Draft Genome Sequence of Firmicutes Strain S0AB, a Heterotrophic Iron/Sulfur-Oxidizing Extreme Acidophile.

The draft whole-genome sequence of the extremely acidophilic and novel Firmicutes strain S0AB is reported. The genome comprises 3.3 Mbp and has a GC content of 43.72%. In total, 3,240 protein-coding genes, 56 tRNA genes, and 11 rRNA genes were predicted.

Draft Genome Sequence of the Novel, Moderately Thermophilic, Iron- and Sulfur-Oxidizing Firmicute Strain Y002, Isolated from an Extremely Acidic Geothermal Environment.

We report the draft genome sequence of the Firmicute strain Y002, a facultatively anaerobic, acidophilic bacterium that catalyzes the dissimilatory oxidation of iron and sulfur and the reduction of ferric iron. Analysis of the genome (2.9 Mb; G+C content, 46 mol%) provided insights into its ability to grow in extremely acidic geothermal environments.

Chromium (VI) Inhibition of Low pH Bioleaching of Limonitic Nickel-Cobalt Ore.

Limonitic layers of the regolith, which are often stockpiled as waste materials at laterite mines, commonly contain significant concentrations of valuable base metals, such as nickel, cobalt, and manganese. There is currently considerable demand for these transition metals, and this is projected to continue to increase (alongside their commodity values) during the next few decades, due in the most part to their use in battery and renewable technologies. Limonite bioprocessing is an emerging technology that often uses acidophilic prokaryotes to catalyse the oxidation of zero-valent sulphur coupled to the reduction of Fe (III) and Mn (IV) minerals, resulting in the release of target metals. Chromium-bearing minerals, such as chromite, where the metal is present as Cr (III), are widespread in laterite deposits. However, there are also reports that the more oxidised and more biotoxic form of this metal [Cr (VI)] may be present in some limonites, formed by the oxidation of Cr (III) by manganese (IV) oxides. Bioleaching experiments carried out in laboratory-scale reactors using limonites from a laterite mine in New Caledonia found that solid densities of ∼10% w/v resulted in complete inhibition of iron reduction by acidophiles, which is a critical reaction in the reductive dissolution process. Further investigations found this to be due to the release of Cr (VI) in the acidic liquors. X-ray absorption near edge structure (XANES) spectroscopy analysis of the limonites used found that between 3.1 and 8.0% of the total chromium in the three limonite samples used in experiments was present in the raw materials as Cr (VI). Microbial inhibition due to Cr (VI) could be eliminated either by adding limonite incrementally or by the addition of ferrous iron, which reduces Cr (VI) to less toxic Cr (III), resulting in rates of extraction of cobalt (the main target metal in the experiments) of >90%.

Isolation and characterization of a novel acidophilic zero-valent sulfur- and ferric iron-respiring Firmicute.

A novel, obligately anaerobic, acidophilic bacterium (strain I2511), isolated from sediment in an abandoned copper mine, was shown to couple the oxidation of organic electron donors to the reduction of both zero-valent sulfur and ferric iron in acidic media. The isolate was an obligate heterotroph that used a variety of organic compounds as electron donors and required yeast extract for growth. Alternative electron acceptors (sulfate, tetrathionate, thiosulfate and nitrate) were not used by the novel isolate. The strain grew as motile, endospore-forming rods, and was mesophilic and moderately acidophilic, with a growth rate of 0.01 h-1 at optimum pH (3.7) and temperature (35 °C). Analysis of its 16S rRNA gene sequence placed strain I2511 within the phylum Firmicutes, distantly related to validated species. Phylogenetic analysis and physiological traits indicate that the novel strain represents a species of a candidate novel genus. Strain I2511 was included in a microbial consortium in a low pH "hybrid" sulfidogenic bioreactor designed to remove chalcophilic metals from metal-contaminated liquors and was present in >50% relative abundance when bioreactor was operated at pH ∼ 2.0. Results indicate that the novel isolate could be applied in biotechnologies to treat acidic and neutral pH, metal-rich effluents.

Characteristics of an Iron-Reducing, Moderately Acidophilic Actinobacterium Isolated from Pyritic Mine Waste, and Its Potential Role in Mitigating Mineral Dissolution in Mineral Tailings Deposits.

