Chemosphere 88 (2012) 204–210

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Chemosphere

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Application of phosphate-solubilizing bacteria for enhancing bioavailabilityand phytoextraction of cadmium (Cd) from polluted soil

Seulki Jeong a, Hee Sun Moon b,⇑, Kyoungphile Nam a, Jae Young Kim a, Tae Sung Kim c

a Dept. of Civil and Environmental Engineering, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-742, South Koreab School of Earth and Environmental Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-742, South Koreac Haechun ETS Co. Ltd., 169-12 Geumam-dong, Gyeryongsi, Chungnam 321-900, South Korea

a r t i c l e i n f o

Article history:Received 21 October 2011Received in revised form 2 March 2012Accepted 2 March 2012Available online 1 April 2012

Keywords:PhytoremediationPhosphate-solubilizing bacteriaBrassica junceaAbutilon theophrastiCadmium

0045-6535/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.chemosphere.2012.03.013

⇑ Corresponding author. Tel.: +82 2 880 6645; fax:E-mail address: hmoon@snu.ac.kr (H.S. Moon).

a b s t r a c t

In this study, phosphate-solubilizing bacteria (PSB), Bacillus megaterium, were used to enhance Cd bio-availability and phytoextractability of Cd from contaminated soils. This strain showed a potential fordirectly solubilizing phosphorous from soils more than 10 folds greater than the control without inocu-lation. The results of pot experiments revealed that inoculation with B. megaterium significantly increasedthe extent of Cd accumulation in Brassica juncea and Abutilon theophrasti by two folds relative to the unin-oculated control. The maximum Cd concentrations due to inoculation were 1.6 and 1.8 mg Cd g�1 plantfor B. juncea and A. theophrasti after 10 wk, respectively. The total biomass of A. theophrasti was not sig-nificantly promoted by the inoculation treatment, yet the total biomass of B. juncea increased from 0.087to 0.448 g. It is also worth to mention that B. juncea predominantly accumulates Cd in its stems (39%)whereas A. theophrasti accumulates it in its leaves (68%) after 10 wk. The change of the Cd speciation indi-cated that inoculation of B. megaterium as PSB increased the bioavailabilty of Cd and consequentlyenhanced its uptake by plants. The present study may provide a new insight for improving phytoreme-diation using PSB in the Cd-contaminated soils.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Soil contamination with heavy metals is becoming one of themost severe environmental issues as they cannot be degraded,but can persist in soil environments (Jiang et al., 2008; Ma et al.,2009). Among heavy metals, Cd is one of the most toxic contami-nants in soils and is a non-essential element for biological meta-bolic functions in plants and animals (Sheng et al., 2008a). Thus,the accumulation of Cd in soils can pose a threat to food safetyand is a potential health risk (Dell’Amico et al., 2005).

To date, many methods have been developed to remediate Cd-con-taminated soils, including a range of conventional physical and chem-ical engineering technologies that are often expensive and involvesubstantial excavation and transportation (Suresh and Ravishankar,2004). These processes often employ stringent physicochemicalagents, which have a negative impact on an ecosystem (Lasat, 2002).

Phytoremediation has been considered as a novel environment-friendly technology, which uses plants to remove or immobilizeheavy metals (Suresh and Ravishankar, 2004). Phytoremediation of-fers benefits over physical and chemical approaches for removingheavy metals from soils in terms of cost and safety to humans andenvironments (Suza et al., 2008). Several studies have been

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demonstrated to remediate Cd from soil using phytoextraction(Chen et al., 2010; Luo et al., 2011). However, most plants that couldaccumulate high concentrations of heavy metals (i.e., hyperaccu-mulator) are not suitable for field applications due to their smallbiomass and slow growth (Sheng et al., 2008a). In addition, low bio-availability of heavy metals in soils may also limit the efficiency ofphytoremediation (Sheng and Xia, 2006; Jiang et al., 2008).

