Diversity of 16S ribosomal DNA-defined bacterial population in acid rock drainage from Japanese pyrite mine

doi:10.1263/jbb.100.644

Journal of Bioscience and Bioengineering

Volume 100, Issue 6, December 2005, Pages 644-652

https://doi.org/10.1263/jbb.100.644 Get rights and content

Four acidophilic bacteria (YARDs1-4) were isolated from an acid rock drainage (ARD) from Yanahara mine, Okayama prefecture, Japan. The physiological and 16S rDNA sequence analyses revealed that YARD1 was closely affiliated with Acidithiobacillus ferrooxidans, YARD2 was an Acidiphilium-like bacterium, and YARD3 and YARD4 were sulfur-oxidizing bacteria with a relatively close relationship to A. ferrooxidans in the phylogenetic analysis. A molecular approach based on the construction of a 16S rDNA clone library was used to investigate the microbial population of the ARD. Small-subunit rRNA genes were PCR amplified, subsequently cloned and screened for variation by a restriction fragment length polymorphism (RFLP) analysis. A total of 284 clones were grouped into 133 operational taxonomic units (OTUs) by the RFLP analysis. Among them, an OTU showing the same RFLP pattern as those of the isolates from the ARD was not detected. The phylogenetic analysis based on the 16S rDNA sequences from 10 major OTUs and their close relatives revealed that 4 OTUs containing 32.1% of the total clones were loosely affiliated with Verrucomicrobia, 2 OTUs containing 6.6% of the total clones were loosely affiliated with Chloribi, and other OTUs were affiliated with Actinobacteria, Nitrospirae, and β-Proteobacteria.

Section snippets

Environmental samples of ARD and physico-chemical analysis of samples

Water samples were collected from the ARD of Yanahara mine in October 2003. Physico-chemical analyses of the water samples were carried out at the Research Institute for Bioresources, Okayama University. The analytical methods used are listed in Table 1. The water samples were treated within 6 h of collection.

Isolation of acidophilic bacteria

Isolation of acidophilic bacteria from ARD samples from the Yanahara mine was carried out using five different media. Ferrous sulfate (3%) in 9K-medium [0.3% (NH₂) ₂SO₄, 0.05% K₂HPO₄,

Properties of acidophilic bacteria isolated from ARD

The physico-chemical properties of the ARD from the Yanahara mine are summarized in Table 1. The water was acidic due to the presence of sulfuric ions and had a high iron concentration. The concentrations of nitrate and phosphate ions were very low. In addition to iron, the concentrations of manganese, aluminum, nickel, and cobalt were relatively high. The temperature of the water is kept at approximately 22°C all year round. The high concentration of ferrous iron and the low concentration of

Acknowledgments

We thank Mr. Toshiyuki Takeshima, Dowa Mining Co., Ltd., Tokyo, for his cooperation in sampling ARD from the Yanahara mine. We thank Prof. Isao Aoyama and Mr. Hisao Nishizaki, Research Institute for Bioresources, Okayama University, for their help in physico-chemical analyses of the ARD from the Yanahara mine. This research was supported by the Yakumo Foundation for Environmental Science.

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Iron- and sulfur-oxidizing bacteria in a treatment plant of acid rock drainage (ARD) from a pyrite mine in Yanahara, Okayama prefecture, Japan, were analyzed using the gene (cbbL) encoding the large subunit of ribulose-1, 5-bisphosphate carboxylase/oxygenase (RubisCO). Analyses of partial sequences of cbbL genes from Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans and Acidithiobacillus caldus strains revealed the diversity in their cbbL gene sequences. In contrast to the presence of two copies of form I cbbL genes (cbbL1 and cbbL2) in A. ferrooxidans genome, A. thiooxidans and A. caldus had a single copy of form I cbbL gene in their genomes. A phylogenetic analysis based on deduced amino acid sequences from cbbL genes detected in the ARD treatment plant and their close relatives revealed that 89% of the total clones were affiliated with A. ferrooxidans. Clones loosely affiliated with the cbbL from A. thiooxidans NB1-3 or Thiobacillus denitrificans was also detected in the treatment plant. cbbL gene sequences of iron- or sulfur-oxidizing bacteria isolated from the ARD and the ARD treatment plant were not detected in the cbbL libraries from the treatment plant, suggesting the low frequencies of isolates in the samples.

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Diversity of 16S ribosomal DNA-defined bacterial population in acid rock drainage from Japanese pyrite mine

Section snippets

Environmental samples of ARD and physico-chemical analysis of samples

Isolation of acidophilic bacteria

Properties of acidophilic bacteria isolated from ARD

Acknowledgments

J. Microbiol. Methods

FEMS Microbiol. Ecol.

Adv. Appl. Microbiol.

Syst. Appl. Microbiol.

Syst. Appl. Microbiol.

FEMS microbiol. Ecol.

Syst. Appl. Microbiol.

FEMS Microbiol. Ecol.

FEMS Microbiol. Lett.

FEMS Microbiol. Ecol.

FEMS Microbiol. Ecol.

Mine-water chemistry: the good, the bad and the ugly

Environ. Geol.

Acid mine drainage and acid rock drainage processes in the environment of Herrerías mine (Iberian pyrite belt, Huelve-Spain) and impact on the Andevalo dum

Environ. Geol.

Bioleaching review part A: progress in bioleaching: fundamentals and mechanism of bacterial metal sulfide oxidation

Appl. Microbiol. Biotechnol.

(Bio)chemistry of bacterial leaching — direct vs indirect bioleaching

Hydrometallurgy

Mechanism of pyrite dissolution in the presence of Thiobacillus ferrooxidans

Appl. Environ. Microbiol.

Characteristics and adaptability of iron- and sulfur-oxidizing microorganisms used for the recovery of metals from minerals and their concentrates

Microb. Cell Fact.

Molecular characterization of Acidithiobacillus ferrooxidans and A. thiooxidans strains isolated from mine wastes in Brazil

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Phylogenetic heterogeneity of the species Acidithiobacillus ferrooxidans

Int. J. Syst. Evol. Microbiol.

Sulfurisphaera ohwakuensis gen. nov., sp. nov., a novel extremely thermophilic acidophile of the order Sulfolobales

Int. J. Syst. Bacteriol.

Ferroplasma acidiphilum gen. nov., sp. nov., an acidophilic, autotrophic, ferrous-iron-oxidizing, cell-wall-lacking, mesophilic member of the Ferroplasmaceae fam. nov., comprising a distinct lineage of the Archaea

Int. J. Syst. Evol. Microbiol.

An archaeal iron-oxidizing extreme acidophile important in acid mine drainage

Science

The acidophilic Thiobacilli and other bacteria that share their habitat

Annu. Rev. Microbiol.

Cultural and phylogenetic analysis of mixed microbial populations found in natural and commercial bioleaching environments

Appl. Environ. Microbiol.