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1 Bioresource Technology 12 (211) Contents lists vilble t ScienceDirect Bioresource Technology journl homepge: Arsenic-resistnt bcteri solubilized rsenic in the growth medi nd incresed growth of rsenic hyperccumultor Pteris vittt L. Piys Ghosh, Bl Rthinsbpthi b, Len Q. M, Soil nd Wter Science Deprtment, University of Florid, Ginesville, FL 32611, United Sttes b Horticulturl Sciences Deprtment, University of Florid, Ginesville, FL 32611, United Sttes rticle info bstrct Article history: Received 5 April 211 Received in revised form 14 July 211 Accepted 19 July 211 Avilble online 24 July 211 Keywords: Pteris vittt Rhizosphere Fluorescent bcteri As solubiliztion Plnt growth promotion The role of rsenic-resistnt bcteri (ARB) in rsenic solubiliztion from growth medi nd growth enhncement of rsenic-hyperccumultor Pteris vittt L. ws exmined. Seven ARB (tolernt to 1 mm rsente) were isolted from the P. vittt rhizosphere nd identified by 16S rrna sequencing s Pseudomons sp., Commons sp. nd Stenotrophomons sp. During 7-d hydroponic experiments, these bcteri effectively solubilized rsenic from the growth medi spiked with insoluble FeAsO 4 nd AlAsO 4 minerls (from <5 lgl 1 to mg L 1 As) nd enhnced plnt rsenic uptke (from to mg kg 1 As in the fronds). Production of (1) pyochelin-type siderophores by ARB (fluorescent under ultrviolet illumintion nd chrcterized with thin lyer chromtogrphy) nd (2) root exudte (dissolved orgnic C) by P. vittt my be responsible for As solubiliztion. Increse in P. vittt root biomss from to g/plnt dw by ARB nd by rsenic ws ssocited with rsenic-induced plnt P uptke. Arsenic resistnt bcteri my hve potentil to enhnce phytoremedition of rsenic-contminted soils by P. vittt. Published by Elsevier Ltd. 1. Introduction Arsenic (As) pollution cuses public helth problems in millions of people round the world (Nordstrom, 22). Chronic exposure to As cn cuse skin nd lung cncers nd other non-crcinogenic symptoms such s dibetes, skin diseses, nd nervous nd crdiovsculr problems (Vhter, 27). The helth hzrds ssocited with rsenic-contminted soil coupled with the high remedition cost mkes it necessry to develop cost-effective technologies to restore polluted lnd. Phytoremedition is cost effective technique, which employs plnts to remedite polluted soils nd to improve soil properties. It is effective in removing metls of low concentrtions from surfce soils nd wter, lthough longer tretment times re required compred to other conventionl techniques such s soil excvtion nd disposl to lndfill. The rsenic hyperccumultor, Pteris vittt L. (Chinese Brke fern), is cpble of ccumulting up to 2.3% As in its dry biomss (M et l., 21), mking it vible cndidte for phytoremedition of As-contminted sites. It is cpble of extrcting As from both soluble nd insoluble forms (Tu et l., 24), nd hs been utilized in field pplictions to remedite As-contminted soils (Shelmerdine et l., 29; Kertulis-Trtr et l., 26) nd groundwter (Ntrjn et l., 29). Arsenic uptke in P. vittt minly occurs vi the roots, so soil rhizosphere chrcteristics significntly ffect its As ccumultion. Corresponding uthor. E-mil ddress: lqm@ufl.edu (L.Q. M). The vilbility of nutrients nd As re likely impcted by the microbil community in the rhizosphere of P. vittt. Free-living s well s symbiotic plnt growth promoting rhizobcteri (PGPR) cn enhnce plnt growth directly by providing vilble phosphte (P) for plnt uptke, lowering plnt ethylene levels, nd sequestering Fe for plnts by producing Fe chelting substnces (Hung et l., 24). The Fe solubiliztion ctivity of PGPR cn impct As levels in soils. In erobic soils, rsente (AsV) is present s HAsO 2 4 nd H 2 AsO 4, nd is often bound to Fe/Al