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Isolation and Characterization of Pb-Solubilizing Bacteria and Their Effects on Pb Uptake by Brassica juncea: Implications for Microbe-Assisted Phytoremediation
1Department of Soil Science, College of Agriculture, Isfahan University of Technology, Isfahan 84156-83111, Iran, 2Institute of Agricultural Sciences ICA-CSIC, 28006 Madrid, Spain, 3Centro de Estudios Avanzados en Zonas Áridas (CEAZA), La Serena 1700000, Chile, 4Department of Agricultural Biotechnology, College of Agriculture, Isfahan University of Technology, Isfahan 84156-83111, Iran
J. Microbiol. Biotechnol. 2018; 28(7): 1156-1167
Published July 28, 2018 https://doi.org/10.4014/jmb.1712.12038
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
Introduction
The growing industrialization and urbanization worldwide have resulted in serious environmental hazards due to the wide array of pollutants, including heavy metals, released into the soil environment. Although trace concentrations of certain heavy metals such as Fe, Mn, Zn, and Cu are essential for living cells, others like Cd, Hg, and Pb have no known biological or physiological functions and are even considered to be toxic not only to humans but to plants and microorganisms as well [1]. Among these toxic metals, Pb has the greatest detrimental impact on human health. Most Pb concentrations found in the environment are connected to such human activities as metal mining and smelting, lead battery production, leaded petroleum consumption, agricultural use of sewage sludge, and uncontrolled disposal of industrial wastes [2]. Chronic exposure to Pb increases the risks of high blood pressure, diseases of the nervous system and kidneys, hyperactivity, anemia, and infertility [2].
Accumulation of heavy metals in soils enhances their risk of entering the food chain via crop uptake. Humans and animals may also be exposed to these metals through inhalation of contaminated dust and consumption of contaminated waters [3]. To avoid the harmful effects of heavy metals, it is necessary to remediate metal-contaminated sites. Although effective in remediating soils contaminated with heavy metals, particularly those contaminated with high heavy-metal concentrations, traditional cleanup technologies (
Metal hyperaccumulators are mostly slow-growing plants with a small biomass, limiting the efficacy of phytoextraction in remediating metal-contaminated sites [6]. Plant growth and biomass production in contaminated soils, and thereby phytoextraction efficiency, can be improved by utilizing plant growth-promoting rhizobacteria (PGPR). PGPR have positive effects on plant performance via such varied mechanisms as phosphorus mobilization, atmospheric nitrogen fixation, iron sequestration by siderophores, and production of phytohormones including auxins like indole acetic acid (IAA) [7-9].
Phytoremediation efficiency is not only limited by the slow growth and low biomass production of hyperaccumulator plants but also by the low bioavailability of heavy metals in many contaminated soils [9, 10]. A large proportion of heavy metals present in soils is, indeed, either bound to the organic and inorganic soil constituents or precipitated as sparingly soluble minerals. Carbonates, hydroxides, and phosphates are the most important minerals that control the solubility of metals in soils within a pH range of 5.5-7.5 [11]. Adding chelating agents to soil has been suggested as a means of increasing metal availability to plants [12]. However, application of these expensive compounds has faced increasing criticism on the grounds of their phyto-and microorganism-toxicity traits [13, 14]. One promising alternative proposed to overcome this limitation is the bioaugmentation of contaminated soils with microorganisms that are able to increase metal solubility while also enhancing plant growth and biomass production, and thereby overall metal uptake by plants [8-10]. Jiang
Lots of works have been carried out using PGPR to improve metal phytoextraction, but studies on the application of those PGPR strains capable of metal solubilization to overcome low metal availability in soils, as a bottleneck in phytoremediation efficiency, are rare. Microbial strains with both metal-solubilizing and plant growth-promoting abilities may effectively be used for bioaugmentation-phytoremediation of heavy metal-contaminated soils. This study was designed and implemented in pursuit of the following objectives: (i) to isolate and characterize Pb-resistant bacteria from metal contaminated mine soils that are capable of both solubilizing PbCO3 and PbO as sparingly soluble Pb compounds, (ii) to examine the plant growth-promoting characteristics of the isolated Pb-solubilizing bacteria, and finally (iii) to evaluate the inoculation effects of the isolated bacteria on the growth and Pb uptake of Indian mustard (
Materials and Methods
Site Description and Soil Sampling
Two study sites located inside metal mining areas in Isfahan Province, Iran, were selected for soil sampling. Three soil samples were collected from the Bama Pb/Zn Mine (51° 32’ E and 32° 28’ N) and two from the Nakhlak Pb Mine (53° 50’ E and 33° 34’ N). A portion of the soil samples was kept in sterile zip-lock plastic bags and stored at 4°C for bacterial isolation and characterization procedures. Another portion was air-dried, passed through a sieve (2 mm), and analyzed for important soil properties. The soil pH and electrical conductivity (EC) were determined in 1:2 (soil:water) extracts with a glass electrode (Cyberscan 2100) and a conductivity meter (Elmetron CC-501), respectively. The soil organic carbon content was determined using the potassium dichromate-sulfuric acid method [18], total Pb and Zn concentrations were measured via digesting soil samples in 6N HNO3 [19], and bioavailable Cd, Cu, Pb, and Zn concentrations were estimated using the diethylenetriaminepentaacetic acid (DTPA)-CaCl2-triethanolamine extraction method [20]. Metal concentrations in the extracts were determined with a flame atomic absorption spectrophotometer (FAAS, Z-5300). Some of the properties and metal concentrations of the soils are listed in Table 1.
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Table 1 . Some characteristics of the soils that the Pb-solubilizing bacteria were isolated from.
Soila pHb ECb (dS m) OC (%) DTPA-extractable metals (mg kg) Total concentration of metals (mg kg) Cu Cd Pb Zn Pb Zn B1 7.4 0.86 1.27 2.70 0.72 696 225 5489 4200 B2 7.8 0.47 1.10 3.80 0.99 727 352 7909 4387 B3 7.6 1.68 2.85 1.00 0.45 174 79.80 1343 2325 N1 8.5 11.70 0.22 0.92 0.32 294 645 5128 3850 N2 7.6 17.90 0.08 0.75 0.17 688 11.10 1866 887 aB and N represent the Bama and Nakhlak mine soils, respectively.
bThe pH and EC were measured in a 1:2 soil:water suspension.
EC, electrical conductivity; OC, organic carbon; DTPA, diethylenetriaminepentaacetic acid.
Isolation and Purification of Pb-Tolerant Bacteria
The Pb-resistant bacteria were isolated using the Tris-buffered low-phosphate agar (TLP) medium containing sucrose as the carbon source to minimize Pb complexation to phosphate [21]. The medium was sterilized by autoclaving at 120°C for 20 min and subsequently supplemented with a filter-sterilized Pb(NO3)2 solution containing 50 mg-Pb/l. To prevent soil fungi growth, the media were supplemented with 10 mg/l of cycloheximide after autoclaving. The soil samples were then serially diluted with a sterile saline solution (0.85% NaCl) before 0.1 ml of each dilution was plated on the surface of the TLP medium. The bacterially inoculated plates were incubated at 30°C for up to 4 days, and individual colonies of different morphological characteristics, including color, size, form, opacity, and elevation, were selected and streaked on the same medium 3 to 4 times until pure cultures were obtained. The bacterial colonies were then regrown on Luria-Bertani (LB) medium and stored for further studies. A total of 171 bacterial isolates showing different morphological appearances on the agar medium were selected for studying the solubilization of Pb carbonate and oxide minerals.
Isolation of Pb-Solubilizing Bacteria
In the initial screening test for Pb tolerance, 110 Pb-resistant strains from the Bama Mine soils and 61 Pb-resistant strains from the Nakhlak Mine soils were isolated using a spread plate procedure and the pH-neutral TLP medium. These strains were subsequently screened to assess their potential to solubilize sparingly soluble Pb compounds (PbO and PbCO3). This ability was revealed by the appearance of clear haloes of solubilization around the colonies in the medium containing sucrose as the carbon source. For this purpose, inocula of the Pb-resistant bacterial isolates were prepared by centrifugation of 2 ml of logarithmic phase grown bacteria at 10,062 ×
Quantification of Pb Solubilization
Into 250-ml Erlenmeyer flasks containing 100 ml of the sterile liquid SLP, 500 mg/l of Pb as PbCO3 or 1,115 mg/l of Pb as PbO was added, and the medium was then inoculated with 1 ml of the logarithmic phase inoculum at approximately 108 CFU/ml. Media without metal addition and without bacterial inoculation were used as controls. The test flasks were incubated in a rotary shaker in the dark at 180 rpm and 30°C. After 10 days of incubation, 25 ml of the culture was centrifuged at 8,000 ×
Plant Growth-Promoting Properties of the Isolates
IAA production. The Pb-solubilizing bacterial strains were cultured in flasks containing 25 ml of LB medium supplemented with 0.5 mg/ml of L-tryptophan (filter-sterilized) at 30°C on an orbiter shaker at 150 rpm. After incubation for 24, 48, and 96 h, 1 ml of the cell-free suspension was mixed vigorously with 2 ml of Salkowski’s reagent [22] and allowed to stand at room temperature for 20 min. The absorbance of the pink color developed after 25 min of incubation was read at 530 nm. The IAA concentration in the culture was determined using a calibration curve of pure IAA.
Siderophore production. Siderophore produced by the isolated strains was detected according to the chrome azurol S (CAS) agar method described in Alexander and Zuberer [23]. The bacterial strains were grown twice in the Fe-free SLP medium before the inocula were prepared and adjusted to about 108 CFU/ml by suspending in a sterile saline solution. Subsequently, 40 µl of the inocula was spotted on CAS-agar plates and incubated in the dark for 10 days. Orange-yellow haloes around the colonies on the blue agar were interpreted as siderophore excretion.
Phosphate-solubilizing ability. The phosphate solubilization ability of the strains was determined in Pikovskaya’s Agar medium containing 0.5% tricalcium phosphate [24]. Forty microliters of each bacterial inoculum (about 108 CFU/ml) was spotted in Pikovskays’s Agar medium and incubated in the dark for 10 days. A clear halo around the colony was interpreted as the bacteria’s phosphate solubilization ability.
Genetic Identification
Characterization of each selected metal-solubilizing bacterium at the genus level was accomplished by partial sequencing of the 16S ribosomal DNA gene. Genomic DNA was extracted with the UltraClean Microbial DNA Isolation Kit (MO BIO Laboratories, Inc., USA) and amplified by the polymerase chain reaction (PCR) using the universal bacterial primers fD1 (5’-AGAGTTTGATCC TGGCTCAG-3’) and rP2 (5’-ACGGCTACCTTGTTACGACTT-3’)[25]. Each PCR contained 1 µl of gDNA and 0.4 µM (final concentration) of each primer in a final volume of 50 µl. The following PCR conditions were used: initial denaturing time of 5 min at 95°C, followed by 35 PCR cycles of 95°C for 1 min; 55°C, 1 min; and 72°C, 2 min; and a final extension step at 72°C for 10 min. Five microliters of the PCR products was subjected to electrophoretic investigation on a 1% TAE agarose gel and purified with the Ultra Clean PCR Clean-up Kit (MO BIO Laboratories, Inc.,) according to the manufacturer’s instructions. The purified PCR products were sequenced by Secugen (Madrid, Spain). Pairwise alignments were conducted using DNASTAR Lasergene (ver. 7.00) and the 16S rRNA gene sequences thus obtained were compared with those in the GenBank Database using the BLAST program from the NCBI website to determine the percent similarity.
Maximum Tolerance Concentration (MTC) of the Isolates
The maximum tolerance of the selected isolates against increasing concentrations of heavy metals (Zn, Pb, Cd, and Cu) was evaluated on SLP medium at pH 6.8 until the strains were unable to grow colonies on the agar plates. The maximum concentration of heavy metals at which the bacteria still presented growth was designated as the MTC. For this purpose, 10 µl of the logarithmic phase bacterial culture at approximately 108 CFU/ml was drop-inoculated onto Petri dishes in three replicates. A 100 mM stock solution of each metal was prepared using CdCl2•2H2O, CuSO4•5H2O, ZnSO4•7H2O, and Pb(NO3)2 and filtered through a 0.22 µm Millipore filter. The plates were incubated at 28°C for up to 10 days. This experiment was repeated twice in three replicates [26].
