Microbial stratification in low pH oxic and suboxic macroscopic growths along an acid mine drainage

Abstract

Macroscopic growths at geographically separated acid mine drainages (AMDs) exhibit distinct populations. Yet, local heterogeneities are poorly understood. To gain novel mechanistic insights into this, we used OMICs tools to profile microbial populations coexisting in a single pyrite gallery AMD (pH ∼2) in three distinct compartments: two from a stratified streamer (uppermost oxic and lowermost anoxic sediment-attached strata) and one from a submerged anoxic non-stratified mat biofilm. The communities colonising pyrite and those in the mature formations appear to be populated by the greatest diversity of bacteria and archaea (including 'ARMAN' (archaeal Richmond Mine acidophilic nano-organisms)-related), as compared with the known AMD, with ∼44.9% unclassified sequences. We propose that the thick polymeric matrix may provide a safety shield against the prevailing extreme condition and also a massive carbon source, enabling non-typical acidophiles to develop more easily. Only 1 of 39 species were shared, suggesting a high metabolic heterogeneity in local microenvironments, defined by the O2 concentration, spatial location and biofilm architecture. The suboxic mats, compositionally most similar to each other, are more diverse and active for S, CO2, CH4, fatty acid and lipopolysaccharide metabolism. The oxic stratum of the streamer, displaying a higher diversity of the so-called 'ARMAN'-related Euryarchaeota, shows a higher expression level of proteins involved in signal transduction, cell growth and N, H2, Fe, aromatic amino acids, sphingolipid and peptidoglycan metabolism. Our study is the first to highlight profound taxonomic and functional shifts in single AMD formations, as well as new microbial species and the importance of H2 in acidic suboxic macroscopic growths.

Figure 1

Gallery scheme, showing the location of the main macroscopic growths (env1 microenvironment, where B1A and B1B develop, and env2, where B2 upwellings appear) found along the Los Rueldos AMD system. Main geochemical characteristics of the drainage are shown; metal and sulphate concentrations are represented in mg l⁻¹. Corresponding DO profiles to each microenvironment are displayed. Photographs illustrating the general aspect of the main microbial formations within the Los Rueldos AMD are included for further clarification. The reappearance of the B1 morphology at the end of the gallery is illustrated. The discontinuous line on the top represents the gallery ceiling with the presence of stalactites.

Figure 2

Maximum-likelihood phylogenies for the bacterial 16S rRNA full-length sequence amplicons retrieved from the constructed gene clone libraries. Scale bars represent changes per site or the ( × 100) % difference in nucleotide sequences. The main phylogenetic clusters are highlighted in grey. Bootstrap values are labelled at their corresponding nodes with circles: >85%, closed circles; >50%, open circles. The contribution of each OPU to the library is represented in the accompanying graphs. Representative sequences for each sample are differentiated by a colour code: B1A, red; B1B, blue; B2, green.

Figure 3

Venn diagram showing the common and distinct distribution of the number of (a) bacterial, archaeal, ‘ARMAN'-related and total OPU-based species (in this order from the left to the right), (b) potential protein-coding genes as assigned to pyrosequences, (c) proteins being expressed as found in the metaproteomes and (d) metabolic reactions associated to proteins being expressed.

Figure 4

Maximum-likelihood phylogenies for the archaeal 16S rRNA full-length sequence amplicons retrieved from the constructed gene clone libraries. Scale bars represent changes per site or the ( × 100) % difference in nucleotide sequences. The main phylogenetic clusters are highlighted in grey. Bootstrap values are indicated at the corresponding nodes with circles: >85%, closed circles; >50%, open circles. The contribution of each OPU to the library is represented in the accompanying graphs. Representative sequences for each sample are differentiated by a colour code: B1A, red; B1B, blue; B2, green.

Figure 5

The main differences at the level of microbial diversity between the Los Rueldos macroscopic growths and similar macroscopic structures previously reported from other AMD sites. (a) Mynydd Parys mine streamer (Hallberg et al., 2006). (b) Tinto River macroscopic floating filaments (López-Archilla et al., 2004). (c) Los Rueldos mine streamer/mats (this study). (d) Iron Mountain mine acidic biofilm (Tyson et al., 2004).

Figure 6

KEGG profiling analysis of the Los Rueldos and Richmond (AMD.R) AMD community genomes. Hierarchical clustering and heatmap colours are based on functional compositions of KEGGs as determined by the relative percentages of KEGG Orthology (KO) identifiers assigned to particular KEGG categories. The colour scale quantifies the relative percentages from one sample to the other, and goes from red (highly abundant) to black (abundant) to light green (low abundant).

Figure 7

Heat map and clustering of predicted genes encoding enzymes for the sulphur, nitrogen, CO₂, H₂ and iron metabolism in the Los Rueldos AMD macroscopic growths. The heatmap colours represent the relative percentages of distinct presumptive reactions assigned to particular enzymes within each sample. Hierarchical clustering based on relative percentage of predicted pooled genes encoding proteins involved in specifically transformations is shown. Note: a total of 438 (or 0.76%) of the total ORF fragments (B1A: 185; B1B: 145 and B2: 108) were identified as showing close sequence similarity to genes that encode enzymes known to be involved in the turnover of S, N, CO₂, H₂ and Fe and, accordingly, presumable functionalities were established and accounted in this figure. Colour scale as in Figure 6.

Figure 8

Scheme illustrating the presumably predominant metabolisms in the Los Rueldos microenvironments. Hypothetical initial steps of colonisation are indicated for the establishment of the B1 streamer as well as main metabolic transformations that give origin to auto- and heterotrophic metabolisms. AMD flow is indicated by the arrows at the right side of each.