Reactive pyritic mine tailings can be populated by chemolithotrophic prokaryotes that enhance the solubilities of many metals, though iron-reducing heterotrophic microorganisms can inhibit the environmental risk posed by tailings by promoting processes that are the reverse of those carried out by pyrite-oxidising autotrophic bacteria. A strain (IT2) of Curtobacterium ammoniigenes, a bacterium not previously identified as being associated with acidic mine wastes, was isolated from pyritic mine tailings and partially characterized. Strain IT2 was able to reduce ferric iron under anaerobic conditions, but was not found to catalyse the oxidation of ferrous iron or elemental (zero-valent) sulfur, and was an obligate heterotrophic. It metabolized monosaccharides and required small amounts of yeast extract for growth. Isolate IT2 is a mesophilic bacterium, with a temperature growth optimum of 30 °C and is moderately acidophilic, growing optimally at pH 4.0 and between pH 2.7 and 5.0. The isolate tolerated elevated concentrations of many transition metals, and was able to grow in the cell-free spent medium of the acidophilic autotroph Acidithiobacillus ferrooxidans, supporting the hypothesis that it can proliferate in acidic mine tailings. Its potential role in mitigating the production of acidic, metal-rich drainage waters from mine wastes is discussed.

Salt Stress-Induced Loss of Iron Oxidoreduction Activities and Reacquisition of That Phenotype Depend on rus Operon Transcription in Acidithiobacillus ferridurans.

The type strain of the mineral-oxidizing acidophilic bacterium Acidithiobacillus ferridurans was grown in liquid medium containing elevated concentrations of sodium chloride with hydrogen as electron donor. While it became more tolerant to chloride, after about 1 year, the salt-stressed acidophile was found to have lost its ability to oxidize iron, though not sulfur or hydrogen. Detailed molecular examination revealed that this was due to an insertion sequence, ISAfd1, which belongs to the ISPepr1 subgroup of the IS4 family, having been inserted downstream of the two promoters PI and PII of the rus operon (which codes for the iron oxidation pathway in this acidophile), thereby preventing its transcription. The ability to oxidize iron was regained on protracted incubation of the culture inoculated onto salt-free solid medium containing ferrous iron and incubated under hydrogen. Two revertant strains were obtained. In one, the insertion sequence ISAfd1 had been excised, leaving an 11-bp signature, while in the other an ∼2,500-bp insertion sequence (belonging to the IS66 family) was detected in the downstream inverted repeat of ISAfd1 The transcriptional start site of the rus operon in the second revertant strain was downstream of the two ISs, due to the creation of a new "hybrid" promoter. The loss and subsequent regaining of the ability of A. ferriduransT to reduce ferric iron were concurrent with those observed for ferrous iron oxidation, suggesting that these two traits are closely linked in this acidophile.IMPORTANCE Iron-oxidizing acidophilic bacteria have primary roles in the oxidative dissolution of sulfide minerals, a process that underpins commercial mineral-processing biotechnologies ("biomining"). Most of these prokaryotes have relatively low tolerance to chloride, which limits their activities when only saline or brackish waters are available. The study showed that it was possible to adapt a typical iron-oxidizing acidophile to grow in the presence of salt concentrations similar to those in seawater, but in so doing they lost their ability to oxidize iron, though not sulfur or hydrogen. The bacterium regained its capacity for oxidizing iron when the salt stress was removed but simultaneously reverted to tolerating lower concentrations of salt. These results suggest that the bacteria that have the main roles in biomining operations could survive but become ineffective in cases where saline or brackish waters are used for irrigation.

Comparative Genome Analysis Provides Insights into Both the Lifestyle of Acidithiobacillus ferrivorans Strain CF27 and the Chimeric Nature of the Iron-Oxidizing Acidithiobacilli Genomes.