minerls, mking it insoluble (Tu et l., 24); however, Fe solubiliztion lso releses the As from the minerls, mking it biovilble to plnts nd soil microorgnisms. Thus As-resistnt microorgnisms might hve selective dvntge to survive under these conditions. Although some bcteri ssocited with P. vittt hve been identified nd chrcterized to limited extent (Hung et l., 21), their effects on As uptke hve not been exmined. In the current study, seven As-resistnt bcteri from the rhizosphere soil were isolted nd identified, nd their As trnsformtion bility to improve plnt growth nd As uptke vi solubilizing Fe nd As from insoluble minerls ws determined. 2. Methods 2.1. Soil collection nd chrcteriztion The soil smples were collected from res ner Ginesville, Florid where P. vittt grow nturlly, nd they were collected from Archer Feed store (AF), Archer Ministorge (AM), Rinbow /$ - see front mtter Published by Elsevier Ltd. doi:1.116/j.biortech

2 P. Ghosh et l. / Bioresource Technology 12 (211) Springs (RS) nd Crystl River Qurries (CQ). The sites AF nd AM re locted in Archer, FL, nd RS site in Rinbow Springs Stte Prk, Dunnellon, FL, nd CQ site in Crystl River Qurries, FL, which is dolomite mining site. P. vittt in the AF site grew under n rsenic-treted wood structure. During rin the As my be wshed out of the treted wood (Chirenje et l., 23), which my be the source of As in this site. The AM site is close to n re, which hd previous history of As contmintion (Jng et l., 22). The RS site is locted in prk nd hs no record of As pollution, but P. vittt grew very close to n old wooden bridge. So this my be nother exmple of As pollution from rsenic-treted wood. From tht perspective, the site CQ is unique. The fern grew on the shdy slopes of the mine tilings t the edge of n bndoned open-cut dolomite mining site. The soil smples were collected from the surfce 4 5 in. from the rhizosphere of P. vittt nd from bulk soil, which ws 1 feet wy from the plnt where no other plnts were growing. The soil smples were stored in refrigertor before use. Soil ph ws mesured fter mixing the soil with wter t 1:1 (w/w) for 2 d. Rhizosphere nd bulk soil were evluted for wter-soluble As nd Fe in 1:4 soil to wter mixtures obtined fter shking for 1 h, centrifugtion t 35g for 15 min, nd filtrtion through.45 lm membrne filter. Arsenic in solution ws determined by grphite furnce tomic bsorption spectrophotometry (GFAAS, Vrin AA24Z, Wlnut Creek, CA). Soil smples were digested using modified EPA Method 35B for the Hot Block Digestion System (Environmentl Express, Mt. Plesnt, SC) nd As ws determined using GFAAS vi EPA Method SW-846 (Tu et l., 24) As trnsformtion in soils Soil smples (.2 g; rhizosphere nd bulk), which were stored in refrigertor for week prior to incubtion with 3 ml of hlf-strength Luri Bertni medium with 2 mm glucose (LEB) contining either 1 mm AsV or 1 mm AsIII. The bcteril medium components were purchsed from Sigm Chemicl Co. (St. Louis, Missouri). Sodium rsente (N 2 HAsO 4 7H 2 O) nd sodium rsenite (NAsO 2 ) were from Mllinckrodt Bker Inc. (Phillipsburg, New Jersey). The suspensions were incubted for 24 h t 3 C shking t 2 rpm. The superntnts were nlyzed for totl As nd for specition of AsV nd AsIII fter seprtion using the Sep-Pk Ò Plus C18 rsenic-crtridge (Wter Corportion, Milford, MA; Srivstv et l., 26). Concentrtions of AsV nd AsIII were determined using GFAAS Isoltion nd sequencing of 16S rrna of As-resistnt bcteri DNA M r mrker, Tq polymerse, dntps nd pcr 2.1-TOPO cloning kit were from Invitrogen (Crlsbd, Cliforni). Oligonucleotide primers were synthesized using the custom primer synthesis unit of IDT DNA Technologies (Corlville, Iow). Soil (1 g) ws incubted with 1 ml.9% (w/v) sline solution contining 1 mm of