Isolate Tolerance to Acid and Alkaline pH and Sodium Chloride
Experiments were performed to monitor the ranges of pH and NaCl concentration at which the isolated bacterial strains maintained their ability to grow on the SLP agar medium [27]. For this purpose, the medium was buffered with 0.1 M sodium acetate/acetic acid at pH 4.5, 25 mM MES at pH 5.5 and 6.5, 25 mM HEPES at pH 7.5, 25 mM Tris-HCl at pH 8.5, and 25 mM sodium carbonate at pH 9.5 and 10.5. Subsequently, 10 µl of the exponential phase growing bacteria in the LB medium, at approximately 108 CFU/ml, was drop-inoculated on the medium and incubated at 28°C for up to 10 days. In the case of increasing salt concentration, NaCl from 3% up to 10% was added to the medium before autoclaving and the same amount of bacterial inoculum was used each time. These assays were repeated twice in three plates for each treatment.
Influence of Selected Isolates on the Growth and Pb Uptake of B. juncea
Preparation of bacterial inocula. Two isolated strains,
Soil. Soil collected from a metal-contaminated site around the Bama Pb/Zn Mine (51° 33’ 47/69’’E and 32° 30’ 45/79’’N) was air-dried and sieved to finer than 4 mm. It was characterized as a calcareous silty soil (15.6% sand, 83.5% silt, 0.9% clay) with pH 7.4 and an EC equal to 0.72 dS/m. The total Pb, Zn, and Cd concentrations of the soil were 446, 413, and 6.2 mg/kg, respectively, as determined by FAAS after soil digestion in 6N HNO3 [19].
Pot experiment.
Plant analysis. Plant growth parameters including shoot length as well as shoot and root dry weights were measured. The maximum shoot height was measured before harvesting on day 55 of the growth period. Subsequently, roots were separated and washed extensively with 1 mM Ca(NO3)2•4H2O and then with distilled water in order to remove surface-adsorbed Pb ions. Shoot and root dry matters were recorded after oven-drying at 75°C for 72 h.
The oven-dried plant samples were digested in a mixture of concentrated HNO3-HCl (70%) and H2O2 (30%) [28], and the Pb concentration in the extracts was determined using FAAS. Total Pb uptake by the plant shoots was calculated as the product of the shoot dry weight and Pb concentration.
Statistical Analysis
One-way analysis of variance followed by the least significant difference (LSD) test (
Results and Discussion
General Characteristics of the Metal-Contaminated Soils
The properties of the soils from which the bacteria were isolated are reported in Table 1. Soil pH values ranged from 7.4 to 8.5. The soils collected from the Bama Mine had EC values in the range of 0.47−1.68 dS/m whereas those of the Nakhlak Mine soil ranged from 11.7 to 17.9 dS/m. The total Pb concentration in the soils ranged from 1,343 to 7,909 mg/kg and the available (DTPA-CaCl2 extractable) fraction of Pb in the soils varied from 174 to 727 mg/kg. As judged from these values, all the soils were contaminated with Pb [29]. The values for total and available metals indicate that the soils were also contaminated with Zn and Cd [29].
Plant Growth-Promoting Characteristics of the Pb-Solubilizing Strains
IAA production. Plant growth enhancement by IAA is mostly due to its stimulating effect on root development, plant cell elongation, and cell division [30-32]. The root system enhancement, in turn, leads to a higher uptake of ions as a result of the larger soil volume made available to the roots.
All the Pb-solubilizing isolates, except YSP66 and YSP40n, were found capable of producing IAA in the LB medium supplemented with L-tryptophan (Table 2). The strains YSP40 and YSP24 recorded the highest IAA production rates (Table 2). Moreover, the strains YSP18, YSP24, YSP36, YSP40, and YSP151 produced IAA in the culture medium lacking L-tryptophan (Table 2). Since there are several tryptophan-independent pathways for IAA synthesis [33], it is possible that the latter strains were able to produce IAA without its major precursor, tryptophan. The strains YSP24, YSP40, YSP69, and YSP110 recorded reductions in their IAA content in the medium supplemented with tryptophan after 48-96 h (Table 2). This might have been due to the degradation of IAA by IAA oxidase secreted by the bacteria or to the storage of IAA in the bacterial cells as previously suggested [34]. The production of IAA has been reported to be common in
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Table 2 . Indole acetic acid (IAA) production by the Pb-solubilizing isolates after 24, 48, and 96 h of incubation.a
Isolate IAA (mg/l) LB medium LB medium+L-tryptophan (500 mg/l) 24h 48h 96h 24h 48h 96h YSP15 ND ND ND 0.67±0.09 pq 0.53±0.14 q 0.49±0.12 q YSP17 0.46±0.18 hj 0.39 hk 0.43±0.17 hk 4.69±0.43 hi 4.2±0.17 hk 4.25±0.29 hk YSP18 1.14±0.11 df 1.19±0.11 d 1.16±0.22 de 1.10±0.14 nq 1.61±0.13 lq 1.05±0.14 nq YSP24 ND b 1.03±0.11 dg 2.33±0.30 b 0.58±0.09 pq 72.72±2 b 58.88±2.78 d YSP36 1.24±0.33 d 0.88±0.10 g 0.88±0.09 g 0.92±0.30 pq 1.08±0.30 nq 1.03±0.32 nq YSP40 2.58±0.54 a 2.85±0.35 mn 1.51±0.15 n 6.64±0.68 g 77.71±3.33 a 61.65±5.49 c YSP40n ND 0.07±0.05 ln ND ND 0.12±0.03 q ND YSP66 ND ND 0.49±0.13 hi ND ND 0.97±0.24 oq YSP69 ND 0.19±0.06 kn ND ND 3.94±0.11 hk 1.14±0.18 nq YSP95 0.55±0.04 h 0.48±0.10 hi 0.41±0.08 hk 0.55±0.07 q 1.60±0.16 lq 1.51±0.17 mq YSP104 0.37±0.18 hk ND ND 3.41±0.42 hl 3.94±0.16 hk 3.89±0.37 hk YSP110 0.51±0.15 hi 0.47±0.15 hi 0.46±0.10 hj 1.39±0.20 nq 5.23±0.23 gh 3.78±0.12 hk YSP143 0.24±0.06 jm 0.21±0.06 kn 0.31±.03 il 2.41±0.11 kp 3.24±0.30 im 4.04±0.29 hk YSP149 0.45±0.13 hj 0.42±0.08 hk 0.43±0.12 hk 2.85±0.35 in 3.25±0.37 im 2.80±0.25 jo YSP151 0.93±0.20 eg 0.92±0.24 fg 1.62±0.14 c 4.45±0.41 hj 9.21±0.78 f 11.41±0.94 e aData represent the average of three replicates ± standard deviation. Means in each medium followed by the same letter are not significantly different according to LSD (
p < 0.05). ND: not detected.
Siderophore production. Bioaugmentation with siderophore-producing bacteria has been proposed as an efficient strategy to enhance plant growth and metal uptake in phytoremediation practices [35]. In addition, chelation of heavy metals by siderophores has been found to promote bacterial IAA synthesis through reduced oxidative degradation of IAA by free heavy metals [36].
Ten out of the 15 Pb-solubilizing strains were able to produce siderophores (Table 3). Siderophore production levels by the different bacterial strains were compared on the basis of the ratio of colony + haloe diameter to colony diameter. The strains YSP151 and YSP149 exhibited the highest capability for siderophore production (Table 3).
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Table 3 . Siderophore production and P solubilization abilities of the Pb-solubilizing isolates.
Isolate YSP15 YSP17 YSP18 YSP24 YSP36 YSP40 YSP40n YSP66 YSP69 YSP95 YSP104 YSP110 YSP143 YSP149 YSP151 Siderophore production a 0 1.12 0 0 1.92 1.55 0 0.8 1.67 1.00 0.33 1.41 0 2.25 3.83 P solubilizationb – – – + – – – – – + – – – – – aData represent the colony + haloes diameter/colony diameter ratio on CAS-agar medium.
b+, Clear halo produced on Pikovskaya’s medium; –, No clear halo on Pikovskaya’s medium.
Production of metal-chelating substances, such as organic acids and siderophores, plays a special role in metal detoxification, and thereby in increasing metal tolerance in bacteria. These chelating agents may form complexes with heavy metals that are considered to be less toxic so as to inactivate and minimize the cytological impacts of free metal ions [37]. Abou-Shanab
Phosphate solubilization. Only two Pb-solubilizing strains (YSP24 and YSP95) were able to produce clear haloes on Pikovskaya’s medium containing sparingly soluble Ca3(PO4)2 as the sole source of phosphate (Table 3). Low P availability may retard plant growth and metal uptake in phytoremediation practices of contaminated soils [24]. Therefore, selection of bacterial strains capable of increasing P solubility in these soils would be favorable for improving plant growth and, in turn, enhancing metal uptake from these soils [7].
Lead Solubilization Ability of the Isolates
Only 15 (8.8%) out of the 171 Pb-resistant isolates showed clear halos in agar plates amended with PbO or PbCO3. Hence, this ability was not a common feature among the isolated bacteria. Colonies that formed zones of clearance around the colony were selected for further studies.
The isolated strains that were able to form zones of clearance around the colony on the solid medium containing PbO or PbCO3 were also able to dissolve these minerals in the liquid medium. The results of Pb solubilization by the isolates and the associated pH changes in the liquid (SLP) medium are reported in Table 4. Clearly, maximum values of PbCO3 solubilization in the SLP medium were recorded in the presence of strain YSP17 (4.28 mg-Pb/l), followed by YSP104 (2.64 mg-Pb/l) and YSP69 (2.16 mg-Pb/l), which were 26.7, 16.5, and 13.5 times higher than that of the control, respectively (Table 4). In the PbO-containing SLP medium, the maximum value of Pb concentration was recorded with inoculation of the strains YSP69, YSP40n, and YSP110 (8.99, 8.73, 8.42 mg-Pb/l, respectively), which were more than 30 times higher than that of the control. The bacterial isolates might have increased dissolution of the Pb-bearing minerals through such mechanisms as ligand-promoted dissolution induced by the bacterial metabolites and proton-promoted dissolution triggered by a pH decrease [39, 40]. Nevertheless, the magnitude of metal solubilization by a bacterial isolate varies depending on the mineral composition and solubilization mechanism(s) exploited by the isolate. Li
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Table 4 . Total soluble Pb and final pH measured in PbCO3- and PbO-containing SLP broth medium after inoculation with Pbsolubilizing bacterial isolates.
Bacterial Isolate Pb liberateda (mg/l) Final pH of the medium PbCO3 PbO PbCO3 PbO Controlb YSP15 1.48±0.26 e 0.91±0.08 g 5.95±0.08 b 6.98±0.07 b 6.76±0.18 ab YSP17 4.28±0.15 a 2.13±0.44 ef 4.04±0.12 g 6.60±0.26 c 4.54±0.06 h YSP18 0.36±0.02 i 1.81±0.15 ef 5.71±0.12 b 5.60±0.17 e 6.85±0.09 a YSP24 1.98±0.18 cd 1.74±0.10 f 4.27±0.50 efg 4.01±0.16 h 4.81±0.07 g YSP36 1.65±0.38 de 6.73±0.47 b 4.46±0.14 de 4.21±0.03 h 5.72±0.17 c YSP40 1.61±0.20 de 2.35±0.25 de 4.94±0.14 c 6.26±0.13 d 6.53±0.08 b YSP40n 1.02±0.02 fg 8.73±0.38 a 4.38±0.11 def 4.27±0.05 h 5.45±0.06 de YSP66 1.45±0.39 e 0.84±0.08 gh 4.07±0.08 fg 6.01±0.13 d 5.26±0.14 ef YSP69 2.16±0.02 c 8.99±0.26 a 4.43±0.19 de 4.27±0.14 h 4.49±0.13 h YSP95 0.21±0.11 i 2.18±0.72 ef 5.93±0.18 b 5.12±0.16 g 5.1±0.16 f YSP104 2.64±0.41 b 2.86±0.20 d 4.63±0.37 cd 5.54±0.11 ef 5.73±0.15 c YSP110 0.81±0.02 gh 8.42±0.76 a 4.15±0.13 efg 4.20±0.19 h 5.26±0.09 ef YSP143 0.47±0.03 hi 0.67±0.08 gh 4.46±0.17 de 5.65±0.30 e 5.58±0.05 cd YSP149 1.98±0.06 cd 0.81±0.21 gh 4.60±0.15 d 6.59±0.05 c 4.19±0.18 i YSP151 1.36±0.42 ef 5.82±0.52 c 4.43±0.11 de 5.29±0.17 fg 5.18±0.20 f Controlc 0.16±0.10 i 0.27±0.05 h 7.56±0.06 a 7.36±0.18 a 6.90±0.10 a aSLP medium supplemented with 1,115 mg/l PbO or 500 mg/l PbCO3 and inoculated with different bacterial strains.
bSLP medium inoculated with different bacterial strains without Pb salts.
cSLP medium supplemented with 1,115 mg/l PbO or 500 mg/l PbCO3 without bacterial inoculation.
Means in each column followed by the same letter are not significantly different according to LSD (
p < 0.05).
