Isolation, Identification, and Characterization of an Aluminum-Tolerant Bacterium Burkholderia sp. SB1 from an Acidic Red Soil

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Title: Isolation, Identification and Characterization of an Aluminum-Tolerant Bacterium Burkholderia sp. SB1 from Acidic Red Soil

Author: HUANG Shoucheng, WANG Xiaodong, LIU Xu, HE Genhe, WU Jichun

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Please cite this article as: HUANG Shoucheng, WANG Xiaodong, LIU Xu, HE Genhe, WU Jichun, Isolation, Identification and Characterization of an Aluminum-Tolerant Bacterium Burkholderia sp. SB1 from Acidic Red Soil, Pedosphere (2017), 10.1016/S1002-0160(17)60390-4.

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ACCEPTED MANUSCRIPT PEDOSPHERE Pedosphere ISSN 1002-0160/CN 32-1315/P

doi:10.1016/S1002-0160(17)60390-4

Isolation, Identification and Characterization of an Aluminum-Tolerant Bacterium Burkholderia sp. SB1 from Acidic Red Soil

HUANG Shoucheng 2, WANG Xiaodong 3, LIU Xu1, HE Genhe 1*, WU Jichun 4 1

School of Life Sciences, Key Laboratory of Biology diversity and Ecological Engineering of Jiangxi Province, Jinggangshan University,

Ji′an,343009, China; 2

School of Life Sciences, Anhui Science and Technology University, Fengyang, 233100, China.

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Jiangxi Academy of Forestry, Nanchang, 330013, China;

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Department of Hydrosciences, State Key Laboratory of Pollution Control and Resources Reuse, Nanjing University, Nanjing, 210093, China.

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∗Corresponding author. E-mail: [email protected].

ABSTRACT

An aluminum (Al)-tolerant bacterium strain, SB1, was isolated from the acidic red soil on Chingkang Mountain, located in the Jiangxi province of China. Polyphasic analysis, including a 16S rDNA phylogenetic tree as well as morphological and physicochemical properties, revealed that the isolate was a gram-negative, rod-shaped bacterium, which was recognized as Burkholderia sp. SB1 had extreme acidity tolerance (pH 2.2) and excellent Al resistance (270 mg/ L Al3+). It could remove Al

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up to 97.7% at a concentration of 54 mg/ L Al3+. The behavior of strain SB1 under different temperatures and antibiotics was also examined. We noticed that SB1 preferred moderate temperature conditions, ranging from 25 to 37℃, and exhibited notable

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resistance to multiple antibiotics (including ampicillin, streptomycin and tetracycline), but was sensitive to chloramphenicol. Therefore, as the first reported bacterium to possess favorable Al resistance and excellent Al removal rate, Burkholderia sp. SB1 can potentially be used as an agent for the bioremediation of Al toxicity in acidic red soil.

INTRODUCTION

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Key Words: Aluminum tolerance; Bacteria; Burkholderia sp.; Acidic red soil.

Aluminum (Al) is one of the most abundant trivalent metal elements in the Earth’s crust and is usually immobilized in all

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kinds of mineral forms in mildly acidic or neutral pH environments (Kochian et al., 2004). As the soil pH decreases, however, this element adopts a soluble state and is released into the soil, with detrimental effects on the growth of plants and microorganisms that are sensitive to Al toxicity (Amonette et al., 2003; Zheng et al., 2004). Plants have various mechanisms to perform Al detoxification (Panhwar et al. 2015). Microorganisms have also devised a variety of defenses against Al toxicity, including the thickening of the cell wall to immobilize Al (Wang et al., 2013), and the induction of organic acids to chelate Al extracellularly (Suzuki et al., 2007), among others. As we know, microorganisms play essential roles in the soil-plant ecosystem, where they act as a bridge for the adaptation of plants in severely disturbed soils (Aroca and Ruiz-Lozano, 2009). Moreover, microorganisms can contribute greatly to the dissipation of contaminants and thus improve the soil environment (García-Delgado et al., 2015; Liu et al., 2015). It has been reported that, in Al- contaminated soils, the application of particular microbes can facilitate the growth of plants; for example, the inoculation of plant growth- promoting bacteria could effectively ameliorate Al toxicity in rice (Panhwar et al., 2015). For this reason, the isolation and identification of stress-related microorganisms in extreme environments is of great importance for bioremediation. Acidic soil, which accounts for over forty percent of global arable land, presents a frustrating problem for plant growth (Kochian, 1995). Interestingly, extremely stressful environments usually contain undiscovered microorganisms that exhibit exceptionally high resistance to the corresponding stress. Thus far, a lot of microbes that exhibit high Al resistance, such as