The iron-oxidizing species Acidithiobacillus ferrivorans is one of few acidophiles able to oxidize ferrous iron and reduced inorganic sulfur compounds at low temperatures (<10°C). To complete the genome of At. ferrivorans strain CF27, new sequences were generated, and an update assembly and functional annotation were undertaken, followed by a comparative analysis with other Acidithiobacillus species whose genomes are publically available. The At. ferrivorans CF27 genome comprises a 3,409,655 bp chromosome and a 46,453 bp plasmid. At. ferrivorans CF27 possesses genes allowing its adaptation to cold, metal(loid)-rich environments, as well as others that enable it to sense environmental changes, allowing At. ferrivorans CF27 to escape hostile conditions and to move toward favorable locations. Interestingly, the genome of At. ferrivorans CF27 exhibits a large number of genomic islands (mostly containing genes of unknown function), suggesting that a large number of genes has been acquired by horizontal gene transfer over time. Furthermore, several genes specific to At. ferrivorans CF27 have been identified that could be responsible for the phenotypic differences of this strain compared to other Acidithiobacillus species. Most genes located inside At. ferrivorans CF27-specific gene clusters which have been analyzed were expressed by both ferrous iron-grown and sulfur-attached cells, indicating that they are not pseudogenes and may play a role in both situations. Analysis of the taxonomic composition of genomes of the Acidithiobacillia infers that they are chimeric in nature, supporting the premise that they belong to a particular taxonomic class, distinct to other proteobacterial subgroups.

Indirect Redox Transformations of Iron, Copper, and Chromium Catalyzed by Extremely Acidophilic Bacteria.

Experiments were carried out to examine redox transformations of copper and chromium by acidophilic bacteria (Acidithiobacillus, Leptospirillum, and Acidiphilium), and also of iron (III) reduction by Acidithiobacillus spp. under aerobic conditions. Reduction of iron (III) was found with all five species of Acidithiobacillus tested, grown aerobically on elemental sulfur. Cultures maintained at pH 1.0 for protracted periods displayed increasing propensity for aerobic iron (III) reduction, which was observed with cell-free culture liquors as well as those containing bacteria. At. caldus grown on hydrogen also reduced iron (III) under aerobic conditions, confirming that the unknown metabolite(s) responsible for iron (III) reduction were not (exclusively) sulfur intermediates. Reduction of copper (II) by aerobic cultures of sulfur-grown Acidithiobacillus spp. showed similar trends to iron (III) reduction in being more pronounced as culture pH declined, and occurring in both the presence and absence of cells. Cultures of Acidithiobacillus grown anaerobically on hydrogen only reduced copper (II) when iron (III) (which was also reduced) was also included; identical results were found with Acidiphilium cryptum grown micro-aerobically on glucose. Harvested biomass of hydrogen-grown At. ferridurans oxidized iron (II) but not copper (I), and copper (I) was only oxidized by growing cultures of Acidithiobacillus spp. when iron (II) was also included. The data confirmed that oxidation and reduction of copper were both mediated by acidophilic bacteria indirectly, via iron (II) and iron (III). No oxidation of chromium (III) by acidophilic bacteria was observed even when, in the case of Leptospirillum spp., the redox potential of oxidized cultures exceeded +900 mV. Cultures of At. ferridurans and A. cryptum reduced chromium (VI), though only when iron (III) was also present, confirming an indirect mechanism and contradicting an earlier report of direct chromium reduction by A. cryptum. Measurements of redox potentials of iron, copper and chromium couples in acidic, sulfate-containing liquors showed that these differed from situations where metals are not complexed by inorganic ligands, and supported the current observations of indirect copper oxido-reduction and chromium reduction mediated by acidophilic bacteria. The implications of these results for both industrial applications of acidophiles and for exobiology are discussed.

Iron Kinetics and Evolution of Microbial Populations in Low-pH, Ferrous Iron-Oxidizing Bioreactors.

Iron-rich, acidic wastewaters are commonplace pollutants associated with metal and coal mining. Continuous-flow bioreactors were commissioned and tested for their capacities to oxidize ferrous iron in synthetic and actual acid mine drainage waters using (initially) pure cultures of the recently described acidophilic, iron-oxidizing heterotrophic bacterium Acidithrix ferrooxidans grown in the presence of glucose and yeast extract. The bioreactors became rapidly colonized by this bacterium, which formed macroscopic streamer growths in the flowing waters. Over 97% of ferrous iron in pH 2.0-2.2 synthetic mine water was oxidized (at up to 225 mg L(-1) h(-1)) at dilution rates (D) of 0.6 h(-1). Rates of iron oxidation decreased with pH but were still significant, with influent liquors as low as pH 1.37. When fed with actual mine water, >90% of ferrous iron was oxidized at D values of 0.4 h(-1), and microbial communities within the bioreactors changed over time, with Atx. ferrooxidans becoming increasingly displaced by the autotrophic iron-oxidizing acidophiles Ferrovum myxofaciens, Acidithiobacillus ferrivorans, and Leptospirillum ferrooxidans (which were all indigenous to the mine water), although this did not have a negative impact on net ferrous-iron oxidation. The results confirmed the potential of using a heterotrophic acidophile to facilitate the rapid commissioning of iron-oxidizing bioreactors and illustrated how microbial communities within them can evolve without compromising the performances of the bioreactors.