Bacterial-induced metal solubilization of minerals has frequently been attributed to ligand-promoted dissolution of minerals caused by bacterial metabolites such as sidero-phores, carboxylic organic acid anions, and biosurfactants [41]. Abou-Shanab
The pH of the SLP medium inoculated with the Pb-solubilizing bacterial strains decreased both in the presence and absence of Pb minerals (Table 4), suggesting the role of the bacterial strains in producing H+ probably through organic acid secretion [7, 15]. Bacteria may also acidify their environment by the export of protons for the maintenance of charge balance [42]. The greatest drops in pH level (about 3 units) in the control SLP medium were induced by the strains YSP149 and YSP69. In the PbO-containing medium, the largest decrease in pH was measured in the presence of YSP24, whereas YSP17 and YSP66 gave rise to the largest pH decreases in the PbCO3-containing medium. These results are in agreement with those reported by Ji
Molecular Identification of the Pb-Solubilizing Bacteria
Table 5 presents the isolated Pb-solubilizing strains identified on the basis of 16S rRNA gene sequences. The size of the PCR products generated with the selected bacterial isolates ranged from approximately 784 to 1,351 bp. Based on a sequence identity of 97% or greater, the closest matches of the isolates were determined using BLAST. The results show a preponderance of the
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Table 5 . Identification of isolated Pb-solubilizing bacteria.
Isolates Source soil Length of 16S rRNA gene sequenced (bp) GenBank Accession No. Most closely related organism Species (strain) Accession No. % Gene identity YSP17 Nakhlak mine 1173 KY887777 Bacillus mojavensis (strain ifo 15718 )NR_118290.1 97 YSP24 Nakhlak mine 1346 KY887778 Paenibacillus illinoisensis (strain NBRC 15959)NR_113828.1 99 YSP36 Nakhlak mine 1065 KY887779 Bacillus simplex (strain RD36)KJ_534463.1 97 YSP40 Nakhlak mine 784 KY887780 Brevibacterium frigoritolerans (strain DSM 8801)NR_117474.1 99 YSP40n Nakhlak mine 1351 KY887781 Staphylococcus pasteuri (strain ATCC51129)NR_024669.1 100 YSP69 Bama mine 1313 KY887782 Bacillus cereus (strain CCM2010 )NR_115714.1 99 YSP104 Bama mine 1158 KY887783 Bacillus altitudinis (strain 41KF2b)NR_042337.1 100 YSP110 Bama mine 1156 KY887784 Bacillus filamentosus (strain SGD-14)NR_134701.1 99 YSP149 Bama mine 1219 KY887785 Bacillus licheniformis (strain ATCC14580)NR_074923.1 99 YSP151 Bama mine 1012 KY887786 Bacillus paralicheniformis (strain kj-16)NR_137421.1 99
Tolerance to Heavy Metals, NaCl, and pH
Among the 15 Pb-solubilizing isolates, 10 strains were selected for further experiments according to their best performance in Pb solubilization and PGPR traits. Mine soils most often contain more than one metal, and tolerance to several heavy metals is a desirable trait for bacteria with potential use in phytoremediation. All the isolates tested were found to exhibit multiple tolerance to high concentrations of metals, especially to Pb and Zn (Table 6). The highest MTC values were detected for Pb (≥207.2 mg/l), indicating that the isolates were highly resistant to Pb. YSP69, YSP104, and YSP110 were the most tolerant to Pb, but also to Zn, Cu, and Cd. Sheng
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Table 6 . Maximum tolerance concentration of the Pb-solubilizing isolates against heavy metals and NaCl and ability of the isolates to grow in different pH ranges.
Isolate Pb (mg/l) Zn (mg/l) Cu (mg/l) Cd (mg/l) NaCl (%) pH range YSP17 207.20 65.38 – 22.48 9 5.5–10.5 YSP24 207.20 65.38 – 11.24 5 5.5–10.5 YSP36 207.20 13.07 – – 3 5.5–10.5 YSP40 207.20 65.38 6.35 – 3 5.5–10.5 YSP40n 207.20 163.45 – 11.24 8 5.5–9.5 YSP69 414.40 130.76 12.71 224.82 8 5.5–10.5 YSP104 414.40 196.14 6.35 112.41 8 5.5–10.5 YSP110 414.40 130.76 6.35 112.41 10 5.5–10.5 YSP149 207.20 65.38 6.35 112.41 10 5.5–10.5 YSP151 207.20 68.38 6.35 11.24 9 5.5–10.5 –, No bacterial growth observed.
The Pb-solubilizing bacterial strains tested displayed a notable tolerance to salt stress while they were also able to grow well in a pH range of 5.5–10.5, except for YSP40n that did not grow at pH levels higher than 9.5 (Table 6). Bioaugmentation by salt-tolerant bacteria can both promote plant growth and help extract heavy metals in phytoremediation practices of salt-affected, metal-contaminated soils of arid regions [49]. The pH and salt tolerance of the Pb-solubilizing strains tested points to the feasibility of using them in contaminated soils also affected by moderately acid or alkaline pH levels and/or elevated salinity.
Effects of Pb-Solubilizing Strains on the Growth and Pb Uptake of B. juncea
Effects of
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Table 7 . Inoculation effects of
Brevibacterium frigoritolerans YSP40 andBacillus paralicheniformis YSP151 strains and their consortium on the growth and Pb absorption ofBrassica juncea .Inoculated strain Shoot length (cm) Shoot dry weight (g/pot) Root dry weight (g/pot) Shoot Pb concentration (mg/kg) Root Pb concentration (mg/kg) Pb uptake by shoot (μg/pot) Non (control) 12.30±2.08 b 1.84±0.79 c 0.16±0.19 a 38.23±2.9 c 96.93±6 b 69.01±24.73 c YSP40 34.83±7.09 a 3.99±0.59 ab 0.39±0.05 a 55.31±10.1 b 79.55±6.5 b 224.1±67.36 b YSP151 35.68±2.17 a 4.38±0.55 a 0.82±0.82 a 73.6±13.6 a 141.7±35.2 a 319.7±48.62 a YSP40+YSP151 32.30±1.88 a 2.89±0.36 bc 0.38±0.06 a 67.16±5.3 ab 80.94±5.7 b 193.1±14.82 b Values are the mean ± standard deviation. Means in each column followed by the same letter are not significantly different according to LSD (
p < 0.05).
Table 7 reports Pb accumulation levels in the roots and shoots of
The total Pb uptake by plant shoots was 3.2 and 4.6 times higher than that of the control when the plants were inoculated with
The present study highlighted the role of bacterial strains isolated from mine soils in increasing soil metal availability and uptake by plants, for remediating metal-contaminated sites more efficiently. Since the potential of a plant for use as a phytoremediator in a metal-contaminated soil relies on the total metal quantity taken up by the harvestable part of the plant biomass, inoculation of the metal-solubilizing and plant growth-promoting strains, such as
Acknowledgments
This work was supported by grants from Isfahan University of Technology and MINECO (AGL2013-40758-R).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
References
- Nagajyoti PC, Lee KD, Sreekanth TVM. 2010. Heavy metals, occurrence and toxicity for plants: a review.
Environ. Chem. Lett. 8 : 199-216. - Thornton I, Rautiu R, Brush S. 2001. Lead: The Facts. Ian Allan Printing Ltd., London.
- Triantafyllidou S, Edwards M. Lead (Pb) in U.S. drinking water: school case studies, detection challenges and public health considerations. Your Drinking Water: Challenges and Solutions for the 21st Century. Drinking Water Symposium, April 20th & 21st, 2009. Yale University.
- Dermont G, Bergeron M, Mercier G, Richer-Laflèche M. 2008. Soil washing for metal removal: a review of physical/chemical technologies and field applications.
J. Hazard. Mater. 152 : 1-31. - Jabeen R, Ahmad A, Iqbal M. 2009. Phytoremediation of heavy metals: physiological and molecular mechanisms.
Bot. Rev. 75 : 339-364. - Shao HB, Chu LY, Ni FT, Guo DG, Li H, Li WX. 2010. Perspective on phytoremediation for improving heavy metal-contaminated soils, pp. 227-244.
In: Ashraf M, Ozturk M, Ahmad MSA (eds),Plant Adaptation and Phytoremediation . Springer, Dordrecht. - Jiang CY, Sheng XF, Qian M, Wang QY. 2008. Isolation and characterization of a heavy metal-resistant Burkholderia sp. from heavy metal-contaminated paddy field soil and its potential in promoting plant growth and heavy metal accumulation in metal-polluted soil.
Chemosphere 72 : 157-164. - Sheng XF, Jiang CY, He LY. 2008. Characterization of plant growth-promoting
Bacillus edaphicus NBT and its effect on lead uptake by Indian mustard in a lead-amended soil.Can. J. Microbiol. 54 : 417-422. - Sheng XF, Xia JJ. 2006. Improvement of rape (
Brassica napus ) plant growth and cadmium uptake by cadmium-resistant bacteria.Chemosphere 64 : 1036-1042. - Sheng X-F, Xia JJ, Jiang CY, He LY, Qian M. 2008. Characterization of heavy metal-resistant endophytic bacteria from rape (
Brassica napus ) roots and their potential in promoting the growth and lead accumulation of rape.Environ. Pollut. 156 : 1164-1170. - Lindsay WL. 1979. Chemical Equilibrium in Soils. Wiley Interscience Publishers, New York.
- Zhao S, Jia L, Duo L. 2013. The use of biodegradable chelators for enhanced phytoextraction of heavy metals by
Festuca arundinacea from municipal solid waste compost and associated heavy metal leaching.Bioresour. Technol. 129 : 249-255. - Evangelou MWH, Bauer U, Ebel M, Schaeffer A. 2007. The influence of EDDS and EDTA on the uptake of heavy metals of Cd and Cu from soil with tobacco,
Nicotiana tabacum .Chemosphere 68 : 345-353. - Usman ARA, Almaroai YA, Ahmad M, Vithanage M, Ok YS. 2013. Toxicity of synthetic chelators and metal availability in poultry manure amended Cd, Pb and As contaminated agricultural soil.
J. Hazard. Mater. 262 : 1022-1030. - Fasim F, Ahmed N, Parsons R, Gadd GM. 2002. Solubilization of zinc salts by a bacterium isolated from the air environment of a tannery.
FEMS Microbiol. Lett. 213 : 1-6. - Long XX, Chen XM, Chen YG, Wong JW-C, Wei ZB, Wu QT. 2011. Isolation and characterization endophytic bacteria from hyperaccumulator
Sedum alfredii Hance and their potential to promote phytoextraction of zinc polluted soil.World J. Microbiol. Biotechnol. 27 : 1197-1207. - Braud A, Jézéquel K, Vieille E, Tritter A, Lebeau T. 2006. Changes in extractability of Cr and Pb in a poly contaminated soil after bioaugmentation with microbial producers of biosurfactants, organic acids and siderophores.
Water Air Soil Pollut. 6 : 261-279. - Nelson DW, Sommers LE. 1996. Total carbon, organic carbon, and organic matter, pp. 961-1010.
In: Sparks DL (ed),Method of Soil Analysis, Part 3, Chemical Methods . Soil Science Society of America, Inc., American Society of Agronomy, Madison, WI. - Zheljazkov VD, Nielsen NE. 1996. Effect of heavy metals on peppermint and cornmint.
Plant Soil 178 : 59-66. - Lindsay WL, Norvell WA. 1978. Development of a DTPA soil test for zinc, iron, manganese, and copper.
Soil Sci. Soc. Am. J. 42 : 421-428. - Mergeau MD, Nies HG, Schlegel J, Gerits P, Charles van Gijsegem F. 1985.
Alcaligenes eutrophus CH34 is a facultative chemolitotroph with plasmid-bound resistance to heavy metals.J. Bacteriol. 162 : 328-334. - Gordon SA, Weber RP. 1951. Colorimetric estimation of indole acetic acid.
Plant Physiol. 26 : 192-195. - Alexander DB, Zuberer DA. 1991. Use of chrome Azurol-S reagents to evaluate siderophore production by rhizosphere bacteria.
Biol. Fertil. Soils 12 : 39-45. - Zaidi S, Usmani S, Singh BR, Musarrat J. 2006. Significance of
Bacillus subtilis strain SJ-101 as a bio inoculant for concurrent plant growth promotion and nickel accumulation inBrassica juncea .Chemosphere 64 : 991-997. - Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 1991. 16S ribosomal DNA amplification for phylogenetic study.
J. Bacteriol. 173 : 697-703. - Affan QA, Shoeb E, Badar U, Akhtar J. 2009. Isolation and characterization of bacterial isolates having heavy metal tolerance.