AsV for 24 h t 3 C shking t 2 rpm. To isolte As-resistnt bcteri (ARB),.2 ml of the sline solution ws plted on LEB gr contining hlf-strength LB with 2 mm glucose t ph 7 contining 1 mm AsV. Individul colonies were isolted fter five rounds of single colony purifiction. Genomic DNA ws isolted from the bcteril isoltes using the cetyltrimethylmmonium bromide technique (Smbrook et l., 1989). PCR rections, 5 ll totl, contined 1 ng of genomic DNA from the bcteril strins, 2 pmol PCR primer, 4 nmole dntps, 125 nmol MgCl 2, 25 lg bovine serum lbumin, nd 1 unit Tq polymerse in 1 PCR buffer (Invitrogen). The primers were ribo1492r 5 -GGTTACCTTGTTACGACTT-OH-3 nd ribo27f 5 -AGA- GTTTGATCCTGGCTCAG-OH-3 (Gremion et l., 23). Following 3 PCR cycles ech of 94 C for 1 min, 5 C for 45 s, nd 72 C for 2 min, the rection product ws nlyzed using n grose gel electrophoresis. The PCR product ws cloned into pcr 2.1- TOPO vector nd sequenced (Smbrook et l., 1989). Cloned PCR products from totl of seven ARBs were sequenced. The 16S rrna coding DNA sequences determined in this study hve been deposited in GenBnk (Tble 2). Multiple sequence of 16S rrna DNA ws compred with sequences vilble in the GenBnk dtbse using BLAST (Alstchul et l., 199) Arsenic solubiliztion nd plnt growth enhncement by Asresistnt bcteri Arsenic solubiliztion nd plnt growth by ARB FeAsO 4 nd AlAsO 4 minerls were synthesized (Tu et l., 24), wshed, nd dried t 3 C for 3 h. Six-month old P. vittt plnts, which were purchsed from Milestone (Apopk, FL), were cclimted in 1 L of.25 Fe-free Hoglnd medium t ph 7 for 2 weeks. Fe-free medium ws used to simulte the low-fe growth hbitt of P. vittt in the environment (Bondd nd M, 22). Then.25 g of pre-wshed FeAsO 4 or AlAsO 4 minerl ws dded to the medium. The seven ARB isoltes were grown overnight seprtely nd then mixed together such tht equl cfu/ml of ech isolte ws represented in volume of 12 ml t density of OD 6 vlue of 1.5. One ml of this mixture ws dded to 1 L of growth medium. After 7 d, As concentrtion ws mesured in the plnt tissue nd the growth medium, nd fresh plnt biomss of P. vittt ws recorded fter wshing the plnt roots in DI wter nd drying on blotting pper. The plnt smples were dried in n oven t 8 C for 2 d, digested with H 2 SO 4 /H 2 O 2 (Tu nd M, 23), nd nlyzed for As using GFAAS. In ddition to As, totl Fe nd Al concentrtions in the plnts were mesured in the sme extrcts. To determine the mount of Fe/Al precipitting on root surfces, plnt roots (.2 g) were wshed in 5 ml 1:1 v/v HNO 3 nd wter for 2 min nd Fe nd Al concentrtions in the superntnts nd plnt digest were determined using inductively-coupled plsm emission spectroscopy (PerkinElmer 53DV, Wlthm, MA) vi EPA Method 27. To confirm the role of root exudte in solubilizing As from FeAsO 4 nd AlAsO 4 minerls, dissolved orgnic C (DOC) in the spent growth medium ws nlyzed using totl crbon nlyzer (TOC-55A, Shimdzu) equipped with n utosmpler (Tu et l., 24) Arsenic solubiliztion nd plnt growth effects of ARB nd Bcillus sp. To further test the efficiency of ARB in As solubiliztion, the experiment described in Section ws repeted with soil Tble 1 ph, wter soluble Fe nd As, nd totl rsenic concentrtions (mg kg 1 ) in four rhizosphere nd bulk soils of P. vittt. Vlues re men ± SE of three replictes. The detection limit for rsenic ws.5 mg L 1 nd for iron ws.2 mg L 1. ph Totl As Wter soluble As Wter soluble Fe Rhizosphere soil * AF 7.97 ± ±.23.5 ±.34.2 ±. AM 7.29 ± ± ± ±.3 CQ 7.6 ± ± ± ±.7 RS 7.25 ± ± ±.33.2 ±. Bulk soil AF 7.56 ± ±.73.2 ±.1.2 ±. AM 6.81 ± ±.5.12 ±.4.57 ±.1 CQ 6.82 ± ±.3.2 ±.3.2 ±. RS 6.92 ±.3.7 ±.2.1 ±.1.2 ±. * Smples AF, AM, CQ nd RS were collected from Archer Feed Store, Archer Ministorge, Crystl River Qurry, nd Rinbow Springs.