The bioavailability of metals can be enhanced by using chemicalchelates such EDTA, however, these compounds can increase themetal leaching risk and have negative effects on soil fertility or soilstructures (Khan et al., 2000). To avoid these problems, the applica-tion of plant growth promoting rhizobacteria (PGPR) can be consid-ered as an important phytoremediation technology for enhancingbiomass production as well as tolerance of the plants to heavy met-als (Chen et al., 2010; Luo et al., 2011). A number of studies havedemonstrated the importance of bacterial inoculation for plant sur-vival in heavy metal-polluted environments (Compant et al., 2005;Sheng et al., 2008b; Chen et al., 2010; Luo et al., 2011). The PGPRsstimulate plant growth by producing growth hormones, solubilizingphosphate, fixing nitrogen, and producing siderophore (Compantet al., 2005; Chakraborty et al., 2006). In particular, phosphate-sol-ubilizing bacteria (PSB) are often used to facilitate P dissolutionfor plant growth from inorganic and organic pools of total soil Pby means of phosphate enzyme and organic acid production (Hildaand Fraga, 1999; Chen et al., 2006).

S. Jeong et al. / Chemosphere 88 (2012) 204–210 205

In this study, PSB, Bacillus megaterium, was used for heavy metal re-moval from soils to enhance Cd bioavailability in soils as well as plantgrowth for a successful phytoremediation scheme. The hypothesis ofthis research is that inoculation of B. megaterium increases the solubi-lization of insoluble P compounds, thereby enhance plant growth. Inaddition, organic acid production during P solubilzation may resultin the increase of Cd bioavailability resulting from changing Cd speci-ation and mobility in soil. Although many phytoremediation re-searches have tried to enhance the efficiency of heavy metal uptakeby plant or heavy metal immobilization using bacterial inoculationin heavy metal-contaminated soil (Park et al., 2011a,b), there are littlestudies reported to examine the effect of PSB inoculation on Cd phy-toextraction efficiency and Cd bioavailablity. The objective of thisstudy was to investigate the effects of inoculation of B. megateriumon plant growth and Cd uptake efficiency by Brassica juncea and Abu-tilon theophrasti. In addition, the changes of bioavailable Cd-speciationover time in soils with PSB inoculation and its effects on Cd uptakewere discussed.

2. Materials and methods

2.1. Soils and plants

The uncontaminated surface soil (0–30 cm) was collected froma small field area at Seoul National University. After the collectedsoil samples were air-dried and ground to pass through a 2 mmmesh, the soil samples were artificially contaminated with anaqueous solution of CdCl2 to have the final concentration of80 mg Cd kg�1 soil. This prepared soil samples were aged for 150 d.

The seeds of B. juncea and A. theophrasti procured from the Na-tional Academy of Agricultural Science (NAAS, South Korea) werepreviously sown in pots containing uncontaminated soil and usedfor the pot experiment using Cd-contaminated soils. B. juncea andA. theophrasti are well known as Cd hyperaccumulators, which cangrow in Cd-contaminated soils up to 250 and 200 mg kg�1 soil,respectively (Singh and Tewari, 2003; Fotiadis et al., 2009). Espe-cially, the plants belonging to Brassicaceae families have shownthe high Cd accumulation potential in Cd-contaminated soil whichis up to 100 mg kg�1 soil (Ghosh and Singh, 2005; Gill et al., 2011).

2.2. Bacterial strain and growth conditions

B. megaterium (DSM Number: 3228) obtained from DeutscheSammlung von Mikroorganismen und Zellkulturen (DSMZ, GermanCollection of Microorganisms and Cell Cultures, Germany) isknown as one of the PSB (Chakraborty et al., 2006; Wu et al.,2007). B. megaterium was grown in the nutrient broth in a shaker(160 rpm) at 30 �C for 12 h. The cells in the exponential phase wereharvested by centrifugation at 3500 rpm for 20 min and washedthree times with phosphate buffer saline and recentrifuged. Bacte-rial inoculum was prepared by resuspending the cells in a 10 mLmineral salt basal medium to obtain an inoculum density of about108 cells mL�1. The mineral salt basal medium contained (mg L�1):1000 (NH4)2SO4, 800 K2HPO4, 200 KH2PO4, 200 MgSO4�7H2O, 100CaCl2�2H2O, 5 FeCl3�6H2O, 1 (NH4)6Mo7O24�4H2O and pH adjust-ment to 7 with 0.1 N HCl.