Draft Genome Sequence of a Novel Acidophilic Iron-Oxidizing Firmicutes Species, "Acidibacillus ferrooxidans" (SLC66T).

Here, we present the draft genome sequence of the type strain of "Acidibacillus ferrooxidans," a mesophilic, heterotrophic, and acidophilic bacterium that was isolated from mine spoilage subjected to accelerated weathering in humidity cell tests carried out by the former U.S. Bureau of Mines in Salt Lake City, UT.

Acidithiobacillus ferriphilus sp. nov., a facultatively anaerobic iron- and sulfur-metabolizing extreme acidophile.

The genus Acidithiobacillus includes three species that conserve energy from the oxidation of ferrous iron, as well as reduced sulfur, to support their growth. Previous work, based on multi-locus sequence analysis, identified a fourth group of iron- and sulfur-oxidizing acidithiobacilli as a potential distinct species. Eleven strains of 'Group IV' acidithiobacilli, isolated from different global locations, have been studied. These were all shown to be obligate chemolithotrophs, growing aerobically by coupling the oxidation of ferrous iron or reduced sulfur (but not hydrogen) to molecular oxygen, or anaerobically by the oxidation of reduced sulfur coupled to ferric iron reduction. All strains were mesophilic, although some were also psychrotolerant. Strain variation was also noted in terms of tolerance to extremely low pH and to elevated concentrations of transition metals. One strain was noted to display far greater tolerance to chloride than reported for other iron-oxidizing acidithiobacilli. All of the strains were able to catalyse the oxidative dissolution of pyrite and, on the basis of some of the combined traits of some of the strains examined, it is proposed that these may have niche roles in commercial mineral bioprocessing operations, such as for low temperature bioleaching of polysulfide ores in brackish waters. The name Acidithiobacillus ferriphilus sp. nov. is proposed to accommodate the strains described, with the type strain being M20T ( = DSM 100412T = JCM 30830T).

Genome Sequence of the Acidophilic Ferrous Iron-Oxidizing Isolate Acidithrix ferrooxidans Strain Py-F3, the Proposed Type Strain of the Novel Actinobacterial Genus Acidithrix.

Extremely acidophilic iron-oxidizing Gram-positive bacteria comprise species within the phyla Firmicutes and Actinobacteria. Here, we report the 4.02-Mb draft genome of Acidithrix ferrooxidans Py-F3, which was isolated from a stream draining an abandoned copper mine and proposed as the type species of a new genus of Actinobacteria.

Genome Sequence of the Acidophilic Iron Oxidizer Ferrimicrobium acidiphilum Strain T23T.

Extremely acidophilic iron-oxidizing bacteria have largely been characterized for the phyla Proteobacteria and Nitrospira. Here, we report the draft genome of an iron-oxidizing and -reducing heterotrophic mesophile of the Actinobacteria, Ferrimicrobium acidiphilum, which was isolated from an abandoned pyrite mine. The genome sequence comprises 3.08 Mb.

Acidithrix ferrooxidans gen. nov., sp. nov.; a filamentous and obligately heterotrophic, acidophilic member of the Actinobacteria that catalyzes dissimilatory oxido-reduction of iron.

A novel acidophilic member of the phylum Actinobacteria was isolated from an acidic stream draining an abandoned copper mine in north Wales. The isolate (PY-F3) was demonstrated to be a heterotroph that catalyzed the oxidation of ferrous iron (but not of sulfur or hydrogen) under aerobic conditions, and the reduction of ferric iron under micro-aerobic and anaerobic conditions. PY-F3 formed long entangled filaments of cells (>50 µm long) during active growth phases, though these degenerated into smaller fragments and single cells in late stationary phase. Although isolate PY-F3 was not observed to grow below pH 2.0 and 10 °C, harvested biomass was found to oxidize ferrous iron at relatively fast rates at pH 1.5 and 5 °C. Phylogenetic analysis, based on comparisons of 16S rRNA gene sequences, showed that isolate PY-F3 has 91-93% gene similarity to those of the four classified genera and species of acidophilic Actinobacteria, and therefore is a representative of a novel genus. The binomial Acidithrix ferrooxidans is proposed for this new species, with PY-F3 as the designated type strain (=DSM 28176(T), =JCM 19728(T)).

