Microbial diversity in a brazilian acid moderate drainage and experimental nickel bioleaching system

L. Alves1, a, C. Bernardelli2, b, V. A. Leão1,c, M. C. Teixeira1, d, E. Donati2,e 1Federal University of Ouro Preto - Rua Costa Sena, 171. Ouro Preto, MG, Brazil. 35400-000 2Centro de Investigación y Desarrollo en Fermentaciones Industriales, CINDEFI (CONICET La

Plata, UNLP). Calle 50 y 115, (1900) La Plata, Argentina. alucienezinha@yahoo.com.br, bcecibe@quimica.unlp.ar, cva.leao@uol.com.br,

dteixeira@ef.ufop.br, edonati@quimica.unlp.ar

Keywords: Acid mine drainage (AMD), DGGE, FISH, Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans, Burkholderia Abstract. The aim of this work was to determine the microbial diversity of the acid mine drainage (AMD) material collected at an abandoned pyrite mine in Ouro Preto, Brazil. AMD samples were compared to a nickel sulfide column bioleaching pregnant solution which was used as reference. Fluorescent in situ hybridization analyses (FISH) and Denaturing Gradient Gel Electrophoresis (DGGE) were used. FISH analysis was carried out using specific 16S rRNA probes. The extracted DNA was amplified using universal primers for bacterial 16S rRNA genes and analyzed by DGGE. Acidithiobacillus. ferrooxidans was not detected in AMD samples. However, the presence of Acidithiobacillus thiooxidans was confirmed. In other hand, in the bioleaching tanks samples studied, both bacteria species were detected. The non-identified DNA bands were cloned and sequenced for complete characterization.

Introduction

AMD is the most severe environmental problem in the mining industry and is the result of chemical and biological oxidation of sulfide minerals like pyrite (FeS2) [1]. Classical microbial ecology analysis is limited by the unavoidable need for isolation of the microorganisms prior to their characterization [2,3]. Furthermore, culture-based methods are time consuming and are often selective [4] while culture-independent methods reveal a higher diversity degree when a bacterial community's composition is studied [5].In microbial molecular ecology it is well established that Denaturing Gradient Gel Electrophoresis (DGGE) offers a rapid culture-independent way for detecting and identifying predominant PCR-targeted populations [6,7]. Besides DGGE, Fluorescent in situ hybridization analyses (FISH), a quick and sensitivity technique, is a powerful tool for phylogenetic, ecologic and environmental studies in microbiology [2]. It detects nucleic acids sequences by a fluorescent labeled probe that hybridizes specifically to its complementary target sequence within the intact cell [2]. In this work, we studied and compared the community’s composition of an AMD in Ouro Preto, Minas Gerais State, Brazil with the community of nickel sulfide bioleaching system by DGGE, FISH and culture methods.

Materials and methods

Environmental samples from an AMD site and a bioleaching column system. AMD sampling was carried out in February, 2008 at an abandoned pyrite mine in Ouro Preto, Minas Gerais State, Brazil. Superficial water samples were collected using glass bottles. Pyrite and sediments were collected using plastic bags. Similar procedure was applied for column samples. The pregnant solution of one pilot scale nickel sulfide concentrate bioleaching system [8] was collected and filtered using paper (8µm) before been processed for DNA extraction. Temperature, pH and Eh were measured using a portable pHmeter.

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Studies of enrichment and isolation Experiments were carried out at CINDEFI in La Plata, Argentina. All experiments were replicated and done with analytical grade reagents and, if required, in sterile conditions. For chemiolithotrophic microorganisms sterile 50 ml flasks (Labotest) containing 40ml 9K medium pH 1.8 or iron-free 9K medium (0K medium) pH 3.0 were inoculated with solid and liquid samples (10%w/v or v/v). Flasks were kept at 32oC in orbital agitators (180 rpm). Cell number was determined by direct counting. For heterotrophic bacteria solid FeSo and Yeo media [9] and R3A media [10] supplied with 760µl of cycloheximide, were adopted. Studies of molecular characterization Experimental protocol for FISH was according to Amann, 1995 [2]. The oligonucleotides used were rRNA-targeted probes NON338 (negative control 5’-ACTCCTACGGGAGGCAGC-3’), ATT223 (5’-AGACGTAGGCTCCTCTTC-3’) LF665 (5’-CGCTTCCCTCTCCCAGCCT-3’) and TF539 (5’- CAGACCTAACGTACCGCC–3’) specific for At. thiooxidans, Leptospirillum ferrooxidans and At. ferrooxidans, respectively. Hybridized cells were stained with 4’6’-diamidino-2-phenylindole (DAPI) solution, washed with ethanol 80% and distilled water and slides were visualized by epifluorescence microscopy. For DGGE analysis, cells were washed and incubated with lysis buffer (0,05M NaOH, 0,025% SDS) at 95 oC, for 15 min and centrifuged at 13,000Xg, 2 min. DNA containing extract was stored at -20oC. Polymerase chain reaction (PCR) was performed using a Taq DNA Polymerase reagent kit (Invitrogen/Brazil) and GoTaq DNA Polymerase kit reagent (Promega/USA). PCR amplification of 16S rDNA was carried out using bacterial universal primers 341F-GC: 5’ – CCTACGGGAGGCAGCAG – 3’ and 907R: 5’ – CCTTCAATTCMTTTGAGTTT – 3’. The 16S rRNA fragment was obtained for subsequent DGGE analysis as previously described [6]. Bacterial PCR products were run at 60oC, 100V for 16h. Gels were stained with SYBR-Gold, bands were excised from gels, re-amplified, purified with QIAquick PCR purification Kit (QIAGEN Tecnolab/Argentina), cloned with P-GEM-T Easy Vector Systems (Promega/USA) and sequenced.