ACCEPTED MANUSCRIPT Flavobacterium sp. and Penicillium janthineleum F-13, have been isolated from various soil environments (Konishi et al., 1994; Zhang et al., 2002), suggesting that such environments harbor microorganisms that are endowed with high Al tolerance. Some researchers have shown that fungi and yeasts are generally more tolerant to acidity than bacteria (Myrold and Nason, 1992; Kawai et al., 2000), but attempts to isolate Al-resistant bacteria continue to this day. It can be hypothesized that microbial communities in extreme stress conditions are quite different from those found in favorable soils and that Al tolerance is much more important for the survival of microorganisms in these stressful soils. Acidic red soils cover an extensive area of China. Due to significant and long-term acid rain erosion, these soils exhibit gradually lower pH values, but high levels of exchangeable Al. In spite of the fact that low pH and Al toxicity are the two major environmental stressors in these soils(Lu et al., 2011), little is known about microbial tolerance to this type of soils. Therefore, in the present study, we report the isolation, identification and characterization of an Al-tolerant bacterium from the typical acidic red soil of the Chingkang Mountain, located in t h e Jiangxi province of China. The objectives of the present work were to: (i) isolate and identify Al-tolerant bacterium from acidic red soils, (ii) investigate the morphological and physiochemical characteristics of the bacteria under different conditions, MATERIALS AND METHODS Soil sampling

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and (iii) study their capacity for Al resistance.

The soil samples represent a typical forest acidic red soil and were collected from Chingkang Mountain, in the middle section of the Lohsiao mountain range of the Jiangxi province, Eastern China (27° 06′ N, 115°01′ E). Details of the soil’s physicochemical

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properties can be found in our previous work(He et al., 2012). Culture conditions and isolation

The GM medium was used to investigate the effects of Al on the isolated microorganisms with a few modifications. The medium was composed of 1.0% glucose, 0.05% peptone, 0.02% yeast extract, and 0.02% MgSO4·7H2O, with the pH adjusted to 3.5 with HCl. The medium was autoclaved at 120℃ for 20 min. Al chloride hexahydrate (AlCl3·6H2O) solution was

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filter-sterilized and then added to the medium to a final Al3+ concentration of 50 µmol/L. A fungal inhibitor (Fungicidin, 50 mg/L) was also added. Culture was carried out in a rotary shaker at 37℃ and 220 rpm. After 24 h, when significant growth was obtained, one loopful of the growth medium was transferred to a medium containing 0.1 mmol/L of Al3+ for subsequent incubation, until

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significant growth was observed. This process was repeated up to a final Al3+ concentration of 4 mmol/L. When significant growth was observed in the presence of 4 mmol/L Al3+, the culture suspension was serially diluted and streaked onto agar plates for incubation at 37℃ for 48 h, until typical colonies had formed (Chantratita et al., 2007). Eight colonies were purified by further

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subculture on Ashdown agar medium and preserved in 30% glycerol stock at −20℃ for further use. Identification by PCR-RFLP and phylogenetic analysis All eight isolates were identified by PCR-RFLP based on amplification of t h e 16S rDNA gene. PCR was performed in a final

