Bacterial, Archaeal, and Eukaryotic Diversity across Distinct Microhabitats in an Acid Mine Drainage

Abstract

Acid mine drainages are characterized by their low pH and the presence of dissolved toxic metallic species. Microorganisms survive in different microhabitats within the ecosystem, namely water, sediments, and biofilms. In this report, we surveyed the microbial diversity within all domains of life in the different microhabitats at Los Rueldos abandoned mercury underground mine (NW Spain), and predicted bacterial function based on community composition. Sediment samples contained higher proportions of soil bacteria (AD3, Acidobacteria), as well as Crenarchaeota and Methanomassiliicoccaceae archaea. Oxic and hypoxic biofilm samples were enriched in bacterial iron oxidizers from the genus Leptospirillum, order Acidithiobacillales, class Betaproteobacteria, and archaea from the class Thermoplasmata. Water samples were enriched in Cyanobacteria and Thermoplasmata archaea at a 3-98% of the sunlight influence, whilst Betaproteobacteria, Thermoplasmata archaea, and Micrarchaea dominated in acid water collected in total darkness. Stalactites hanging from the Fe-rich mine ceiling were dominated by the neutrophilic iron oxidizer Gallionella and other lineages that were absent in the rest of the microhabitats (e.g., Chlorobi, Chloroflexi). Eukaryotes were detected in biofilms and open-air water samples, and belonged mainly to clades SAR (Alveolata and Stramenopiles), and Opisthokonta (Fungi). Oxic and hypoxic biofilms displayed higher proportions of ciliates (Gonostomum, Oxytricha), whereas water samples were enriched in fungi (Paramicrosporidium and unknown microbial Helotiales). Predicted function through bacterial community composition suggested adaptive evolutive convergence of function in heterogeneous communities. Our study showcases a broad description of the microbial diversity across different microhabitats in the same environment and expands the knowledge on the diversity of microbial eukaryotes in AMD habitats.

Keywords: Archaea; Bacteria; Eukarya; acid mine drainage; biofilm; ore; sediment; stalactite.

FIGURE 1

Maps of the Los Rueldos emplacement in Asturias (NW Spain) (A). Depiction of the sampling locations along the AMD (B).

FIGURE 2

Spatial variation of geochemical variables [conductivity, Cond.; redox potential, Eh; pH; T, °C; and outside light (%)] of the AMD samples across collection sites. Symbols (dots or triangles) and vertical error bars represent the mean of the three measurements at each sampling location and their corresponding standard errors, respectively.

FIGURE 3

(A) Rarefaction curves at a 3% of dissimilarity cut-off among sequences. (B) Alpha diversity metrics (observed species, Chao1, Shannon, and Simpson indices) for Bacteria, Archaea, and Eukarya in Los Rueldos AMD samples.

FIGURE 4

Taxonomy profiles displaying the microbial diversity in the AMD samples, revealed by high throughput sequencing of the 16S/18S rRNA genes. (A) Bar plots displaying the relative abundances at the phylum level within Bacteria, Archaea, and Eukarya. (B) Heatmaps showing the main classes detected within each phyla.

FIGURE 5

Principal coordinates analysis (PCoA) based on the weighted UniFrac distance metric for Bacteria, Archaea, and Eukarya. PCoA on the phylogenetic distances among samples revealing the main taxa contributing to differences in their microbial diversity (A). PCoA to visualize similarities in the microbial composition (B).

FIGURE 6

Phylogeny of 18S rRNA full-length genes recovered from biofilm (B1A and B2) and water samples (WOUT and WEN). All clades from which microbial eukaryotes had been reported in AMD ecosystems were included. The groups containing newly detected microbial signatures are highlighted in yellow. Bar graphs represent number of sequences per OPU. Bubble charts are proportional to the abundance of each specified taxa. Scale bar represents changes per site (%).

FIGURE 7

Summarized model integrating diversity and predicted function in Los Rueldos microhabitats (mineral, water, and biofilm). Leptospirillum spp., Acidithiobacillales, and Betaproteobacteria are probable drivers of iron and sulfur oxidation in mineral (S and ST), water (WOUT, WEN, and WIN), and biofilm samples (BS, B1A, B1B, B2, and BF). The distribution of the neutrophilic iron oxidizer Gallionella is restricted to the stalactite ST. Carbon fixers are predictively photosynthetic in water ponds outside the gallery and are mainly represented by Cyanobacteria. Leptospirillum spp. are suggested to perform inorganic carbon and nitrogen fixation in all microhabitats inside of the cave. Decomposition of organic matter in the drainage accumulating outside of the gallery (WEN) would support the life of heterotrophic community members (Fungi, Protozoa, Proteobacteria, and Archaea) in this compartment. The extracellular polymeric matrix of the biofilms would constitute the main carbon source inside of the gallery, supporting the heterotrophic lifestyle of most abundant microbial groups present in the cave (Ciliates, Proteobacteria, and Archaea). The stagnant nature of the AMD would favor the existance of marked microhabitats along the drainage. Biofilm ciliates could have an impact on the population structure of bacterial members of the community, and the presence of different genera in distinct biofilm strata likely relates to differential bacterial/archaeal community composition, suggesting adaptation to distinctive environmental variables (oxic/hypoxic, water/mineral fraction).