3 8758 P. Ghosh et l. / Bioresource Technology 12 (211) Tble 2 Phylogenetic identifiction nd fluorescent compound production of rsenic-resistnt isoltes from the rhizosphere of P. vittt. Isolte (GenBnk ccession number) bcteri Bcillus sp. (GenBnk ccession number JN132398) s tretment, which ws grown to n OD 6 of 1.5. We selected Bcillus sp. s our control bcterium becuse it is commonly found soil bcterium isolted from non rsenic-contminted soil, nd there hs been no report of P. vittt being infected by ny bcteril pthogen present in soil. After 7-d of growth, plnt biomss (dw) ws determined fter drying in n oven t 8 C for 2 d. To determine the potentil role of P in enhncing plnt growth, dried plnt smples were digested using H 2 SO 4 /H 2 O 2 to nlyze P using modified molybdenum blue method. Briefly the ph of the digestion solution ws djusted to 5 with NOH nd HCl. Ten milliliter of the solution ws pipetted into 2 ml glss test tube, nd to this.5 ml of L-cysteine (5% w/v in.6 M HCl) ws dded. The test tube ws cpped tightly to llow AsV reduction to AsIII for 5 min t 8 C. The solution ws cooled to room temperture, nd P ws determined by the molybdenum blue method Plnt growth enhncement by rsenic To seprte the effect of As from tht of Fe, six-month old P. vittt plnts were cclimted in 1 L of.25 Fe-free Hoglnd medium t ph 7 for 2 weeks. The tretment hd or 3 mg/l wtersoluble AsV with nd without the ARB consortium. The fresh plnt weight ws recorded fter 7 d of tretment nd corresponding dry weights were mesured fter drying in n oven t 8 C for 2 d Presence of siderophores To test the presence of siderophores in spent growth medium, the medium ws extrcted with chloroform. The wter soluble frction contining the siderophores ws dried by injecting ir through nitrogen evportor (N-EVAP TM, Orgnomtion Assocites Inc., MA, USA) to increse the concentrtion of siderophores in the solution. Then the concentrted extrct ws nlyzed using thin lyer chromtogrphy (TLC) on cellulose pltes developed in solvent mixture of butnol:cetic cid:wter (3:1:2 v/v) system. To test for the presence of pyochelin type siderophores, the fluorescent spots on the TLC were spryed with.1 M FeCl 3 /HCl solution Sttisticl nlysis Nerest phylogenetic neighbor bsed on prtil sequencing of 16S rrna % Identity PG4 (JF345176) Commons testosterone 1 High (CP122.1) PG5 (JF345177) Pseudomons sp. (EU ) 1 High PG6 (JF345178) Pseudomons putid 99 High (GU248219) PG9 (JF345179) Pseudomons putid 1 Low (AM ) PG1 Pseudomons putid 99 Low (JF34518) (AY ) PG12 Pseudomons sp. (FJ ) 1 High (JF345181) PG16 (JF345182) Stenotrophomons rhizophil (GU ) 97 Low Fluorescence Fluorescence were denoted s high or low bsed on the intensity of fluorescence emitted by the strins under UV bench t 254 nm. High ws spotted clerly nd low ws fintly visible. All the experiments followed completely rndomized design (CRD) with three replictes for ech tretment. The nlysis of vrince (ANOVA) nd Tukey s men grouping were used to determine significnce of the interctions between the tretment mens. All sttisticl nlyses were performed with SAS sttisticl softwre (SAS Inst., Cry, North Crolin, USA). 