Metabolically active eukaryotic communities in extremely acidic mine drainage

Abstract

Acid mine drainage (AMD) microbial communities contain microbial eukaryotes (both fungi and protists) that confer a biofilm structure and impact the abundance of bacteria and archaea and the community composition via grazing and other mechanisms. Since prokaryotes impact iron oxidation rates and thus regulate AMD generation rates, it is important to analyze the fungal and protistan populations. We utilized 18S rRNA and beta-tubulin gene phylogenies and fluorescent rRNA-specific probes to characterize the eukaryotic diversity and distribution in extremely acidic (pHs 0.8 to 1.38), warm (30 to 50 degrees C), metal-rich (up to 269 mM Fe(2+), 16.8 mM Zn, 8.5 mM As, and 4.1 mM Cu) AMD solutions from the Richmond Mine at Iron Mountain, Calif. A Rhodophyta (red algae) lineage and organisms from the Vahlkampfiidae family were identified. The fungal 18S rRNA and tubulin gene sequences formed two distinct phylogenetic groups associated with the classes Dothideomycetes and Eurotiomycetes. Three fungal isolates that were closely related to the Dothideomycetes clones were obtained. We suggest the name "Acidomyces richmondensis" for these isolates. Since these ascomycete fungi were morphologically indistinguishable, rRNA-specific oligonucleotide probes were designed to target the Dothideomycetes and Eurotiomycetes via fluorescent in situ hybridization (FISH). FISH analyses indicated that Eurotiomycetes are generally more abundant than Dothideomycetes in all of the seven locations studied within the Richmond Mine system. This is the first study to combine the culture-independent detection of fungi with in situ detection and a demonstration of activity in an acidic environment. The results expand our understanding of the subsurface AMD microbial community structure.

FIG. 1.

Phylogenetic correlations of AMD fungal 18S rRNA gene and beta-tubulin sequences belonging to the phylum Ascomycota. (A) Unrooted tree generated from a DNA alignment by the FastDNAMl ML method (22), with the Zygomycota and Basidiomycota fungal phyla as outgroups (not shown). The RT clones are from Rio Tinto AMD (8). Phylogenetic analyses of beta-tubulin protein sequences (generated with Protein_ML in ARB) associated with the Dothideomycetes (B) and Eurotiomycetes (C) classes from the mine are also shown. Tree B is a subset of taxa from tree C and was generated with the same sequences. In both B and C, the diplomonads Giardia intestinalis and Hexamita inflata were used as outgroups. Correlations between environmental sequences of the two molecules are shaded, and their classes are shown with brackets. Bayesian posterior probabilities, distance bootstrap values, and percentages of occurrence in the quartet-puzzling tree (in that order) are given at the corresponding nodes. The specificities of 18S rRNA probes are shown with brackets. Bar, 0.1 changes per site or 10% sequence difference.

FIG. 2.

Phylogeny (ML) of protozoan beta-tubulin proteins, including the IMTB41 and IMTB53 (in bold) sequences from the Richmond Mine. The taxonomic affiliations of the reference sequences are labeled on the right. The mine clones were statistically supported to be within the Rhodophyta, or red algae, group. Bayesian posterior probabilities, distance bootstrap values, and percentages of occurrence in the quartet-puzzling tree (in that order) are shown at the corresponding nodes. The diplomonads Giardia lamblia and Hexamita were used as outgroups. Bar, 0.1 changes per site.

FIG. 3.

FISH analyses of five-way (August 2002) community, using Doh299 (Cy3 labeled, highlighting IM group II [Dothideomycetes] in red), and Eur1108 (fluorescein isothiocyanate labeled, highlighting IM group I [Eurotiomycetes] in green) rRNA probes. The image is a collage of four images to the same scale, and the filaments are all <5 μm thick. There is significant nonspecific fluorescence in these images from the minerals (rounded objects) in the sample. Note that the Eurotiomycetes (in green) are branched in several places and that the Eurotiomycetes are more abundant than the Dothideomycetes.

FIG. 4.

Micrographs of protists from the Richmond Mine imaged with eukaryotic domain-level rRNA FISH probes. (A and B) The EUKb-mix probes (32) were labeled with Cy3 (red), and the DNAs were labeled with DAPI (blue). (C) The Euk502 probe labeled with Cy3 and DAPI is shown in green. Note that some of the protists were not targeted by this probe. N, nuclei of the eukaryotes; R, cytoplasmic staining of FISH probes corresponds to the cellular location of rRNA. Bars, ∼5 μm.