J. Park et al.

: Immobilization of Pb by phosphate-solubilizing bacteria

67

Isolation of Phosphate-Solubilizing Bacteria and Characterization of

Their Effects on Lead Immobilization
Jinhee PARK*,1,2, Nanthi BOLAN1,2, Mallavarapu MEGHARAJ1,2 and Ravi NAIDU1,2
1
2

Centre for Environmental Risk Assessment and Remediation (CERAR), University of South Australia, SA-5095
Cooperative Research Centre for Contaminants Assessment and Remediation of the Environment (CRC CARE),
University of South Australia, SA-5095
Keywords: Lead immobilization, Phosphate solubilizing bacteria (PSB), Phosphate amendment, Phosphate solubilization,
NH4NO3-extractable Pb

Abstract
Phosphate compounds have been used for the immobilization of heavy metals in contaminated environments. In this
study, phosphate-solubilizing bacteria (PSB) were isolated from phosphate-amended and lead (Pb)-contaminated soils, and
their effects on phosphate-induced immobilization of Pb were examined. Two isolated PSB strains, CS2-B1 and SM1-B1,
which were putatively identified as Pantoea sp. and Enterobacter sp., respectively, were able to solubilize phosphorous (P)
from insoluble P sources through the release of organic acids. Strains CS2-B1 and SM1-B1 solubilized 479 mg/L and 293
mg/L, respectively, of P from tricalcium phosphate. The addition of either CS2-B1 or SM1-B1 and various P amendments
into Pb-contaminated soil reduced the NH4NO3-extractable Pb concentration, with a 51% increase in the immobilization of Pb
observed in rock phosphate-amended soil containing SM1-B1over control soil lacking added PSB. This study indicates that
PSB increase the solubility of P in soil, thereby leading to effective immobilization of Pb in contaminated soils.

1. Introduction

there has been increasing interest in the use of slow release

phosphate fertilizers, such as rock phosphate (Rajan et al.,

In soil, phosphorus (P) is sequestered by adsorption

1996; Chen et al., 2006a). Insoluble phosphate compounds

to the surface of soil particles and through precipitation

can be solubilized by organic acids and phosphatase

reactions with soil cations, particularly iron, aluminum,

enzymes produced by plants and microorganisms (Kucey,

and calcium (Harris et al., 2006). For this reason, a

1983; Duponnois et al., 2005). For example, phosphate-

large amount of soluble P fertilizer is commonly applied

solubilizing bacteria (PSB) have been shown to enhance

to agricultural soils in order to increase plant growth,

the solubilization of insoluble P compounds through the

which is likely to adversely affect both the environment

release of low molecular weight organic acids (Sahu and

and economy. In many countries, there has been a steady

Jana, 2000).

increase in the use of P fertilizer (Syers et al., 2008), which is

Phosphate fertilizers can also be used to immobilize

considered a major source of heavy metal contamination in

heavy metals in soil (McGowen et al., 2001; Bolan et al.,

agricultural soils (McLaughlin et al., 1996; Bolan et al., 2003).

2003; Basta and McGowen., 2004). Various mechanisms

In addition, excess amounts of P fertilizer often leach from

have

soil and cause eutrophication of surface and groundwater

immobilization of metals, which include direct metal

sources (He et al., 2003; Sharpley et al., 2003). Therefore,

adsorption by P compounds, phosphate anion-induced

been

proposed

*Corresponding author: Jinhee Park, E-mail: JinHee.Park@postgrads.unisa.edu.ac, Tel: +61-8-8302-6244