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volume of 25 μL using 0.1 μL bacterial suspension as the template, 0.5 μL each of the primers (10 μmol/L), 2 μL of dNTP mixture (2.5 mmol/L), 1.5 μL MgCl2 (25 mmol/L), 2.5 μL of 10× PCR reaction buffer and 0.2 μL of Taq DNA Polymerase (5 units/μL, Takara). The primers were: 27f (5’-AGAGT TTGAT CCTGG CTCAG-3’) and 1492r (5’-TACCT TGTTA CGACT T-3’). The thermocycler program included an initial denaturation at 94°C for 10 min, followed by 30 cycles at 94℃ for 30 sec, 55℃ for 40 sec and 72℃ for 1 min, with a final elongation at 72℃ for 10 min. The PCR products were visualized on a 1% ethidium bromide (EB) agarose gel. The PCR products were digested using two restriction enzymes, Hha I and Rsa I. The digested RFLP fragments were separated on a 2% (w/v) agarose gel and photographed. Clones with the same RFLP patterns were considered to represent a single operational taxonomic unit (OTU). A representative clone from each OTU was selected and sequenced. Subsequently, the sequences were analyzed using the NCBI Blast tool (http: //blast.ncbi.nlm.nih.gov/Blast.cgi) and RDP classifier (http:// rdp.cme.msu.edu/). A dendrogram based on 16S rDNA sequences was constructed by the neighbor joining (NJ) method, using MEGA 5.0 analytical software (Tamura et al., 2011). Characterization of Al-tolerant bacteria The identification of Al-tolerant bacteria was performed according to Bergey’s Manual of Determinative Bacteriology (Holt and Krieg, 1994). First, we studied the morphological characteristics of the Al-tolerant bacteria by streaking the strain on solid GM media. We also cultured the strain in liquid GM media for 28 h and observed morphological features using a scanning electron microscope (Zeiss, Supra55). Then, we assessed the effects of Al, temperature, pH and antibiotics on the growth of Al-tolerant

ACCEPTED MANUSCRIPT bacteria by spectrophotometry (OD600). Aluminum removal assay SB1 cells were individually inoculated into solutions containing initial Al3+ concentrations of 27 mg/L, 54 mg/L, 108 mg/L, 162 mg/L and 216 mg/L (in biological triplicates) and incubated at 37°C and 200 rpm for 24 h. After the incubation period, the culture was centrifuged at 8000×g for 15 min. The supernatants were carefully transferred into sterile and dry vials. The concentration of Al3+ in the supernatants was determined using an Atomic Adsorption Spectrophotometer (Perkin-Elmer Plasma 400 Emission Spectrometer, Perkin-Elmer Inc, USA). Statistical analysis All data were collected for three replicates and analyzed using SPSS 19.0 software for Windows (IBM Corporation, United States). The differences in all measurements were compared using one-way analysis of variance (ANOVA), followed by a least significant difference test (LSD, p≤0.05). RESULTS AND DISCUSSION

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Isolation and molecular identification of Al tolerant bacteria

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Eight strains of acid- and Al-tolerant bacteria were isolated and purified from the agar plates containing 4 mmol/L of Al3+ at pH 3.5. A single clone was picked from each strain and was then used for PCR amplification of the 16S rDNA gene (Fig. 1a). The eight sequences were digested with the two restriction enzymes (HhaI and RsaI) and a single unique OTU was identified (Fig. 1b, c, d). The sequence was submitted into DDBJ and was given accession number AB711470. According to the BLAST

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analysis of partial 16SrDNA sequence of strain SB1, SB1 clustered in a phylogenetic branch that contained Burkholderia species. Strain SB1 showed a high degree of similarity (99%) to the sequence of Burkholderia silvatlantica strain 41 16S ribosomal RNA (NCBI Accession No. KP974795), which was the first discovery that this bacterial species can resist Al toxicity. A phylogenetic tree based on related Burkholderia species is shown in Fig. 2. The new aluminum-tolerant strain SB1 was then referred to as

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Burkholderia sp. strain SB1.

