The microbial composition of acid streamers (macroscopic biofilms) in acidic, metal-rich waters in two locations (an abandoned copper mine and a chalybeate spa) in north Wales was studied using cultivationbased and biomolecular techniques. Known chemolithotrophic and heterotrophic acidophiles were readily isolated from disrupted streamers, but they accounted for only <1 to 7% of the total microorganisms present. Fluorescent in situ hybridization (FISH) revealed that 80 to 90% of the microbes in both types of streamers were ␤-Proteobacteria. Terminal restriction fragment length polymorphism analysis of the streamers suggested that a single bacterial species was dominant in the copper mine streamers, while two distinct bacteria (one of which was identical to the bacterium found in the copper mine streamers) accounted for about 90% of the streamers in the spa water. 16S rRNA gene clone libraries showed that the ␤-proteobacterium found in both locations was closely related to a clone detected previously in acid mine drainage in California and that its closest characterized relatives were neutrophilic ammonium oxidizers. Using a modified isolation technique, this bacterium was isolated from the copper mine streamers and shown to be a novel acidophilic autotrophic iron oxidizer. The ␤-proteobacterium found only in the spa streamers was closely related to the neutrophilic iron oxidizer Gallionella ferruginea. FISH analysis using oligonucleotide probes that targeted the two ␤-proteobacteria confirmed that the biodiversity of the streamers in both locations was very limited. The microbial compositions of the acid streamers found at the two north Wales sites are very different from the microbial compositions of the previously described acid streamers found at Iron Mountain, California, and the Rio Tinto, Spain.Microorganisms that inhabit the most extreme acidic niches on our planet (acidophiles) are highly diverse in terms of their physiologies and phylogenetic relationships (4, 18). There have been many reports of macroscopic microbial growth in extremely acidic (pH Ͻ3) metal-rich environments (mostly associated with mines and mine drainage waters) following the initial description by Lackey (26). These growths may occur as gelatinous filaments in flowing mine waters (acid streamers), as stalactite-like forms hanging from pit props and underground roofs (microbial pipes and snotites), or as thick biofilms. One of the first attempts to isolate bacteria from acid streamers was the attempt of Dugan et al. (11), although all of the isolates obtained in that study were neutrophilic rather than acidophilic. In contrast, acidophilic chemolithotrophic and heterotrophic acidophilic bacteria were isolated by McGinness and Johnson from acid streamers found in an abandoned pyrite mine (Cae Coch) in north Wales (32). These researchers considered acid streamers to be mixed communities of iron-and sulfur-oxidizing acidophiles, which acted as the primary producers in the mine, and heterotrophic bacteria, which utilized lysates and exuda...

Nineteen characterized strains and isolates of acidophilic heterotrophic bacteria were screened for their abilities to catalyse the reductive dissolution of the ferric iron mineral schwertmannite, under oxygen-limiting conditions. Acidocella facilis, Acidobacterium capsulatum, and all of the Acidiphilium, Acidocella and Acidobacterium-like isolates that grew in liquid cultures were able to reduce iron. In contrast, neither Acidisphaera rubrifaciens nor three Acidisphaera-like isolates tested were found to have the capacity for dissimilatory iron reduction. One of two iron-oxidizing Frateuria-like isolates also reduced iron under oxygen-limiting conditions. Microbial dissolution of schwertmannite was paralleled with increased concentrations of soluble ferrous iron and sulfate in microbial cultures, together with increased pH values and decreased redox potentials. While dissimilatory ferric iron reduction has been described previously for Acidiphilium spp., this is this first report of this capacity in Acidocella and the moderate acidophile Acidobacterium. The finding has significant implications for understanding of the biogeochemistry of acidic environments.

