Microbial Communities Associated With Passive Acidic Abandoned Coal Mine Remediation

Abstract

Acid mine drainage (AMD) is an environmental issue that can be characterized by either acidic or circumneutral pH and high dissolved metal content in contaminated waters. It is estimated to affect roughly 3000 miles of waterways within the state of Pennsylvania, with half being acidic and half being circumneutral. To negate the harmful effects of AMD, ∼300 passive remediation systems have been constructed within the state of Pennsylvania. In this study, we evaluated the microbial community structure and functional capability associated with Middle Branch passive remediation system in central PA. Sediment and water samples were collected from each area within the passive remediation system and its receiving stream. Environmental parameters associated with the remediation system were found to explain a significant amount of variation in microbial community structure. This study revealed shifts in microbial community structure from acidophilic bacteria in raw AMD discharge to a more metabolically diverse set of taxa (i.e., Acidimicrobiales, Rhizobiales, Chthoniobacteraceae) toward the end of the system. Vertical flow ponds and the aerobic wetland showed strong metabolic capability for sulfur redox environments. These findings are integral to the understanding of designing effective passive remediation systems because it provides insight as to how certain bacteria [sulfate reducing bacteria (SRBs) and sulfur oxidizing bacteria (SOBs)] are potentially contributing to a microbially mediated AMD remediation process. This study further supports previous investigations that demonstrated the effectiveness of SRBs in the process of removing sulfate and heavy metals from contaminated water.

Keywords: 16S rRNA; acid mine drainage; passive remediation system; shotgun-metagenomics; sulfate reducing bacteria.

FIGURE 1

Relative abundance bar chart of OTUs at the phylum taxonomic rank within each sampling location. Relative abundance outputs were generated from an unrarified OTU table picked using the USEARCH sequence analysis tool. The x-axis displays all sampling locations in chronological order, and the y-axis represents phyla abundance. The eight phyla that constituted the greatest percentage of total sample community composition across all sample locations within each sample matrix are plotted, and all remaining phyla were grouped into the “other” category.

FIGURE 2

Principal coordinates analysis (PCoA) plots were used to visualize differences in weighted Unifrac distances of instream, treatment ponds, and raw AMD input samples. Points clustered closely together are similar in terms of phylogenetic distance, whereas points that are distant from each other are phylogenetically distinct. Communities from the raw input from a distinct cluster from the rest of the sites, which include upstream, downstream, and the treatment system (ANOSIM P = 0.001). As indicated by the figure legend, points were colored by site (raw AMD, treatment system, upstream, and downstream) and shaped based on sample type (sediment and water).

FIGURE 3

A weighted principal coordinates axes (PCoA) plot and heatmap were generated to visualize community differences between water samples throughout Middle Branch. (A) The PcoA plot exhibits water samples which are colored based on sulfate concentration. Sites within the system and the receiving stream are clustered together toward the left. The raw AMD samples are highest in sulfate concentration and distinctly cluster separately from the treatment system and receiving stream. (B) Heatmap of SRB and SOB relative abundance throughout the remediation system. Darker shades of red indicate higher relative abundance whereas, lighter shades of color indicate lower relative abundance.

FIGURE 4

Relative abundance bar graphs of the top 10 OTUs at the order taxonomic rank within SAPS locations. The x-axis displays all sampling locations in chronological order, and the y-axis represents order abundance. The top 10 taxonomic order constituted the greatest percentage of total sample community composition across SAPS samples. Sample type (water and sediment) were separated into unique 100% bar graphs, where sediment samples are displayed on the right and water samples on the left half of the figure.

FIGURE 5

Heatmap of relative abundance levels of genes (KO numbers) related to sulfur metabolism throughout Middle Branch remediation system. Darker shades of red represent a higher level of abundance while lighter shades signify lower expression levels. Clustering along the top of the heatmap represent genes associated with dissimilatory sulfate reduction/oxidation. Clustering at the bottom represent genes associated with assimilatory sulfate reduction/oxidation. Genes with log transformed RPKM Counts below 1.5 were removed from the heatmap.