2.3. Phosphorus solubilization assay

The potential of B. megaterium for solubilizing phosphate fromtricalcium phosphate (TCP) and the soils was quantitatively mea-sured in 250 mL Wheaton bottles with 1 mL of B. megaterium at30 �C for 13 d on a rotary shaker at 160 rpm. The P solubilization testfrom TCP was carried out in a 100 mL Pikovskaya medium (Pikovs-kaya, 1948) (in g): 10 glucose, 0.5 (NH4)2SO4, 0.1 MgSO4�7H2O, 0.5

yeast extract, 0.2 KCl, 0.2 NaCl, 0.002 FeSO4�7H2O, 0.002 Mn FeS-O4�H2O, 5 Ca3(PO4)2 and 1000 mL distilled water (pH 7). The P solu-bilization test from the soils was performed using 5 g soils in 100 mLnutrient broth (NB, 5 g peptone, 3 g beef extract, and 1000 mL dis-tilled water; pH 7). The Pikovskaya medium and the NB broth with-out B. megaterium were used as controls. The culture samples wereremoved periodically from the bottles and filtered (0.4 lm). The sol-ubilized phosphate (water-soluble phosphorous) from TCP or soilsin filtrate by B. megaterium was extracted using 20 mL of 0.03 NNH4F and 0.025 N HCl (Sparks, 1996) and was analyzed using ionchromatography equipped with a CD20 conductivity detector anda Dionex IonPac AS14-4 mm column (DX-500, Dionex, USA). Theexperiment was conducted in triplicate.

2.4. Pot experiment

The effect of B. megaterium on Cd uptake by the two plants (i.e.,B. juncea and A. theophrasti) were studied using a series of pots(9 cm diameter and 10 cm height) containing 300 g Cd-contami-nated soils (80 ± 2 mg kg�1). Four 1-month-old seedlings of eachplant were transplanted into each pot. The physicochemical prop-erties of the contaminated soils used for the pot experiment arelisted in Table 1. The plants were grown in a growth chamber(E15, Conviron, Canada) under constant environmental conditions(25 ± 1 �C, 18 h photoperiod) and the soils were watered with50 mL tap water every day.

After 4 wk from transplantation, half of the pots were inocu-lated with 2 mL of B. megaterium suspension (108 cells mL�1)which was prepared as mentioned earlier, and reinoculated onthe 6th and 10th wk from transplantation to achieve sufficientbacterial activity with soil to enhance the plant growth. The plantswere harvested after 4, 6, 10, 12 (B. juncea only), and 15 wk(A. theophrasti only) of growth for further analysis. At each timeinterval, four seedlings in each pot were harvested all together toobtain sufficient biomass. For the analysis, both plants in the inoc-ulated and the uninoculated pots were carefully removed from thesoils; the roots and the shoots were separated and washed withdistilled water to remove soil particles.

2.5. Analysis of Cd in plants and soils

To determine the dry weight of the plants, the shoots and rootswere oven-dried separately at 70 �C for 24 h. Oven-dried root andshoot samples were ground into fine powder for Cd contents anal-ysis. The samples of shoots and roots were digested with a solutionof HNO3, H2O2, and distilled H2O (9:1:1, v/v/v) using a microwavedigester (MSP1000, CEM, USA) according to the EPA 3052 methodto extract the Cd. After digestion, the volume of each sample wasadjusted to 25 mL with distilled water.

When harvesting the plants, the soils taken from each pot wereair-dried at room temperature. The changes of Cd speciation in thesoils over time were determined by extracting from triplicate soilsamples (1 g each) using the Tessier’s sequential extraction meth-od (Tessier et al., 1979). The concentrations of Cd in plants andsoils were determined by an atomic absorption spectrometry(AA7000, Shimadzu, Japan). The standard error of Cd speciationanalysis ranged from 0.4 to 6.0 mg kg�1 depending on fraction step.The differences between specific pairs of mean Cd concentrationswere identified by the student’s t-test (p < 0.05).

3. Results and discussion

3.1. Potential of B. megaterium for solubilizing phosphrous in soils

Previous studies have observed the ability of B. megaterium tosolubilize insoluble inorganic phosphate compounds such as TCP,

Table 1Physicochemical properties of the artificially contaminated soils used in this study.