Fig. 1 PCR amplification of the 16S rDNA gene of eight bacterial strains (a) and RFLP fingerprints of their 16S rDNA sequences using Hha I and/or Rsa I, respectively (b, c, d). Marker, DL2000 DNA ladder; Control, ddH2O.

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Fig. 2 Phylogenetic tree derived from 16S rDNA sequence of strain SB1 (The tree was evaluated by bootstrap analysis of the neighbor-joining method with 1,000 resamplings using MEGA 5.0) Morphological characteristics of strain SB1

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Colonies of strain SB1 on GM plates appeared thin, flat, faint yellow, opaque, round with smooth edges and approximately 1.2~3 mm in diameter (Fig. 3a). The ultrastructure of strain SB1 under scanning electron microscopy showed short, rod-like organisms 1.2~1.5×1.9~3.7 μm in size (Fig. 3b). Gram staining tests showed that strain SB1 is a Gram-negative bacterium (Fig. 3c). The

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morphological characteristics, therefore, confirmed that the isolate was similar to Burkholderia sp.. Fig. 3 Morphological identification of strain SB1. (a) the colony morphology, (b) the ultrastructure using scanning electron microscopy and (c) the Gram staining of strain SB1. Al tolerance of strain SB1 The growth curves of SB1 under different Al concentrations at pH 3.5 are displayed in Fig. 4. Al had a significant impact on the growth of SB1: as the Al concentration increased from 0 to 12 mmol/L, the growth rate of strain SB1 obviously decreased. Higher concentrations of Al (6~10 mmol/L) had stronger inhibitory effects on the growth of SB1. Moreover, we found that 12 mmol/L Al was lethal to SB1, as it could not grow at all, suggesting that this is the limiting concentration for the survival of SB1. Still, strain SB1 was able to grow in 10 mmol/L Al3+ at pH 3.5, a concentration t h a t is very toxic for most bacteria (Pina and Cervantes, 1996). Therefore, the results showed that SB1 has a very high tolerance to Al, this is in accordance with the previous finding that Burkholderia genera is one of predominant Al-resistant microorganisms in acidic forest soil (Kunito et al. 2012). To date, several Al-resistant bacteria have been reported, including the consortium bacteria (Desulfovibrio desulfuricans, Proteus sp. and Ralstonia sp.) (Martins et al., 2012), Providencia rettgeri (Abo-Amer et al., 2013), Rhizobium strains (Avelar Ferreira et al., 2012), Acidiphilium cryptum (Fischer et al., 2002), Flavobacterium sp. (Konishi et al., 1994) and Anoxybacillus sp.(Lim et al., 2014). Some of these bacteria, such as Acidiphilium cryptum, are able to grow in medium containing 17100 mg/L of Al2(SO4)3

ACCEPTED MANUSCRIPT without a lag phase and have inducible Al resistance (Fischer et al., 2002). Flavobacterium sp., has been reported to be Al-tolerant up to 2000 mg/L, while the maximum Al tolerance concentration of Arthrobacter sp. is approximately 300 mg/L (P and Mutschlechner, 2004). Another bacterium, Anoxybacillus sp. SK 3-4 was able to grow in medium containing 200 mg/L~800 mg/L Al with relative growth rates ranging from 77% to 100%. In our study, we found that Burkholderia sp. SB1 was able to grow in 10 mmol/L Al3+ (about 270 mg/L), suggesting that it has moderate Al tolerance in acidic red soil. Fig. 4 The turbidity growth curves of strain SB1 under different Al concentration and a continuous period incubation. Acid tolerance of strain SB1 Because Al mainly exists as inorganic monomeric Al at pH below 5.0, Al-tolerant microorganisms a l s o have to be acid-tolerant (Kawai et al., 2000). A previous report found a Al tolerant Burkholderia species, named Burkholderia acidipaludis sp. nov.,

demonstrated strong tolerance to acidity (Aizawa et al., 2010). The effects of pH values on the growth of strain SB1 under 0 or

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4 mmol/L Al were therefore studied. After the bacteria were cultured for 14 h, we measured the OD600 of SB1 (shown in Fig.

