 0.67

0.01 (3)% Nitrogen 0.08

0.01 (3)Carbon:nitrogen 8.37

0.18 (4)Trace metal (mg kg

soil)Ni 24

2.21 (8)

Percent soil texture values were determined on a dry mass basis.

Whitﬁeld (1974).

The ‘expandable’ component in this sample is a random mixed-layer illite/ smectite.75

A.C. Singer et al. / Environmental Pollution 147 (2007) 74

The contaminated soils were mixed by rolling within a closed barrel twiceweekly for seven weeks to ensure homogeneous contamination and completeremoval of solvent vapours. Approximately 0.280 kg of spiked soil wasamended to each of 88 experimental columns (see Section2.4), which wereconstructed of polyvinylchloride (PVC) pipes (5.08 cm I.D.

20 cm length).Columns were initially ﬁlled with pristine soil at the bottom (approx. 3 cm) tominimise gas diffusion from beneath the soil proﬁle and to trap leached chem-icals, denoted inFig. 1as the pristine soil barrier (Singer et al., 2001). The contaminated soil was added to the columns to a depth of 10 cm(1.43 g soil cm

) after the addition of a glass-ﬁbre ﬁlter barrier to separatethe contaminated and pristine soils.

2.2. Planted soil column design

Forty columns were each sown with four

A. lesbiacum

seeds (obtainedfrom Lesvos, Greece, and kindly provided by A.J.M. Baker) on the soil surfaceand maintained in a glasshouse with natural solar illumination. The soil col-umns were irrigated daily (

15 mL tap water) by an automated overheadsprinkler system for the full length of the study (14 weeks from sowing of seeds). Fourteen days after sowing, columns were restocked with seedlingsto compensate for poor germination and mortality, ensuring at least two plantswithin each planted column at this point. The different amendments were thenapplied and no further transplants were made, so as to minimize disruption tothe soil proﬁle. However, this procedure led to some treatments containing rep-licate columns with fewer than the desired two plants each (Table 2).

2.3. Bacterial inoculum

A Ni-tolerant histidine utilizing bacterium was previously isolated fromthe experimental soil by batch enrichment on 3.22 mM

-histidine and3.22 mM NiSO

in minimal salts medium (MSM) (Gilbert and Crowley,1997) adjusted to pH 7.0 with NaOH. The major species of Ni in solution,as determined by MINEQL

(Environmental Research Software, Hallowell,

0-5 cm depth5-10 cm depthglass-fiber filter pristine soil barrier

Fig. 1.

Alyssum lesbiacum

and soil proﬁle after removal from PVC pipe.Evidence of dense rooting zone, particularly in the 5 to 10 cm depth.

T a b l e 2 P l a n t b i o m a s s a n d n i c k e l p h y o t o e x t r a c t i o n s u m m a r y ( m e a n

S D ,

4 ) N u m b e r T r e a t m e n t F o c u s e d c o n t r a s t s

M a n i p u l a t e d

P l a n t s c o l u m n

S h o o t c o l u m n

R o o t c o l u m n

B i o m a s s c o l u m n

B i o m a s s p l a n t

k g

s h o o t k g

r o o t S h o o t N i p l a n t

A v e r a g e d r y b i o m a s s ( g ) A v e r a g e N i ( m g ) 1 B a S a S o H i s 1 v s . 2 B a 1 . 5 0