Received 19 January 2010; accepted 24 March 2010

for

the

phosphate-induced

68

J. Park et al. / Pedologist (2010) 67-75

metal adsorption, direct precipitation of metals with P

concentrations of Pb (Mergeay et al., 1985). The Tris

in solution as metal phosphates, precipitation through

medium contained 6.06 g of Tris, 4.68 g of NaCl, 1.49 g of

the liming action of rock phosphate, and rhizosphere

KCl, 1.07 g of NH4Cl, 0.43 g of Na2SO4, 0.2 g of MgCl2 6H2O,

modification

mycorrhizal

0.03 g of CaCl2 2H2O, 0.23 g of Na2HPO4 12H2O, 0.005 g

association (Bolan et al., 2003). The immobilization of Pb

through

acidification

and

of Fe(III)(NH4) citrate, and 1 mL of trace element solution

by the addition of P compounds to contaminated water

SL 7 of Biebl and Pfennig [1 mL HCl (25%), 70 mg ZnCl2,

and soil is increasingly becoming a preferred remediation

100 mg MnCl2 4H2O, 60 mg H3BO3, 200 mg CoCl2 6H2O,

technology (USEPA, 2001; Takeuchi et al., 1988). For

20 mg CuCl2 2H2O, 20 mg NiCl2 6H2O, and 40 mg Na2MoO4

example, the use of P as a best management practice of the

2H2O in 1000 mL Milli-Q water] in 1000 mL of Milli-Q

United States Environmental Protection Agency (USEPA)

water. For the determination of the minimum inhibition

has been applied at numerous Pb-contaminated sites,

concentration (MIC; the lowest concentration of metal at

including shooting range sites (USEPA, 2001). Based on

which zero growth occurs), 0.2% glucose supplemented

these reports, we hypothesized that if PSB solubilized P

Tris-buffered low-phosphate medium containing 0, 250,

from insoluble P sources, the immobilization of Pb would

300, 350, 400, and 450 mg/L of Pb was used. The bacterial

be enhanced in soils contaminated with this heavy metal.

growth was determined in 50 mL tubes containing 20 mL

The objectives of this study were to (i) isolate PSB from

medium, 200 L of inoculums (ca. 2.9109 CFU/mL for

P-amended and Pb-contaminated soils, (ii) characterize

CS2-B1 and 2.8109 CFU/mL for SM1-B1) and different

PSB in relation to P solubilization, and (iii) examine the

concentration of Pb after incubation for 7 days at 25

effects of PSB on P-induced immobilization of Pb.

at 125 rpm shaking incubator. Growth was monitored by

measuring optical density at 600 nm wavelength over the

2. Materials and Methods

Isolation and characterization of PSB

course of 7 days.
Solubilization of P

Phosphate fertilizer-amended agricultural soil and

The solubilization of P by PSB was quantified using

Pb-contaminated soil from Zn and Pb smelters were used

insoluble tricalcium phosphate [Ca3(PO4)2] (5 g/L) in

to isolate PSB. To extract bacteria from soil, 0.5 g of soil

NBRIP broth medium. Phosphorous concentrations were

was mixed with 50 mL of sterile 0.2 % NaCl solution (soil:

measured using a spectrophotometer (Synergy HT, BIO-

solution = 1:100, w:v) and shaken for 16 h in an end-over-

TEK, USA) at 880 nm by the phosphomolybdate method

end shaker (Illmer and Schinner, 1992). The isolation of

(Murphy and Riley, 1962). The acid phosphatase activity

PSB was performed using National Botanical Research

in the culture media was measured by the -nitrophenol

Institutes phosphate (NBRIP) medium containing (per

reaction method (Tabatabai and Bremner, 1969). The

L) 10 g glucose, 5 g Ca3(PO4)2, 5 g MgCl26H2O, 0.25 g

organic acid content of the culture media was measured

MgSO47H2O, 0.2 g KCl, 0.1 g (NH4)2SO4, and 1.5% agar

using an ion chromatograph (IC, ICS-2000, Dionex, USA)

(pH 7.0). The colonies formed on the NBRIP plates with

with an AS 19 column (Dionex, USA) at a flow rate of 0.25

clear halos were considered PSB (Nautiyala, 1999). The

mL/min and a multi-step gradient of KOH. Low molecular

PSB strains that were most effective for the solubilization

weight organic acids, including lactate, acetate, propionate,

of P were then identified using crude DNA extractions

pyruvate, malonate, maleate, tartarate, oxalate, succinate,

followed by PCR analysis (Bell et al., 1998). Sequencing of

fumarate, citrate, and trans-aconite, were analyzed.