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5). The results indicated that the growth of strain SB1 was significantly retarded as the pH level decreased, regardless of the presence of Al. In the presence of Al, strain SB1 could hardly grow at pH 2.2, but it exhibited favorable growth performance at the same pH in the absence of Al, suggesting that the presence of Al exacerbates the inhibitory effect of acidity on the growth of SB1. Therefore, SB1 had excellent acid resistance, especially in the absence of Al,

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Although a small amount of yeast extract and peptone were included in the GM medium in the present study, their influence is likely to be negligible (Kawai et al., 2000). Therefore, our results demonstrate that strain SB1 has good acid resistance. Moreover, we found that acidity exacerbated the growth-inhibiting effect of Al on SB1. This is not consistent with the view that the Al-tolerant bacteria should mainly be influenced by Al itself. In contrast, the pH seems to be the more important factor previously reported for Al-sensitive species such as Arthrobacter sp. (P and Mutschlechner, 2004).

Removal of Al by strain SB1

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Fig. 5 Effects of pH values and Al on the growth of strain SB1

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Al-resistant bacteria usually have the ability to remove Al3+ to some degree, which alleviates the Al toxicity in soil environments. To evaluate the potential ability of strain SB1 to clean up the Al in contaminated environments, its capacity to remove Al3+ at a wide range of initial concentrations (27 mg/L, 54 mg/L, 108 mg/L, 162 mg/L and 216 mg/L) was tested (Fig. 6). The highest Al3+

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removal rate (up to 97.7%) was observed at an initial Al concentration of 54 mg/L. The removal rate varied from 74.4% to 84.4% at higher concentrations of Al3+. It has previously been reported that Providencia rettgeri can remove 97% of Al at an initial Al3+ concentration of 50 mg/L (Abo-Amer et al., 2013). A mixed culture of Desulfovibrio desulfuricans, Proteus sp. and Ralstonia sp. was able to remove 78% of Al at an initial Al3+ concentration of 173 mg/L after 27 days of incubation (Martins et al., 2012). In the

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present study, strain SB1 seems to be more adaptive to Al-containing environments and shows high efficiency in its ability to reduce Al toxicity. Considering t hat the Al concentration in our tested soil sample was not so high (11.5 mg/Kg), strain SB1 should have a excellent ability to remove Al3+.

Fig. 6 Removal rate of Al by strain SB1 at different initial concentrations of Al3+

Antibiotic resistant and temperature adaptability of strain SB1 Bacterial resistance refers to the ability of microorganisms to keep growing in a cytotoxic environment. Environmental microorganisms are successful creators of new biosynthetic pathways that generate new bioactive compounds (D'Costa et al., 2006). Antibiotic resistance in bacteria is more frequently associated with and strongly correlated with metal resistance (Calomiris et al., 1984). We evaluated the effect of some common antibiotics (Ampicillin, Streptomycin sulfate, Tetracycline and Chloramphenicol) on the growth of strain SB1 under 4 mmol/L Al at pH 3.5. After the bacteria were cultured for 14 h, we measured the OD600 of SB1 (Table 1). We found that, except for sensitivity to Chloramphenicol, strain SB1 was highly resistant to Ampicillin, Streptomycin sulfate and Tetracycline, and it even showed combined resistance to these three antibiotics.. The development of resistance may be due to a nonspecific mechanism with gene regulation of plasmids and chromosomes, which may be heritable or transferable due to the presence of a resistance factor (Silver and Walderhang, 1992). Jorquera et al reported the occurrence of Al tolerance plasmids in bacterial populations. Interestingly, the plasmids were also responsible for metal and antibiotic resistance (Jorquera et al., 2010). We found that SB1 not only had good acid and Al resistance but also had

