Promotion of growth and Cu accumulation of bio-energy crop (Zea mays) by bacteria: Implications for energy plant biomass production and phytoremediation

Journal of Environmental Management 103 (2012) 58e64

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Promotion of growth and Cu accumulation of bio-energy crop (Zea mays) by bacteria: Implications for energy plant biomass production and phytoremediation Xiafang Sheng*, Leni Sun, Zhi Huang, Linyan He, Wenhui Zhang, Zhaojin Chen Key Laboratory of Agricultural Environment Microbiology, Ministry of Agriculture, College of Life Science, Nanjing Agricultural University, Nanjing 210095, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 December 2011 Received in revised form 18 February 2012 Accepted 23 February 2012 Available online 28 March 2012

Three metal-resistant and plant growth-promoting bacteria (Burkholderia sp. GL12, Bacillus megaterium JL35 and Sphingomonas sp. YM22) were evaluated for their potential to solubilize Cu2 (OH)2CO3 in solution culture and their plant growth promotion and Cu uptake in maize (Zea mays, an energy crop) grown in a natural highly Cu-contaminated soil. The impacts of the bacteria on the Cu availability and the bacterial community in rhizosphere soils of maize were also investigated. Inductively coupled-plasma optical emission spectrometer analysis showed variable amounts of water-soluble Cu (ranging from 20.5 to 227 mg L
1) released by the bacteria from Cu2 (OH)2CO3 in solution culture. Inoculation with the bacteria was found to significantly increase root (ranging from 48% to 83%) and above-ground tissue (ranging from 33% to 56%) dry weights of maize compared to the uninoculated controls. Increases in Cu contents of roots and above-ground tissues varied from 69% to 107% and from 16% to 86% in the bacterial-inoculated plants compared to the uninoculated controls, respectively. Inoculation with the bacteria was also found to significantly increase the water-extractive Cu concentrations (ranging from 63 to 94%) in the rhizosphere soils of the maize plants compared to the uninoculated controls in pot experiments. Denaturing gradient gel electrophoresis and sequence analyses showed that the bacteria could colonize the rhizosphere soils and significantly change the bacterial community compositions in the rhizosphere soils. These results suggest that the metal-resistant and plant growth-promoting bacteria may be exploited for promoting the maize (energy crop) biomass production and Cu phytoremediation in a natural highly Cu-contaminated soil. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Metal-resistant and plant growthpromoting bacteria Energy plant Bacterial communities Phytoextraction Maize

1. Introduction Copper tailings, produced from extraction and processing of copper ores have caused severe damage to ecosystems including high levels of heavy metal-contamination in soils near the copper mines, leading to economic losses and negative impacts on human food chain and health (Wong, 2003; Andreazza et al., 2010; Meers et al., 2010). Such Cu-polluted areas are not suitable for the cultivation of food and feed crops due to the presence of high levels of heavy metals and require remediation to reduce environmental health risk to living organisms. Heavy metal-contaminated land utilization for the production of energy crops is an important consideration. The cultivation of energy plants is often considered as a very promising renewable energy option for the future (Van Ginneken et al., 2007; Mleczek et al., 2010). Furthermore,

* Corresponding author. Tel.: þ86 25 84395125; fax: þ86 25 84396326. E-mail addresses: [email protected], [email protected] (X. Sheng). 0301-4797/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.jenvman.2012.02.030

ecological remediation of heavy metal-polluted sites has received much attention around the world because it provides an ecologically sound and safe method for restoration and remediation (Wu et al., 2006). It can be expected that the energy plant cultivation in the heavily metal-contaminated soils will become more important for both energy generation and phytoremediation of heavy metal-contaminated soils. Phytoextraction offers significantly more benefits than conventional technology to accumulate heavy metals from the soil. Low availability of heavy metals in soils and small biomass and slow growth of most hyperaccumulators identified so far limit the efficiency of phytoextraction (Chen et al., 2004), while chemically induced hyperaccumulation is impaired by the high cost of some synthetic chelators and various environmental risks (Meers et al., 2010). Many microorganisms in soils are tolerant to heavy metals and play important roles in mobilization of heavy metals (Idris et al., 2004). The presence of rhizosphere bacteria increased concentration of Zn in Thlaspi caerulescens (Whiting et al., 2001) and Cd in Brassica napus (Sheng and Xia, 2006). In addition, bacteria producing indole acetic acid