16S rRNA was conducted at the Flinders DNA Sequencing

Facility (Adelaide). Acquired 16S rRNA sequences were
assessed through the Greengenes website (http://
greengenes.lbl.gov/).

Phosphate-induced immobilization of Pb
The effect of PSB on phosphate-induced immobilization
of Pb was examined using three insoluble P sources:

The bacterial resistance to Pb was tested in Tris-

tricalcium phosphate (Fluka), hydroxyapatite (Riedel

buffered low-phosphate medium (pH 7.0) with increasing

de Han), and rock phosphate (Nutri-Tech Solution).

J. Park et al.: Immobilization of Pb by phosphate-solubilizing bacteria

69

The solubility of these P compounds was determined

Five grams of soil samples in 50 mL centrifuge tubes

by measuring the amount of water-soluble and citric

were treated with tricalcium phosphate (0.0025 g), hy-

acid-soluble P formed in solution. The water-soluble

droxyapatite (0.005 g), or rock phosphate (0.01 g) in the

P concentration of P amendments was anlayzed by

presence and absence of PSB (0.5 mL bacterial suspen-

extracting 0.4 g of the P compounds with 40 mL of water.

sion (ca. 1 109 CFU/mL)), and incubated for 14 days at

Citric acid solubility is often used to compare the reactivity

25o C without shaking. Samples inoculated with sterile

of insoluble P compounds, such as rock phosphate

water instead of bacterial suspension served as controls.

(Braithwaite et al., 1990). To evaluate citric acid solubility,

Bacterial suspensions were prepared by the following

0.4 g of each P compound was extracted with 40 mL of 2%

method. Bacteria grown in 100 mL of Trypticase soy yeast

citric acid solution for 30 min in an end-over-end shaker.

(TSY) medium, which contained 30 g/L Trypticase soy

After shaking, samples were centrifuged at 3000 rpm for

broth and 3 g/L yeast extract, for 48 h were harvested by

10 min and filtered through Whatman No. 41 filter paper.

centrifugation at 4000 rpm for 20 min. The cells were re-

The filtrate was analyzed for P by the phosphomolybdate

suspended in a sterile saline solution (8.5 g NaCl per liter

method using ascorbic acid as the reducing agent (Murphy

Milli-Q water), washed twice with sterile saline and twice

and Riley, 1962; Clescerl et al., 1999).

with sterilized water, and were finally resuspended in 100

The Pb-contaminated soil was collected from a

mL of sterilized water.

shooting range in South Korea, dried at room temperature

After incubation, the population of PSB was estimat-

(25C), and then passed through a 2-mm sieve. The soil

ed by the plate counting method and the pH of the soil was

sample was analyzed for pH, electrical conductivity (EC),

measured. Immobilization of Pb was monitored by mea-

and levels of total Pb, NH4NO3-extractable Pb, and Olsen

suring NH4NO3-extractable Pb. Soils were extracted with

P. To measure pH and EC, 5 g of each air dried soil sample

a 1 M NH4NO3 solution for 2 h in an end-over-end shaker

was placed into a 50 mL centrifuge tube and 25 mL of Milli-

and extractable Pb concentrations were analyzed by In-

Q water was then added. Samples were shaken in an end-

ductively Coupled Plasma-Optical Emission Spectroscopy

over-end shaker for 1 h and pH and EC were measured

(ICP-OES, Agilent, Japan).

with a pH and EC electrode after calibration.