ACCEPTED MANUSCRIPT multi-antibiotic resistance; the underlying relationship between acid/Al resistance and antibiotic resistance should be further studied. TABLE 1 Multiple antibiotic resistance of strain SB1 Several parameters (e.g., temperature and pH level) are known to interfere with Al toxicity (P and Mutschlechner, 2004). After 14 h of culture, we assessed the effects of temperature on the growth of strain SB1 under 4 mmol/L Al at pH3.5. The results indicated that strain SB1 is more adaptive to mild temperatures ranging from 25℃ to 37℃; it could hardly survive at a temperature of 55℃ (Table 1). It has been reported that increased temperatures increased Al toxicity in Arthrobacter sp. (P and Mutschlechner, 2004); however, whether temperature can affect Al toxicity in SB1 needs further investigation. In addition, the effects of temperature on the growth of SB1 were not correlated with the presence or absence of antibiotics, suggesting that the temperature and the antibiotic impact the growth of strain SB1 independently. TABLE 2 The effect of different temperatures on the growth of strain SB1 CONCLUSIONS In summary, the present work reports the isolation, identification and characterization of Al-tolerant bacteria from acidic red

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soil in Jiangxi province of China. Polyphasic analysis indicates that strain SB1 is gram-negative and rod-shaped, and it was

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identified as a Burkholderia sp. bacterium. Strain SB1 exhibits high resistance to Al and acidity. The combination of Al and acidity exaggerates the inhibitory effects on the growth of SB1. SB1 seems to be more adaptive to moderate temperatures (25~37℃) and is endowed with resistance to multiple antibiotics. Interestingly, as the first identified Burkholderia sp. bacterium that is capable

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of removing Al3+ up to a extreme percentage, strain SB1 might be a candidate for bioremediation in acidic red soil. ACKNOWLEDGEMENTS

This work was supported by the Natural Science Foundation of China (No.41462008) and the PhD research startup foundation

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of Jinggangshan University (No.JZB1307).

REFERENCES

Abo-Amer A, Ramadan A, Abo-State M, Abu-Gharbia M.2013. Biosorption of aluminum, cobalt, and copperions by Providencia

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rettgeri isolated from wastewater. Journal of Basic Microbiology. 53:477-488 Aizawa T, Bao Ve N, Vijarnsorn P, Nakajima M, Sunairi M. 2010. Burkholderia acidipaludis sp. nov., aluminium-tolerant bacteria isolated from Chinese water chestnut (Eleocharis dulcis) growing in highly acidic swamps in South-East Asia.

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International Journal of Systematic and Evolutionary Microbiology, 60: 2036-2041. Amonette JE, Russell CK, Carosino KA, Robinson NL, Ho JT.2003. Toxicity of Al to Desulfovibrio desulfuricans. Applied and Environmental Microbiology. 69:4057-4066.

Aroca R, Ruiz-Lozano JM. 2009.Induction of Plant Tolerance to Semi-arid Environments by Beneficial Soil Microorganisms – A

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Review. In: Lichtfouse E (ed) Climate Change, Intercropping, Pest Control and Beneficial Microorganisms. Springer Netherlands, pp 121-135

Avelar Ferreira P, Bomfeti C, Lima Soares B, de Souza Moreira F.2012.Efficient nitrogen-fixing Rhizobium strains isolated from amazonian soils are highly tolerant to acidity and aluminium. World Journal of Microbiology and Biotechnology. 28:1947-1959. Calomiris JJ, Armstrong JL, Seidler RJ.1984.Association of metal tolerance with multiple antibiotic resistance of bacteria isolated from drinking water. Applied and Environmental Microbiology. 47:1238-1242 Chantratita N Wuthiekanun V, Boonbumrung K, Tiyawisutsri R, Vesaratchavest M, Limmathurotsakul D, Chierakul W, Wongratanacheewin S, Pukritiyakamee S, White NJ, Day NPJ, Peacock SJ. 2007.Biological Relevance of Colony Morphology and Phenotypic Switching by Burkholderia pseudomallei. Journal of Bacteriology .189:807-817. D'Costa VM, McGrann KM, Hughes DW, Wright GD.2006. Sampling the Antibiotic Resistome. Science. 311:374-377 Fischer J, Quentmeier A, Gansel S, Sabados V, Friedrich C.2002.Inducible aluminum resistance of Acidiphilium cryptum and aluminum tolerance of other acidophilic bacteria. Archives of Microbiology. 178:554-558. García-Delgado C, Yunta F, Eymar E. 2015. Bioremediation of multi-polluted soil by spent mushroom (Agaricus bisporus) substrate: Polycyclic aromatic hydrocarbons degradation and Pb availability. Journal of Hazardous Materials. 300:281-288.