J. Basic Appl. Sci. 5 : 55-60. - Nonnoi F, Chinnaswamy A, de la Torre VSG, de la Peña TC, Lucas MM, Pueyo JJ. 2012. Metal tolerance of rhizobial strains isolated from nodules of herbaceous legumes (
Medicago spp. andTrifolium spp.) growing in mercurycontaminated soils.Appl. Soil Ecol. 61 : 49-59. - Jones JB Jr, Case VW. 1990. Sampling, handling, and analyzing plant tissue samples, pp. 389-447.
In: Westerman RL (ed),Soil Testing and Plant Analysis . Soil Science Society of America, Inc., Madison, WI. - NYS. New York State Brownfield Cleanup Program Development of Soil Cleanup Objectives Technical Support Document, Albany, NY: New York State Department of Environmental Conservation and New York State Department of Health; 2006. Available from http://www.dec.ny.gov/chemical/34189.html.
- Davies PJ. 2010. The Plant Hormones: Their Nature, Occurrence and Functions, 3rd Ed. Kluwer Academic Publishers, New York.
- Malhotra M, Srivastava S. 2009. Stress-responsive indole-3-acetic acid biosynthesis by
Azospirillum brasilense SM and i ts ability to modulate plant growth.Eur. J. Soil Biol. 45 : 73-80. - Patten CL, Glick BR. 2002. The role of bacterial indole acetic acid in the development of the host plant root system.
Appl. Environ. Microbiol. 68 : 3795-3801. - Ribeiro CM, Cardoso EJBN. 2012. Isolation, selection and characterization of root-associated growth promoting bacteria in Brazil Pine (
Araucaria angustifolia ).Microbiol. Res. 167 : 69-78. - Ji LY, Zhang WW, Yu D, Cao YR, Xu H. 2012. Effect of heavy metal-solubilizing microorganisms on zinc and cadmium extractions from heavy metal contaminated soil with
Tricholoma lobynsis .World J. Microbiol. Biotechnol. 28 : 293-301. - Rajkumar M, Ae N, Narasimha M, Prasad V, Freitas H. 2009. Potential of siderophore-producing bacteria for improving heavy metal phytoextraction.
Trends Biotechnol. 28 : 142-149. - Dimkpa CO, Merten D, Svatoš A, Büchel G, Kothe E. 2009. Metal-induced oxidative stress impacting plant growth in contaminated soil is alleviated by microbial siderophores.
Soil Biol. Biochem. 41 : 154-162. - Gao Y, Miao C, Mao L, Zhou P, Jin Z, Shi W. 2010. Improvement of phytoextraction and antioxidative defense in
Solanum nigrum L. under cadmium stress by application of cadmium-resistant strain and citric acid.J. Hazard. Mater. 181 : 771-777. - Abou-Shanab RA, Ghozlan H, Ghanem K, Moawad H. 2005. Behavior of bacterial populations isolated from rhizosphere of
Diplachne fusca dominant in industrial sites.World J. Microbiol. Biotechnol. 21 : 1095-1101. - Li WC, Ye ZH, Wong MH. 2010. Metal mobilization and production of short-chain organic acids by rhizosphere bacteria associated with a Cd/Zn hyperaccumulating plant, Sedum alfredii.
Plant Soil 326 : 453-467. - Ma Y, Prasad MNV, Rajkumar M, Freitas H. 2011. Plant growth promoting rhizobacteria and endophytes accelerate phytoremediation of metalliferous soils.
Biotechnol. Adv. 29 : 248-258. - Sessitsch A, Kuffner M, Kidd P, Vangronsveld J, Wenzel WW, Fallmann K,
et al . 2013. The role of plant-associated bacteria in the mobilization and phytoextraction of trace elements in contaminated soils.Soil Biol. Biochem. 60 : 182-194. - Abou-Shanab RA, Ghanem K, Ghanem N, Al-Kolaibe A. 2008. The role of bacteria on heavy-metal extraction and uptake by plants growing on multi-metal-contaminated soils.
World J. Microbiol. Biotechnol. 24 : 253-262. - Braud A, Jézéquel K, Bazot S, Lebeau T. 2009. Enhanced phytoextraction of an agricultural Cr- and Pb-contaminated soil by bioaugmentation with siderophore-producing bacteria.
Chemosphere 74 : 280-286. - Dimkpa CO, Merten D, Svatoš A, Büchel G, Kothe E. 2009. Siderophores mediate reduced and increased uptake of cadmium by
Streptomyces tendae F4 and sunflower (Helianthus annuus ), respectively.J. Appl. Microbiol. 107 : 1687-1696. - Dhal B, Thatoi H, Das N, Pandey BD. 2010. Reduction of hexavalent chromium by
Bacillus sp. isolated from chromite mine soils and characterization of reduced product.J. Chem. Technol. Biotechnol. 85 : 1471-1479. - Govarthanan M, Lee KJ, Cho M, Kim JS, Kamala-Kannan S, Oh BT. 2013. Significance of autochthonous
Bacillus sp. KK1 on biomineralization of lead in mine tailings.Chemosphere 90 : 2267-2272. - Seralathan K, Kui JL. 2008. Metal tolerance and antibiotic resistance of
Bacillus species isolated from Sunchon Bay, South Korea.Biotechnology 7 : 149-152. - Sheng XF, He LY, Wang QY, Ye HS, Jiang CY. 2008. Effects of inoculation of biosurfactant-producing
Bacillus sp. J119 on plant growth and cadmium uptake in a cadmium-amended soil.J. Hazard. Mater. 155 : 17-22. - Teixeira C, Almeida CMR, Nunes da Silva M, Bordalo AA, Mucha AP. 2014. Development of autochthonous microbial consortia for enhanced phytoremediation of salt-marsh sediments contaminated with cadmium.
Sci. Total Environ. 493 : 757-765. - Kumar KV, Singh N, Behl NH, Srivastava S. 2008. Influence of plant growth promoting bacteria and its mutant on heavy metal toxicity in
Brassica juncea grown in fly ash amended soil.Chemosphere 72 : 678-683. - Tangahu BV, Abdullah SRS, Basri H, Idris M, Anuar N, Mukhlisin MA. 2011. Review on heavy metals (As, Pb, and Hg) uptake by plants through phytoremediation.
Int. J. Chem. Eng. 2011 : 939161. - Kumari B, Singh SN. 2011. Phytoremediation of metals from fly ash through bacterial augmentation.
Ecotoxicology 20 : 166-176. - Burd GI, Dixon DG, Glick BR. 1998. A plant growth-promoting bacterium that decreases nickel toxicity in seedlings.
Appl. Environ. Microbiol. 64 : 3663-3668.
Related articles in JMB

Article
Research article
J. Microbiol. Biotechnol. 2018; 28(7): 1156-1167
Published online July 28, 2018 https://doi.org/10.4014/jmb.1712.12038
Copyright © The Korean Society for Microbiology and Biotechnology.
Isolation and Characterization of Pb-Solubilizing Bacteria and Their Effects on Pb Uptake by Brassica juncea: Implications for Microbe-Assisted Phytoremediation
Zahra Yahaghi 1, 2, Mehran Shirvani 1*, Farshid Nourbakhsh 1, Teodoro Coba de la Peña 2, 3, José J. Pueyo 2 and Majid Talebi 4
1Department of Soil Science, College of Agriculture, Isfahan University of Technology, Isfahan 84156-83111, Iran, 2Institute of Agricultural Sciences ICA-CSIC, 28006 Madrid, Spain, 3Centro de Estudios Avanzados en Zonas Áridas (CEAZA), La Serena 1700000, Chile, 4Department of Agricultural Biotechnology, College of Agriculture, Isfahan University of Technology, Isfahan 84156-83111, Iran
Abstract
The aim of this study was to isolate and characterize lead (Pb)-solubilizing bacteria from
heavy metal-contaminated mine soils and to evaluate their inoculation effects on the growth
and Pb absorption of Brassica juncea. The isolates were also evaluated for their plant growthpromoting
characteristics as well as heavy metal and salt tolerance. A total of 171 Pb-tolerant
isolates were identified, of which only 15 bacterial strains were able to produce clear haloes in
solid medium containing PbO or PbCO3, indicating Pb solubilization. All of these 15 strains
were also able to dissolve the Pb minerals in a liquid medium, which was accompanied by
significant decreases in pH values of the medium. Based on 16S rRNA gene sequence analysis,
the Pb-solubilizing strains belonged to genera Bacillus, Paenibacillus, Brevibacterium, and
Staphylococcus. A majority of the Pb-solubilizing strains were able to produce indole acetic acid
and siderophores to different extents. Two of the Pb-solubilizing isolates were able to
solubilize inorganic phosphate as well. Some of the strains displayed tolerance to different
heavy metals and to salt stress and were able to grow in a wide pH range. Inoculation with
two selected Pb-solubilizing and plant growth-promoting strains, (i.e., Brevibacterium
frigoritolerans YSP40 and Bacillus paralicheniformis YSP151) and their consortium enhanced the
growth and Pb uptake of B. juncea plants grown in a metal-contaminated soil. The bacterial
strains isolated in this study are promising candidates to develop novel microbe-assisted
phytoremediation strategies for metal-contaminated soils.
Keywords: Metal-solubilizing bacteria, phytoremediation, Lead, contaminated soils, PGPR
Introduction
The growing industrialization and urbanization worldwide have resulted in serious environmental hazards due to the wide array of pollutants, including heavy metals, released into the soil environment. Although trace concentrations of certain heavy metals such as Fe, Mn, Zn, and Cu are essential for living cells, others like Cd, Hg, and Pb have no known biological or physiological functions and are even considered to be toxic not only to humans but to plants and microorganisms as well [1]. Among these toxic metals, Pb has the greatest detrimental impact on human health. Most Pb concentrations found in the environment are connected to such human activities as metal mining and smelting, lead battery production, leaded petroleum consumption, agricultural use of sewage sludge, and uncontrolled disposal of industrial wastes [2]. Chronic exposure to Pb increases the risks of high blood pressure, diseases of the nervous system and kidneys, hyperactivity, anemia, and infertility [2].
Accumulation of heavy metals in soils enhances their risk of entering the food chain via crop uptake. Humans and animals may also be exposed to these metals through inhalation of contaminated dust and consumption of contaminated waters [3]. To avoid the harmful effects of heavy metals, it is necessary to remediate metal-contaminated sites. Although effective in remediating soils contaminated with heavy metals, particularly those contaminated with high heavy-metal concentrations, traditional cleanup technologies (
Metal hyperaccumulators are mostly slow-growing plants with a small biomass, limiting the efficacy of phytoextraction in remediating metal-contaminated sites [6]. Plant growth and biomass production in contaminated soils, and thereby phytoextraction efficiency, can be improved by utilizing plant growth-promoting rhizobacteria (PGPR). PGPR have positive effects on plant performance via such varied mechanisms as phosphorus mobilization, atmospheric nitrogen fixation, iron sequestration by siderophores, and production of phytohormones including auxins like indole acetic acid (IAA) [7-9].
Phytoremediation efficiency is not only limited by the slow growth and low biomass production of hyperaccumulator plants but also by the low bioavailability of heavy metals in many contaminated soils [9, 10]. A large proportion of heavy metals present in soils is, indeed, either bound to the organic and inorganic soil constituents or precipitated as sparingly soluble minerals. Carbonates, hydroxides, and phosphates are the most important minerals that control the solubility of metals in soils within a pH range of 5.5-7.5 [11]. Adding chelating agents to soil has been suggested as a means of increasing metal availability to plants [12]. However, application of these expensive compounds has faced increasing criticism on the grounds of their phyto-and microorganism-toxicity traits [13, 14]. One promising alternative proposed to overcome this limitation is the bioaugmentation of contaminated soils with microorganisms that are able to increase metal solubility while also enhancing plant growth and biomass production, and thereby overall metal uptake by plants [8-10]. Jiang
Lots of works have been carried out using PGPR to improve metal phytoextraction, but studies on the application of those PGPR strains capable of metal solubilization to overcome low metal availability in soils, as a bottleneck in phytoremediation efficiency, are rare. Microbial strains with both metal-solubilizing and plant growth-promoting abilities may effectively be used for bioaugmentation-phytoremediation of heavy metal-contaminated soils. This study was designed and implemented in pursuit of the following objectives: (i) to isolate and characterize Pb-resistant bacteria from metal contaminated mine soils that are capable of both solubilizing PbCO3 and PbO as sparingly soluble Pb compounds, (ii) to examine the plant growth-promoting characteristics of the isolated Pb-solubilizing bacteria, and finally (iii) to evaluate the inoculation effects of the isolated bacteria on the growth and Pb uptake of Indian mustard (
Materials and Methods
Site Description and Soil Sampling
Two study sites located inside metal mining areas in Isfahan Province, Iran, were selected for soil sampling. Three soil samples were collected from the Bama Pb/Zn Mine (51° 32’ E and 32° 28’ N) and two from the Nakhlak Pb Mine (53° 50’ E and 33° 34’ N). A portion of the soil samples was kept in sterile zip-lock plastic bags and stored at 4°C for bacterial isolation and characterization procedures. Another portion was air-dried, passed through a sieve (2 mm), and analyzed for important soil properties. The soil pH and electrical conductivity (EC) were determined in 1:2 (soil:water) extracts with a glass electrode (Cyberscan 2100) and a conductivity meter (Elmetron CC-501), respectively. The soil organic carbon content was determined using the potassium dichromate-sulfuric acid method [18], total Pb and Zn concentrations were measured via digesting soil samples in 6N HNO3 [19], and bioavailable Cd, Cu, Pb, and Zn concentrations were estimated using the diethylenetriaminepentaacetic acid (DTPA)-CaCl2-triethanolamine extraction method [20]. Metal concentrations in the extracts were determined with a flame atomic absorption spectrophotometer (FAAS, Z-5300). Some of the properties and metal concentrations of the soils are listed in Table 1.