3. Results nd discussion 3.1. Soil chrcteriztion The four soils contined different levels of totl As (Tble 1). The RS soil contined the highest As t 235 mg kg 1, followed by CQ, AM nd AF soils ( mg kg 1 ). In comprison, the bulk soil, tken 1 feet wy from the plnt, hd very low As concentrtions ( mg kg 1 ). It is possible tht P. vittt my preferbly grow in res with high As concentrtion or the presence of P. vittt helped concentrte As ner the rhizosphere. The wter-soluble Fe content in the rhizosphere of CQ (1.88 mg kg 1 ) nd AM (1.27 mg kg 1 ) sites ws higher thn tht in the corresponding bulk soils (6.57 mg kg 1 )(Tble 1). This increse in vilble Fe in the rhizosphere soil my be due to the presence of Fe-solubilizing siderophore produced by rhizobcteri nd root exudte by P. vittt. The other two soils hd very low wter soluble Fe (Tble 1). The ph in the rhizosphere soils rnged from 7 to 8, which were higher thn the typicl cidic soils in Florid ( ; Chen et l., 1999). This is consistent with the fct tht P. vittt prefers to grow in lkline soils (Bondd nd M, 22). The ph in the bulk soil ws , but slightly lower thn those in the rhizosphere ( ; Tble 1). This is consistent with Gonzg et l. (26) who observed ph increse from 7.15 in bulk soil to 7.65 in the rhizosphere of P. vittt fter growing for 8 weeks in As-contminted soils. At ph of 7 8, most of the nutrients become unvilble with the exception of phosphorus nd As, which becomes reltively more vilble t this ph (Bgyoko et l., 2) AsIII oxidtion nd AsV reduction in the rhizosphere nd bulk soils When fresh soil ws incubted in LEB medium contining 1 mm AsV or AsIII, both AsV reduction nd AsIII oxidtion occurred. However, their rtes differed between the rhizosphere nd bulk soils nd mong the four soils. While both soils hd high levels of AsV reduction (67 82 mg/kg; Fig. 1A), more AsIII oxidtion occurred in the bulk soils thn rhizosphere soils. This difference ws most striking with AF soil, which contined 68 mg/kg As in bulk soil compred to 4 mg/kg As in rhizosphere soil (Fig. 1B). This finding my indicte tht more As oxidizing microbes were present in the bulk soils thn in the rhizosphere soils wheres AsV reducing microbes existed in both rhizosphere nd bulk soils. Compred to the bulk soils ( mg kg 1 As), As concentrtions in the rhizosphere soils ( mg kg 1 As) were much greter (Tble 1). Similr trends were observed in wter-soluble As. The higher As levels in the rhizosphere of P. vittt my support different microflor from those in bulk soils. For typicl soils, both AsV reducing nd AsIII oxidizing bcteri exist. However, in n Asenriched soil, bcteri with the cpcity of detoxifying AsV (vi reducing to AsIII), the predominnt form of As in soils, my be selected Isoltion nd identifiction of As-resistnt bcteri (ARB) Three ARB were isolted from the RS nd CQ soils nd one from AM soil. None ws obtined from the AF soil likely becuse the AF rhizosphere soil hd the lowest totl As concentrtion (Tble 1) nd AF bulk soil hd the highest AsIII oxidtion rte (Fig. 1B). Phylogenetic nlyses of the strins suggested tht the strins belonged to the group gmm-proteobcteri, nd three gener Pseudomons, Commons nd Stenotrophomons sp. (Tble 2).

4 P. Ghosh et l. / Bioresource Technology 12 (211) A AsV reduced to AsIII (mg/kg) B AsIII oxidized to AsV (mg/kg) RS CQ AM AR AF Soil smple RS Rhizosphere soil Bulk soil CQ AM Soil smple AF AR Fig. 1. AsV reduction (A) nd AsIII oxidtion (B) in four rhizosphere nd bulk soils of P. vittt..2 g soil ws mixed with 3 ml LEB medium contining 1 mm AsV or AsIII for 24 h. The brs represent men ± SE. Mens indicted by sme letters re not significntly different t =.5 using Tukey s men seprtion test Solubiliztion of As from FeAsO 4 nd AlAsO 4 by P. vittt nd Asresistnt bcteri Both P. vittt nd As-resistnt bcteri were effective in solubilizing As from insoluble FeAsO 4 nd AlAsO 4 minerls dded to the hydroponic growth medium (Tble 3). In the bsence of P. vittt nd ARB (no plnt nd no ARB), neither FeAsO 4 nor AlAsO 4 minerl ws solubilized (As =.5 mg L 1 ). In the presence of P. vittt (P. vittt with no ARB), substntil mount of As ws solubilized, with more