Comparison of acid mine drainage microbial communities in physically and geochemically distinct ecosystems

Abstract

This study presents population analyses of microbial communities inhabiting a site of extreme acid mine drainage (AMD) production. The site is the inactive underground Richmond mine at Iron Mountain, Calif., where the weathering of a massive sulfide ore body (mostly pyrite) produces solutions with pHs of approximately 0.5 to approximately 1.0. Here we used a suite of oligonucleotide probes, designed from molecular data recently acquired from the site, to analyze a number of microbial environments by fluorescent in situ hybridization. Microbial-community analyses were correlated with geochemical and mineralogical data from those environments. The environments investigated were within the ore body and thus at the site of pyrite dissolution, as opposed to environments that occur downstream of the dissolution. Few organism types, as defined by the specificities of the oligonucleotide probes, dominated the microbial communities. The majority of the dominant organisms detected were newly discovered or organisms only recently associated with acid-leaching environments. "Ferroplasma" spp. were detected in many of the communities and were particularly dominant in environments of lowest pH and highest ionic strength. Leptospirillum spp. were also detected in many slime and pyrite-dominated environments. In samples of an unusual subaerial slime, a new uncultured Leptospirillum sp. dominated. Sulfobacillus spp. were detected as a prominent inhabitant in warmer ( approximately 43 degrees C) environments. The information gathered here is critical for determining organisms important to AMD production at Iron Mountain and for directing future studies of this process. The findings presented here also have relevance to the microbiology of industrial bioleaching and to the understanding of geochemical iron and sulfur cycles.

FIG. 1

Schematic map of the Richmond five-way section of the mine. Asterisks mark locations of environmental measurements and sampling, and the numbers correspond to sample descriptions (Table 2).

FIG. 2

Images of biofilm material present at the Iron Mountain mine. (A) Submerged slime streamers (biofilm) in a B-drift stream (approximately 1 m across) that are anchored to the sediment. The three drifts sampled all had macroscopically similar slime streamers. Reprinted with permission from the American Association for the Advancement of Science (14) (http://www.sciencemag.org). (B) Pendulous biofilms (termed snottites) hanging from the tunnel wall directly above the slump in the A drift.

FIG. 3

Environmental conditions of pH (□), temperature (○), and conductivity (▵) measured at the following sites: the A-drift slump (location 1) (A), the A-drift upper region (location 2) (B), the B-drift upper region (location 3) (C), the C-drift upper region (location 4) (D), and the discharge point (pipe entrance) at the Richmond five-way (location 5) (E). Refer to Fig. 1 for the location points of analytical measurements. Nov, November; Feb, February; Oct, October.

FIG. 4

Microbial-community analysis as determined by FISH cell counts on samples of the A-drift slump slime (A), A-drift snottite (B), B-drift slime (November 1998 sample) (C), B-drift slime (October 1999 sample) (D), B-drift sediment (October 1999 sample) (E), A-drift surface slime (F), C-drift slime (February 1999 sample) (G), and the C-drift slime (October 1999 sample) (H). The values obtained are expressed as percentages of the number of cells detected with the DAPI stain (see Materials and Methods). Only the positive portions of error bars are shown. Values shown as <1 were qualitative estimates only.

FIG. 5

FISH of Iron Mountain mine samples with various oligonucleotide probes. Horizontally paired images represent the same fields of view. On the left are cells detected by DAPI. On the right is the corresponding view of cells detected with Cy3-labeled probes. (A) Spirilla and curved rods detected with probe LF1252 for Leptospirillum group III cells in the slump slime from the A-drift. (B) Cocci attached to a crystal in the B-drift slime, detected with probe FER656, which is specific for the “Ferroplasma” genus. (C) Large rods and smaller curved rods in the C-drift slime, detected with the Bacteria-specific probe EUB338. (D) Pleomorphic cocci present in the C-drift slime as detected with probe TH1187, specific for organisms of the Thermoplasmales order. The size bar in panel D is 10 μm and applies to all panels.

FIG. 6

Transmission electron micrographs of cells in the slump slime (C) and snottite samples (A, B, and D). (A) Range of cell morphologies. (B) Small cell-like structures. (C) Lines of dividing cells. (D) Protein S-layer structure. The size bars in panels A, C, and D are 200 nm. The size bar in panel B is 100 nm.