-
Table 1 . Some characteristics of the soils that the Pb-solubilizing bacteria were isolated from..
Soila pHb ECb (dS m) OC (%) DTPA-extractable metals (mg kg) Total concentration of metals (mg kg) Cu Cd Pb Zn Pb Zn B1 7.4 0.86 1.27 2.70 0.72 696 225 5489 4200 B2 7.8 0.47 1.10 3.80 0.99 727 352 7909 4387 B3 7.6 1.68 2.85 1.00 0.45 174 79.80 1343 2325 N1 8.5 11.70 0.22 0.92 0.32 294 645 5128 3850 N2 7.6 17.90 0.08 0.75 0.17 688 11.10 1866 887 aB and N represent the Bama and Nakhlak mine soils, respectively..
bThe pH and EC were measured in a 1:2 soil:water suspension..
EC, electrical conductivity; OC, organic carbon; DTPA, diethylenetriaminepentaacetic acid..
Isolation and Purification of Pb-Tolerant Bacteria
The Pb-resistant bacteria were isolated using the Tris-buffered low-phosphate agar (TLP) medium containing sucrose as the carbon source to minimize Pb complexation to phosphate [21]. The medium was sterilized by autoclaving at 120°C for 20 min and subsequently supplemented with a filter-sterilized Pb(NO3)2 solution containing 50 mg-Pb/l. To prevent soil fungi growth, the media were supplemented with 10 mg/l of cycloheximide after autoclaving. The soil samples were then serially diluted with a sterile saline solution (0.85% NaCl) before 0.1 ml of each dilution was plated on the surface of the TLP medium. The bacterially inoculated plates were incubated at 30°C for up to 4 days, and individual colonies of different morphological characteristics, including color, size, form, opacity, and elevation, were selected and streaked on the same medium 3 to 4 times until pure cultures were obtained. The bacterial colonies were then regrown on Luria-Bertani (LB) medium and stored for further studies. A total of 171 bacterial isolates showing different morphological appearances on the agar medium were selected for studying the solubilization of Pb carbonate and oxide minerals.
Isolation of Pb-Solubilizing Bacteria
In the initial screening test for Pb tolerance, 110 Pb-resistant strains from the Bama Mine soils and 61 Pb-resistant strains from the Nakhlak Mine soils were isolated using a spread plate procedure and the pH-neutral TLP medium. These strains were subsequently screened to assess their potential to solubilize sparingly soluble Pb compounds (PbO and PbCO3). This ability was revealed by the appearance of clear haloes of solubilization around the colonies in the medium containing sucrose as the carbon source. For this purpose, inocula of the Pb-resistant bacterial isolates were prepared by centrifugation of 2 ml of logarithmic phase grown bacteria at 10,062 ×
Quantification of Pb Solubilization
Into 250-ml Erlenmeyer flasks containing 100 ml of the sterile liquid SLP, 500 mg/l of Pb as PbCO3 or 1,115 mg/l of Pb as PbO was added, and the medium was then inoculated with 1 ml of the logarithmic phase inoculum at approximately 108 CFU/ml. Media without metal addition and without bacterial inoculation were used as controls. The test flasks were incubated in a rotary shaker in the dark at 180 rpm and 30°C. After 10 days of incubation, 25 ml of the culture was centrifuged at 8,000 ×
Plant Growth-Promoting Properties of the Isolates
IAA production. The Pb-solubilizing bacterial strains were cultured in flasks containing 25 ml of LB medium supplemented with 0.5 mg/ml of L-tryptophan (filter-sterilized) at 30°C on an orbiter shaker at 150 rpm. After incubation for 24, 48, and 96 h, 1 ml of the cell-free suspension was mixed vigorously with 2 ml of Salkowski’s reagent [22] and allowed to stand at room temperature for 20 min. The absorbance of the pink color developed after 25 min of incubation was read at 530 nm. The IAA concentration in the culture was determined using a calibration curve of pure IAA.
Siderophore production. Siderophore produced by the isolated strains was detected according to the chrome azurol S (CAS) agar method described in Alexander and Zuberer [23]. The bacterial strains were grown twice in the Fe-free SLP medium before the inocula were prepared and adjusted to about 108 CFU/ml by suspending in a sterile saline solution. Subsequently, 40 µl of the inocula was spotted on CAS-agar plates and incubated in the dark for 10 days. Orange-yellow haloes around the colonies on the blue agar were interpreted as siderophore excretion.
Phosphate-solubilizing ability. The phosphate solubilization ability of the strains was determined in Pikovskaya’s Agar medium containing 0.5% tricalcium phosphate [24]. Forty microliters of each bacterial inoculum (about 108 CFU/ml) was spotted in Pikovskays’s Agar medium and incubated in the dark for 10 days. A clear halo around the colony was interpreted as the bacteria’s phosphate solubilization ability.
Genetic Identification
Characterization of each selected metal-solubilizing bacterium at the genus level was accomplished by partial sequencing of the 16S ribosomal DNA gene. Genomic DNA was extracted with the UltraClean Microbial DNA Isolation Kit (MO BIO Laboratories, Inc., USA) and amplified by the polymerase chain reaction (PCR) using the universal bacterial primers fD1 (5’-AGAGTTTGATCC TGGCTCAG-3’) and rP2 (5’-ACGGCTACCTTGTTACGACTT-3’)[25]. Each PCR contained 1 µl of gDNA and 0.4 µM (final concentration) of each primer in a final volume of 50 µl. The following PCR conditions were used: initial denaturing time of 5 min at 95°C, followed by 35 PCR cycles of 95°C for 1 min; 55°C, 1 min; and 72°C, 2 min; and a final extension step at 72°C for 10 min. Five microliters of the PCR products was subjected to electrophoretic investigation on a 1% TAE agarose gel and purified with the Ultra Clean PCR Clean-up Kit (MO BIO Laboratories, Inc.,) according to the manufacturer’s instructions. The purified PCR products were sequenced by Secugen (Madrid, Spain). Pairwise alignments were conducted using DNASTAR Lasergene (ver. 7.00) and the 16S rRNA gene sequences thus obtained were compared with those in the GenBank Database using the BLAST program from the NCBI website to determine the percent similarity.
Maximum Tolerance Concentration (MTC) of the Isolates
The maximum tolerance of the selected isolates against increasing concentrations of heavy metals (Zn, Pb, Cd, and Cu) was evaluated on SLP medium at pH 6.8 until the strains were unable to grow colonies on the agar plates. The maximum concentration of heavy metals at which the bacteria still presented growth was designated as the MTC. For this purpose, 10 µl of the logarithmic phase bacterial culture at approximately 108 CFU/ml was drop-inoculated onto Petri dishes in three replicates. A 100 mM stock solution of each metal was prepared using CdCl2•2H2O, CuSO4•5H2O, ZnSO4•7H2O, and Pb(NO3)2 and filtered through a 0.22 µm Millipore filter. The plates were incubated at 28°C for up to 10 days. This experiment was repeated twice in three replicates [26].
Isolate Tolerance to Acid and Alkaline pH and Sodium Chloride
Experiments were performed to monitor the ranges of pH and NaCl concentration at which the isolated bacterial strains maintained their ability to grow on the SLP agar medium [27]. For this purpose, the medium was buffered with 0.1 M sodium acetate/acetic acid at pH 4.5, 25 mM MES at pH 5.5 and 6.5, 25 mM HEPES at pH 7.5, 25 mM Tris-HCl at pH 8.5, and 25 mM sodium carbonate at pH 9.5 and 10.5. Subsequently, 10 µl of the exponential phase growing bacteria in the LB medium, at approximately 108 CFU/ml, was drop-inoculated on the medium and incubated at 28°C for up to 10 days. In the case of increasing salt concentration, NaCl from 3% up to 10% was added to the medium before autoclaving and the same amount of bacterial inoculum was used each time. These assays were repeated twice in three plates for each treatment.
Influence of Selected Isolates on the Growth and Pb Uptake of B. juncea
Preparation of bacterial inocula. Two isolated strains,
Soil. Soil collected from a metal-contaminated site around the Bama Pb/Zn Mine (51° 33’ 47/69’’E and 32° 30’ 45/79’’N) was air-dried and sieved to finer than 4 mm. It was characterized as a calcareous silty soil (15.6% sand, 83.5% silt, 0.9% clay) with pH 7.4 and an EC equal to 0.72 dS/m. The total Pb, Zn, and Cd concentrations of the soil were 446, 413, and 6.2 mg/kg, respectively, as determined by FAAS after soil digestion in 6N HNO3 [19].
Pot experiment.
Plant analysis. Plant growth parameters including shoot length as well as shoot and root dry weights were measured. The maximum shoot height was measured before harvesting on day 55 of the growth period. Subsequently, roots were separated and washed extensively with 1 mM Ca(NO3)2•4H2O and then with distilled water in order to remove surface-adsorbed Pb ions. Shoot and root dry matters were recorded after oven-drying at 75°C for 72 h.
The oven-dried plant samples were digested in a mixture of concentrated HNO3-HCl (70%) and H2O2 (30%) [28], and the Pb concentration in the extracts was determined using FAAS. Total Pb uptake by the plant shoots was calculated as the product of the shoot dry weight and Pb concentration.
Statistical Analysis
One-way analysis of variance followed by the least significant difference (LSD) test (
Results and Discussion
General Characteristics of the Metal-Contaminated Soils
The properties of the soils from which the bacteria were isolated are reported in Table 1. Soil pH values ranged from 7.4 to 8.5. The soils collected from the Bama Mine had EC values in the range of 0.47−1.68 dS/m whereas those of the Nakhlak Mine soil ranged from 11.7 to 17.9 dS/m. The total Pb concentration in the soils ranged from 1,343 to 7,909 mg/kg and the available (DTPA-CaCl2 extractable) fraction of Pb in the soils varied from 174 to 727 mg/kg. As judged from these values, all the soils were contaminated with Pb [29]. The values for total and available metals indicate that the soils were also contaminated with Zn and Cd [29].
Plant Growth-Promoting Characteristics of the Pb-Solubilizing Strains
IAA production. Plant growth enhancement by IAA is mostly due to its stimulating effect on root development, plant cell elongation, and cell division [30-32]. The root system enhancement, in turn, leads to a higher uptake of ions as a result of the larger soil volume made available to the roots.
All the Pb-solubilizing isolates, except YSP66 and YSP40n, were found capable of producing IAA in the LB medium supplemented with L-tryptophan (Table 2). The strains YSP40 and YSP24 recorded the highest IAA production rates (Table 2). Moreover, the strains YSP18, YSP24, YSP36, YSP40, and YSP151 produced IAA in the culture medium lacking L-tryptophan (Table 2). Since there are several tryptophan-independent pathways for IAA synthesis [33], it is possible that the latter strains were able to produce IAA without its major precursor, tryptophan. The strains YSP24, YSP40, YSP69, and YSP110 recorded reductions in their IAA content in the medium supplemented with tryptophan after 48-96 h (Table 2). This might have been due to the degradation of IAA by IAA oxidase secreted by the bacteria or to the storage of IAA in the bacterial cells as previously suggested [34]. The production of IAA has been reported to be common in
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Table 2 . Indole acetic acid (IAA) production by the Pb-solubilizing isolates after 24, 48, and 96 h of incubation.a.