X. Sheng et al. / Journal of Environmental Management 103 (2012) 58e64

(IAA), siderophores and 1-aminocyclopropane-1-carboxylate (ACC) deaminase are capable of stimulating plant growth and protecting the plants against heavy metal toxicity in heavy metal-contaminated soils (Belimov et al., 2005; Madhaiyan et al., 2007). Rhizosphere microorganisms can enhance biomass production and tolerance of plants to heavy metals in stress environment (Sheng and Xia, 2006). Previous work with Burkholderia sp. GL12, Bacillus megaterium JL35 and Sphingomonas sp. YM22 showed that the heavy metal-resistant and plant growth-promoting bacteria (PGPB) could promote the growth and Cu accumulation of rape plants grown in vermiculite containing 4 mg kg
1 of Cu (Sun et al., 2010). Although a number of studies have demonstrated the importance of bacterial inoculation for plant growth and heavy metal accumulation in heavy metal-polluted environments (Idris et al., 2004; Jiang et al., 2008), to our knowledge, this is the first research report elucidating the impacts of the heavy metal-resistant and plant growth-promoting bacteria (Burkholderia sp. GL12, B. megaterium JL35 and Sphingomonas sp. YM22) on Cu availability, energy plant (maize) biomass production with concurrent Cu accumulation by maize, and the bacterial community in rhizosphere soils of maize plants grown in natural highly Cu-contaminated soil, which may be crucial for establishing close interactions between bacteria and energy plant and for accelerating the efficiency of biomass production and phytoremediation of natural highly Cu-contaminated soils. The objectives of the study were to evaluate the effects of the metal-resistant and PGPB (Burkholderia sp. GL12, B. megaterium JL35 and Sphingomonas sp. YM22) on the availability of Cu in solution culture and in soils, the plant growth, and the Cu uptakes of maize plants, to analyze the colonization of the bacteria in the rhizosphere soils, and to examine the impact of the bacteria on the native bacterial communities in the rhizosphere soils of the maize plants.

2. Materials and methods 2.1. Soil and plant The top soils (0e20 cm) (silty loam) for the pot experiments were collected from an abandoned farmland (vegetables were mainly cultivated on the farmland earlier) near a copper mine wasteland in Nanjing, East China (31140 e32 70 N and 118 220 e119140 E). The soils were immediately transported back to the laboratory and all visible roots and fresh litter materials were removed from the soil samples, then the soil samples were thoroughly mixed and sieved (2 mm). The basic properties of the soils were: pH (1:1 w/v water) 7.11; organic matter 9.44 g kg
1; cation exchange capacity 14.5 cmol kg
1; the total soil Cu, Pb, Mn, Zn, and Cd were 1068, 35, 535, 133, and 4.2 mg kg
1 respectively; the available P, K, Ca, Mg, and Cu were 10.68, 105.5, 540, 258, and 165 mg kg
1 respectively; Soil organic matter was determined titrimetrically following the method described in the Physical Chemical Analysis of Soils (SSICA, 1980). Soil total Cu, Pb, Mn, Zn, and Cd were extracted with HFeHClO4 (SSICA, 1980). P and K available in the soil were extracted with 0.5 M Na2CO3 and 2 M HNO3, respectively, and soil available Ca, Mg, and Cu were extracted with 0.05 M diethylene triamine penlaacetic acid (DTPA) (SSICA, 1980). The above available P, K, Ca, Mg, and Cu contents in the extracts were determined using an inductively coupled-plasma optical emission spectrometer (ICP-OES) (Optimal 2100 DV, Perkin Elmer). Maize (Zea mays L.) cv. zhendan-985 was used in the inoculation experiment because it is a fast growing and large biomass and energy producing plant.

