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An acid mine drainage (pH 2.5-2.7) stream biofilm situated 250 m below ground in the low-temperature (6-10 degrees 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.

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.

Psychrotolerant acidophiles have gained increasing interest because of their importance in biomining operations in environments where the temperature falls well below 10°C during large parts of the year.

Acidithiobacillus ferrivorans is the only characterized acidophile with the ability to live a psychrotrophic lifestyle and is able to oxidize ferrous iron and inorganic sulfur compounds at low temperature. The A. ferrivorans SS3 genome sequence mirrors its low temperature chemolithotrophic lifestyle and indicates ecological flexibility. Two enzyme systems for the synthesis of the protective molecule trehalose as well as multiple cold shock proteins suggest its psychrotolerance. In addition to genes coding for ferrous iron and inorganic sulfur compound oxidation enzymes, candidate genes for molecular hydrogen utilization were identified. Also, A. ferrivorans SS3 was suggested to have the ability to switch between nitrogen fixation and nitrogen uptake. Characterization of ferrous iron and inorganic sulfur compound oxidation during low temperature growth showed that both substrates were efficiently oxidized and revealed the potential of using ferric iron as an alternative electron acceptor. Gene transcript analyses also revealed constitutive expression of genes involved in ferrous iron oxidation and their rapid increase in expression when ferrous iron became available. Growth experiments further suggested ferrous iron was preferred over inorganic sulfur compounds during bioleaching. This phenomenon was especially evident during A. ferrivorans-mediated bioleaching of chalcopyrite as sulfur accumulated and eventually inhibited further leaching. A potential way to alleviate this problem is addition of low temperature, obligate inorganic sulfur compound oxidizing acidophiles. Although, these microorganisms have not been identified, analysis of a cold, acidic biofilm from Kristineberg mine suggested additional psychrotolerant inorganic sulfur compound oxidation oxidizers might be present. Psychrotolerant acidophiles from Kristineberg mine have also been demonstrated to remove inorganic sulfur compounds from mining process water at in situ temperatures. This use of indigenous microorganisms for removal of environmental pollutants is a big step towards greener mining.

Syra-älskande bakterier, s.k. acidofiler, är mikroorganismer som lever i ofta metallrika miljöer med väldigt lågt pH. Dom används industriellt i en process kallad biolakning där metaller utvinns från mineral genom den katalyserande förmågan hos dessa mikroorganismer.

Acidithiobacillus ferrivorans är en nyligen karaktäriserad acidofil som oxiderar järn- och svavelföreningar vid låga temperaturer, den första och hittills enda av sitt slag. Den har visat sig väldigt användbar vid biolakning i kalla miljöer, såsom i Sveriges norra delar där medeltemperaturen sjunker under 10°C under stora delar av året. En inblick i den genetiska potentialen hos denna mikroorganism avspeglar dess livsstil samt avslöjar en bred ekologisk flexibilitet. Ett flertal gener kan sammankopplas med dess förmåga att överleva vid låga temperaturer, såsom gener för tillverkning av s.k. köldchockproteiner. Vid sidan om gener som kodar för järn- och svaveloxiderande enzymer återfinns potentialen att oxidera vätgas. A. ferrivorans verkar även kunna växla mellan att fixera kvävgas och ta upp kvävejoner ifrån sin omgivande miljö, beroende på den tillgängliga kvävekällan. Vidare karaktärisering av järn- och svaveloxidering vid låga temperaturer visade att båda typer av substrat kan oxideras effektivt men avslöjar även att svaveloxidering kan sammankopplas med reducering av järn istället för den traditionella syrgasen. Analys av genuttryck visade att gener för järnoxidering var kontinuerligt uttryckta och att en signifikant uppreglering sker så snart järn introduceras. Det visade sig även att järn verkar vara det föredragna substratet vid biolakning. Detta fenomen är speciellt uppenbart under biolakningsexperiment med A. ferrivorans då elementärt svavel succesivt ackumuleras och så småningom hindrar vidare frigörelse av metaller. En lösning på detta problem skulle kunna vara att tillsätta köldtoleranta mikroorganismer med enbart svaveloxiderande förmåga. Idag finns inte några sådana bakterier beskrivna, men analys utav en kontinuerligt kyld och sur biofilm isolerad ifrån Kristinebergsgruvan indikerar emellertid förekomsten av svaveloxiderande bakterier. Köldtoleranta acidofiler från Kristinebergsgruvan har även visat sig användbara vid industriellt avlägsnande av svavelföreningar från gruvindustriellt processvatten. Att använda naturligt förekommande mikroorganismer vid rening av miljöfarligt avfall är ett stort steg i riktning mot en grönare gruvindustri.

Acidithiobacillus ferrivorans SS3 is a psychrotolerant acidophile capable of growth in the range of 5° to 30°C (optimum, ≈25°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.

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. Biotechnol. Bioeng. 2011; 108:1251-1259. © 2011 Wiley Periodicals, Inc.

Mesophilic iron and sulfur-oxidizing acidophiles are readily found in acid mine drainage sites and bioleaching operations, but relatively little is known about their activities at suboptimal temperatures and in cold environments. The purpose of this work was to characterize the oxidation of elemental sulfur (S(0)), tetrathionate (S4O6(2-)) and ferrous iron (Fe2+) by the psychrotolerant Acidithiobacillus strain SS3. The rates of elemental sulfur and tetrathionate oxidation had temperature optima of 20 degrees and 25 degrees C, respectively, determined using a temperature gradient incubator that involved narrow (1.1 degrees C) incremental increases from 5 degrees to 30 degrees C. Activation energies calculated from the Arrhenius plots were 61 and 89 kJ mol(-1) for tetrathionate and 110 kJ mol(-1) for S(0) oxidation. The oxidation of elemental sulfur produced sulfuric acid at 5 degrees C and decreased the pH to approximately 1. The low pH inhibited further oxidation of the substrate. In media with both S(0) and Fe2+, oxidation of elemental sulfur did not commence until all available ferrous iron was oxidized. These data on sequential oxidation of the two substrates are in keeping with upregulation and downregulation of several proteins previously noted in the literature. Ferric iron was reduced to Fe2+ in parallel with elemental sulfur oxidation, indicating the presence of a sulfur:ferric iron reductase system in this bacterium.