For determining the total heavy metal content, 0.5

3. Results and Discussion

g of air dried soil was added into Teflon microwave soil

digestion vessels and 5 mL of aqua regia (HNO3:HCl =

Isolation and characterization of PSB

1:3, v:v) was then added. Vessels were assembled and

The two bacterial strains which showed the highest

digested in a microwave digestion system (MARSX,

P solubilization capacity, CS2-B1 and SM1-B1, were

CEM, USA). All digestions were conducted in triplicate

putatively identified as Pantoea sp. and Enterobacter sp.,

and each batch included a blank and standard reference

respectively, and the obtained sequences were deposited

material (Montana Soil SRM 2711, certificated by National

into Genbank under accession numbers GQ414734 and

Institute of Standards and Technology). The recovery

GQ414735, respectively. These bacterial genera have

was greater than 90%. To measure the bioavailable Pb

been reported to include PSB by others. For example,

concentration, 5 g of soil sample was extracted with 12.5

Chung et al. (2005) isolated bacteria from rhizosphere

mL of 1 M NH4NO3 solution for 2 h in an end-over-end

soils of various crops in South Korea and reported the

shaker and extractable Pb concentrations were analyzed

genera Enterobacter and Pantoea as PSB. In another study,

by Inductively Coupled Plasma Mass Spectroscopy (ICP-

Prez et al. (2007) isolated PSB from iron-rich acidic soil

MS, Agilent, Japan). Olsen P was measured by extracting

in Venezuela and identified the genera Burkholderia,

soil with 0.5M NaHCO3 (soil: solution=1: 20, w:v) at

Serratia, Ralstonia, and Pantoea among the isolates based

pH 8.5. Phophorus concentration was measured with a

on partial sequencing analysis of 16S rRNA genes.

spectrophotometer using the phosphomolybdate method

(Murphy and Riley, 1962; Clescerl et al., 1999).

Bacterial resistance to heavy metals is an important

consideration when bacteria are to be introduced into soils

70

J. Park et al. / Pedologist (2010) 67-75

for enhancing bioremediation of metal contaminated soils.

of P in soils. Although the mechanism of P solubilization

The present study indicated that the MIC of Pb was 372

by organic acids is not well understood, the pH decrease

mg/L for CS2-B1 and 462 mg/L for SM1-B1. Roane (1999)

resulting from the release of organic acids could be related

isolated Pb-resistant Pseudomonas marginalis and Bacillus

to P solubilization (Fuentes et al., 2009) (Eq. 1).

megaterium from metal-contaminated soils with estimated

MICs of 518 mg/L and 124 mg/L, respectively, for Pb.

Ca10(PO4)6F2 + 12H+ 10Ca2+ + 6H2PO4- + 2F- (1)

The higher level of resistance to Pb by P. marginalis was

For example, the acid produced by microorganisms or

attributed to greater production of extracellular polymer

other abiotic processes in soils has been shown to dissolve

which serves to immobilize and sequester Pb. In another

rock phosphate. This is one reason why rock phosphate

study to examine heavy metal resistance, Bar et al. (2007)

is highly effective as a nutrient source in acidic soils

isolated Klebsiella pneumonia from polluted river with a

(pH < 6.5) (Bolan and Hedley, 1990; Wang et al., 2007)

MIC of 41 mg/L for Pb in citrate mineral medium.

and as a metal-immobilizing agent in acid mine drainage

(Evangelou and Zhang, 1995; Harris and Lottermoser,

Solubilization of P

2006). Similarly, Kumar et al. (2008) isolated a metal-

The two isolated PSB strains were characterized

tolerant, plant growth-promoting bacteria (Enterobacter

with respect to halo formation, medium pH, solubilized P

sp.) which decreased the pH of the growth medium from

concentration, acid phosphatase activity, and production of

7 to 2, thereby achieving the maximum solubilization of

organic acids in culture medium (Table 1). Strain CS2-B1

P (229 mg/L). Chen et al. (2006b) also showed that the P

and SM1-B1 solubilized 479 and 293 mg/L, respectively,

solubilizing activity of isolated strains was related to the

of P from insoluble tricalcium phosphate. Depending on

release of organic acids and the subsequent pH reduction

the composition of the bacterial medium and final pH of

in the medium. In the present study, a significant inverse

the cultured medium, bacterial P solubilization reported

relationship between P solubilization and pH was observed

in the literature ranges from 31.5 mg/L to 898 mg/L

(data was not shown). The effect of organic acids on the

(Chen et al., 2006b; Chung et al., 2005; Son et al., 2006;

solubilization of P was demonstrated by Bolan et al. (1994).