ACCEPTED MANUSCRIPT He GH, Wu JC, Liu Q, Zhang JF. 2012. Microbial and enzyme properties of acidic red soils under aluminum stress. Fresenius Environmental Bulletin.21:2818-2825 Holt JG, Krieg NR. 1994. Bergey's Manual of Determinative Bacteriology(9th ed.). Baltimore: Lippincott Williams & Wilkins Jorquera MA, Hernández M, Martínez O, Marschner P, Mora ML. 2010. Detection of aluminium tolerance plasmids and microbial diversity in the rhizosphere of plants. European Journal of Soil Biology. 46:255-263 Kawai F, Zhang D, Sugimoto M. 2000. Isolation and characterization of acid- and Al-tolerant microorganisms. FEMS Microbiol Lett. 189:143-147. Kochian LV.1995. Cellular mechanisms of aluminum toxicity and resistance in plants. In: Jones RL (ed) Annual Review of Plant Physiology and Plant Molecular Biology, pp 237-260 Kochian LV, Magalhaes JV, Garvin DF, Wang YH, Sorrells ME, Klein PE, Schaffert RE, Li L. 2004. Comparative mapping of a major aluminum tolerance gene in sorghum and other species in the Poaceae. Genetics. 167:1905-1914 . Konishi S, Souta I, Takahashi J, Ohmoto M, Kaneko S. 1994. Isolation and characteristics of acid-tolerant and aluminum-tolerant

t

bacterium. Bioscience Biotechnology and Biochemistry. 58:1960-1963.

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Kunito T, Owaki M, Ihyo Y, Sumi H, Toda H, Fukuda D, Park HD. 2012. Genera Burkholderia and Lipomyces are predominant aluminum-resistant microorganisms isolated from acidic forest soils using cycloheximide-amended growth media. Ann. Microbiol. 62:1339-1344.

Lim JC, Goh KM, Shamsir MS, Ibrahim Z, Chong CS. 2014. Characterization of aluminum resistant Anoxybacillus sp.SK 3-4

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isolated from a hot spring. Journal of Basic Microbiology. 54: (4):514-519.

Liu H, Guo S, Jiao K, Hou J, Xie H, Xu H. 2015. Bioremediation of soils co-contaminated with heavy metals and 2,4,5-trichlorophenol by fruiting body of Clitocybe maxima. Journal of Hazardous Materials. 294:121-127. Lu W, Ma Y, Lin C. 2011. Status of aluminium in environmental compartments contaminated by acidic mine water. Journal of Hazardous Materials. 189:700-709.

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Martins M, Taborda R, Silva G, Assunção A, Matos A, Costa M. 2012. Aluminum and sulphate removal by a highly Al-resistant dissimilatory sulphate-reducing bacteria community. Biodegradation 23:693-703. (Mitchell, R., Ed.):59-81

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Myrold D, Nason G. 1992. Effect of acid rain on soil microbial processes. Wiley-Liss, New York: Environmental Microbiology P PI, Mutschlechner W. 2004. Effect of temperature and pH on the toxicity of aluminum towards two new, soil born species of Arthrobacter sp. Journal of Basic Microbiology. 44:98-105

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Panhwar QA, Naher UA, Radziah O, Shamshuddin J, Razi IM. 2015.Eliminating aluminum toxicity in an acid sulfate soil for rice cultivation using plant growth promoting bacteria. Molecules 20:3628-3646. Pina RG, Cervantes C.1996. Microbial interactions with aluminium. Bio Metas. 9:311-316 Silver S, Walderhang M.1992. Gene regulation of Plasmid and chromosomes determined inorganic ion transport in bacteria.