Results and discussion

AMD and bioleaching column characterization. Sample Eh, pH and temperature were 412 mV, 4.3 and 24ºC, respectively for the AMD. For the column system, the measured pH and Eh were 1.8 and 412mV, respectively. Enrichment and cultivation of microorganisms. No microbial growth was detected in the solid media for autotrophic microorganisms used in our studies. However, the presence of iron and sulfur oxidizing microorganisms was confirmed in the bioleaching samples for the growth of these microorganisms in 9K and 0K medium, respectively. Heterotrophic bacteria were not detected for the column system. In other hand, for AMD samples, only sulfur-oxidizing bacteria were detected using 0K liquid medium. Bacterial growth was also detected in solid media Yeo and R3A thus confirming the presence of heterotrophic organisms for AMD samples. The DNA obtained from the white creaming colonies was submitted to PCR and DGGE analysis. Obtained data were compared to those produced for natural (not enriched) samples and band profiles were similar. FISH, DGGE and sequencing. For FISH experiments, the positive controls using pure bacterial strains hybridized as expected. The probe NON338 was used as negative control and results shown that there was not a nonspecific fluorescence. Amplification of 16S rRNA gene with bacterial primers was successful in most of the samples resulting in reproducible DGGE fingerprints with a relatively small number of bands (fig. 2). Most bands were excised from the DGGE fingerprints, re-amplified, purified, cloned, sequenced and identified by using the BLAST program (www.ncbi.nlm.nih.gov). Samples from the bioleaching columns hybridized with the ATT223 and TF539 probes (fig.2), showing the presence of At. thiooxidans and At. ferrooxidans, respectively. It is important to notice that the column pH (1.8) and the previous enrichment of column inoculum in 0K and 9K media should have favored this bacterial population profile which was confirmed by DGGE analysis. The sequencing and analysis of the obtained clones showed 100% of similarity for

118 Biohydrometallurgy 2009

At. ferrooxidans. Liquid AMD material hybridized with the ATT223 probes (fig.1) thus showing the presence of At. thiooxidans. This result was confirmed after sequencing and shown 99% of similarity with At. thiooxidans. The absence of At. ferrooxidans can be explained by the high pH value (4.3) observed at the sampling point (4.3) as a consequence of the rainy season. Usually At. thiooxidans can grow up to higher pH valuest than At. ferrooxidans. It is also likely that, under those conditions, ferric iron ions could precipitate and iron-oxidizers cells could be adsorbed on its surface. Another bacterium, with 98% of similarity to the Burkholderia group, was also found. Some species of Burkholderia show some pathogenicity but others are involved into environmentally friendly technologies. This bacterial group has been studied for both phosphorous solubilization from high-phosphorous containing iron ores and arsenite bioremediation [11,12]. Some species from that group were also isolated in moderate acid mines drainage [13]. Valverde et al (2006) isolated a bacterial strain from a high-phosphorous iron ore from Minas Gerais State, Brazil, that, based on its genotypic and phenotypic characterization, was classified within a novel species of the genus Burkholderia, designated Burkholderia ferrariae [14]. The role of this bacterium in AMD requires further studies. Studies on pyrite usin DGGE showed band profile similar to that corresponding to At. thiooxidans. That was confirmed by sequencing (99% of similarity with At. thiooxidans, fig.2). DGGE is a powerful tool for community studies, but fails to notice less abundant populations, therefore the combination of different techniques (FISH, cloning, sequencing and culture) was very important in our study.

Fig.1. Epifluorescence micrographs of bacteria. Fluorescent in situ hybridization of bioleaching columns and AMD samples. DAPI stained cells in bioleaching tanks (A and C) and AMD (E). Samples hybridized with TF539 (B) and ATT223 probes (D and F) specific for group of At. ferrooxidans and At. thiooxidans, respectively. The presence of At. ferrooxidans and At. thiooxidans was confirmed for the bioleaching columns (B and D, respectively). At. thiooxidans was detected in all the AMD samples (F).

Fig. 2. Syber-Gold stained DGGE gels (A and B). 16S rRNA gene fragments amplified by PCR using universal primers for bacteria. A: 1, 2– Pyrite solids (bands a and b); 3– AMD (bands c, d and e); 4– Bioleaching columns; 5– At. thiooxidans; 6– At ferrooxidans. B: 1– At ferrooxidans; 3– Pyrite (b); 4– Pyrite; 5– Pyrite (a); 6– Pyrite; 7– At. thiooxidans.

B

A C

D

E

FB

A C

D

E

FB

A C

D

E

F

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Advanced Materials Research Vols. 71-73 119

Conclusion

In this work, we used a combination between culture independent and culture based methods to analyze the bacterial community of two different systems. For the bioleaching system, the presence of the At. ferrooxidans and At. thiooxidans was confirmed. Nevertheless, in AMD samples the usually expected microorganisms L. ferrooxidans and At. ferrooxidans, were not found. This fact could be explained by the relatively high pH values of the samples studied which may be responsible for the precipitation of ferrous iron thus causing the adsorption of iron-oxidizing microbes onto iron precipitate surfaces. Therefore, bacteria could become undetectable causing false negative results. Moreover, another interesting feature was observed, the occurrence of At. thiooxidans associated with Burkholderiaceae group (and, probably other still non identified species).

Acknowledgements

The financial assistance provided by Red Alfa BIOPROAM “Bioprocesos: tecnologías limpias para la protección y sustentabilidad del medio ambiente” (Contract N AML/190901/06/18414/II-0548-FC-FA) is gratefully appreciated. Authors thank ANPCYT (PICT 2006) for financial support.

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