ENVIRONMENTAL SCIENCE AND TECHNOLOGY 2006 (I)

ARSENIC RESISTANT BACTERIA IN MINING WASTES FROM SHANGRAO COAL MINE OF CHINA

Jinbo Xiong, Wenming Wang, Haoxin Fan, Lin Cai and Gejiao Wang State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, P. R. of China

ABSTRACT: A total of 32 arsenic resistant bacterial strains were isolated and identified from the mining wastes of Shangrao coal mine of China. Twelve were isolated using arsenite enrichment cultivation method and twenty were identified using the culture-independent 16S rDNA library technique. Analysis of 16S rDNA revealed that they belong to 4 different phylogenetic Clades. They are Rhodococcus, Arthrobacter, Cupriaridus, Acinetobacter, Pseudomonas, Agrobacterium, Sinorhizobium, Bradyrhizobium, Pseudomonas, Rhodobium, Bacillus, Clostridium and some uncultured environmental clones. Strain C13 showed the highest arsenite resistant level. This strain was able to oxidize arsenite to arsenate aerobically and was identified as Agrobacterium sp.. Another two strains, C8 and C14, were able to reduce arsenate to arsenite and were identified as Arthrobacter sp. and Cupriavidus sp., respectively.

INTRODUCTION Arsenic (As) is one of the most prevalent toxic metalloid, occurring primarily as the inorganic species oxyanion arsenate (H3AsO4) [As(V)] and arsenite (H3AsO3) [As(III)]. Arsenic is a side waste of coal mining, thus the coal mines are generally considered as arsenic contaminated environments. Microorganisms are ubiquitous in arsenic geochemical environments and influence the biochemical cycle of As through conversion to As forms with different solubility, mobility, bioavailability, and toxicity (Silver and Phung, 2005). Several bacteria involved in arsenic transformation processes (through reduction, oxidation and methylation mechanisms) that belong to different phylogenetic groups have been reported (Stolz and Oremland, 2001). Bacterial oxidation of arsenite to arsenate has long been recognized. These bacteria were isolated from arsenic-impacted environments and identified as genera of Achromobacter, Pseudomonas, Alcaligene, Thiobacillus, Thiobacillus and Agrobacterium etc.. Even though chemolithotrophic bacteria have been isolated (Santini et al., 2000), most of the arsenite oxidation bacteria are heterotrophic; they do not gain energy through arsenite oxidation. Several genes encoding arsenite oxidases have been cloned and characterized, and the crystal structures of the arsenite oxidases have also been studied (Ellis et al., 2001). The reduction of arsenate by bacteria has been shown via two mechanisms: dissimilatory reduction and detoxification. Dissimilatory reductions of arsenate have been carried out by microbes, either strict anaerobic or facultative anaerobic, that couple anaerobic heterotrophic growth with arsenate as the terminal electron acceptor. The arsenic detoxification mechanisms have been investigated in various microorganisms of both anaerobes and aerobes (Niggemyer et al., 2001). Arsenate reduction bacteria; including Dusulfomicrobium, Clostridium, Bacillus, Sulfurospirillum, Citrobacter and Wolinella etc.; were identified (Oremland and Stolz, 2003). Even though some arsenite oxidation bacteria and arsenate reduction bacteria have been isolated, the arsenic resistant microbial population of coal mine environments has not been well studied so far. Thus the objective of this study was to identify such arsenic resistant micro-organisms from mining wastes using both cultivation and uncultured methods.

MATERIALS AND METHODS

Collection of coal mine wastes and isolation of arsenic resistant bacteria. The coal mine waste powder was collected from the surface horizon (0-15 cm) of the Shangrao coal mine of Jiangxi province, China. One hundred grams of the mining wastes were amended with Na-arsenite (NaAsO2) to a final concentration of 500 mg/Kg and kept for one week at 27°C. Isolation of arsenic resistant bacteria was performed by adding 1 g (triplicates) of the above treated mining wastes (dry wt.) to 9 ml 0.85% NaCl, vortexing for 10 min, diluting the extraction solution serially, plating the dilutions onto ISBN 0-9768853-6-0 © 2006 American Science Press

ENVIRONMENTAL SCIENCE AND TECHNOLOGY 2006 (I)

chemically defined CDM plates (Weeger and Lievremont, 1999) containing 100 mg/L Na-arsenite and incubating the plates at 27°C for 1 week.