59

2.2. Bacteria Burkholderia sp. GL12, B. megaterium JL35 and Sphingomonas sp. YM22 were isolated from copper-tolerant species Elsholtzia splendens and Commelina communis (Sun et al., 2010). According to Sun et al. (2010), the strains were able to produce IAA, siderophore and ACC deaminase and possess resistance to Cu (0.5 mM), Pb (5 mM), and Zn (10 mM). The three strains (GL12, JL35, and YM22) exhibited antibiotic resistance to streptomycin (50 mg mL
1) and spectinomycin (100 mg mL
1). The nucleotide sequences of strains GL12, JL35, and YM22 have been deposited in the NCBI database under accession numbers EU418711, EU418718, and EU418723 respectively. 2.3. Activation of poorly soluble copper by the bacteria Inocula of bacteria were prepared by using 18 h logarithmic phase cells grown in Luria-Bertani’s (LB) medium (Wang et al., 2007). The composition of the growth medium was as follows: sucrose 1%; (NH4)2SO4 0.1%; K2HPO4 0.2%; MgSO4$7H2O 0.05%; NaCl 0.01%; yeast extract 0.05%; CaCO3 0.05%; Cu2(OH)2CO3 5%; pH 7.2. 5 mL (8.0  108 cfu mL
1) of each inoculum of Burkholderia sp. GL12, B. megaterium JL35 and Sphingomonas sp. YM22 were respectively inoculated in different 250 mL Erlenmeyer flasks (in triplicates) containing 100 mL of medium (autoclaved at 121  C for 30 min). Uninoculated Cu-amended medium (containing Cu2(OH)2CO3, 5%) or inoculated Cu-free medium were made as the controls to determine the abiotic influences on Cu solubility or the presence of Cu on the pH in the medium. Control flasks, previously autoclaved at 121  C for 30 min, and experimental flasks were incubated at 30  C on the rotary shaker at 150 rpm. After 72 h, the bacterial growth was monitored by measuring the optical density (OD) (1 OD z 5.0  108 cfu mL
1) at 600 nm. The harvested spent culture medium was centrifuged at 925g for 20 min at 6  C and then filtered through a 0.22-mm Millipore filter (Shanghai Xingya Purification Material Factory, China) for pH and water-soluble Cu determinations. The pH was determined with pH meter (PHS-3CT, China). The Cu concentrations in the supernatants were determined using the ICP-OES. 2.4. Pot experimental design In the pot experiment, a completely randomized design was used to investigate the effects of three heavy metal-resistant and PGPB on the maize growth as well as the Cu uptake of the plants grown in the natural heavily Cu-contaminated soils. Triplicate pots were used for each treatment. The heavy metal-contaminated soil was thoroughly mixed with fertilizers before addition to pots. Each kg of potted soil received 0.44 g urea and 0.88 g KH2PO4 (Jiang et al., 2008). Each pot (20 cm in diameter and 12 cm in height) contained 1.5 kg of above heavy metal-contaminated soil. The seeds of maize were surface-sterilized with a mixture of ethanol and 30% H2O2 (1:1) for 10 min and washed with sterile water (Jiang et al., 2008). Five surface-sterilized seeds were placed in each pot at a 2 cm depth. After germination (10 days), plants were thinned to two plants per pot. For inoculation, Burkholderia sp. GL12 (spectinomycin resistance), B. megaterium JL35 (spectinomycin resistance) and Sphingomonas sp. YM22 (streptomycin resistance) were grown in LB medium. Cells in the exponential phase were collected by centrifugation at 12,000 rpm for 10 min, washed with sterile distilled water, and recentrifuged. Bacterial inoculum was prepared by resuspending pelleted cells in sterile distilled water to get an inoculum density of 108 cfu mL
1. Bacterial suspensions (10 mL pot
1) were sprayed on the soil surfaces around the

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X. Sheng et al. / Journal of Environmental Management 103 (2012) 58e64

rhizosphere 3 weeks after seedling emergence. Sterile distilled water was sprayed on the soil surfaces as an uninoculated control. The inoculated and uninoculated planted pots were placed outdoors and moved the pots indoors before raining in order to protect them from the rainfall. During cultivation, minimal and maximal temperature ranged from 16  C to 20  C and 25  C to 32  C, respectively. The plants were watered as required. The plants were harvested 5 weeks after inoculation. The soil adhering loosely to the roots was removed by shaking the plants. Three replicate soils firmly adhering to the roots, designated as rhizosphere soil, were collected from each inoculated or uninoculated treatments by brushing respectively. Soil sampling tools were sterilized and the collected rhizosphere soils were placed in sterile bags to prevent cross-contamination of the soil samples. The fresh moist soil samples were passed through a 2-mm sieve and well dispersed for the analyses of the soil pH, extractable Cu concentrations, bacterial communities, and the colonization of the introduced bacteria. Soil pH was measured in distilled water with a ratio of soil-to-solution of 1:2.5 (He et al., 2010). Roots and above-ground tissues were separated and washed, first in several changes of 0.01 M EDTA and then in distilled water to remove any nonspecifically bound Cu and oven-dried for 30 min at 105  C, then at 55  C, until they reached constant weights before determining the root and above-ground tissue dry weights. The oven-dried samples were ground using a stainless steel mill (FZ102, Tianjing, China) to 0.5 mm for analysis. Subsamples of above-ground samples (200 mg) and root samples (200 mg) were then digested in a mixture of concentrated HNO3 and HClO4 (4:1, v/v) (Chen et al., 2004). The volume of each sample was adjusted to 10 mL using double deionized water. The concentrations of Cu in the samples were determined using the ICP-OES. Reagent blank and analytical duplicates were used where appropriate to ensure accuracy and precision in the analysis. The available Cu concentrations in the rhizosphere soils of the maize plants extracted by distilled water, 1 M NH4OAc and 0.05 M DTPA respectively were determined by the ICP-OES. 2.5. Total DNA extraction, purification, and PCR amplification Total DNA was extracted from 10 g of the above rhizosphere soil samples according to the method of Zhou et al. (1996), and then purified by DNA quick midi purification kit (TIANGEN Biotechnology Limited Company, Beijing). A 230-bp DNA fragment in the V3 region of the small subunit ribosomal RNA gene from the purified genomic DNA was amplified by using PCR with a thermocycler (2720 Thermal Cycler, Applied Biosystems). The primer set 341f and 534r as described by Muyzer et al. (1993) were used. The cycling program was as follows: an initial denaturation for 5 min at 95  C, followed by 28 cycles of amplification consisting of (1) denaturation at 93  C for 30 s (2) touchdown annealing (9 cycles at 65  C, 9 cycles at 60  C, 10 cycles at 55  C) for 40 s, and extension at 72  C for 1 min. The program ended with an extension step at 72  C for 10 min. Amplification products were analyzed by electrophoresis in 1.5% (w/v) agarose gels and by ethidium bromide staining. 2.6. Denaturing gradient gel electrophoresis (DGGE) DGGE was performed with a Code Universal Mutation Detection System (Bio-Rad Laboratories, Hercules, CA, USA). 20 mL of the PCR products (approximately 250 ng) were loaded on to 8% (w/v) acrylamide gel (acrylamide /bis solution, 37.5:1; Sigma) containing a linear chemical gradient ranging from 45 to 75% denaturant [7 M urea and 40% (v/v) formamide]. Electrophoresis ran 10 min at 200 Voltage at first, and then 16 h at 80 Volts at a temperature of 62  C

