pH gradient-induced heterogeneity of Fe(III)-reducing microorganisms in coal mining-associated lake sediments

Abstract

Lakes formed because of coal mining are characterized by low pH and high concentrations of Fe(II) and sulfate. The anoxic sediment is often separated into an upper acidic zone (pH 3; zone I) with large amounts of reactive iron and a deeper slightly acidic zone (pH 5.5; zone III) with smaller amounts of iron. In this study, the impact of pH on the Fe(III)-reducing activities in both of these sediment zones was investigated, and molecular analyses that elucidated the sediment microbial diversity were performed. Fe(II) was formed in zone I and III sediment microcosms at rates that were approximately 710 and 895 nmol cm(-3) day(-1), respectively. A shift to pH 5.3 conditions increased Fe(II) formation in zone I by a factor of 2. A shift to pH 3 conditions inhibited Fe(II) formation in zone III. Clone libraries revealed that the majority of the clones from both zones (approximately 44%) belonged to the Acidobacteria phylum. Since Acidobacterium capsulatum reduced Fe oxides at pHs ranging from 2 to 5, Acidobacteria might be involved in the cycling of iron [corrected]. PCR products specific for species related to Acidiphilium revealed that there were higher numbers of phylotypes related to cultured Acidiphilium or Acidisphaera species in zone III than in zone I. From the PCR products obtained for bioleaching-associated bacteria, only one phylotype with a level of similarity to Acidithiobacillus ferrooxidans of 99% was obtained. Using primer sets specific for Geobacteraceae, PCR products were obtained in higher DNA dilutions from zone III than from zone I. Phylogenetic analysis of clone libraries obtained from Fe(III)-reducing enrichment cultures grown at pH 5.5 revealed that the majority of clones were closely related to members of the Betaproteobacteria, primarily species of Thiomonas. Our results demonstrated that the upper acidic sediment was inhabited by acidophiles or moderate acidophiles which can also reduce Fe(III) under slightly acidic conditions. The majority of Fe(III) reducers inhabiting the slightly acidic sediment had only minor capacities to be active under acidic conditions.

FIG. 1.

Vertical biogeochemical characterization of the sediment of acidic coal mining-associated Lake 77. One representative core obtained in November 2001 was used. (A) Symbols: ○, Fe(II); ▪, sulfate. (B) Symbols: ▴, pH; ▵, cell counts. The cell count data are the means ± standard deviations for three replicate sediment samples.

FIG. 2.

Frequencies of bacterial phylogenetic lineages detected in 16S rRNA gene-based clone libraries derived from zones I and III. Calculations were based on the total number of clones associated with phylotypes of sequenced representatives.

FIG. 3.

Effect of pH on microbe-catalyzed Fe(II) formation in zones I (A) and III (B). Sediment from replicate cores obtained in July 2003 was used. Symbols: •, Fe(II) formation at the in situ pH (pH 2.9 in zone I and pH 5.3 in zone III); ○, Fe(II) formation under altered pH conditions. The data are the means ± standard deviations of three replicates.

FIG. 4.

Effect of pH on microbe-catalyzed Fe(II) formation from schwertmannite by A. ferrooxidans DSM 583 at pH 3 (•), pH 4 (○), and pH 5 (▪) with elemental sulfur (10 mM) as the electron donor (A) and by A. capsulatum DSM 11244 at pH 5 (filled symbols) and pH 3 (open symbols) from FeOOH (• and ○) or goethite (▴ and ▵) or from soluble Fe(III)₂(SO₄)₃ at pH 2.2 (□) with glucose (2 mM) as the electron donor (B). The data are the means ± standard deviations of three replicates.

FIG. 5.

Phylogenetic tree of Acidiphilium-related 16S rRNA gene sequences (indicated by boldface type) determined by phylogenetic distance analysis with a maximum likelihood algorithm.

FIG. 6.

Phylogenetic tree of Deltaproteobacteria-related 16S rRNA gene sequences determined by phylogenetic distance analysis with a maximum-likelihood algorithm. The designations for clone sequences derived from dilution PCR analysis with group-specific primers begin with “D-PCR,” and the designations for clone sequences from sediment community clone libraries begin with “Common.”

FIG. 7.

Frequencies of bacterial phylogenetic lineages detected in 16S rRNA gene clone libraries from four MPN enrichment cultures of Fe(III)-reducing bacteria derived from zone III.