Physical properties Nitrogen Phosphorus Cadmium

pH TOC(%)

OrganicMatter (%)

Texture Sand(%)

Silt(%)

Clay(%)

Total(mg kg�1)

NH4–N(mg kg�1)

NO3–N(mg kg�1)

Total(mg kg�1)

Water-soluble(mg kg�1)

Total(mg kg�1)

6.3 1.0 1.6 Sandyloam

71.5 19.5 9.0 11.0 NDa 0.1 258 9.3 80

a Not detected.

0

10

20

30

40

50

60

TCPTCP + PSB

(a)

le p

hosp

horu

s (m

g L-1

)

10 (b)

206 S. Jeong et al. / Chemosphere 88 (2012) 204–210

dicalcium phosphate, hydroxyapatite, or rock phosphate (RP)(Cunningham and Kuiack, 1992; Chen et al., 2006), however, thephosphate solubilization activity of B. megaterium from soils hasnot been reported yet. Therefore, the ability of B. megaterium forsolubilizing phosphate from insoluble phosphorous, i.e., TCP orsoils was investigated as the sole source of phosphate in this study.Inoculation with B. megaterium showed a significant increase ofphosphate in the medium containing TCP compared with the unin-oculated control and the phosphorous solubilization continuedthroughout the incubation period and the final phosphate concen-tration in TCP medium was 45.5 mg L�1 (Fig. 1a).

For soils, the maximum solubilization was achieved after 5 d(8.2 mg L�1) while 0.6 mg L�1 was obtained from the soils underthe uninoculated condition (Fig. 1b). These results indicate thatB. megaterium has the potential for solubilizing inorganic phospho-rus from soils as well as TCP and the bacteria may have helpedplant growth by the enhancement of the uptake of soil mineralssuch as P.

Karunai Selvi et al. (2011) reported that Bacillus sp. could sol-ubilize P up to 68.5 mg from TCP, 5.7 mg from AlPO4 and 22.1 mgfrom FePO4 on the 9th d and 13.8 mg from RP on the 12th d, andthe phosphate solubilization activities were decreased after that.Park et al. (2011a) also investigated that Enterobacter sp. as PSBsolubilized 17.5% of RP in the growth medium. It is generallyknown that the mechanism of mineral phosphate solubilizationby the PSB strain is associated with the production of lowmolecular weight organic acids, which through their hydroxyland carboxyl groups chelate the cation bound to phosphate, thelatter being converted to soluble forms (Sagoe et al., 1998). B.megaterium can produce organic acid such as propionic acid, citricacid, succinic acid, and lactic acid (Chen et al., 2006). In thisstudy, idole-3-acetic acid significantly increased in soil over timeand other organic acids such as lactic acid and succinic acid werenot detected. These organic acids produced by PSB can lead tosolubilize P in soil which can enhance plant growth providingnutrients. However, phosphate solubilization by PSB in soils is acomplex phenomenon, which can depend on many factors suchas nutritional, physiological, and growth conditions (Reyes et al.,2001).

Time (d)0 2 4 6 8 10 12 14

Wat

er s

olub

0

2

4

6

8

SoilSoil + PSB

Fig. 1. Solubilization of phosphorus from tricalcium phosphate (TCP) or soils byBacillus megaterium, phosphate-solubilizing bacteria.

3.2. Plants growth promotion by B. megaterium inoculation

Although several PSB are present in soils, usually their numbersare not high enough to compete with other bacteria commonlyestablished in the rhizosphere (Rodríguez and Fraga, 1999). There-fore, inoculation of the target microorganism at a much higher con-centration than that normally found in soils is necessary to takeadvantage of the property of phosphate solubilization for plantyield enhancement. In this study, 2 mL of B. megaterium suspension(108 cells mL�1) was periodically inoculated to maintain a suffi-cient PSB population and activities in the soils after 4, 6 and10 wk for enhancing plant growth.