in a DGGE chamber containing approximately of 1  TAE buffer (0.04 M Tris base, 0.02 M sodium acetate, and 10 mM EDTA; pH adjusted to 7.4). After electrophoresis, the gel was stained for 40 min with SYBR GREENⅠDNA stain solution, and photographed using a UV transluminator (Bio-Rad).

2.7. Recovery of bands form DGGE gels and sequence analysis Characteristic bands on the DGGE gel were excised, transferred to clean Eppendorf tubes and smashed to release the DNA into 40 mL of sterile deionized water. With 2 mL of the eluted DNA as the template, PCR was performed to generate more target DNA for cloning. The thermal cycling parameters were as followed: 5 min at 95  C, 30 cycles of 45 s at 95  C, 45 s at 52  C and 90 s at 72  C, followed by final extension at 72  C for 10 min. The method for the examination of the PCR products was as described above. The amplification products were cloned into the pMD19-T vector (TakaRa Biotechnology Limited Company, Japan) and sequenced with an ABI model 3730 DNA sequencer (Invitrogen, Shanghai, China). The sequences were aligned with published sequences from the GenBank database using the NCBI BLAST comparison software (Altschul et al., 1997).

2.8. Analysis of DGGE band pattern To evaluate the bacterial community in different rhizosphere soil samples, the positions and signal intensities of detected bands in each gel track were determined with a gel documentation system, Quantity One 4.4.0 (Bio-Rad, USA). Lane background was subtracted by using the rolling disk size at 8 (Salles et al., 2004). Band-matching function with tolerance and optimization setting at 0.75% was used to identify and compare the bands present in each lane (Salles et al., 2004). We calculated the relative intensity of band to eliminate the bias caused by different PCR product amounts loaded in the gel (Salles et al., 2004). Cluster analysis of banding pattern was performed with the Quantity One 4.4.0 (Bio-Rad, USA) software package using the unweighted pair-group method analysis (UPGMA). Microbial community banding profiles on DGGE gels were analyzed using the Quantity One software package (Bio-Rad Laboratories, Hercules, CA, USA), and the correlations between individual band in PCR-DGGE fingerprints and environmental variables (bacterial inoculation) were determined by multivariate analysis using Canoco for Windows 4.5 software (Biometris, Wageningen, The Netherlands). 2.9. Colonization of metal and antibiotic resistant bacteria after inoculation For determination of the above rhizosphere soil colonization, 1 g soil was shaken with 10 mL sterile water and 1% fungicidin (USP, Amresco, USA) solution for 30 min. The resulting suspensions were evaluated for cfu according to the dilution-plate method on 1/5-strength LB agar with addition of 1 mM Cu (as Cu2SO4) for the Cu-resistant bacterial analysis and with addition of 1 mM Cu (as Cu2SO4) þ 100 mg mL
1 spectinomycin or 50 mg mL
1 streptomycin for the three introduced Cu and antibiotic resistant bacteria. By adding fungicidin and spectinomycin or streptomycin, the native fungal and bacterial flora were excluded from the plates. After incubation for 7 days at 28  C, the reisolated, spectinomycin or streptomycin-resistant strains were identified for colony characteristics, heavy metal resistance against the parent strains.

X. Sheng et al. / Journal of Environmental Management 103 (2012) 58e64

2.10. Statistical analysis

61

Cu in above-ground tissue Cu in root

Analysis of variance and the Tukey’s test (p < 0.05) were used to compare treatment means. All the statistical analyses were carried out using SPSS 10.0.

