An acid mine drainage (pH 2.5-2.7) stream biofilm situated 250 m below ground in the low-temperature (6-10°C) Kristineberg mine, northern Sweden, contained a microbial community equipped for growth at low temperature and acidic pH. Metagenomic sequencing of the biofilm and planktonic fractions identified the most abundant microorganism to be similar to the psychrotolerant acidophile, Acidithiobacillus ferrivorans. In addition, metagenome contigs were most similar to other Acidithiobacillus species, an Acidobacteria-like species, and a Gallionellaceae-like species. Analyses of the metagenomes indicated functional characteristics previously characterized as related to growth at low temperature including cold-shock proteins, several pathways for the production of compatible solutes and an anti-freeze protein. In addition, genes were predicted to encode functions related to pH homeostasis and metal resistance related to growth in the acidic metal-containing mine water. Metagenome analyses identified microorganisms capable of nitrogen fixation and exhibiting a primarily autotrophic lifestyle driven by the oxidation of the ferrous iron and inorganic sulfur compounds contained in the sulfidic mine waters. The study identified a low diversity of abundant microorganisms adapted to a low-temperature acidic environment as well as identifying some of the strategies the microorganisms employ to grow in this extreme environment.

Acidithiobacillus ferrivorans SS3 is a psychrotolerant acidophile capable of growth in the range of 5°to 30°C (optimum, Ϸ25°C). It gains energy from the oxidation of ferrous iron and inorganic sulfur compounds and obtains organic carbon from carbon dioxide. Here, we present the draft genome sequence of A. ferrivorans SS3 that will permit investigation of genes involved in growth in acidic environments at low temperatures.Psychrotolerant acidophiles from the Acidithiobacillus genus constitute a new species, Acidithiobacillus ferrivorans (4). This sequenced strain, A. ferrivorans SS3 (originally named Acidithiobacillus ferrooxidans SS3), was isolated from Norilsk, Russia, and was noted for its growth and oxidation of ferrous iron and inorganic sulfur compounds (ISCs) at low temperatures (2,7,8). Strains of A. ferrivorans catalyze low-temperature metal solubilization in the biotechnological process "bioleaching," and it is the dominant species during complex multi-metal sulfide mineral dissolution at 7°C (2). In addition, a mixed culture dominated by A. ferrivorans is being investigated for low-temperature ISC removal from mining process waters (9).DNA preparation, genome sequencing, and draft assembly. A. ferrivorans SS3 was colony streaked 3 times on agarose plates (5), and a single colony was inoculated into pH 2.5 mineral salts medium (3) and incubated at 6°C. Cells were pretreated with 200 g proteinase K ml Ϫ1 in Tris buffer for 10 min at 50°C and lysed with 2% (wt/vol) sodium dodecyl sulfate. High-quality DNA was prepared and then checked via the genomic DNA QC protocol (http://my.jgi.doe.gov/general/). Libraries were constructed for 454 (standard libraries sequenced to ϳ10-fold coverage) and Illumina draft sequencing (ϳ50-fold coverage). Finally, 454 paired-end libraries were constructed with a nominal insert size of 8 kb (sequenced to ϳ15-fold sequence depth). Gene prediction and annotation was performed as described previously (11,12).Genome analysis. The A. ferrivorans SS3 draft genome is 3,152,659 bp distributed in 61 contigs (Ն10 reads and Ն2 kbp), with an average coverage of 30-fold. The sequence has a GC content of 56.5% and, according to the JGI sequence annotation pipeline, contains 3,257 candidate protein-encoding gene models. The genome exhibits genes potentially encoding CO 2 fixation by the Calvin-Benson-Bassham cycle, rus and petI operons involved in iron oxidation, genes for assimilatory sulfur reduction, and a complete repertoire of genes for nitrogen metabolism. A suite of genes potentially encoding ISC metabolizing enzymes were identified (also present in other acidithiobacilli), including tetrathionate hydrolase (tth), sulfide quinone reductase (sqr), and thiosulfate quinone oxidoreductase (doxD). However, in contrast to sequenced A. ferrooxidans strains, A. ferrivorans SS3 contains genes potentially encoding the sulfur oxidation complex SOX (soxYZ-hypB) and a sulfur oxygenase:reductase gene (sor) similar to those in Acidithiobacillus caldus (10, 12). Unexpectedly, a full set of genes is p...

The psychrotolerant acidophile Acidithiobacillus ferrivorans has been identified from cold environments and has been shown to use ferrous iron and inorganic sulfur compounds as its energy sources. A bioinformatic evaluation presented in this study suggested that Acidithiobacillus ferrivorans utilized a ferrous iron oxidation pathway similar to that of the related species Acidithiobacillus ferrooxidans. However, the inorganic sulfur oxidation pathway was less clear, since the Acidithiobacillus ferrivorans genome contained genes from both Acidithiobacillus ferrooxidans and Acidithiobacillus caldus encoding enzymes whose assigned functions are redundant. Transcriptional analysis revealed that the petA1 and petB1 genes (implicated in ferrous iron oxidation) were downregulated upon growth on the inorganic sulfur compound tetrathionate but were on average 10.5-fold upregulated in the presence of ferrous iron. In contrast, expression of cyoB1 (involved in inorganic sulfur compound oxidation) was decreased 6.6-fold upon growth on ferrous iron alone. Competition assays between ferrous iron and tetrathionate with Acidithiobacillus ferrivorans SS3 precultured on chalcopyrite mineral showed a preference for ferrous iron oxidation over tetrathionate oxidation. Also, pure and mixed cultures of psychrotolerant acidophiles were utilized for the bioleaching of metal sulfide minerals in stirred tank reactors at 5 and 25°C in order to investigate the fate of ferrous iron and inorganic sulfur compounds. Solid sulfur accumulated in bioleaching cultures growing on a chalcopyrite concentrate. Sulfur accumulation halted mineral solubilization, but sulfur was oxidized after metal release had ceased. The data indicated that ferrous iron was preferentially oxidized during growth on chalcopyrite, a finding with important implications for biomining in cold environments.

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Process water and effluents from mining operations treating sulfide rich ores often contain considerable concentrations of metastable inorganic sulfur compounds such as thiosulfate and tetrathionate. These species may cause environmental problems if released to downstream recipients due to oxidation to sulfuric acid catalyzed by acidophilic microorganisms. Molecular phylogenic analysis of the tailings pond and recipient streams identified psychrotolerant and mesophilic inorganic sulfur compound oxidizing microorganisms. This suggested year round thiosalt oxidation occurs. Mining process waters may also contain inhibiting substances such as thiocyanate from cyanidation plants. However, toxicity experiments suggested their expected concentrations would not inhibit thiosalt oxidation by Acidithiobacillus ferrivorans SS3. A mixed culture from a permanently cold (4-6 °C) low pH environment was tested for thiosalt removal in a reactor design including a biogenerator and a main reactor containing a biofilm carrier. The biogenerator and main reactors were successively reduced in temperature to 5-6 °C when 43.8% of the chemical oxidation demand was removed. However, it was found that the oxidation of thiosulfate was not fully completed to sulfate since low residual concentrations of tetrathionate and trithionate were found in the discharge. This study has demonstrated the potential of using biotechnological solutions to remove inorganic sulfur compounds at 6°C and thus, reduce the impact of mining on the environment.