Plant biomass is an important factor for the successful applica-tion of phytoremediation to heavy metal-contaminated soils sinceits effectiveness depends on both the plant biomass and the metal

concentration in a plant (Yu and Zhou, 2009). The biomass of B.juncea was improved in the Cd-contaminated soils was improvedafter B. megaterium inoculation compared with that in the uninoc-ulated soils (p < 0.05) (Fig. 2a). The distribution of the dry weightsof each tissue for B. juncea is shown in Supplementary Material(SM), Table SM-1. The highest biomass of leaves was observed atthe 12th wk in the pot with B. megaterium inoculation. The biomassof the above-ground tissues (i.e., stems, leaves, flowers, and fruits)mainly increased in the inoculated soils, while root growth was notsignificantly enhanced by inoculation. Interestingly, the bacteriainoculation seems to promote the growth of B. juncea, whichshowed flowers 1 month earlier compared to the uninoculatedsoils. Even though the slightly high water-soluble phosphorouswas observed in PSB inoculated soil at some growth period, therewas not any significant trend due to complexity of interactionamong phosphate solubilization, soil chemistry and plant(Fig. SM-1).

The effect of B. megaterium inoculation on the growth of Cd ex-posed B. juncea was also investigated during the pot experiment.The total dry weights of B. juncea were increased over time in boththe Cd-contaminated soils and the uncontaminated soils, however,

(a) Brassica juncea

Tota

l dry

wei

ghts

of

plan

ts (

g)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Contaminated soil with PSBContaminated soil without PSB

(b) Abutilon theophrasti

Time (wk)0 2 4 6 8 10 12 14 16

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Fig. 2. Influence of inoculation with Bacillus megaterium (phosphate-solubilizingbacterium, PSB) on total plant biomass with time: (a) Brassica juncea and (b)Abutilon theophrasti.

(a) Brassica juncea

0.0

0.5

1.0

1.5

2.0

2.5

Contaminated soil with PSBContaminated soil without PSB

(b) Abutilon theophrasti

Time (wk)

0 2 4 6 8 10 12 14 16 18

Cd

conc

entr

atio

ns in

pla

nt (

mg

Cd

g-1 p

lant

)

0.0

0.5

1.0

1.5

2.0

2.5

Fig. 3. Influence of inoculation with Bacillus megaterium (phosphate-solubilizingbacteria, PSB) on cadmium uptake by plants with time: (a) Brassica juncea and (b)Abutilon theophrasti.

S. Jeong et al. / Chemosphere 88 (2012) 204–210 207

the exposure of Cd to B. juncea without inoculation of B. megateri-um resulted in a severe decrease in dry weights (Table SM-1).Accordingly, the bacteria seem to play a role not only in improvingplant growth and nutrition but also in protecting the host plantagainst heavy metal toxicity. For A. theophrasti, there was no signif-icant difference in the growth of the total biomass (Fig. 2b). How-ever, the growth of stems was significantly promoted during thefinal growth period compared to the uninoculated control (TableSM-2). A similar result was also observed by Afzal and Bano(2008), who showed that the PSB had similar effects on the growthof root and shoot. These results indicated that the phosphate-solubilizing bacteria, B. megaterium, have helped the uptake of soilminerals such as P by the plants, especially in B. juncea.

3.3. Effect of B. megaterium inoculation on Cd uptake by plant

Fig. 3 shows the Cd uptake by B. juncea and A. theophrasti in theinoculated and the uninoculated soils. For both B. juncea andA. theophrasti, significant increases of accumulated Cd concentra-tions in plants were observed when the soils were inoculated withB. megaterium compared to the uninoculated soils (p < 0.05). Thehighest Cd concentration in plants was 1.6 and 1.8 mg Cd g�1 plantfor B. juncea and A. theophrasti at the 10 wk, respectively.

The Cd accumulation by B. juncea increased up to 175% when B.megaterium was inoculated for 6 wk while no further Cd uptake inthe uninoculated soils was observed after 4 wk (Fig. 3a) and mostof the Cd uptake occurred during the initial period (until 6 wk),showing no more Cd uptake after the flower formation of B. juncea.In A. theophrasti, Cd was accumulated by more than two times afterinoculating B. megaterium compared with the uninoculated control

(Fig. 3b). Unlike B. juncea, Cd was continuously accumulated in A.theophrasti by the 10 wk. This result showed that the inoculationof B. megaterium positively affected the Cd uptake in both B. junceaand A. theophrasti, even though the growth of A. theophrasti was notsignificantly enhanced (Fig. 2b). This means that the metal solubi-lization by B. megaterium may be an important process in the up-take of Cd by the two plants.