Identification of arsenite oxidation and arsenate reduction strains. A single colony of each arsenic resistant isolate was inoculated into a 10 ml liquid CDM medium and incubated at 27°C with 200 rpm shaking. When the OD600 reached to 0.2, each culture was divided equally into two tubes. . Na-arsenite or Na-arsenate (Na3AsO4 12H2O) was added to each tube to a final concentration of 100 mg/L and the tubes were kept shaking for another 8 hrs. Five hundred microliters of each culture were transferred to a microcentrifuge tube and centrifuged for 5 min. Each supernatant was mixed with 25 l of 0.01 M KMnO4 gently. Purple precipitate represents the presence of arsenate in the culture supernatant, while the light brown precipitate represents the presence of arsenite.

Determination of minimal inhibition concentrations (MICs) of the arsenic resistant bacteria. A single colony of each arsenic resistant isolate was inoculated in triplicates into liquid CDM medium supplemented with increasing concentrations of sodium arsenite and shaken at 27°C for one week. The MICs, defined as the lowest As (III) concentration that completely inhibite the growth of each bacterium, were determined (Lim and Cooksey, 1993).

Detection of efficiencies of bacterial arsenite oxidation and arsenate reduction. This was performed using the molybdene blue method to measure arsenate quantum (Veronique et al., 2003). Phosphate was removed from the medium since it disturbs the absorbance of arsenate. Arsenate 3- reacts with MoO4 to form an arsenate-molybdate complex. This complex reacts with ascorbic acid to produce a blue color liquid that can be measured at 846 nm using a spectrophotometer. A single colony of the tested bacterium was inoculated to 100 ml of CDM medium (no phosphate) containing 125 mg/L of Na-arsenite and incubated at 27°C with rigorous shaking. Five milliliters of the culture were taken every hour. Three milliliters were used to check the OD values and the rests were centrifuged. Three hundred microliters of the supernatant were mixed with 5 ml ddH2O, 400 l of 50H2SO4, 200 l of 3% ascorbic acid, 400 l of 3% Na3MoO4 and boiled at 100°C for 10 min. This mixture was then brought to 10 ml with ddH2O and measured for the OD value at 846 nm. A standard curve with arsenate concentration from 0 to 3 mg/L was used. A similar method was used to detect the arsenate reduction efficiency. The differences were adding Na-arsenate (125 mg/L) to the medium instead of Na-arsenite and sampling the culture every 8 hrs.

Identification of uncultured arsenic resistant bacteria using 16S rDNA library method. The coal mine wastes from five randomly selected samples were mixed and DNA was extracted according to the manufacture’s recommendations (Fast DNA SPIN Kit, Q-BIOgene, USA). PCR amplification of the total 16S rDNA of the coal mine samples was performed using 16S rDNA universal primer 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’). PCR reaction was initiated at 94°C for 5 min followed by 32 cycles of 94°C for 1 min, 49°C for 1 min and 72°C for 1 min. The PCR products (∼1460 bp) were separated on 1% agarose gels and purified using the Gel Extraction kit (Watson Biotechnologies, China). The purified PCR products were ligated with pGEM-T (Promega, Madison, WI, USA) and transformed into E. coli DH5α by electroporation. The transformants were subsequently grown for 24 hrs on LB agar plates containing ampicillin, X-Gal and IPTG according to the manufacture’s recommendations. PCR-RFLP analysis was conducted for 92 randomly selected clones from the 16S rDNA library. Each PCR product was digested with the restriction enzyme AluI at 37°C for 4 hrs. The digested DNA fragments were separated on 2% agarose gels and grouped according to their DNA fingerprinting patterns.