0 . 5 8 1 . 1 1

0 . 6 0 0 . 5 6

0 . 4 6 1 . 6 6

1 . 0 6 1 . 1 1

0 . 5 1 1 8 9 8

5 2 3 2 8 6

6 9 1 3 9 8

3 2 5 2 S a S o H i s 1 . 2 5

0 . 5 0 0 . 6 3

0 . 2 4 0 . 3 0

0 . 1 3 0 . 9 3

0 . 3 6 0 . 7 4

0 . 3 5 1 7 0 1

2 8 7 5 7 5

4 0 3 8 6 1

4 5 1 3 S a S o H i s - N i 2 v s . 3 N i 2 . 0 0

1 . 1 5 0 . 7 3

0 . 2 9 0 . 2 6

0 . 1 2 0 . 9 8

0 . 4 1 0 . 4 9

0 . 2 3 1 0 0

4 9 8 8

5 5 3 6

3 0 4 S a S o H i s - P A H 2 v s . 4 P A H 2 . 0 0

0 . 0 0 1 . 4 8

0 . 3 1 1 . 3 2

1 . 0 9 2 . 7 9

1 . 1 0 1 . 4 0

0 . 5 5 1 5 8 3

1 6 7 9 6

1 0 5 1 1 6 7

3 2 3 5 S a S o 2 v s . 5 H i s 2 . 5 0

0 . 5 8 1 . 0 5

0 . 3 2 0 . 6 3

0 . 4 0 1 . 6 8

0 . 6 6 0 . 6 7

0 . 4 3 2 4 5 6

1 6 9 1 2 7

1 3 3 1 0 2 7

5 2 4 6 S a H i s 2 v s . 6 S o 2 . 0 0

0 . 8 2 1 . 1 0

0 . 1 9 0 . 5 0

0 . 2 1 1 . 6 0

0 . 3 0 0 . 8 0

0 . 3 0 1 8 6 0

6 0 2 2 9 8

8 9 1 0 2 1

7 1 1 7 S o H i s 2 v s . 7 S a 2 . 5 0

1 . 2 9 1 . 5 5

0 . 4 4 1 . 0 1

0 . 3 2 2 . 5 6

0 . 6 6 1 . 0 2

0 . 8 7 1 6 1 2

1 2 2 1 6 3

7 3 9 9 8

8 0 3 8 S a 5 & 6 v s . 8 S o / S a 2 . 2 5

0 . 9 6 0 . 8 3

0 . 7 6 0 . 2 8

0 . 3 5 1 . 1 1

1 . 1 0 0 . 4 9

0 . 3 5 2 0 2 9

6 3 3 2 9 2

7 9 7 5 1

4 2 3 9 S o 5 & 7 v s . 9 S o / H i s 2 . 2 5

0 . 5 0 1 . 3 0

0 . 3 7 0 . 6 7

0 . 3 1 1 . 9 7

0 . 6 6 0 . 8 8

0 . 2 0 2 0 8 8

1 9 5 2 6 4

1 2 8 1 2 0 4

3 5 5 1 0 H i s 6 & 7 v s . 1 0 H i s / S a 3 . 2 5

0 . 9 6 1 . 8 9

0 . 7 2 0 . 8 2

0 . 5 0 2 . 7 1

1 . 1 6 0 . 8 3

0 . 3 1 1 6 5 9

4 2 0 1 8 5

1 3 5 9 6 6

2 3 6 B a

b a c t e r i a l i n o c u l u m , S a

s a l i c y l i c a c i d , S o

s o r b i t a n t r i o l e a t e , H i s

h i s t i d i n e . A l l t r e a t m e n t s c o n t a i n e d N i a n d P A H e x c e p t t h o s e d e n o t e d b y - N i

w i t h o u t N i , o r - P A H

w i t h o u t P A H .

F o c u s e d c o n t r a s t s d e n o t e

a p r i o r i

h y p o t h e s i s a b o u t t r e a t m e n t e f f e c t s ; t r e a t m e n t n u m b e r s i n t h e ﬁ r s t c o l u m n a r e u s e d t o d e n o t e t r e a t m e n t c o n t r a s t s b e i n g m a d e .

‘ M a n i p u l a t e d ’ d e n o t e s t h e t r e a t m e n t v a r i a b l e s t h a t a r e h i g h l i g h t e d i n t h e f o c u s e d c o n t r a s t .

A.C. Singer et al. / Environmental Pollution 147 (2007) 74

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Category:	Research > Science
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Upload Date:	07/01/2008
Copyright:	Attribution Non-commercial
Tags:	Education metal phytoremediation ligand chelate hyperaccumulator Education metal phytoremediation ligand chelate hyperaccumulator (fewer)

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