Isolate IAA (mg/l) LB medium LB medium+L-tryptophan (500 mg/l) 24h 48h 96h 24h 48h 96h YSP15 ND ND ND 0.67±0.09 pq 0.53±0.14 q 0.49±0.12 q YSP17 0.46±0.18 hj 0.39 hk 0.43±0.17 hk 4.69±0.43 hi 4.2±0.17 hk 4.25±0.29 hk YSP18 1.14±0.11 df 1.19±0.11 d 1.16±0.22 de 1.10±0.14 nq 1.61±0.13 lq 1.05±0.14 nq YSP24 ND b 1.03±0.11 dg 2.33±0.30 b 0.58±0.09 pq 72.72±2 b 58.88±2.78 d YSP36 1.24±0.33 d 0.88±0.10 g 0.88±0.09 g 0.92±0.30 pq 1.08±0.30 nq 1.03±0.32 nq YSP40 2.58±0.54 a 2.85±0.35 mn 1.51±0.15 n 6.64±0.68 g 77.71±3.33 a 61.65±5.49 c YSP40n ND 0.07±0.05 ln ND ND 0.12±0.03 q ND YSP66 ND ND 0.49±0.13 hi ND ND 0.97±0.24 oq YSP69 ND 0.19±0.06 kn ND ND 3.94±0.11 hk 1.14±0.18 nq YSP95 0.55±0.04 h 0.48±0.10 hi 0.41±0.08 hk 0.55±0.07 q 1.60±0.16 lq 1.51±0.17 mq YSP104 0.37±0.18 hk ND ND 3.41±0.42 hl 3.94±0.16 hk 3.89±0.37 hk YSP110 0.51±0.15 hi 0.47±0.15 hi 0.46±0.10 hj 1.39±0.20 nq 5.23±0.23 gh 3.78±0.12 hk YSP143 0.24±0.06 jm 0.21±0.06 kn 0.31±.03 il 2.41±0.11 kp 3.24±0.30 im 4.04±0.29 hk YSP149 0.45±0.13 hj 0.42±0.08 hk 0.43±0.12 hk 2.85±0.35 in 3.25±0.37 im 2.80±0.25 jo YSP151 0.93±0.20 eg 0.92±0.24 fg 1.62±0.14 c 4.45±0.41 hj 9.21±0.78 f 11.41±0.94 e aData represent the average of three replicates ± standard deviation. Means in each medium followed by the same letter are not significantly different according to LSD (
p < 0.05). ND: not detected..
Siderophore production. Bioaugmentation with siderophore-producing bacteria has been proposed as an efficient strategy to enhance plant growth and metal uptake in phytoremediation practices [35]. In addition, chelation of heavy metals by siderophores has been found to promote bacterial IAA synthesis through reduced oxidative degradation of IAA by free heavy metals [36].
Ten out of the 15 Pb-solubilizing strains were able to produce siderophores (Table 3). Siderophore production levels by the different bacterial strains were compared on the basis of the ratio of colony + haloe diameter to colony diameter. The strains YSP151 and YSP149 exhibited the highest capability for siderophore production (Table 3).
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Table 3 . Siderophore production and P solubilization abilities of the Pb-solubilizing isolates..
Isolate YSP15 YSP17 YSP18 YSP24 YSP36 YSP40 YSP40n YSP66 YSP69 YSP95 YSP104 YSP110 YSP143 YSP149 YSP151 Siderophore production a 0 1.12 0 0 1.92 1.55 0 0.8 1.67 1.00 0.33 1.41 0 2.25 3.83 P solubilizationb – – – + – – – – – + – – – – – aData represent the colony + haloes diameter/colony diameter ratio on CAS-agar medium..
b+, Clear halo produced on Pikovskaya’s medium; –, No clear halo on Pikovskaya’s medium..
Production of metal-chelating substances, such as organic acids and siderophores, plays a special role in metal detoxification, and thereby in increasing metal tolerance in bacteria. These chelating agents may form complexes with heavy metals that are considered to be less toxic so as to inactivate and minimize the cytological impacts of free metal ions [37]. Abou-Shanab
Phosphate solubilization. Only two Pb-solubilizing strains (YSP24 and YSP95) were able to produce clear haloes on Pikovskaya’s medium containing sparingly soluble Ca3(PO4)2 as the sole source of phosphate (Table 3). Low P availability may retard plant growth and metal uptake in phytoremediation practices of contaminated soils [24]. Therefore, selection of bacterial strains capable of increasing P solubility in these soils would be favorable for improving plant growth and, in turn, enhancing metal uptake from these soils [7].
Lead Solubilization Ability of the Isolates
Only 15 (8.8%) out of the 171 Pb-resistant isolates showed clear halos in agar plates amended with PbO or PbCO3. Hence, this ability was not a common feature among the isolated bacteria. Colonies that formed zones of clearance around the colony were selected for further studies.
The isolated strains that were able to form zones of clearance around the colony on the solid medium containing PbO or PbCO3 were also able to dissolve these minerals in the liquid medium. The results of Pb solubilization by the isolates and the associated pH changes in the liquid (SLP) medium are reported in Table 4. Clearly, maximum values of PbCO3 solubilization in the SLP medium were recorded in the presence of strain YSP17 (4.28 mg-Pb/l), followed by YSP104 (2.64 mg-Pb/l) and YSP69 (2.16 mg-Pb/l), which were 26.7, 16.5, and 13.5 times higher than that of the control, respectively (Table 4). In the PbO-containing SLP medium, the maximum value of Pb concentration was recorded with inoculation of the strains YSP69, YSP40n, and YSP110 (8.99, 8.73, 8.42 mg-Pb/l, respectively), which were more than 30 times higher than that of the control. The bacterial isolates might have increased dissolution of the Pb-bearing minerals through such mechanisms as ligand-promoted dissolution induced by the bacterial metabolites and proton-promoted dissolution triggered by a pH decrease [39, 40]. Nevertheless, the magnitude of metal solubilization by a bacterial isolate varies depending on the mineral composition and solubilization mechanism(s) exploited by the isolate. Li
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Table 4 . Total soluble Pb and final pH measured in PbCO3- and PbO-containing SLP broth medium after inoculation with Pbsolubilizing bacterial isolates..
Bacterial Isolate Pb liberateda (mg/l) Final pH of the medium PbCO3 PbO PbCO3 PbO Controlb YSP15 1.48±0.26 e 0.91±0.08 g 5.95±0.08 b 6.98±0.07 b 6.76±0.18 ab YSP17 4.28±0.15 a 2.13±0.44 ef 4.04±0.12 g 6.60±0.26 c 4.54±0.06 h YSP18 0.36±0.02 i 1.81±0.15 ef 5.71±0.12 b 5.60±0.17 e 6.85±0.09 a YSP24 1.98±0.18 cd 1.74±0.10 f 4.27±0.50 efg 4.01±0.16 h 4.81±0.07 g YSP36 1.65±0.38 de 6.73±0.47 b 4.46±0.14 de 4.21±0.03 h 5.72±0.17 c YSP40 1.61±0.20 de 2.35±0.25 de 4.94±0.14 c 6.26±0.13 d 6.53±0.08 b YSP40n 1.02±0.02 fg 8.73±0.38 a 4.38±0.11 def 4.27±0.05 h 5.45±0.06 de YSP66 1.45±0.39 e 0.84±0.08 gh 4.07±0.08 fg 6.01±0.13 d 5.26±0.14 ef YSP69 2.16±0.02 c 8.99±0.26 a 4.43±0.19 de 4.27±0.14 h 4.49±0.13 h YSP95 0.21±0.11 i 2.18±0.72 ef 5.93±0.18 b 5.12±0.16 g 5.1±0.16 f YSP104 2.64±0.41 b 2.86±0.20 d 4.63±0.37 cd 5.54±0.11 ef 5.73±0.15 c YSP110 0.81±0.02 gh 8.42±0.76 a 4.15±0.13 efg 4.20±0.19 h 5.26±0.09 ef YSP143 0.47±0.03 hi 0.67±0.08 gh 4.46±0.17 de 5.65±0.30 e 5.58±0.05 cd YSP149 1.98±0.06 cd 0.81±0.21 gh 4.60±0.15 d 6.59±0.05 c 4.19±0.18 i YSP151 1.36±0.42 ef 5.82±0.52 c 4.43±0.11 de 5.29±0.17 fg 5.18±0.20 f Controlc 0.16±0.10 i 0.27±0.05 h 7.56±0.06 a 7.36±0.18 a 6.90±0.10 a aSLP medium supplemented with 1,115 mg/l PbO or 500 mg/l PbCO3 and inoculated with different bacterial strains..
bSLP medium inoculated with different bacterial strains without Pb salts..
cSLP medium supplemented with 1,115 mg/l PbO or 500 mg/l PbCO3 without bacterial inoculation..
Means in each column followed by the same letter are not significantly different according to LSD (
p < 0.05)..
Bacterial-induced metal solubilization of minerals has frequently been attributed to ligand-promoted dissolution of minerals caused by bacterial metabolites such as sidero-phores, carboxylic organic acid anions, and biosurfactants [41]. Abou-Shanab
The pH of the SLP medium inoculated with the Pb-solubilizing bacterial strains decreased both in the presence and absence of Pb minerals (Table 4), suggesting the role of the bacterial strains in producing H+ probably through organic acid secretion [7, 15]. Bacteria may also acidify their environment by the export of protons for the maintenance of charge balance [42]. The greatest drops in pH level (about 3 units) in the control SLP medium were induced by the strains YSP149 and YSP69. In the PbO-containing medium, the largest decrease in pH was measured in the presence of YSP24, whereas YSP17 and YSP66 gave rise to the largest pH decreases in the PbCO3-containing medium. These results are in agreement with those reported by Ji
Molecular Identification of the Pb-Solubilizing Bacteria
Table 5 presents the isolated Pb-solubilizing strains identified on the basis of 16S rRNA gene sequences. The size of the PCR products generated with the selected bacterial isolates ranged from approximately 784 to 1,351 bp. Based on a sequence identity of 97% or greater, the closest matches of the isolates were determined using BLAST. The results show a preponderance of the
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Table 5 . Identification of isolated Pb-solubilizing bacteria..
Isolates Source soil Length of 16S rRNA gene sequenced (bp) GenBank Accession No. Most closely related organism Species (strain) Accession No. % Gene identity YSP17 Nakhlak mine 1173 KY887777 Bacillus mojavensis (strain ifo 15718 )NR_118290.1 97 YSP24 Nakhlak mine 1346 KY887778 Paenibacillus illinoisensis (strain NBRC 15959)NR_113828.1 99 YSP36 Nakhlak mine 1065 KY887779 Bacillus simplex (strain RD36)KJ_534463.1 97 YSP40 Nakhlak mine 784 KY887780 Brevibacterium frigoritolerans (strain DSM 8801)NR_117474.1 99 YSP40n Nakhlak mine 1351 KY887781 Staphylococcus pasteuri (strain ATCC51129)NR_024669.1 100 YSP69 Bama mine 1313 KY887782 Bacillus cereus (strain CCM2010 )NR_115714.1 99 YSP104 Bama mine 1158 KY887783 Bacillus altitudinis (strain 41KF2b)NR_042337.1 100 YSP110 Bama mine 1156 KY887784 Bacillus filamentosus (strain SGD-14)NR_134701.1 99 YSP149 Bama mine 1219 KY887785 Bacillus licheniformis (strain ATCC14580)NR_074923.1 99 YSP151 Bama mine 1012 KY887786 Bacillus paralicheniformis (strain kj-16)NR_137421.1 99
Tolerance to Heavy Metals, NaCl, and pH
Among the 15 Pb-solubilizing isolates, 10 strains were selected for further experiments according to their best performance in Pb solubilization and PGPR traits. Mine soils most often contain more than one metal, and tolerance to several heavy metals is a desirable trait for bacteria with potential use in phytoremediation. All the isolates tested were found to exhibit multiple tolerance to high concentrations of metals, especially to Pb and Zn (Table 6). The highest MTC values were detected for Pb (≥207.2 mg/l), indicating that the isolates were highly resistant to Pb. YSP69, YSP104, and YSP110 were the most tolerant to Pb, but also to Zn, Cu, and Cd. Sheng
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Table 6 . Maximum tolerance concentration of the Pb-solubilizing isolates against heavy metals and NaCl and ability of the isolates to grow in different pH ranges..
Isolate Pb (mg/l) Zn (mg/l) Cu (mg/l) Cd (mg/l) NaCl (%) pH range YSP17 207.20 65.38 – 22.48 9 5.5–10.5 YSP24 207.20 65.38 – 11.24 5 5.5–10.5 YSP36 207.20 13.07 – – 3 5.5–10.5 YSP40 207.20 65.38 6.35 – 3 5.5–10.5 YSP40n 207.20 163.45 – 11.24 8 5.5–9.5 YSP69 414.40 130.76 12.71 224.82 8 5.5–10.5 YSP104 414.40 196.14 6.35 112.41 8 5.5–10.5 YSP110 414.40 130.76 6.35 112.41 10 5.5–10.5 YSP149 207.20 65.38 6.35 112.41 10 5.5–10.5 YSP151 207.20 68.38 6.35 11.24 9 5.5–10.5 –, No bacterial growth observed..