As from AlAsO 4 (5.4 mg L 1 ) thn FeAsO 4 tretment (2.58 mg L 1 ) being solubilized (p =.1; Tble 3). Similrly, ARB inocultion (no plnt with ARB) ws effective in solubilizing As in the medium, with more from AlAsO 4 thn FeAsO 4 tretment (8.2 vs mg L 1 ). There were two possible resons for higher As solubiliztion from AlAsO 4 thn from FeAsO 4 tretment. The substntilly higher solubility of AlAsO 4 thn FeAsO 4 probbly mkes it more soluble in the medium (Ksp = vs ; Ptnik, 24). In ddition, it is possible tht, under Fe-deficient conditions in the AlAsO 4 tretment (.25 Fe-free Hoglnd solution), the plnts nd ARB exuded more orgnic cids to obtin Fe. This ide ws supported by the 12 76% more DOC in the AlAsO 4 thn the FeAsO 4 contining medium (Tble 3). P. vittt produced 1.84 mg L 1 DOC in the FeAsO 4 compred to 3.24 mg L 1 in the AlAsO 4 tretment (Tble 3), which is consistent with the dt by Tu et l. (24). Both P. vittt nd ARB were effective in solubilizing As; however, ARB ws 59 6% more effective thn P. vittt in As solubiliztion (Tble 3). Though no bcteri were dded to the P. vittt tretment, bcteri were present on the roots of P. vittt; however, these bcteri were not s effective s ARB in As solubiliztion. Inocultion of ARB in P. vittt tretment further incresed As concentrtions, especilly in the AlAsO 4 tretment, which incresed by 46% (Tble 3). Significntly higher mount of As solubilized by ARB thn P. vittt my be due to the fct tht more siderophores were produced by ARB under Fe-strvtion conditions to complex with Fe or Al, mking As more soluble. The presence of siderophores ws verified by exmintion of fluorescence of medi extrcts. The nlysis using thin lyer chromtogrphy indicted tht there were two UV-positive spots with R f vlues of.44 nd.. These spots when spryed with.1 M FeCl 3 /HCl generted ornge red colortion (dt not shown), suggesting the presence of t lest two compounds similr to pyochelin type siderophores (Ankenbuer et l., 1991; Cox et l., 1981) Effect of As-resistnt bcteri on uptke nd trnsloction of As, Fe nd Al by P. vittt In ddition to being effective in As solubiliztion from FeAsO 4 nd AlAsO 4 minerls, P. vittt ws lso ble to tke up As from the growth medium (Tble 4). However, As concentrtions in the plnt were low with only smll mount of As being trnslocted from the roots to fronds, resulting in the As trnsloction fctor (TF; concentrtion rtio in fronds to roots) of.9 in FeAsO 4 nd.5 in AlAsO 4 tretments. Unlike As (Tble 3), neither Fe nor Al ws detected in the growth medium (dt not shown). However, P. vittt ws ble to tke up some Fe nd Al, with frond nd root concentrtions being 19.8 nd 385 mg kg 1 Fe in FeAsO 4 tretment, nd 2. nd 39.2 mg kg 1 Al in AlAsO 4 tretment (Tble 4). Apprently, P. vittt ws ineffective in tking up or trnslocting Al or Fe (TF <.8; Tble 4). Inocultion with ARB significntly incresed As concentrtions in P. vittt (Tble 4). While substntilly more As ws ccumu- Tble 3 Effect of As-resistnt bcteri (ARB) nd P. vittt on As nd dissolved orgnic C (DOC) concentrtions in.25 Fe-free Hoglnd medium spiked with.25 g of insoluble FeAsO 4 nd AlAsO 4 fter growing P. vittt for 7 d. Tretment As concentrtions (mg L 1 ) DOC concentrtions (mg L 1 ) No ARB With ARB No ARB With ARB FeAsO 4 No plnt.5 ±. A 4.14 ±.19BC.4 ±.1.57 ±.6b P. vittt 2.58 ±.39B 2.89 ±.21CB 1.84 ±.22d 3.82 ±.37b AlAsO 4 No plnt.5 ±.A 8.2 ±.38D.3 ±..64 ±.5b P. vittt 5.4 ±.65C 7.37 ± 1.64C,D 3.24 ±.18c 4.62 ±.26e The vlues re men ± SE of three replictes. Sme letters following mens for As or DOC concentrtions indicte no significnt difference bsed on Tukey s men grouping t =.5. The detection limit for rsenic ws 5 lgl 1.