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Microbiological Reviews. 56:195-228

Suzuki T, Tamura S, Nakanishi H, Tashiro M, Nishizawa N, Yoshimura E.2007). Reduction of Aluminum Toxicity by 2-Isopropylmalic Acid in the Budding Yeast Saccharomyces cerevisiae. Biological Trace Element Research. 120:257-263.

Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S.2011.MEGA5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Molecular Biology and Evolution. 28:2731-2739. Wang C, Zhao XQ, Aizawa T, Sunairi M, Shen R-F.2013.High Aluminum Tolerance of Rhodotorula sp. RS1 is Associated with Thickening of the Cell Wall Rather than Chelation of Aluminum Ions. Pedosphere. 23:29-38. Zhang D, Duine J, Kawai F.2002. The extremely high Al resistance of Penicillium janthineleum F-13 is not caused by internal or external sequestration of Al. Biometals. 15:167-174. Zheng S, Lin X, Yang J, Liu Q, Tang C .2004.The kinetics of aluminum adsorption and desorption by root cell walls of an aluminum resistant wheat (Triticum aestivum L.) cultivar. Plant and Soil. 261:85-90.

Figure captions

ACCEPTED MANUSCRIPT Fig. 1 PCR amplification of the 16S rDNA gene of eight bacterial strains (a) and RFLP fingerprints of their 16S rDNA sequences using Hha I and/or Rsa I, respectively (b, c, d). Marker, DL2000 DNA ladder; Control, ddH2O. Fig. 2 Phylogenetic tree derived from 16S rDNA sequence of strain SB1 (The tree was evaluated by bootstrap analysis of the neighbor-joining method with 1,000 resamplings using MEGA 5.0). Fig. 3 Morphological identification of strain SB1. (a) the colony morphology, (b) the ultrastructure using scanning electron microscopy and (c) the Gram staining of strain SB1. Fig. 4 The turbidity growth curves of strain SB1 under different Al concentration and a continuous period incubation. Fig. 5 Effects of pH values and Al on the growth of strain SB1. Fig. 6 Removal rate of Al by strain SB1 at different initial concentrations of Al3+ Tables Al concentrations (mmol) Amp Str Tm 0 Amp Str Tm 4 + Amp 4 + Str 4 + Tm 4 + Cm 4 + + + Amp Str Tm 4

Inital pH Antibiotics concentrations SB1 growth (µg/mL) (OD600 values) 3.50 0 0.703+0.05 3.50 0 0.562+0.06 3.50 50 0.562+0.04 3.50 30 0.514+0.02 3.50 30 0.651+0.09 3.50 34 0.002+0.00 3.50 50+30+30 0.572+0.05

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Antibiotics

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Table 1. Bacterial drug resistance analysis

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Amp: Ampicillin; Str: Streptomycin sulfur; Tm: Tetracycline; Cm: Chloramphenicol. Table 2. The Effect of different temperature on the growth of bacteria

Inital pH

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Temperature Al concentrations (ºC) (mmol) 25 4 25 4 37 4 37 4 45 4 45 4 55 4 55 4

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3.50 3.50 3.50 3.50 3.50 3.50 3.50 3.50

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Amp-Str-TmAmp+ Str +Tm+ Amp-Str-TmAmp+ Str +Tm+ Amp-Str-TmAmp+ Str +Tm+ Amp-Str-TmAmp+Str +Tm+

SB1 growth (OD600 values) 0.546+0.04 0.538+0.03 0.562+0.06 0.572+0.05 0.272+0.03 0.226+0.02 0.021+0.004 0.026+0.008

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