Above-ground tissue weight Root weight

1400

10 9

Burkholderia sp. GL12, B. megaterium JL35 and Sphingomonas sp. YM22 have the ability to solubilize Cu2(OH)2CO3. After 3 days of incubation, a decrease in the pH (4.11e6.10) of the medium inoculated with the strains was observed compared to the control (6.93). In addition, the associated pH drop corresponded to the increase in the cell growth (Table 1). The high quantities of biomass produced by the heavy metal-resistant strains GL12 and YM22 were accompanied with high levels of water-soluble Cu in the medium. Significant increase (p < 0.05) in the water-soluble Cu (20.5e227 mg L
1) released in the medium by the heavy metal-resistant strains was observed as compared to the control (14.8 mg L
1). The greatest Cu (p < 0.05) release was obtained by the strain YM22 (Table 1). Changes of water-soluble Cu (mg L
1), cell growth and pH in the liquid SMY medium supplemented with Cu2 (OH)2CO3 after 3 days of incubation 3.2. Plant growth promotion in pot experiment Fig. 1 showed that significant increases of above-ground tissue and root dry weights of maize plants were observed when the high level of Cu-contaminated soil was inoculated with the three strains, compared to the control. Inoculation with Burkholderia sp. GL12, B. megaterium JL35 and Sphingomonas sp. YM22 was found to significantly increase the above-ground tissue (ranging from 33% to 56%) and root (ranging from 48% to 83%) dry weights (p < 0.05) compared to the control (Fig. 1). The greatest above-ground tissue (p < 0.05) biomass was obtained with the strain JL35 (Fig. 1). 3.3. Mobilization of Cu in soil Table 2 showed that the water-extractive Cu contents in the rhizosphere soils of maize plants inoculated with Burkholderia sp. GL12, B. megaterium JL35 and Sphingomonas sp. YM22 were increased from 63% to 94% (p < 0.05) compared to the control. Inoculation with strains GL12 and JL35 was also found to significantly increase the NH4OAc-extractive Cu contents in the rhizosphere soils compared to the control. However, there were no significant changes in the DTPA-extractive Cu contents in the rhizosphere soils between the inoculated and uninoculated-maize plants (Table 2). Also, the pH in the rhizosphere soils did not change significantly between the inoculated and uninoculatedmaize plants (Table 2).

Table 1 Changes of water-soluble Cu (mg L
1), cell growth and pH in the liquid SMY medium supplemented with Cu2 (OH)2CO3 after 3 days of incubation. Strain

Water-soluble Cu

OD600

pH

Control Burkholderia sp. GL12 Bacillus megaterium JL35 Sphingomonas sp. YM22

15  1.7d 66  5.0b 21  2.4c 227  9.0aa

1.53  0.14 0.44  0.01 1.64  0.01

6.93  0.13aa 4.11  0.10c 6.10  0.23b 4.17  0.04c

a Means followed by the same letter within a column are not significantly different (P < 0.05) according to Tukey’s test.

8 1000

7

800

6 5

600

4 3

400

Dry weight (g pot -1)

3.1. Bacterial solubilization of copper in solution

Total Cu uptake (µg tissue -1)

1200

3. Results

2

200

1

0

0 Control

GL12

JL35

YM22

Bacterial strains added Fig. 1. The influence of the strains on the total Cu uptake (mg tissue
1) and dry weight (g pot
1) of maize plants grown in heavy metal-contaminated soils. Error bars are standard deviation (n ¼ 3).

The influence of the strains on available Cu contents (mg kg
1) and pH in the rhizosphere soils of maize plants grown in heavy metal-contaminated soils. Significant increases of total Cu uptake (ranging from 69% to 107%) in the roots of the maize plants inoculated with Burkholderia sp. GL12, B. megaterium JL35 and Sphingomonas sp. YM22 were observed compared to the control (Fig. 1). Inoculation with B. megaterium JL35 was also found to significantly increase the above-ground tissue total Cu uptakes (86%) of the maize plants compared to the control. In addition, in the bacterial-inoculated maize plants, total Cu uptakes in the roots were increased by 6.6-fold to 8.6-fold compared to that in the above-ground tissues. 3.4. Bacterial community profiles revealed by DGGE As shown in Fig. 2a, samples #1, #2, #3, #4, #5, and #6 from the inoculated rhizosphere soils of the maize plants showed a more complex DGGE pattern with average thirty three (33  2) visible bands than that of samples #7 and #8 from the uninoculated rhizosphere soils of the maize plants with average twenty eight (28  1.4) visible bands, indicating the presence of a higher number of different bacterial taxa in the inoculated rhizosphere soils of the maize plants. Except for the common bands between the inoculated and uninoculated rhizosphere soils of the maize plants, the special bands (Bands 2, 5, 6, 7, and 9) were found in the DGGE profiles of the inoculated rhizosphere soils of the maize plants. Clustering of the profiles revealed the differences among the eight rhizosphere soil samples (Fig. 2b). The greatest difference was

