Biofilm bacterial community structure in streams affected by acid mine drainage

Abstract

We examined the bacterial communities of epilithic biofilms in 17 streams which represented a gradient ranging from relatively pristine streams to streams highly impacted by acid mine drainage (AMD). A combination of automated ribosomal intergenic spacer analysis with multivariate analysis and ordination provided a sensitive, high-throughput method to monitor the impact of AMD on stream bacterial communities. Significant differences in community structure were detected among neutral to alkaline (pH 6.7 to 8.3), acidic (pH 3.9 to 5.7), and very acidic (pH 2.8 to 3.5) streams. DNA sequence analysis revealed that the acidic streams were generally dominated by bacteria related to the iron-oxidizing genus Gallionella, while the organisms in very acidic streams were less diverse and included a high proportion of acidophilic eukaryotes, including taxa related to the algal genera Navicula and Klebsormidium. Despite the presence of high concentrations of dissolved metals (e.g., Al and Zn) and deposits of iron hydroxide in some of the streams studied, pH was the most important determinant of the observed differences in bacterial community variability. These findings confirm that any restoration activities in such systems must focus on dealing with pH as the first priority.

FIG. 1.

Ratio of the concentration of 16S rRNA genes to the concentration of 18S rRNA genes, comparing of the cycle threshold values for qPCR performed with biofilm DNA extract from each stream. The error bars indicate standard errors for five replicates per stream site. The dashed line indicates a 1:1 ratio. The values above the bars are average concentrations of bacterial 16S rRNA-primed DNA (black) and average concentrations of 18S rRNA-primed DNA (gray).

FIG. 2.

Differences in bacterial community profiles for biofilm samples from five rocks for 17 different streams. The plot is a nonmetric MDS plot of ARISA traces derived from a Bray-Curtis distance matrix of samples. 2D Stress = 0.25.

FIG. 3.

Differences in bacterial community profiles for rocks in different streams. Each plot was constructed from identical data using nonmetric MDS of ARISA traces derived from a Bray-Curtis distance matrix of samples. The data points are average traces for five bacterial community profiles for each site. 2D Stress = 0.18. The sizes of the gray circles reflect the relative values for the monitored water quality parameters recorded for each site. For plots (a and b), the data are grouped according to site category (a) (categories are described in Table 1) or pH (b) (N, neutral to alkaline [pH >6.7]. A, acidic [pH 3.9 to 5.7]; VA, very acidic [pH <3.5]).

FIG. 4.

Compositions of bacterial communities in the biofilms of a stream with a neutral pH (BR), an acidic stream (DU), and a very acidic stream (OB). Groups were identified by sequence analysis of 16S rRNA genes and are indicated as follows: black sections, alphaproteobacteria; white sections, betaproteobacteria; dark gray sections, gammaproteobacteria; light gray sections, deltaproteobacteria; dotted sections, other bacteria identified to the phylum level; sections with horizontal stripes, other bacteria not identified to the phylum level; sections with vertical stripes, eukaryotes. The smaller graphs show the same data but include only bacteria identified to the phylum level. The clone sequence accession numbers are FJ203732 to FJ203994.

FIG. 5.

Species accumulation curves for 16S rRNA gene sequences derived from clone libraries of DNA from biofilms in a stream with a neutral pH (BR), an acidic stream (DU), and a very acidic stream (OB), amplified using universal bacterial primers PB36 and PB38. A new species was considered to be present if the amplified DNA sequence shared <99% similarity with any other sequence analyzed for the same clone library.