A novel bacterium, strain b6(T) (T=type strain), was isolated from a disused mine site by growth using arsenite [As(III)] as energy source in a simple mineral medium. Cells of strain b6(T) were rod-shaped, Gram-negative, non-sporulating and motile. Optimum growth occurred at temperatures between 20 and 30 degrees C, and at pH between 4.0 and 7.5. Strain b6(T) grew chemoautotrophically on As(III), sulphur and thiosulphate, and also heterotrophically on yeast extract and a variety of defined organic compounds. Several other Thiomonas strains, including the type species Thiomonas (Tm.) intermedia, were able to oxidize As(III), though only strain b6(T) and strain NO115 could grow using As(III) as sole energy source in the absence of any organic compound. The G+C content of the DNA of strain b6(T) was 65.1 mol %. Comparative small subunit (SSU) ribosomal RNA (rRNA) analysis indicated that strain b6(T) belongs to the genus Thiomonas in the beta-subdivision of the Proteobacteria. It was closely related to an unnamed Thiomonas strain (NO115) isolated from a Norwegian mining site, though sequence identities between strain b6(T) and characterized Thiomonas species were less than 95%. DNA-DNA hybridization between strain b6(T) and the type species of the genus Tm. intermedia showed less than 50% homology. On the basis of phylogenetic and phenotypic characteristics, strain b6(T) (DSM 16361(T), LMG 22795(T)) is proposed as the type strain of the new species Thiomonas arsenivorans, sp. nov.

A nine year study was carried out on the evolution of macroscopic “acid streamer” growths in acidic, metal-rich mine water from the point of construction of a new channel to drain an abandoned underground copper mine. The new channel became rapidly colonized by acidophilic bacteria: two species of autotrophic iron-oxidizers (Acidithiobacillus ferrivorans and “Ferrovum myxofaciens”) and a heterotrophic iron-oxidizer (a novel genus/species with the proposed name “Acidithrix ferrooxidans”). The same bacteria dominated the acid streamer communities for the entire nine year period, with the autotrophic species accounting for ~80% of the micro-organisms in the streamer growths (as determined by terminal restriction enzyme fragment length polymorphism (T-RFLP) analysis). Biodiversity of the acid streamers became somewhat greater in time, and included species of heterotrophic acidophiles that reduce ferric iron (Acidiphilium, Acidobacterium, Acidocella and gammaproteobacterium WJ2) and other autotrophic iron-oxidizers (Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans). The diversity of archaea in the acid streamers was far more limited; relatively few clones were obtained, all of which were very distantly related to known species of euryarchaeotes. Some differences were apparent between the acid streamer community and planktonic-phase bacteria. This study has provided unique insights into the evolution of an extremophilic microbial community, and identified several novel species of acidophilic prokaryotes.

Acidic, metal‐rich water that had accumulated within two abandoned, adjacent copper mines in north Wales was removed to prevent its possible catastrophic release. About 274 000 m3 of acidic (pH ∼2.4) mine water was pumped out of the mines over a 14‐week period. Concentrations of dissolved species (iron, sulfate, aluminium, copper, manganese and zinc) increased as water at lower depths within the mines was accessed. The discharged water flowed through a small wetland area, reaching the sea about 3 km north of the mine site. Analysis of the water at three sampling stations revealed that there was very little removal of aluminium and most of the heavy metals present except (ferrous) iron, which was partially removed as a result of oxidation and hydrolysis of the resulting ferric iron. The dominant bacterium in the subterranean mine water, which was essentially devoid of oxygen, was the iron‐ and sulfur‐oxidizer Acidithiobacillus ferrooxidans. The microbial populations in the pumped mine water were monitored using combined cultivation‐dependent (isolation on solid media) and cultivation‐independent (terminal restriction fragment length polymorphism and clone library) techniques. Other bacteria detected in the mine water included other iron‐oxidizers (Leptospirillum spp., ‘Ferrimicrobium acidiphilum’ and Gallionella‐like organisms) and heterotrophic acidophiles (Acidiphilium, Acidisphaera and Acidobacterium). Archaeal clones were also detected; most of these were related to methanogens. Owing to the absence of an effective remediation strategy, an estimated 7.5 tonnes of copper, 3.1 tonnes of manganese, 14.8 tonnes of zinc and 15.3 tonnes of aluminium was discharged into the Irish Sea as a consequence of the dewatering of the mines.

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