Phylogeny of Microorganisms Populating a Thick, Subaerial, Predominantly Lithotrophic Biofilm at an Extreme Acid Mine Drainage Site

Abstract

An unusually thick (ϳ1 cm) slime developed on a slump of finely disseminated pyrite ore within an extreme acid mine drainage site at Iron Mountain, near Redding, Calif. The slime was studied over the period of 1 year. The subaerial form of the slime distinguished it from more typical submerged streamers. Phylogenetic analysis of 16S rRNA genes revealed a diversity of sequences that were mostly novel. Nearest relatives to the majority of sequences came from iron-oxidizing acidophiles, and it appears that iron oxidation is the predominant metabolic characteristic of the organisms in the slime. The most abundant of the 16S rRNA genes detected were from organisms related to Leptospirillum species. The dominant sequence (71% of clones) may represent a new genus. Sequences within the Archaea of the Thermoplasmales lineage were detected. Most of these were only distantly related to known microorganisms. Also, sequences affiliating with Acidimicrobium were detected. Some of these were closely related to "Ferromicrobium acidophilus," and others were affiliated with a lineage only represented by environmental clones. Unexpectedly, sequences that affiliated within the delta subdivision of the Proteobacteria were detected. The predominant metabolic feature of bacteria of this subdivision is anaerobic sulfate or metal reduction. Thus, microenvironments of low redox potential possibly exist in the predominantly oxidizing environments of the slime. These results expand our knowledge of the biodiversity of acid mine drainage environments and extend our understanding of the ecology of extremely acidic systems.Dissolution of sulfide ores exposed to oxygen, water, and microorganisms results in acid production and environmentally detrimental acid mine drainage (AMD) (35). For the aqueous dissolution of sulfide ores dominated by pyrite (FeS 2 ) at low pH, ferric ion is the predominant oxidant. The overall reaction is written: FeS 2 ϩ 14Fe 3ϩ ϩ 8H 2 O315Fe 2ϩ ϩ 2SO 4 2Ϫ ϩ 16 H ϩ . The reaction is limited by the availability of ferric ion. At pH values of less than ϳ3.0, the inorganic rate of ferrous oxidation is slow, and acidophilic organisms can mediate production of ferric iron and conserve energy from this. Thus, it is not surprising that the oxidation of pyrite is greatly increased in the presence of iron-oxidizing species such as Thiobacillus ferrooxidans over the abiotic rate; see Nordstrom and Southham (36) for a discussion. Presently the understanding of biological enhanced pyrite oxidation is incomplete, but it is clear that microbial iron oxidation would replenish ferric ions for the above reaction.The best-studied organism with respect to microbial enhancement of AMD is T. ferrooxidans. Models have been proposed for its energetic characteristics (28) and role in pyrite dissolution (41), and several investigations have studied ironoxidizing and respiratory enzymes (7,11,17). However, numerous microorganisms are known for their acidiphily and iron-oxidizing capabilities, and it is apparent that different microorganisms have...

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