5 876 P. Ghosh et l. / Bioresource Technology 12 (211) Tble 4 Effect of As-resistnt bcteri (ARB) on As, Al nd Fe concentrtions in P. vittt fter growing for 7 d in.25 Fe-free Hoglnd medium spiked with.25 g insoluble FeAsO 4 nd AlAsO 4 minerls. Tretment Tissue No ARB With ARB No ARB With ARB As concentrtions (mg kg 1 ) Fe concentrtions (mg kg 1 ) FeAsO 4 Fronds 18.1 ± 1.2A 35.3 ± 2.6B 19.8 ± 5.53A 21.3 ± 2.83A Roots-internl 19.4 ± 1.8A 45 ± 27C 385 ± 16.8C 662 ± 48.5B Roots-externl 3.34 ±.1D 36.3 ± 1.26B 21.5 ± 11.A 54.2 ± 9.29D TF b AlAsO 4 Fronds 21.9 ± 1.8A 236 ± 17E 2. ±.4A 6.93 ±.42B Roots-internl 43.4 ± 6.4B 18 ± 12E 39.2 ± 3.36D 77.3 ± 5.19C Roots-externl 3.85 ±.3D 9.99 ±.4F 23.9 ±.95E 5.83 ±.35B TF Arsenic solubilized from root surfce (roots-externl) ws obtined by wshing in 1:1 HNO 3. The vlues re men ± SE of three replictes nd the mens of As, Fe nd Al were nlyzed seprtely. Mens followed by sme letters were not significntly different bsed on Tukey s men grouping t =.5. b TF = trnsloction fctor, concentrtion rtio in the fronds to the roots. lted in the roots thn fronds (45 vs mg kg 1 ) in the FeAsO 4 tretment, significnt mount of As ws trnslocted from roots to fronds in P. vittt in AlAsO 4 tretment (18 vs. 236 mg kg 1 ). The higher mount of Fe ccumulted in the roots (662 vs. 385 mg kg 1 ) with ARB tretment my hve hindered As trnsloction in the FeAsO 4 tretment. Only in AlAsO 4 tretment, ARB tretment incresed As trnsloction compred to no ARB tretment, with As TF incresing from.5 to 1.3. The fct tht much higher Fe concentrtion ws observed inside the roots (roots-internl; 85 95%) thn the root surfce (roots-externl; 5 15%) is consistent with siderophore-ssisted Fe uptke by P. vittt (Tble 4) Effect of As-resistnt bcteri nd Bcillus sp. on growth of P. vittt Inocultion of ARB substntilly incresed the concentrtions of As, Fe nd Al in P. vittt, with the impcts being more significnt in the roots thn the fronds (Tble 4). For exmple, ARB incresed the As concentrtions by 2 11 times in the fronds compred to 4 23 times in the roots. A similr pttern ws observed for P. vittt biomss (Tble 5). Little chnge ws observed in frond biomss but ARB incresed root biomss by 89% in the FeAsO 4 tretment nd 13% in the AlAsO 4 tretment. The relese of Fe from the FeAsO 4 + ARB tretment my hve helped P. vittt growth; however, the increse of P. vittt biomss in the AlAsO 4 + ARB tretment my not be due to Al uptke s it is not nutrient. Our dt clerly demonstrted tht ARB ws more effective in promoting P. vittt growth thn the microbes ssocited with P. vittt roots in the no ARB tretment (Tble 5). To better understnd the role of ARB in promoting plnt growth, we repeted the experiment with bcteri Bcillus sp. being dded s tretment. No difference ws observed between the control nd Bcillus Tble 5 Effect of rsenic-resistnt bcteri (ARB) on biomss of P. vittt grown for 7 d in.25 Fe-free Hoglnd medium spiked with.25 g of insoluble FeAsO 4 nd AlAsO 4 minerls. Tretment Biomss (g/plnt dw) No ARB With ARB FeAsO 4 Fronds 6.1 ±.29A 6.34 ±.48A Roots 2.2 ±.17C 4.16 ±.24B AlAsO 4 Fronds 6.23 ±.49A 5.23 ±.54A,B Roots 1.51 ±.36C 3.43 ±.7B The vlues re men ± SE of three replictes. Mens followed by the sme letters were not significntly different bsed on Tukey s men grouping t =.5. sp. tretment (Fig. 2A), but ARB incresed both root nd frond biomss, with lrger increse being observed in the roots thn the fronds nd in the AlAsO 4 thn in the FeAsO 4 tretment. For exmple, the increse in root nd frond biomss ws 47% nd 23% in FeAsO 4 tretment compred to 74% nd 6% in AlAsO 4 tretment, respectively (Fig. 2A). A g /plnt B P concentrtion (mg g -1 dw) Root Frond Root Frond FeAsO4 AlAsO4 Control Bcillus Consortium Root Frond Root Frond FeAsO4 AlAsO4 Fig. 2. Effects of ARB (rsenic-resistnt bcteri) nd Bcillus sp. on biomss (A) nd P concentrtion (B) of P. vittt (control with no bcteri dded) fter growing in.25 Fe-free Hoglnd solution contining either FeAsO 4 or AlAsO 4 for 7 dys. The vlues re men ± SE of three replictes. Mens indicted by the sme letters were not significntly different bsed on Tukey s men grouping t =.5.