Ma et al., 2009; Oliveira et al., 2009). For example, a

They noticed that the addition of organic acids, such as

Gordonia sp. was only capable of solubilizing 31.5 mg/L of

acetic and citric acids, increased the dissolution of North

P in NBRIP medium with a final pH of 6.0 after bacterial

Carolina Phosphate Rock and the difference in the extent

culture, whereas Pantoea agglomerans solubilized 898

of dissolution amongst organic acids was attributed to the

mg/L of P in Pikovskayas medium with a final of pH 2.6.

difference in the supply of protons and the sequestration

Although acid phosphatase activity is unlikely to influence

of dissolution products such as calcium.

mineral P solubilization directly, it may participate in

lowering pH, thereby indirectly contributing to mineral P
solubilization.

Water and citric acid solubility of P amendments

The solubility of P amendments is important because

It is well known that the production of organic acids

the dissolution of P is the primary factor in immobilizing

and acid phosphatase plays a major role in the solubilization

Pb from contaminated soils (Ma and Rao, 1999). We

Table 1. Characterization of the two isolated PSB strains in NBRIP broth medium
Bacterial strain

Halo formation
on solid medium

pH

Solubilized P
(mg/L)

Acid phosphatase activity

(p-nitrophenol (mM))

Organic acid
(mg/L)

Control

7.100.11

6.60.9

0.01440.0018

4913

CS2-B1

Yes
(2 mm)

4.310.07

479.212.4

0.14200.0000

601143

SM1-B1

Yes
(3 mm)

4.600.38

293.10.6

0.14010.0169

31467

J. Park et al.: Immobilization of Pb by phosphate-solubilizing bacteria

71

Table 2. Water and citric acid solubility of tricalcium phosphate, hydroxyapatite, and rock phosphate
P compound

Total P (%)

Water soluble P

Citric acid soluble P

(% w/w)

(% total P)

(% w/w)

(% total P)

N.D.*

N.D. *

12.40.14

62.10.68

18.5

N.D.*

N.D. *

12.50.26

67.51.41

10

N.D. *

N.D. *

3.10.02

31.40.23

Tricalcium phosphate

20.0

Hydroxyapatite
Rock phosphate

*N.D.; Not detected. Detection limit: 0.02 mg P/kg

Table 3. Physicochemical properties of the soil used for Pb immobilization

pH

EC (S/cm)

Total Pb (mg/kg)

NH4NO3-extractable Pb (mg/kg)

Olsen P (mg/kg)

5.880.09

34.70.9

346.154

28.72.7

3.780.52

measured the water solubility of tricalcium phosphate,

hydroxyapatite, and rock phosphate (Table 2) and as
expected, the water soluble P concentration was below
the limit of detection for these three P sources. However,
as indicated by citric acid solubility, hydroxyapatite and
rock phosphate were found to be the most and least
soluble, respectively (Table 2). Based on the 2% citric acid
solubility test for rock phosphates, all P sources used in
this study were considered as reactive (> 30% of total
P soluble in citric acid; (Hedley and Bolan, 1997)) (Table
2). The effectiveness of rock phosphate as a source of P
depends on both the solubility, as measured by citric acid
solubility, and particle size (Szilas et al., 2008). The surface
area of rock phosphate increases with decreasing particle
size, thereby resulting in greater dissolution in soils. The
rock phosphate tested in this study was finely ground (<
200 m), which may be one reason for its high solubility.
Immobilization of Pb by PSB in P amended soil
We next examined the effect of amending soil with
the three insoluble P sources in the presence and absence
of PSB on the immobilization of Pb (Figure 1). The
properties of the soil used for the immobilization tests are
summarized in Table 3. The soil was slightly acidic and
had a high concentration of bioavailable Pb concentration

Figure 1. Immobilization of Pb in contaminated soil amended

with strains CS2-B1(a) and SM1-B1 (b) and three
P compounds (TP: Tricalcium phosphate, HA:
Hydroxyapatite, RP: Rock phosphate) after 14 days.