Table 2 The influence of the strains on available Cu contents (mg kg
1) and pH in rhizosphere soils of maize plants on heavy metal-contaminated soils. Strain

Available Cu

pH

Water-extractive NH4OAc-extractive DTPA-extractive Control GL12 JL35 YM22

0.16  0.01b 0.26  0.05a 0.29  0.04a 0.31  0.06aa

4.86  0.30b 5.55  0.04a 5.39  0.20a 5.42  0.30a

165  10a 169  5a 168  0.6a 173  2.5a

7.30  0.08a 7.38  0.08a 7.30  0.06a 7.35  0.09a

a Means followed by the same letter within a column are not significantly different (P < 0.05) according to Tukey’s test.

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X. Sheng et al. / Journal of Environmental Management 103 (2012) 58e64

Fig. 2. Analysis of the composition of the bacterial community in rhizosphere soil samples of inoculated- and uninoculated-maize plants grown in heavy metal-contaminated soil. (a) DGGE profiles of the rhizosphere soil samples; (b) Similarity of the rhizosphere soil samples based on a UPGMA clustering method. #1e#8 indicate the bacteria inoculated- and uninoculated rhizosphere soil samples. The bands marked with black or white dots and numbers were excised and sequenced.

found between the profiles of the control samples (#7 and #8) and all the inoculated samples (#1, #2, #3, #4, #5, and #6) (Fig. 2b). The profiles of the control samples clustered apart from the inoculated samples, with the similarity of 80%. The profiles of the inoculated samples were separated into two major clusters; profiles of samples #1 and #2 into one and profiles #3, #4, #5, and #6 into another group. However, all the rhizosphere soil samples showed the low similarity (44%) of the bacterial community. Total 14 characteristic bands (bands 1e14) were recovered from the gel, cloned and sequenced. Sequence homologies to known sequences in the NCBI database ranged from 96% to 100%. Phylogenetic affiliations of excised DGGE bands were represented by Acidobacteria (21.4%), Firmicutes (21.4%), a-Proteobacteria (14.3%), g-Proteobacteria (14.3%), d-Proteobacteria (7.1%), Actinobacteria (7.1%), Verrucomicrobia (7.1%), and unclassied bacterium (7.1%) (Table S1). Proteobacteria, Acidobacteria, and Firmicutes were the dominant bacterial groups in these soil samples. B. megaterium (band 6), Bacillus sp. (band 7) and uncultured bacterium (band 2) were observed in the rhizosphere soil samples inoculated with B. megaterium JL35, while Sphingomonas sp. (band 9) and uncultured bacterium (band 5) were observed in the rhizosphere soil samples inoculated with Sphingomonas sp. YM22. However, no special bacterial groups in the rhizosphere soil samples inoculated with Burkholderia sp. GL12 were found. In order to test the effects of bacterial inoculation on the structure of the bacterial community in the rhizosphere soils, the DGGE band profiles obtained from fingerprints were converted into computer digital images using the Quantity One software package (Bio-Rad Laboratories, Hercules, CA, USA), imported into Excel files to resolve individual peaks and quantify, analyzed by canonical correspondence analysis (CCA) using Canoco for Windows 4.5 software (Biometris, Wageningen, The Netherlands) (Fig. 3). The

first axis accounted for 12.3%, the second axis accounted for 8.8% (p ¼ 0.002) of the variation for the soil samples, respectively. The treatments with inoculated strains JL35, GL12 and YM22 replicates clustered together and the uninoculated rhizosphere bacterial

Fig. 3. Canonical correspondence analysis of the DGGE bacterial community profiles from the inoculated and uninoculated pot experiments. This triplot shows the relationship among the bands (represented by numbers in the plot) in the DGGE profiles and the four treatments tested (+ uninoculated control, A JL35-inoculated, : GL12inoculated, and ; YM22-inoculated). In numerical order, beginning at the top of the gel, a number was assigned to each possible vertical location for a band among the 8 lanes analyzed.