Insights into the pathways of iron- and sulfur-oxidation, and biofilm formation from the chemolithotrophic acidophile Acidithiobacillus ferrivorans CF27.

The iron-oxidizing acidithiobacilli cluster into at least four groups, three of which (Acidithiobacillus ferrooxidans, Acidithiobacillus ferridurans and Acidithiobacillus ferrivorans) have been designated as separate species. While these have many physiological traits in common, they differ in some phenotypic characteristics including motility, and pH and temperature minima. In contrast to At. ferrooxidans and At. ferridurans, all At. ferrivorans strains analysed to date possess the iro gene (encoding an iron oxidase) and, with the exception of strain CF27, the rusB gene encoding an iso-rusticyanin whose exact function is uncertain. Strain CF27 differs from other acidithiobacilli by its marked propensity to form macroscopic biofilms in liquid media. To identify the genetic determinants responsible for the oxidation of ferrous iron and sulfur and for the formation of extracellular polymeric substances, the genome of At. ferrivorans CF27 strain was sequenced and comparative genomic studies carried out with other Acidithiobacillus spp.. Genetic disparities were detected that indicate possible differences in ferrous iron and reduced inorganic sulfur compounds oxidation pathways among iron-oxidizing acidithiobacilli. In addition, strain CF27 is the only sequenced Acidithiobacillus spp. to possess genes involved in the biosynthesis of fucose, a sugar known to confer high thickening and flocculating properties to extracellular polymeric substances.

Draft Genome Sequence of the Nominated Type Strain of "Ferrovum myxofaciens," an Acidophilic, Iron-Oxidizing Betaproteobacterium.

"Ferrovum myxofaciens" is an iron-oxidizing betaproteobacterium with widespread distribution in acidic low-temperature environments, such as acid mine drainage streams. Here, we describe the genomic features of this novel acidophile and investigate the relevant metabolic pathways that enable its survival in these environments.

Acidibacter ferrireducens gen. nov., sp. nov.: an acidophilic ferric iron-reducing gammaproteobacterium.

An acidophilic gammaproteobacterium, isolated from a pit lake at an abandoned metal mine in south-west Spain, was shown to be distantly related to all characterized prokaryotes, and to be the first representative of a novel genus and species. Isolate MCF85 is a Gram-negative, non-motile, rod-shaped mesophilic bacterium with a temperature growth optimum of 32-35 °C (range 8-45 °C). It was categorized as a moderate acidophile, growing optimally at pH 3.5-4.0 and between pH 2.5 and 4.5. Under optimum conditions its culture doubling time was around 75 min. Only organic electron donors were used by MCF85, and the isolate was confirmed to be an obligate heterotroph. It grew on a limited range of sugars (hexoses and disaccharides, though not pentoses) and some other small molecular weight organic compounds, and growth was partially or completely inhibited by small concentrations of some aliphatic acids. The acidophile grew in the presence of >100 mM ferrous iron or aluminium, but was more sensitive to some other metals, such as copper. It was also much more tolerant of arsenic (V) than arsenic (III). Isolate MCF85 catalysed the reductive dissolution of the ferric iron mineral schwertmannite when incubated under micro-aerobic or anaerobic conditions, causing the culture media pH to increase. There was no evidence, however, that the acidophile could grow by ferric iron respiration under strictly anoxic conditions. Isolate MCF85 is the designated type strain of the novel species Acidibacter ferrireducens (=DSM 27237(T) = NCCB 100460(T)).

Uncovering a microbial enigma: isolation and characterization of the streamer-generating, iron-oxidizing, acidophilic bacterium "Ferrovum myxofaciens".