(8.3% of total Pb). In general, the NH4NO3-extractable

Pb concentration was lower in the presence than in the

with strain SM1-B1 appearing to have more pronounced

absence of P compounds, indicating that these compounds

effects (Figure 1 (b)) than strain CS2-B1 (Figure 1 (a)).

induced the immobilization of Pb. The inoculation of

As indicated by the decrease in NH4NO3-extractable

PSB into the soil in the presence of P compounds caused

Pb, the addition of CS2-B1 to soil resulted in a 16% and

a greater decrease in the concentration of NH4NO3-

14% increase in the amount of immobilized Pb in the

extractable Pb when compared to P compounds alone,

presence of tricalcium phosphate and rock phosphate,

72

J. Park et al. / Pedologist (2010) 67-75

respectively, but showed no significant effect (p<0.05)

pH of PSB-inoculated soil which contained these two P

with hydroxyapatite. The effect on Pb immobilization was

sources.

much more pronounced for strain SM1-B1, which increased

Ca3(PO4)2 + 4H+ 3Ca2+ + 2H2PO4- 

immobilization of Pb by 39% with tricalcium phosphate,

60% with hydroxyapatite, and 51% with rock phosphate

(2)

Ca5(PO4)3OH + 7H+ 5Ca2+ + 3H2PO4- + H2O (3)

when compared to soil without added bacteria. While the

more soluble tricalcium phosphate and hydroxyapatite P

Immobilization of either dissolved Pb or the

sources are likely to immobilize Pb rapidly, the addition of

Pb present in contaminated soils by apatite through

rock phosphate supplies a constant level of P slowly to soil

precipitation as Pb phosphate minerals has been well

solutions, thereby achieving long-term immobilization of

documented (Bolan et al., 2003). For rock phosphate, the

Pb in the environment (Ma and Rao, 1999).

primary mechanism for Pb immobilization involves the

It should be noted that the soils and P amendments

dissolution of rock phosphate and precipitation of Pb as

were not sterilized before conducting the immobilization

a carbonated fluoropyromorphite-like mineral (Ma et al.,

assays and hence, it was not possible to discriminate

1995). Such precipitates are more commonly identified

the contribution of indigenous bacteria to the P-induced

as hydroxypyromorphite or chloropyromorphite. The

immobilization of Pb. PSB were detected in hydroxyapatite-

reaction of dissolved Pb with apatite is thought to occur

and rock phosphate-amended soils even when the two

by two processes. In the first, Pb2+ can react with apatite

isolated PSB strains were not inoculated (Table 4). It

through hydroxyapatite [Ca10(PO4)6(OH)2] dissolution,

is likely that these indigenous PSB also contributed to

followed

P solubilization and immobilization of Pb in the soil not

[(Pb10(PO4)6(OH)2)], as described by Ma et al. (1993). In

amended with bacteria.

the second process, Pb2+ can substitute directly for Ca2+

by

precipitation

of

hydroxypyromorphite

The inoculation of PSB slightly increased the soil pH

in apatite (Adriano et al., 2004). However, using a range

in the presence of tricalcium phosphate and hydroxyapatite

of spectroscopic techniques, which included XRD, IR,

compared to soil without added PSB (Table 4). The

EXAFS, and SEM, Laperche and Traina (1998) showed

dissolution of phosphate sources, such as tricalcium

that even at low Pb2+ concentrations (24 M), decreases

phosphate and hydroxyapatite, consumes hydrogen ions

of aqueous Pb2+ by apatite resulted from the precipitation

according to Eq. 2 and 3 (Bohner et al., 1997; Bolan et

of pyromorphite and there was no evidence for co-

al., 2003), which may explain the observed increases in

precipitation as (Pb, Ca) apatite. Thus, the dissolution of

Table 4. P
 hosphate solubilizing bacterial population and pH of soil samples after two weeks of
incubation with CS2-B1 and SM1-B1
Sample*