X. Sheng et al. / Journal of Environmental Management 103 (2012) 58e64

community was separated from the inoculated rhizosphere soil bacterial communities. All the inoculated-soil samples (#1, #2, #3 #4, and #6) except sample #5 generally distribute in the first and second quadrants, whereas, uninoculated-soil samples (#7 and #8) generally distribute in the third and fourth quadrants (Fig. 3). CCA revealed a significant effect of bacterial inoculation on the rhizosphere soil bacterial community structure (p ¼ 0.002). 3.5. Cu-resistant bacteria and survival and establishment of introduced bacteria in maize Except for Burkholderia sp. GL12, inoculation with B. megaterium JL35 and Sphingomonas sp. YM22 was found to significantly increase the number of Cu-resistant bacteria in the rhizosphers soils of the maize plants compared to the control (Fig. 4). The number of Cu-resistant bacteria in the rhizospher soils of the maize plants inoculated by B. megaterium JL35 and Sphingomonas sp. YM22 was increased by 42e72% compared to the control. The Cu and spectinomycin-resistant Burkholderia sp. GL12 and B. megaterium JL35 and Cu and streptomycin-resistant Sphingomonas sp. YM22 were tested for their ability to colonize maize rhizosphere soils. The inoculated bacteria were detectable in the maize-rhizophere soils for 5 weeks after inoculation (Fig. 4). However, the survival of the inoculated bacterial strain JL35 was better on maize rhizosphere soil (6.3  104 cfu g
1 of fresh soil) than that of the inoculated bacterial strains GL12 (0.35  104 cfu g
1 of fresh soil) and YM22 (3.5  104 cfu g
1 of fresh soil). 4. Discussion There are many regions where conventional agriculture is affected by the presence of the high level of heavy metals in the soils, causing economic losses and food safety concerns (Meers et al., 2010). Energy plant cultivation in the heavily metal-contaminated soils will be an alternative land use and play an important role in energy plant biomass production and ecological remediation of the heavy metal-polluted sites. Effective phytoextraction depends mainly on the bioavailability of heavy metals in soils and on the plant itself and the interaction of plant roots with bacteria. The study showed that the insoluble Cu was solubilized in the presence of the strains (Burkholderia sp. GL12, B. megaterium JL35 and Sphingomonas sp. YM22) as shown in

Cu-resistant bacteria

12

Introduced bacteria

7 6

*

5

10 *

4

8 3 6 2 4

1

2 0

0 Control

JL35 GL12 Bacterial strains added

YM 22

Introduced bacteria (×10 4 cfu g-1)

Cu-resistant bacteria (×10 6 cfu g-1)

14

-1

Fig. 4. The influence of the introduced strains on the number of Cu-resistant bacteria and the colonization of the introduced strains in the rhizosphere soils of maize grown in the heavy metal-contaminated soil. Error bars are standard deviation (n ¼ 3). An asterisk (*) denotes a value significantly greater than the corresponding control value (p < 0.05).

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Table 1, indicating that strains GL12, JL35, and YM22 have the potential of Cu-resistance and Cu solubilization. Studies have demonstrated that bacterial isolates could significantly increase heavy metal availability in soil (Chen et al., 2005; Abou-Shanab et al., 2006; Sheng and Xia, 2006). The same results were obtained in our pot experiment where the water-soluble Cu contents in the rhizosphere soils of the maize plants were significantly enhanced by the strains. However, non-significant decreases of pH in the rhizosphere soils of the maize plants were observed when the soil was inoculated with the three strains, compared to the control soil (Table 2). Maybe the organic acids in the rhizosphere soils were responsible for the increases in available Cu  ski et al., 1998). contents (Cieslin Rhizobacteria having the characteristics of producing IAA, siderophores and ACC deaminase can stimulate plant growth and protect plants against heavy metals toxicity in heavy metal-contaminated soils (Madhaiyan et al., 2007; Glick, 2010; Rajkumar et al., 2010; Ma et al., 2011; Zhang et al., 2011). Burkholderia sp. GL12, B. megaterium JL35 and Sphingomonas sp. YM22 possess multiple plant growth-promoting characteristics such as IAA, siderophores and ACC deaminase production and heavy metal-solubilizing ability which are responsible for the protection of the maize plants against Cu toxicity and the promotion of the root and above-ground tissue growth and Cu uptake of the maize plants grown in a heavily Cu-contaminated soil compared to the control (Fig. 1). Fan et al. (2011) isolated a root nodule bacterium, Sinorhizobium meliloti CCNWSX0020 from Medicago lupulina grown in mine tailings and found that inoculation with the strain increased the biomass of Medicago lupulina grown in Cu-added medium and the copper concentration inside the plant tissues. Andreazza et al. (2010) also showed that bacterial augmentation of oatmeal plants with rhizosphere isolates substantially improved copper bioaccumulation by oatmeal plants in the copper contaminated soils and a copper mining waste. As successful inoculants, bacteria must be able to rapidly colonize the root system during the growing season (de-Bashan et al., 2010). Although dilution-plate method showed that Burkholderia sp. GL12, B. megaterium JL35 and Sphingomonas sp. YM22 were able to colonize rhizosphere soils of the maize plants (Fig. 4), low survival of the inoculated Burkholderia sp. GL12 was observed on maize rhizosphere soils. In complex microflora, PCR-DGGE does not detect microorganisms present at a level lower than 1% of the total microbial population (Felske et al., 1998), so in the DGGE profiles, only B. megaterium JL35 (corresponding to band 6) and Sphingomonas sp. YM22 (corresponding to band 9) were detected in the rhizosphere soils of the maize plants (Table S1; Fig. 2). The results obtained showed that in the high level Cu-contaminated soils, B. megaterium JL35 and Sphingomonas sp. YM22 were able to colonize rhizosphere soils of the maize plants. Furthermore, DGGE and FISH analyses showed that the PGPB could change the bacterial colonization of the tailings by inducing proliferation of other rhizosphere bacteria on the root surfaces (de-Bashan et al., 2010). Clustering and CCA showed that inoculation with Burkholderia sp. GL12, B. megaterium JL35 and Sphingomonas sp. YM22 significantly changed the rhizosphere soil bacterial community structure in the pot experiment over a period of 35 days (Figs. 2 and 3). Root secretion induced by the PGPB may stimulate the proliferation of other bacteria in the rhizosphere soils (deBashan et al., 2010; Epelde et al., 2010). Rhizosphere fitness is a major condition for the success of any PGPB. Our study suggests that B. megaterium JL35 and Sphingomonas sp. YM22 have sufficient rhizosphere fitness to serve as an effective PGPB. Pot experiment demonstrated that the application of the PGPB could effectively promote the above-ground tissue biomass production and Cu uptake of the energy plants even under