DNA sequencing and phylogenetic analysis. A total of 32 dominant RFLP clones (12 pure isolates and 20 culture-independent clones) were selected for sequencing analysis. The DNA of each isolate was re-amplified by PCR and purified by UltraPureTM PCR kit (SBS Genetech, Shanghai, China). BLASTN searches (http://www.ncbi.nlm.nih.gov/BLAST) were used to compare similarities with the 16S rDNA sequences of GenBank. Sequence alignments were performed using the ClustalW algorithm (Thompson et al., 1994). A phylogenetic tree was generated from alignments by the

53 6 ENVIRONMENTAL SCIENCE AND TECHNOLOGY 2006 (I)

neighbour-joining method and the reliability of the inferred tree was tested with bootstrap test using the MEGA3 program (http://www.megasoftware.net). . RESULTS AND DISCUSSION

Arsenic-resistant bacteria from Shangrao coal mine wastes. The water soluble arsenic concentration of the coal mine wastes was 15 mg/Kg; thus the coal mine sample was considered as a middle level of arsenic contamination. The pH of the mining wastes was 7.8. A total of 21 arsenic- resistant bacteria from the coal mine wastes were isolated using an arsenite enrichment cultivation method. After screening with their morphologies and PCR-RFLP fingerprinting patterns, they were later confirmed as 12 strains (C5 etc., Fig. 1). These bacteria showed different arsenite resistant levels (C5 etc., Fig. 1).). Among them, strain C13 showed the highest arsenite resistant level (1,500 mg/L).

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Fig. 1. MIC test results for arsenite resistant strains. The experiments were performed as described in “Materials and Methods”. C13 is an arsenite oxidation strain.

Identification and phylogenetic analysis of the cultured and uncultured arsenic-resistant bacteria. Ninety-two uncultured 16S rDNA clones were analyzed by PCR-RFLP. A total of 20 representatives of the polymorphic PCR-RFLP fingerprinting groups were selected for 16S rDNA sequencing, together with the 12 cultured arsenic resistant bacteria. Data analysis revealed that these 32 16S rDNA sequences belong to four major phylogenetic Clades (Fig. 2). The majority of the 16S rDNA sequences of Clade 1 (18 isolates) were uncultured bacteria (14 clones, named as UC) and they were similar to the uncultured environmental clones, except for UC115, which was similar to Acidimicrobium. The small cluster located on the top of Clade 1 had four cultivated bacteria (C5, C8, C15 and C16). They were identified as Rhodococcus and Arthrobacter. Clades 2 and 3 contained mainly cultured arsenic-resistant bacteria (C22 etc.) belonging to Cupriaridus, Acinetobacter, Pseudomonas, Agrobacterium and Sinorhizobiu and also three uncultured clones that belong to Bradyrhizobium, Pseudomonas and Rhodobium. Clade 4 contained two uncultured clones that were identified as Bacillus and Clostridium (Fig. 2).

The arsenite oxidation and arsenate reduction bacteria. All of the 12 cultured arsenic-resistant bacteria were tested for their oxidation and reduction abilities with KMnO4 biochemical analysis. Strain C13 showed the ability to oxidize arsenite, and two strains, C8 and C14, were able to reduce arsenate to arsenite. Strains C13 and C8 were chosen to determine their arsenite oxidation and arsenate reduction efficiencies, respectively. Strain C13 was able to oxidize arsenite to arsenate aerobically. The arsenite oxidation started at 10 hrs after inoculation, and after 16 hrs, 100% of Na-arsenite was oxidized to arsenate (Fig. 3). C13 could not grow with As (III) as a sole energy source, and the growth was not enhanced in the presence of arsenite (data not shown). Heterotrophic metabolic conversion of As (III) to As (V) of C13 may couple to oxygen reduction. Biological oxidation of C13 occurred at a maximum speed of 29 M/h during exponential growth. This speed is faster than that of the reported arsenite oxidation strain

53 7 ENVIRONMENTAL SCIENCE AND TECHNOLOGY 2006 (I)

Agrobacterium tumefaciens (16.7 M/h, Kashyap et al., 2004). The generation time of C13 was 10 hrs.

