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Evaluation of copper resistant bacteria from vineyard soils and mining waste for copper biosorption

Vineyard soils are frequently polluted with high concentrations of copper due application of copper sulfate in order to control fungal diseases. Bioremediation is an efficient process for the treatment of contaminated sites. Efficient copper sorption bacteria can be used for bioremoval of copper from contaminated sites. In this study, a total of 106 copper resistant bacteria were examined for resistance to copper toxicity and biosorption of copper. Eighty isolates (45 from vineyard Mollisol, 35 from Inceptisol) were obtained from EMBRAPA (Empresa Brasileira de Pesquisa Agropecuária) experimental station, Bento Gonçalves, RS, Brazil (29°09′53.92″S and 51°31′39.40″W) and 26 were obtained from copper mining waste from Caçapava do Sul, RS, Brazil (30°29′43.48″S and 53′32′37.87W). Based on resistance to copper toxicity and biosorption, 15 isolates were identified by 16S rRNA gene sequencing. Maximal copper resistance and biosorption at high copper concentration were observed with isolate N2 which removed 80 mg L−1 in 24 h. Contrarily isolate N11 (Bacillus pumilus) displayed the highest specific copper biosorption (121.82 mg/L/OD unit in 24 h). GenBank MEGABLAST analysis revealed that isolate N2 is 99% similar to Staphylococcus pasteuri. Results indicate that several of our isolates have potential use for bioremediation treatment of vineyards soils and mining waste contaminated with high copper concentration.

Treatment technologies such as ion exchange adsorption, electrodialyses, precipitation and chemical reduction can be used to remove heavy metals (17). These methods are conversely expensive compared to bioremediation processes.
Biological removal of pollutants is attractive in this technology, and it is considered cost-effective and eco-friendly (4,7,8,13,14,25). Contaminated environments select resistant microorganisms over pollutants (3). Microorganisms that are resistant to toxic and recalcitrant chemicals can be isolated from polluted sites as well as natural soils and used for bioremediation of environments contaminated with specific chemicals to which they are resistant (6,7,15,27,28).
Biosorption is an important bioremediation process for removal of copper and other toxic heavy metals from the environments.
In this study, in search for efficient strains for copper bioremoval, we examined a total of 106 copper resistant bacteria isolated from two copper contaminated vineyard soils and copper mining waste for copper biosorption at high concentration. We employed DNA-based methods to identify promising copper resistant isolates with potential for copper bioremoval from contaminated environments.

Soil sample
Soil samples were collected from three contaminated soils from South of Brazil. Two of them were collected from copper contaminated vineyards areas of EMBRAPA experimental station, Bento Gonçalves, RS, Brazil (29º09'53.92''S and 51º31'39.40''W). These two soils were classified as Inceptisol and Mollisol. The copper mining waste sample was obtained from the copper mining area of Caçapava do Sul, RS, Brazil (30º29'43.48''S and 53'32'37.87W). Soil samples and copper mining waste were characterized. Table 1 presents the physicochemical parameters analyzed in the Laboratory of Soil Analysis from Federal University of Rio Grande do Sul.

Enrichment and Isolation of Copper-Resistant Bacteria
Enrichment of copper resistant bacteria was in 100 mL of nutrient broth (NB) (5 g of Peptone and 3 g of Beef extract) in 250 mL Erlenmeyer flasks to which 300 mg L -1 of Cu(II) as copper sulfate (CuSO 4 .5H 2 O) was added and pH was adjusted to 7.0. NB was sterilized by autoclaving at 121ºC for 20 min.
The soil samples were independently used to inoculate (1%, w/v) sterile medium amended with Cu(II) and incubated for 24 h, with shaking (150 rpm) at 30ºC. Subsequently, 1 mL of enrichment culture was used to inoculate 99 mL of sterile medium amended with Cu(II) and incubated for 24 h, with shaking (150 rpm, 30ºC). This procedure was repeated two times. Cu(II)-resistant bacterial were thereafter purified by repeated streaking on nutrient agar (NA) plates containing Cu(II) (300 mg L -1 ). The isolates were coded with letter C for Mollisol isolates, letter N for Inceptisol isolates and letter R for waste from copper mining area.    (CuBiosor = CuTotal added -CuTotal after growth).

DNA Sequence Similarity and Phylogenetic Analysis
GenBank BLAST (N) was used for homology searches.
Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 4.1 (30). Nucleotide sequence similarity searches were conducted by Genbank BLAST (N).
The ribosomal RNA gene sequences were submitted to the GenBank database under accession numbers ranging from FJ577657 to FJ577671. units at high concentration of Cu(II) (300 mg L -1 ).     and R17 removed similar levels of copper from the culture. Table 4. Biomass levels and Cu(II) bioremoval in cultures of isolates from copper mining waste incubated in NB medium contaminated with 300 mg L -1 of Cu(II) and incubated at 30ºC for 24 h with orbital shaking.

Identity of selected isolates based on 16S rRNA gene sequence
Fifteen bacterial isolates selected on the basis of copper biosorption and resistances to high copper concentration were identified by 16S rRNA gene sequence analysis. Nucleotide sequences in the range of 471-506 nucleic acid bases (Table 5) were used for Genbank blast analysis and construction of phylogenetic tree. Most of the isolates were identified as  Figure 1 presents the phylogenetic relationship among selected isolates from three different copper contaminated areas in study.   High sorptive capacity of prokaryotic (29) and eukaryotic (19) microorganisms are due to components of cell walls that offer an array of functional groups with metal binding capacity.
Metals can also be accumulated in the cell cytoplasm (3), but some studies show that higher copper concentrations are linked to the cell wall than the cytoplasm in one prokaryote specie (29). Copper is one of the metals with great potential for bioremoval from contaminated environments through biosorption. In a comparative study (11) on selective binding of different metals to the cell wall of Pseudomonas sp., Cu(II) had much more affinity than other heavy metals like Ni(II), Co (II) and Cd(II) when evaluated together.
In summary, we evaluated copper biosorption by several environmental isolates of copper resistant bacteria. DNA-based methods were employed to characterize selected highly copper resistant isolates. Our results showed that several of the isolates have good potential for copper bioremoval from complex environments contaminated with copper.