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nonsterile conditions. Although we found that Cu accumulates mainly in roots and not in shoots, inoculation with the strains was found to significantly increase the total Cu uptake in the roots or shoots (with strain JL35) of the maize plants compared to the control, so in the long run a gradual reduction of the Cu pollution levels may be obtained after removing the maize plants. However, the extent of stimulation of the maize plants by the tested bacterial strains and the persistence of plant growth-promoting activity under high level Cu-contaminated field conditions remains unclear. Thus, experiments concerning stimulation of the energy plants and Cu uptake should be followed by investigations under high level Cu-contaminated field conditions. In addition, the maize plant (cultivar Zhendan-985) may be used to produce energy grains with concurrent phytoremediation of Cu under high level Cu-contaminated field condition in the presence of the metal-resistant and PGPB (Burkholderia sp. GL12, B. megaterium JL35 and Sphingomonas sp. YM22). 5. Conclusions The study demonstrated that the heavy metal-resistant and plant growth-promoting Burkholderia sp. GL12, B. megaterium JL35 and Sphingomonas sp. YM22 could increase the availability of Cu both in solution culture and in rhizosphere soils. Pot experiments showed that the bacteria could successfully colonize the rhizosphere soils of maize plants and significantly changed the bacterial communities in the rhizosphere soils, resulting in significant increases in biomass production and Cu uptake of the maize plants grown in the heavily Cu-contaminated soils. With the bacterial innate capability of expressing multiple traits and the high biomass and high capacity of Cu accumulation in maize plants, it might be potential for developing an effective plant-microbe partnership for energy plant biomass production and phytoextraction of Cu from heavily Cu-contaminated soils. A further understanding of the relationship between the energy plant and the heavy metal-resistant and plant growth-promoting bacteria is a critical prerequisite for the effective energy plant biomass production and phytoremediation of heavy metal-contaminated soils in natural ecosystem. Acknowledgments This research was supported by Chinese National Natural Science Foundation (40871127; 21007028), Chinese National Programs for High Technology Research and Development (2006AA10Z404) and the Fundamental Research Funds for the Central Universities (Y0201100263). Appendix. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jenvman.2012.02.030. References Abou-Shanab, R.A.I., Angle, J.S., Chaney, R.L., 2006. Bacterial inoculants affecting nickel uptake by Alyssum murale from low, moderate and high Ni soils. Soil Biol. Biochem. 38, 2882e2889. Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSIBLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389e3402. Andreazza, R., Okeke, B.C., Lambais, M.R., Bortolon, L., de Melo, G.W.B., de Oliveira Camargo, F.A., 2010. Bacterial stimulation of copper phytoaccumulation by bioaugmentation with rhizosphere bacteria. Chemosphere 81, 1149e1154. Belimov, A.A., Hontzeas, N., Safronova, V.I., Demchinskaya, S.V., Piluzza, G., Bullitta, S., Glick, B.R., 2005. Cadmium-tolerant plant growth-promoting bacteria associated with the roots of Indian mustard (Brassica juncea L. Czern.). Soil Biol. Biochem. 37, 241e250.

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