6 P. Ghosh et l. / Bioresource Technology 12 (211) Since plnt biomss increse ws lrger in the AlAsO 4 tretment thn tht in the FeAsO 4 tretment (Fig. 2A), Fe probbly did not ply significnt role s nutrient. We hypothesized tht As-induced P uptke by P. vittt my hve helped its growth. In hydroponic experiment, Luongo nd M (25) observed tht plnt biomss increse fter exposure to 1 mg L 1 As for 2 weeks ws ssocited with substntil increse in P uptke by P. vittt. Compred to the control, P concentrtions incresed from 2.33 to 5.19 g kg 1 in the fronds, nd from.91 to 5.76 g kg 1 in the roots (Luongo nd M, 25). Similr results were obtined by Tu et l. (24b) s well s in this experiment. It is possible tht As stress my hve upregulted P uptke by P. vittt (Fig. 2B). For exmple, ARB incresed root P by 45% in FeAsO 4 tretment nd by 266% in AlAsO 4 tretments. The much greter increse in root P concentrtions (Fig. 2B) ws consistent with greter increse in root biomss (Fig. 2A). The fct tht P concentrtions nd biomss in the roots (r =.64) ws better correlted thn tht in the fronds (r =.44) (p =.5), lso supported our hypothesis tht rsenic-induced P uptke ws probbly responsible for biomss increse in P. vittt. To further exmine this hypothesis, the impct of As on the bility of ARB in promoting plnt growth ws further tested. In this experiment, sme cfu/ml of ARB consortium ws inoculted s the experiment in Section to the growth medium with or without 3. mg/l As. The level of As concentrtion in the growth medium ws similr to the experiment discussed in Section (Tble 3). Consistent with our hypothesis, higher frond nd root biomss were obtined with As (4.8 nd 3.5 g/fronds nd roots dw, respectively) thn without As (3.5 nd 1.9 g/plnt dw) tretments (dt not shown), gin supporting rsenic-induced P uptke. 4. Conclusions P. vittt nd ARB were efficient in solubilizing As from insoluble FeAsO 4 nd AlAsO 4, with ARB being more efficient thn P. vittt. P. vittt ws inefficient in tking up Al, nd high As nd Fe in the roots suggested possible co-precipittion of As nd Fe. ARB were efficient in promoting P. vittt growth. There ws symbiotic reltionship between P. vittt nd the fluorescent ARB in its rhizosphere. In the presence of ARB nd As, the increse in P uptke by P. vittt ws probbly due to rsenic-induced P deficiency, which enbled the plnt to grow better in the As-rich environment. In the future, ARB my be pplied in field pplictions to enhnce As hyperccumultion by P. vittt. Acknowledgements This project is supported in prt by UF-IFAS Innovtion Grnt. The uthors would like to thnk Mr. Benjmin Jffe in Deprtment of Biology t Northern Arizon University for his help in soil smpling nd Dr. Andy Ogrm in Soil nd Wter Science Deprtment t University of Florid for providing Bcillus sp. References Alstchul, S.F., Gish, W., Miller, W., Myers, E.W., Lipmn, D.J., 199. Bsic locl lignment serch tool. J. Mol. 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