Fig. 2. A phylogenetic tree showing the genetic relationship of the coal mine bacteria (bold) based on partial 16S ribosomal RNA gene sequences. Phylogenetic analysis was performed as described in “Materials and Methods”. “C” is for cultured bacteria and “UC” is for uncultured bacterial clones (bold). The others are referent 16S rDNA sequences from the Genbank. Scale bar 0.05 = 5% difference among nucleotide sequences.

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Strain C8 showed the ability to reduce arsenate to arsenite aerobically. The generation time of C8 was 17 hrs. Cells’ growth was concurrent with As (V) reduction. The reduction started after 17 hrs of incubation. After 64 hours, As (V) was transformed completely as the result of microbial activity (Fig. 4). The arsenate reduction speed of C8 was slower than a reported strain Thermus HR13, which reduced the same arsenate quantity in 16 hrs (Gihring and Banfield, 2001).

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CONCLUSION In this study, we identified the arsenic-resistant microbial population of a Chinese coal mine environment. Phylogenetic analysis of 16S rDNA revealed different Clades. The cultivated and uncultured arsenic-resistant bacteria were generally separated in different phylogenetic groups. Strain C13 showed the highest arsenite resistant level and could oxidize arsenite aerobically. Using the full-length 16S rDNA sequence, C13 was identified as Agrobacterium sp.. Another two strains, C8 and C14, were able to reduce arsenate to arsenite and were identified as Arthrobacter sp. and Cupriavidus sp., respectively. The rest arsenic resistant bacteria may contain the general arsenic resistant mechanisms through the function of ars genes, or may grow by using some nutrition from other died bacteria. These arsenic resistant micro-organisms may have the potential for bioremediation of arsenic contaminated environments.

ACKNOWLEDGMENTS We thank Kai Zhao for arsenic redox speciation analysis. This work was supported by the National Natural Science Foundation of China No. 30570058 for G.W.

REFERENCES

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Muller, D., D. Lievremont., D.D. Simeonova., J.C. Hubert., and M.C. Lett. 2003. Arsenite oxidase aox genes from a metal-resistant -proteobacterium.” J. Bacteriol. 185:135–141. Niggemyer, A., S. Spring., E. Stackebrandt., and R.F. Rosenzweig. 2001. “Isolation and characterization of a novel As(V)-reducing bacterium: implications for arsenic mobilization and the genus Desulfitobacterium.” Appl. Environ. Microbiol. 67:5568–5580. Salmassi, T., K. Venkanteswaren., M. Satomi., K.H. Nealson., D.K. Newman., and J.G. Hering. 2001. “Oxidation of arenite by Agrobcterium albertimagni, AOl15, sp.nov., isolated form Hot Creek, California.” Geomicrobiol. J. 19:53-66. Santin, J.M., L.I. Sly., R.D. Schnagl., and J.M. Macy. 2000. “A new chemolithic arsenite-oxidizing bacterium from a gold mine: phylogenetic, physiological and preliminary biochemical studies.” Appl. Environ. Microbiol.66:92-97. Silver, S. and Phung, L.T. 2005. “Genes and enzymes involved in bacterial oxidation and reduction of inoganic arsenic”. Appl. Environ. Microbiol. 71:599-608. Simeonova, D.D., L. Didier., L. Florence., and A.E. Daniel. 2004. “Microplate screening assay for the detection of arsenite-oxidizing and arsenate-reducing bacteria.” Microbiol. Letters 237:249—253. Stolz, J. F., and R. S. Oremland. 1999. “Bacterial respiration of arsenic and selenium.” FEMS Microbiol. Rev. 23:615–627. Thompson, J.D., D.G.Higgins, and T.J. Gibson. 1994. “CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice.” Nucleic Acids Res. 22: 4673–4680. Weeger, W., D. Lievremont., M. Perret., F. Lagarde., J.C. Hubert, M. Leroy, and M.C. Lett. 1999. “Oxidation of arsenite to arsenate by a bacterium isolated from an aquatic environment.” Biometals 12:141–149.

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