The Cd distribution profiles in different plant tissues of B. junceaand A. theophrasti are shown in Fig. 4. Interestingly, B. juncea accu-mulated Cd effectively in their stems, whereas A. theophrasti accu-mulated Cd in their leaves regardless of inoculation of B.megaterium. For B. juncea grown in the inoculated soils, the contentof Cd accumulation in stems varied from 25% to 48% at the 6 and12 wk, respectively. Also, Cd accumulation for A. theophrasti waspredominant in leaves showing more than 70% of the total Cd accu-mulation for the whole growth period.

The translocation factor (TF) is defined as the ratio of metal con-centration in plant shoots to that in plant roots (Zhang et al., 2010).The TF of B. juncea with bacteria inoculation increased from 2.7 to3.2 over time; this can be attributed to the efficient translocation ofCd from the root to the shoot system compared to the uninoculatedplant (Table 2) For A. theophrasti, the TF was not significantly dif-ferent with the bacteria inoculation. The higher TF values were ob-served for A. theophrasti than B. juncea, indicating that A.theophrasti could transfer more Cd to their shoots than B. juncea.

These results showed that B. juncea was more effective in termsof the TF than A. theophrasti for the accumulative effect of Cd by theinoculation with B. megaterium, even though a higher total Cdaccumulation was observed in A. theophrasti. This maydemonstrate that the PSB inoculation was in no way related to

With PSB

0.0

0.3

0.6

0.9

1.2

1.5

1.8

RootStemLeaveFlowerFruit

Without PSB

Time (wk)

0 2 4 6 8 10 120.0

0.3

0.6

0.9

1.2

1.5

1.8

(a) Brassica juncea

Cd

conc

entr

atio

ns in

pla

nts

(mg

Cd

g-1 pla

nt)

With PSB

0.0

0.3

0.6

0.9

1.2

1.5

1.8

RootStemleave

Without PSB

Time (wk)

0 2 4 6 8 10 12 14 16 180.0

0.3

0.6

0.9

1.2

1.5

1.8

(b) Abutilon theophrasti

Cd

conc

entr

atio

ns in

pla

nt (

mg

Cd

g-1 pla

nt)

Fig. 4. Distribution of cadmium concentrations in different plant tissues: (a) Brassica juncea and (b) Abutilon theophrasti.

Table 2Translocation factors of Brassica juncea and Abutilon theophrasti with and withoutBacillus megaterium (PSB) inoculation.

Time (wk) Brassica juncea Abutilon theophrasti

With PSB Without PSB With PSB Without PSB

0 – – – –4 2.2 2.2 0.2 0.26 1.2 5.8 6.7 3.410 4.5 1.0 4.6 8.112 (15) 3.2 2.6 8.4 6.9

208 S. Jeong et al. / Chemosphere 88 (2012) 204–210

the intrinsic accumulative capacity of plants. Basically, A. theophr-asti could accumulate more Cd in their tissues than B. juncea,regardless of bacteria inoculation (Fig. 3).

3.4. Effect of B. megaterium inoculation on Cd bioavailability

The change of Cd speciation in soils, especially the bioavailablefraction of Cd, after inoculation with B. megaterium was trackedusing the Tessier’s sequential extraction during the phytoextrac-tion (Tessier et al., 1979) (Fig. 5). Generally, most of the mobilemetals can be removed in the first fractionation (exchangeablefraction) and then extracted in order of decreasing metal mobility,which means an exchangeable form (fraction 1) can be consideredreadily mobile and easily bioavailable, while a residual form wasconsidered to be the most inactive. The carbonate bound, Fe–Mnoxide bound, and organic matter bound fractions could be consid-ered relatively active depending on the actual physical and chem-ical properties of the soils (Lu et al., 2005). Most of the metals existin a mobile form when soil is artificially spiked with metal and thismobile fraction decreases with time while the amount of metalsorbed into the other fractions gradually increases. This is becauseslow and steady diffusion of the spiked metals toward inert sites inthe soils occur with time (Lock and Janssen, 2003).