The Pb-solubilizing bacterial strains tested displayed a notable tolerance to salt stress while they were also able to grow well in a pH range of 5.5–10.5, except for YSP40n that did not grow at pH levels higher than 9.5 (Table 6). Bioaugmentation by salt-tolerant bacteria can both promote plant growth and help extract heavy metals in phytoremediation practices of salt-affected, metal-contaminated soils of arid regions [49]. The pH and salt tolerance of the Pb-solubilizing strains tested points to the feasibility of using them in contaminated soils also affected by moderately acid or alkaline pH levels and/or elevated salinity.
Effects of Pb-Solubilizing Strains on the Growth and Pb Uptake of B. juncea
Effects of
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Table 7 . Inoculation effects of
Brevibacterium frigoritolerans YSP40 andBacillus paralicheniformis YSP151 strains and their consortium on the growth and Pb absorption ofBrassica juncea ..Inoculated strain Shoot length (cm) Shoot dry weight (g/pot) Root dry weight (g/pot) Shoot Pb concentration (mg/kg) Root Pb concentration (mg/kg) Pb uptake by shoot (μg/pot) Non (control) 12.30±2.08 b 1.84±0.79 c 0.16±0.19 a 38.23±2.9 c 96.93±6 b 69.01±24.73 c YSP40 34.83±7.09 a 3.99±0.59 ab 0.39±0.05 a 55.31±10.1 b 79.55±6.5 b 224.1±67.36 b YSP151 35.68±2.17 a 4.38±0.55 a 0.82±0.82 a 73.6±13.6 a 141.7±35.2 a 319.7±48.62 a YSP40+YSP151 32.30±1.88 a 2.89±0.36 bc 0.38±0.06 a 67.16±5.3 ab 80.94±5.7 b 193.1±14.82 b Values are the mean ± standard deviation. Means in each column followed by the same letter are not significantly different according to LSD (
p < 0.05)..
Table 7 reports Pb accumulation levels in the roots and shoots of
The total Pb uptake by plant shoots was 3.2 and 4.6 times higher than that of the control when the plants were inoculated with
The present study highlighted the role of bacterial strains isolated from mine soils in increasing soil metal availability and uptake by plants, for remediating metal-contaminated sites more efficiently. Since the potential of a plant for use as a phytoremediator in a metal-contaminated soil relies on the total metal quantity taken up by the harvestable part of the plant biomass, inoculation of the metal-solubilizing and plant growth-promoting strains, such as
Acknowledgments
This work was supported by grants from Isfahan University of Technology and MINECO (AGL2013-40758-R).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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Table 1 . Some characteristics of the soils that the Pb-solubilizing bacteria were isolated from..
Soila pHb ECb (dS m) OC (%) DTPA-extractable metals (mg kg) Total concentration of metals (mg kg) Cu Cd Pb Zn Pb Zn B1 7.4 0.86 1.27 2.70 0.72 696 225 5489 4200 B2 7.8 0.47 1.10 3.80 0.99 727 352 7909 4387 B3 7.6 1.68 2.85 1.00 0.45 174 79.80 1343 2325 N1 8.5 11.70 0.22 0.92 0.32 294 645 5128 3850 N2 7.6 17.90 0.08 0.75 0.17 688 11.10 1866 887 aB and N represent the Bama and Nakhlak mine soils, respectively..
bThe pH and EC were measured in a 1:2 soil:water suspension..
EC, electrical conductivity; OC, organic carbon; DTPA, diethylenetriaminepentaacetic acid..
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Table 2 . Indole acetic acid (IAA) production by the Pb-solubilizing isolates after 24, 48, and 96 h of incubation.a.
Isolate IAA (mg/l) LB medium LB medium+L-tryptophan (500 mg/l) 24h 48h 96h 24h 48h 96h YSP15 ND ND ND 0.67±0.09 pq 0.53±0.14 q 0.49±0.12 q YSP17 0.46±0.18 hj 0.39 hk 0.43±0.17 hk 4.69±0.43 hi 4.2±0.17 hk 4.25±0.29 hk YSP18 1.14±0.11 df 1.19±0.11 d 1.16±0.22 de 1.10±0.14 nq 1.61±0.13 lq 1.05±0.14 nq YSP24 ND b 1.03±0.11 dg 2.33±0.30 b 0.58±0.09 pq 72.72±2 b 58.88±2.78 d YSP36 1.24±0.33 d 0.88±0.10 g 0.88±0.09 g 0.92±0.30 pq 1.08±0.30 nq 1.03±0.32 nq YSP40 2.58±0.54 a 2.85±0.35 mn 1.51±0.15 n 6.64±0.68 g 77.71±3.33 a 61.65±5.49 c YSP40n ND 0.07±0.05 ln ND ND 0.12±0.03 q ND YSP66 ND ND 0.49±0.13 hi ND ND 0.97±0.24 oq YSP69 ND 0.19±0.06 kn ND ND 3.94±0.11 hk 1.14±0.18 nq YSP95 0.55±0.04 h 0.48±0.10 hi 0.41±0.08 hk 0.55±0.07 q 1.60±0.16 lq 1.51±0.17 mq YSP104 0.37±0.18 hk ND ND 3.41±0.42 hl 3.94±0.16 hk 3.89±0.37 hk YSP110 0.51±0.15 hi 0.47±0.15 hi 0.46±0.10 hj 1.39±0.20 nq 5.23±0.23 gh 3.78±0.12 hk YSP143 0.24±0.06 jm 0.21±0.06 kn 0.31±.03 il 2.41±0.11 kp 3.24±0.30 im 4.04±0.29 hk YSP149 0.45±0.13 hj 0.42±0.08 hk 0.43±0.12 hk 2.85±0.35 in 3.25±0.37 im 2.80±0.25 jo YSP151 0.93±0.20 eg 0.92±0.24 fg 1.62±0.14 c 4.45±0.41 hj 9.21±0.78 f 11.41±0.94 e aData represent the average of three replicates ± standard deviation. Means in each medium followed by the same letter are not significantly different according to LSD (
p < 0.05). ND: not detected..
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Table 3 . Siderophore production and P solubilization abilities of the Pb-solubilizing isolates..
Isolate YSP15 YSP17 YSP18 YSP24 YSP36 YSP40 YSP40n YSP66 YSP69 YSP95 YSP104 YSP110 YSP143 YSP149 YSP151 Siderophore production a 0 1.12 0 0 1.92 1.55 0 0.8 1.67 1.00 0.33 1.41 0 2.25 3.83 P solubilizationb – – – + – – – – – + – – – – – aData represent the colony + haloes diameter/colony diameter ratio on CAS-agar medium..
b+, Clear halo produced on Pikovskaya’s medium; –, No clear halo on Pikovskaya’s medium..
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Table 4 . Total soluble Pb and final pH measured in PbCO3- and PbO-containing SLP broth medium after inoculation with Pbsolubilizing bacterial isolates..
Bacterial Isolate Pb liberateda (mg/l) Final pH of the medium PbCO3 PbO PbCO3 PbO Controlb YSP15 1.48±0.26 e 0.91±0.08 g 5.95±0.08 b 6.98±0.07 b 6.76±0.18 ab YSP17 4.28±0.15 a 2.13±0.44 ef 4.04±0.12 g 6.60±0.26 c 4.54±0.06 h YSP18 0.36±0.02 i 1.81±0.15 ef 5.71±0.12 b 5.60±0.17 e 6.85±0.09 a YSP24 1.98±0.18 cd 1.74±0.10 f 4.27±0.50 efg 4.01±0.16 h 4.81±0.07 g YSP36 1.65±0.38 de 6.73±0.47 b 4.46±0.14 de 4.21±0.03 h 5.72±0.17 c YSP40 1.61±0.20 de 2.35±0.25 de 4.94±0.14 c 6.26±0.13 d 6.53±0.08 b YSP40n 1.02±0.02 fg 8.73±0.38 a 4.38±0.11 def 4.27±0.05 h 5.45±0.06 de YSP66 1.45±0.39 e 0.84±0.08 gh 4.07±0.08 fg 6.01±0.13 d 5.26±0.14 ef YSP69 2.16±0.02 c 8.99±0.26 a 4.43±0.19 de 4.27±0.14 h 4.49±0.13 h YSP95 0.21±0.11 i 2.18±0.72 ef 5.93±0.18 b 5.12±0.16 g 5.1±0.16 f YSP104 2.64±0.41 b 2.86±0.20 d 4.63±0.37 cd 5.54±0.11 ef 5.73±0.15 c YSP110 0.81±0.02 gh 8.42±0.76 a 4.15±0.13 efg 4.20±0.19 h 5.26±0.09 ef YSP143 0.47±0.03 hi 0.67±0.08 gh 4.46±0.17 de 5.65±0.30 e 5.58±0.05 cd YSP149 1.98±0.06 cd 0.81±0.21 gh 4.60±0.15 d 6.59±0.05 c 4.19±0.18 i YSP151 1.36±0.42 ef 5.82±0.52 c 4.43±0.11 de 5.29±0.17 fg 5.18±0.20 f Controlc 0.16±0.10 i 0.27±0.05 h 7.56±0.06 a 7.36±0.18 a 6.90±0.10 a aSLP medium supplemented with 1,115 mg/l PbO or 500 mg/l PbCO3 and inoculated with different bacterial strains..
bSLP medium inoculated with different bacterial strains without Pb salts..
cSLP medium supplemented with 1,115 mg/l PbO or 500 mg/l PbCO3 without bacterial inoculation..
Means in each column followed by the same letter are not significantly different according to LSD (
p < 0.05)..
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Table 5 . Identification of isolated Pb-solubilizing bacteria..
Isolates Source soil Length of 16S rRNA gene sequenced (bp) GenBank Accession No. Most closely related organism Species (strain) Accession No. % Gene identity YSP17 Nakhlak mine 1173 KY887777 Bacillus mojavensis (strain ifo 15718 )NR_118290.1 97 YSP24 Nakhlak mine 1346 KY887778 Paenibacillus illinoisensis (strain NBRC 15959)NR_113828.1 99 YSP36 Nakhlak mine 1065 KY887779 Bacillus simplex (strain RD36)KJ_534463.1 97 YSP40 Nakhlak mine 784 KY887780 Brevibacterium frigoritolerans (strain DSM 8801)NR_117474.1 99 YSP40n Nakhlak mine 1351 KY887781 Staphylococcus pasteuri (strain ATCC51129)NR_024669.1 100 YSP69 Bama mine 1313 KY887782 Bacillus cereus (strain CCM2010 )NR_115714.1 99 YSP104 Bama mine 1158 KY887783 Bacillus altitudinis (strain 41KF2b)NR_042337.1 100 YSP110 Bama mine 1156 KY887784 Bacillus filamentosus (strain SGD-14)NR_134701.1 99 YSP149 Bama mine 1219 KY887785 Bacillus licheniformis (strain ATCC14580)NR_074923.1 99 YSP151 Bama mine 1012 KY887786 Bacillus paralicheniformis (strain kj-16)NR_137421.1 99
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Table 6 . Maximum tolerance concentration of the Pb-solubilizing isolates against heavy metals and NaCl and ability of the isolates to grow in different pH ranges..
Isolate Pb (mg/l) Zn (mg/l) Cu (mg/l) Cd (mg/l) NaCl (%) pH range YSP17 207.20 65.38 – 22.48 9 5.5–10.5 YSP24 207.20 65.38 – 11.24 5 5.5–10.5 YSP36 207.20 13.07 – – 3 5.5–10.5 YSP40 207.20 65.38 6.35 – 3 5.5–10.5 YSP40n 207.20 163.45 – 11.24 8 5.5–9.5 YSP69 414.40 130.76 12.71 224.82 8 5.5–10.5 YSP104 414.40 196.14 6.35 112.41 8 5.5–10.5 YSP110 414.40 130.76 6.35 112.41 10 5.5–10.5 YSP149 207.20 65.38 6.35 112.41 10 5.5–10.5 YSP151 207.20 68.38 6.35 11.24 9 5.5–10.5 –, No bacterial growth observed..
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Table 7 . Inoculation effects of
Brevibacterium frigoritolerans YSP40 andBacillus paralicheniformis YSP151 strains and their consortium on the growth and Pb absorption ofBrassica juncea ..Inoculated strain Shoot length (cm) Shoot dry weight (g/pot) Root dry weight (g/pot) Shoot Pb concentration (mg/kg) Root Pb concentration (mg/kg) Pb uptake by shoot (μg/pot) Non (control) 12.30±2.08 b 1.84±0.79 c 0.16±0.19 a 38.23±2.9 c 96.93±6 b 69.01±24.73 c YSP40 34.83±7.09 a 3.99±0.59 ab 0.39±0.05 a 55.31±10.1 b 79.55±6.5 b 224.1±67.36 b YSP151 35.68±2.17 a 4.38±0.55 a 0.82±0.82 a 73.6±13.6 a 141.7±35.2 a 319.7±48.62 a YSP40+YSP151 32.30±1.88 a 2.89±0.36 bc 0.38±0.06 a 67.16±5.3 ab 80.94±5.7 b 193.1±14.82 b Values are the mean ± standard deviation. Means in each column followed by the same letter are not significantly different according to LSD (
p < 0.05)..