CS2-B1

SM1-B1

PSB CFU/ g dry soil

pH

PSB CFU/ g dry soil

pH

4.25 x 103

5.710.19

5.800.02

SB

1.93 x 106

5.730.13

4.55 x 106

5.620.00

6.160.08

5.780.01

STPB

1.45 x 106

6.200.10

1.42 x 107

6.080.02

SHA

8.00 x 10

5.910.28

7.80 x 10

5.890.11

SHAB

6.45 x 106

5.920.26

2.70 x 106

6.070.13

SRP

5.00 x 102

5.780.35

5.00 x 104

5.900.21

SRPB

1.18 x 10

5.780.30

1.73 x 10

5.800.17

STP

*S
 , soil only; SB, soil+PSB; STP, soil+tricalcium phosphate; STPB, soil+tricalcium phosphate+PSB;
SHA, soil+hydroxyapatite; SHAB, soil+hydroxyapatite+PSB; SRP, soil+rock phosphate; SRPB,
soil+rock phosphate+PSB

J. Park et al.: Immobilization of Pb by phosphate-solubilizing bacteria

73

apatite is an important initial step in the immobilization

chemical immobilization treatments for reducing

of Pb as pyromorphite and for this reason, PSB play an

heavy metal transport in a smelter-contaminated

important role in the dissolution of phosphate rocks and

soil.Environmental Pollution,127:73-82.

subsequent immobilization of Pb.

Bell, J.M., Paton, J.C. and Turnidge, J. 1998. Emergence

of vancomycin-resistant enterococci in Australia:

Conclusions

Phenotypic and genotypic characteristics of isolates.

Journal of Clinical Microbiology, 36: 2187-2190.

The most effective P solubilizing strains isolated from

Bolan, N.S. and Hedley, M.J. 1990. Dissolution of phosphate

P-amended and Pb-contaminated soils were putatively

rocks in soils. 2. Effect of pH on the dissolution and plant

identified as Pantoea sp. and Enterobacter sp. The major

availability of phosphate rock in soil with pH dependent

mechanism of P solubilization by these PSB is considered

charge. Nutrient Cycling in Agroecosystems, 24: 125-

to involve pH reduction through the production of organic

134.

acids. Rock phosphate addition to Pb-contaminated soils

Bolan, N.S., Naidu, R., Mahimairaja, S. and Baskaran, S.

in the presence of PSB achieved the immobilization of Pb,

1994. Influence of low-molecular-weight organic acids

which represents a promising strategy for controlling the

on the solubilization of phosphates. In: Biology and

quantity of Pb that cycles annually in ecosystems. The

Fertility of Soils, 18: 311-319.

inoculation of PSB with P amendments in Pb-contaminated

Bolan, N.S., Adriano, D.C. and Naidu, R. 2003. Role of

soil enhances Pb immobilization by the solubilization of

phosphorus in (im)mobilization and bioavailability

P and subsequent formation of insoluble Pb phosphate

of heavy metals in the soil-plant system. Reviews on

precipitates. As immobilized Pb is unavailable to plants,

Environmental contamination and Toxicology, 177: 1-

the dietary intake of Pb by humans and herbivorous

44.

animals could be lowered through this bioremediation

Bohner, M., Lematre, J. and Ring, T.A. 1997. Kinetics of

dissolution of -tricalcium phosphate. Journal of Colloid

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Acknowledgements

Braithwaite, A.C., Eaton, A.C. and Groom, P.S. 1990.

Factors affecting the solubility of phosphate rock

This study was sponsored by the Cooperative

Research Centre for Contamination Assessment and

residues in 2% citric acid and 2% formic acid. Nutrient

Cycling in Agroecosystems, 23: 37-42.

Remediation of the Environment (CRC CARE), Australia

Chen, G.C., He, Z.L., Stoffella, P.J., Yang, X.E., Yu, S. and

in collaboration with UniSA. The authors thank Dr. Z.

Calvert, D. 2006a. Use of dolomite phosphate rock

Chen and Dr. M. Rahman for their assistance with ICP

(DPR) fertilizers to reduce phosphorus leaching from

analysis.

sandy soil.Environmental Pollution,139:176-182.

Chen, Y.P., Rekha, P.D., Arun, A.B., Shen, F.T., Lai, W.A.

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