In this study, the soils before planting (aged for 150 d) werecomposed of three fractions: exchangeable (fraction 1), carbonatebound (fraction 2), and Fe–Mn oxide bound (fraction 3), whichwere 65%, 26%, and 9%, respectively (0 wk in Fig. 5). The interest-ing change of Cd speciation was observed in the soils inoculation

with B. megaterium for both B. juncea and A. theophrasti, in whichthe ratio of the exchangeable metal form increased over time.However, the Cd speciation was shown to be the general trendin the uninoculated soils, as mentioned earlier. Especially, the or-ganic matter bound (fraction 4), which were not observed in theinitial period of growth, was formed after 6 and 10 wk for B. jun-cea and A. theophrasti, respectively (Fig. 5). The organic acid suchas indole-3 acetic acid was released and the pH was slightly de-creased from 6 to 5 during the experiment in the inoculated soilswhile there was no change in the uninoculated soils (data notshown).

This study shows that the non-bioavailable and insoluble metalfractions were gradually solubilized by the inoculation of PSB (i.e.,B. megaterium), indicating inoculated bacteria have the potential ofCd-resistance and solubilization ability. The organic acids such asindole-3-acetic acid exudated by B. megaterium lead to acidify soil

With PSB

20

40

60

80

100

Without PSB

Time (wk)0 2 4 6 8 10 12

Cd

conc

ntra

tion

s in

soi

l (m

g kg

-1)

0

20

40

60

80

100

(a) Brassica juncea

With PSB

20

40

60

80

100

Without PSB

Time (wk)0 2 4 6 8 10 12 14 16

0

20

40

60

80

100

(b) Abutilon theophrasti

Cd

conc

ntra

tion

s in

soi

l (m

g kg

-1)

Fig. 5. Changes of cadmium speciation in soils using the 5-step sequential extraction method by inoculation with Bacillus megaterium: (a) Brassica juncea and (b) Abutilontheophrasti.

S. Jeong et al. / Chemosphere 88 (2012) 204–210 209

or can directly solubilize and sequester Cd from the soils. Conse-quently, PSB could enhance the mobilization and bioavailabilityof Cd in soils increasing more mobile Cd speciation such asexchangeable Cd form in our study. Also, the barely soluble metalbound phosphorous can be solubilized by phosphate-solubilizingactive substances by an interaction between bacteria and the rootsof plants. In addition, these mechanisms increase the mobility aswell as phytoavailability of Cd in soils and enhance the total accu-mulation of Cd in plants.

4. Conclusions

Our study demonstrated that B. megaterium as phosphate-solubilizing bacteria (PSB) has a potential for solubilizing phospho-rous from soils itself and could promote plant growth by providingsoil minerals such as P. In addition, this bacterial strain couldreduce the plant stress against Cd, especially for B. juncea. In ourexperiments, the application of PSB (i.e., B. megaterium) in phyto-remediation significantly enhanced the Cd uptake efficiency aswell as plant biomass promotion (only for B. juncea) as the amountof bioavailable (mobile phase) Cd in the soils was increased withbacterial inoculation. From these results, it can be concluded thatPSB may be an important bioresource that increases the solubiliza-tion of insoluble Cd compound.

In that respect, the findings from this study may suggest that asuitable combination or modification of a roots/rhizosphere sys-tem using PSB could promote the metal bioavailability and effi-ciency of phytoextraction.

Acknowledgements

The financial support was provided by Haechun ETS Co. Ltd.,and the National Research Foundation of Korea (NRF) grant fundedby the Korea Government (MEST) (No. 2011-0013835). Also, thisresearch was supported by the Korean Ministry of the Environ-ment, as ‘‘The GAIA Project’’. The additional support for this re-search was provided by Brain Korea 21 Project through theSchool of Earth and Environmental Sciences, and Department of Ci-vil and Environmental Engineering, Seoul National University in2011. This study was technically supported by the Engineering Re-search Institute and Integrated Research Institute of Constructionand Environmental Engineering, Seoul National University.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, in theonline version, at http://dx.doi.org/10.1016/j.chemosphere.2012.03.013.

210 S. Jeong et al. / Chemosphere 88 (2012) 204–210

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