References
- Nagajyoti PC, Lee KD, Sreekanth TVM. 2010. Heavy metals, occurrence and toxicity for plants: a review.
Environ. Chem. Lett. 8 : 199-216. - Thornton I, Rautiu R, Brush S. 2001. Lead: The Facts. Ian Allan Printing Ltd., London.
- Triantafyllidou S, Edwards M. Lead (Pb) in U.S. drinking water: school case studies, detection challenges and public health considerations. Your Drinking Water: Challenges and Solutions for the 21st Century. Drinking Water Symposium, April 20th & 21st, 2009. Yale University.
- Dermont G, Bergeron M, Mercier G, Richer-Laflèche M. 2008. Soil washing for metal removal: a review of physical/chemical technologies and field applications.
J. Hazard. Mater. 152 : 1-31. - Jabeen R, Ahmad A, Iqbal M. 2009. Phytoremediation of heavy metals: physiological and molecular mechanisms.
Bot. Rev. 75 : 339-364. - Shao HB, Chu LY, Ni FT, Guo DG, Li H, Li WX. 2010. Perspective on phytoremediation for improving heavy metal-contaminated soils, pp. 227-244.
In: Ashraf M, Ozturk M, Ahmad MSA (eds),Plant Adaptation and Phytoremediation . Springer, Dordrecht. - Jiang CY, Sheng XF, Qian M, Wang QY. 2008. Isolation and characterization of a heavy metal-resistant Burkholderia sp. from heavy metal-contaminated paddy field soil and its potential in promoting plant growth and heavy metal accumulation in metal-polluted soil.
Chemosphere 72 : 157-164. - Sheng XF, Jiang CY, He LY. 2008. Characterization of plant growth-promoting
Bacillus edaphicus NBT and its effect on lead uptake by Indian mustard in a lead-amended soil.Can. J. Microbiol. 54 : 417-422. - Sheng XF, Xia JJ. 2006. Improvement of rape (
Brassica napus ) plant growth and cadmium uptake by cadmium-resistant bacteria.Chemosphere 64 : 1036-1042. - Sheng X-F, Xia JJ, Jiang CY, He LY, Qian M. 2008. Characterization of heavy metal-resistant endophytic bacteria from rape (
Brassica napus ) roots and their potential in promoting the growth and lead accumulation of rape.Environ. Pollut. 156 : 1164-1170. - Lindsay WL. 1979. Chemical Equilibrium in Soils. Wiley Interscience Publishers, New York.
- Zhao S, Jia L, Duo L. 2013. The use of biodegradable chelators for enhanced phytoextraction of heavy metals by
Festuca arundinacea from municipal solid waste compost and associated heavy metal leaching.Bioresour. Technol. 129 : 249-255. - Evangelou MWH, Bauer U, Ebel M, Schaeffer A. 2007. The influence of EDDS and EDTA on the uptake of heavy metals of Cd and Cu from soil with tobacco,
Nicotiana tabacum .Chemosphere 68 : 345-353. - Usman ARA, Almaroai YA, Ahmad M, Vithanage M, Ok YS. 2013. Toxicity of synthetic chelators and metal availability in poultry manure amended Cd, Pb and As contaminated agricultural soil.
J. Hazard. Mater. 262 : 1022-1030. - Fasim F, Ahmed N, Parsons R, Gadd GM. 2002. Solubilization of zinc salts by a bacterium isolated from the air environment of a tannery.
FEMS Microbiol. Lett. 213 : 1-6. - Long XX, Chen XM, Chen YG, Wong JW-C, Wei ZB, Wu QT. 2011. Isolation and characterization endophytic bacteria from hyperaccumulator
Sedum alfredii Hance and their potential to promote phytoextraction of zinc polluted soil.World J. Microbiol. Biotechnol. 27 : 1197-1207. - Braud A, Jézéquel K, Vieille E, Tritter A, Lebeau T. 2006. Changes in extractability of Cr and Pb in a poly contaminated soil after bioaugmentation with microbial producers of biosurfactants, organic acids and siderophores.
Water Air Soil Pollut. 6 : 261-279. - Nelson DW, Sommers LE. 1996. Total carbon, organic carbon, and organic matter, pp. 961-1010.
In: Sparks DL (ed),Method of Soil Analysis, Part 3, Chemical Methods . Soil Science Society of America, Inc., American Society of Agronomy, Madison, WI. - Zheljazkov VD, Nielsen NE. 1996. Effect of heavy metals on peppermint and cornmint.
Plant Soil 178 : 59-66. - Lindsay WL, Norvell WA. 1978. Development of a DTPA soil test for zinc, iron, manganese, and copper.
Soil Sci. Soc. Am. J. 42 : 421-428. - Mergeau MD, Nies HG, Schlegel J, Gerits P, Charles van Gijsegem F. 1985.
Alcaligenes eutrophus CH34 is a facultative chemolitotroph with plasmid-bound resistance to heavy metals.J. Bacteriol. 162 : 328-334. - Gordon SA, Weber RP. 1951. Colorimetric estimation of indole acetic acid.
Plant Physiol. 26 : 192-195. - Alexander DB, Zuberer DA. 1991. Use of chrome Azurol-S reagents to evaluate siderophore production by rhizosphere bacteria.
Biol. Fertil. Soils 12 : 39-45. - Zaidi S, Usmani S, Singh BR, Musarrat J. 2006. Significance of
Bacillus subtilis strain SJ-101 as a bio inoculant for concurrent plant growth promotion and nickel accumulation inBrassica juncea .Chemosphere 64 : 991-997. - Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 1991. 16S ribosomal DNA amplification for phylogenetic study.
J. Bacteriol. 173 : 697-703. - Affan QA, Shoeb E, Badar U, Akhtar J. 2009. Isolation and characterization of bacterial isolates having heavy metal tolerance.
J. Basic Appl. Sci. 5 : 55-60. - Nonnoi F, Chinnaswamy A, de la Torre VSG, de la Peña TC, Lucas MM, Pueyo JJ. 2012. Metal tolerance of rhizobial strains isolated from nodules of herbaceous legumes (
Medicago spp. andTrifolium spp.) growing in mercurycontaminated soils.Appl. Soil Ecol. 61 : 49-59. - Jones JB Jr, Case VW. 1990. Sampling, handling, and analyzing plant tissue samples, pp. 389-447.
In: Westerman RL (ed),Soil Testing and Plant Analysis . Soil Science Society of America, Inc., Madison, WI. - NYS. New York State Brownfield Cleanup Program Development of Soil Cleanup Objectives Technical Support Document, Albany, NY: New York State Department of Environmental Conservation and New York State Department of Health; 2006. Available from http://www.dec.ny.gov/chemical/34189.html.
- Davies PJ. 2010. The Plant Hormones: Their Nature, Occurrence and Functions, 3rd Ed. Kluwer Academic Publishers, New York.
- Malhotra M, Srivastava S. 2009. Stress-responsive indole-3-acetic acid biosynthesis by
Azospirillum brasilense SM and i ts ability to modulate plant growth.Eur. J. Soil Biol. 45 : 73-80. - Patten CL, Glick BR. 2002. The role of bacterial indole acetic acid in the development of the host plant root system.
Appl. Environ. Microbiol. 68 : 3795-3801. - Ribeiro CM, Cardoso EJBN. 2012. Isolation, selection and characterization of root-associated growth promoting bacteria in Brazil Pine (
Araucaria angustifolia ).Microbiol. Res. 167 : 69-78. - Ji LY, Zhang WW, Yu D, Cao YR, Xu H. 2012. Effect of heavy metal-solubilizing microorganisms on zinc and cadmium extractions from heavy metal contaminated soil with
Tricholoma lobynsis .World J. Microbiol. Biotechnol. 28 : 293-301. - Rajkumar M, Ae N, Narasimha M, Prasad V, Freitas H. 2009. Potential of siderophore-producing bacteria for improving heavy metal phytoextraction.
Trends Biotechnol. 28 : 142-149. - Dimkpa CO, Merten D, Svatoš A, Büchel G, Kothe E. 2009. Metal-induced oxidative stress impacting plant growth in contaminated soil is alleviated by microbial siderophores.
Soil Biol. Biochem. 41 : 154-162. - Gao Y, Miao C, Mao L, Zhou P, Jin Z, Shi W. 2010. Improvement of phytoextraction and antioxidative defense in
Solanum nigrum L. under cadmium stress by application of cadmium-resistant strain and citric acid.J. Hazard. Mater. 181 : 771-777. - Abou-Shanab RA, Ghozlan H, Ghanem K, Moawad H. 2005. Behavior of bacterial populations isolated from rhizosphere of
Diplachne fusca dominant in industrial sites.World J. Microbiol. Biotechnol. 21 : 1095-1101. - Li WC, Ye ZH, Wong MH. 2010. Metal mobilization and production of short-chain organic acids by rhizosphere bacteria associated with a Cd/Zn hyperaccumulating plant, Sedum alfredii.
Plant Soil 326 : 453-467. - Ma Y, Prasad MNV, Rajkumar M, Freitas H. 2011. Plant growth promoting rhizobacteria and endophytes accelerate phytoremediation of metalliferous soils.
Biotechnol. Adv. 29 : 248-258. - Sessitsch A, Kuffner M, Kidd P, Vangronsveld J, Wenzel WW, Fallmann K,
et al . 2013. The role of plant-associated bacteria in the mobilization and phytoextraction of trace elements in contaminated soils.Soil Biol. Biochem. 60 : 182-194. - Abou-Shanab RA, Ghanem K, Ghanem N, Al-Kolaibe A. 2008. The role of bacteria on heavy-metal extraction and uptake by plants growing on multi-metal-contaminated soils.
World J. Microbiol. Biotechnol. 24 : 253-262. - Braud A, Jézéquel K, Bazot S, Lebeau T. 2009. Enhanced phytoextraction of an agricultural Cr- and Pb-contaminated soil by bioaugmentation with siderophore-producing bacteria.
Chemosphere 74 : 280-286. - Dimkpa CO, Merten D, Svatoš A, Büchel G, Kothe E. 2009. Siderophores mediate reduced and increased uptake of cadmium by
Streptomyces tendae F4 and sunflower (Helianthus annuus ), respectively.J. Appl. Microbiol. 107 : 1687-1696. - Dhal B, Thatoi H, Das N, Pandey BD. 2010. Reduction of hexavalent chromium by
Bacillus sp. isolated from chromite mine soils and characterization of reduced product.J. Chem. Technol. Biotechnol. 85 : 1471-1479. - Govarthanan M, Lee KJ, Cho M, Kim JS, Kamala-Kannan S, Oh BT. 2013. Significance of autochthonous
Bacillus sp. KK1 on biomineralization of lead in mine tailings.Chemosphere 90 : 2267-2272. - Seralathan K, Kui JL. 2008. Metal tolerance and antibiotic resistance of
Bacillus species isolated from Sunchon Bay, South Korea.Biotechnology 7 : 149-152. - Sheng XF, He LY, Wang QY, Ye HS, Jiang CY. 2008. Effects of inoculation of biosurfactant-producing
Bacillus sp. J119 on plant growth and cadmium uptake in a cadmium-amended soil.J. Hazard. Mater. 155 : 17-22. - Teixeira C, Almeida CMR, Nunes da Silva M, Bordalo AA, Mucha AP. 2014. Development of autochthonous microbial consortia for enhanced phytoremediation of salt-marsh sediments contaminated with cadmium.
Sci. Total Environ. 493 : 757-765. - Kumar KV, Singh N, Behl NH, Srivastava S. 2008. Influence of plant growth promoting bacteria and its mutant on heavy metal toxicity in
Brassica juncea grown in fly ash amended soil.Chemosphere 72 : 678-683. - Tangahu BV, Abdullah SRS, Basri H, Idris M, Anuar N, Mukhlisin MA. 2011. Review on heavy metals (As, Pb, and Hg) uptake by plants through phytoremediation.
Int. J. Chem. Eng. 2011 : 939161. - Kumari B, Singh SN. 2011. Phytoremediation of metals from fly ash through bacterial augmentation.
Ecotoxicology 20 : 166-176. - Burd GI, Dixon DG, Glick BR. 1998. A plant growth-promoting bacterium that decreases nickel toxicity in seedlings.
Appl. Environ. Microbiol. 64 : 3663-3668.