A betaproteobacterium, shown by molecular techniques to have widespread global distribution in extremely acidic (pH 2 to 4) ferruginous mine waters and also to be a major component of "acid streamer" growths in mine-impacted water bodies, has proven to be recalcitrant to enrichment and isolation. A modified "overlay" solid medium was devised and used to isolate this bacterium from a number of mine water samples. The physiological and phylogenetic characteristics of a pure culture of an isolate from an abandoned copper mine ("Ferrovum myxofaciens" strain P3G) have been elucidated. "F. myxofaciens" is an extremely acidophilic, psychrotolerant obligate autotroph that appears to use only ferrous iron as an electron donor and oxygen as an electron acceptor. It appears to use the Calvin-Benson-Bassham pathway to fix CO2 and is diazotrophic. It also produces copious amounts of extracellular polymeric materials that cause cells to attach to each other (and to form small streamer-like growth in vitro) and to different solid surfaces. "F. myxofaciens" can catalyze the oxidative dissolution of pyrite and, like many other acidophiles, is tolerant of many (cationic) transition metals. "F. myxofaciens" and related clone sequences form a monophyletic group within the Betaproteobacteria distantly related to classified orders, with genera of the family Nitrosomonadaceae (lithoautotrophic, ammonium-oxidizing neutrophiles) as the closest relatives. On the basis of the phylogenetic and phenotypic differences of "F. myxofaciens" and other Betaproteobacteria, a new family, "Ferrovaceae," and order, "Ferrovales," within the class Betaproteobacteria are proposed. "F. myxofaciens" is the first extreme acidophile to be described in the class Betaproteobacteria.

Acidithiobacillus ferridurans sp. nov., an acidophilic iron-, sulfur- and hydrogen-metabolizing chemolithotrophic gammaproteobacterium.

Twelve strains of iron-oxidizing acidithiobacilli isolated from acidic sites throughout the world, including some previously shown by multi-locus sequence analyses and DNA-DNA hybridization to comprise a distinct species, were characterized in terms of their physiologies. The bacteria were shown to be obligately chemolithotrophic, acidophilic and mesophilic, and grew in both oxic and anoxic environments, using ferrous iron, reduced sulfur or hydrogen as electron donors and oxygen or ferric iron as electron acceptors. Some of the strains grew at lower pH than those reported for the two recognized iron-oxidizing Acidithiobacillus species, Acidithiobacillus ferrooxidans and Acidithiobacillus ferrivorans. Tolerance of transition metals and aluminium, and also specific rates of iron oxidation and reduction, were more similar to those of A. ferrooxidans (to which the strains are more closely related) than to A. ferrivorans. The name Acidithiobacillus ferridurans sp. nov. is proposed to accommodate the 12 strains, with the type strain being JCM 18981(T) ( = ATCC 33020(T)).

Anaerobic sulfur metabolism coupled to dissimilatory iron reduction in the extremophile Acidithiobacillus ferrooxidans.

Gene transcription (microarrays) and protein levels (proteomics) were compared in cultures of the acidophilic chemolithotroph Acidithiobacillus ferrooxidans grown on elemental sulfur as the electron donor under aerobic and anaerobic conditions, using either molecular oxygen or ferric iron as the electron acceptor, respectively. No evidence supporting the role of either tetrathionate hydrolase or arsenic reductase in mediating the transfer of electrons to ferric iron (as suggested by previous studies) was obtained. In addition, no novel ferric iron reductase was identified. However, data suggested that sulfur was disproportionated under anaerobic conditions, forming hydrogen sulfide via sulfur reductase and sulfate via heterodisulfide reductase and ATP sulfurylase. Supporting physiological evidence for H2S production came from the observation that soluble Cu(2+) included in anaerobically incubated cultures was precipitated (seemingly as CuS). Since H(2)S reduces ferric iron to ferrous in acidic medium, its production under anaerobic conditions indicates that anaerobic iron reduction is mediated, at least in part, by an indirect mechanism. Evidence was obtained for an alternative model implicating the transfer of electrons from S(0) to Fe(3+) via a respiratory chain that includes a bc(1) complex and a cytochrome c. Central carbon pathways were upregulated under aerobic conditions, correlating with higher growth rates, while many Calvin-Benson-Bassham cycle components were upregulated during anaerobic growth, probably as a result of more limited access to carbon dioxide. These results are important for understanding the role of A. ferrooxidans in environmental biogeochemical metal cycling and in industrial bioleaching operations.