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The extraction of nickel, cobalt, and other metals from laterite ores via bioleaching with sulfur-oxidizing and ferric iron-reducing, autotrophic, acidophilic bacteria (e.g. Acidithiobacillus species) has been demonstrated under anaerobic as well as aerobic conditions in experiments in different laboratories. This study demonstrated the bioleaching of laterites from Brazil with the addition of elemental sulfur in 2-L stirred-tank bioreactors with pure and mixed cultures of Acidithiobacillus and Sulfobacillus species under aerobic conditions. In particular, a potential disturbance of mineral dissolution under aerobic conditions by ferrous iron-oxidizing acidophiles likely introduced as contaminants in an applied bioleaching process was investigated with Leptospirillum ferrooxidans at 30°C and Leptospirillum ferriphilum at 40°C, at maintained pH 1.5 or without maintained pH leading to an increase in acidity (with pH values <1.0) due to the biological production of sulfuric acid. Despite the proportion of ferrous iron to the total amount of extracted iron in the solution being drastically reduced in the presence of Leptospirillum species, there was a negligible effect on the extraction efficiency of nickel and cobalt, which is positive news for laterite bioleaching under aerobic conditions. [ABSTRACT FROM AUTHOR]

The extraction of nickel, cobalt, and other metals from laterite ores via bioleaching with sulfur-oxidizing and ferric iron-reducing, autotrophic, acidophilic bacteria (e.g. Acidithiobacillus species) has been demonstrated under anaerobic as well as aerobic conditions in experiments in different laboratories. This study demonstrated the bioleaching of laterites from Brazil with the addition of elemental sulfur in 2-L stirred-tank bioreactors with pure and mixed cultures of Acidithiobacillus and Sulfobacillus species under aerobic conditions. In particular, a potential disturbance of mineral dissolution under aerobic conditions by ferrous iron-oxidizing acidophiles likely introduced as contaminants in an applied bioleaching process was investigated with Leptospirillum ferrooxidans at 30°C and Leptospirillum ferriphilum at 40°C, at maintained pH 1.5 or without maintained pH leading to an increase in acidity (with pH values

Abstract Background Acidophilic microorganisms like Leptospirillum sp. CF-1 thrive in environments with extremely low pH and high concentrations of dissolved heavy metals that can induce the generation of reactive oxygen species (ROS). Several hypothetical genes and proteins from Leptospirillum sp. CF-1 are known to be up-regulated under oxidative stress conditions. Results In the present work, the function of hypothetical gene ABH19_09590 from Leptospirillum sp. CF-1 was studied. Heterologous expression of this gene in Escherichia coli led to an increase in the ability to grow under oxidant conditions with 5 mM K2CrO4 or 5 mM H2O2. Similarly, a significant reduction in ROS production in E. coli transformed with a plasmid carrying ABH19_09590 was observed after exposure to these oxidative stress elicitors for 30 min, compared to a strain complemented with the empty vector. A co-transcriptional study using RT-PCR showed that ABH19_09590 is contained in an operon, here named the “och” operon, that also contains ABH19_09585, ABH19_09595 and ABH19_09600 genes. The expression of the och operon was significantly up-regulated in Leptospirillum sp. CF-1 exposed to 5 mM K2CrO4 for 15 and 30 min. Genes of this operon potentially encode a NADH:ubiquinone oxidoreductase, a CXXC motif-containing protein likely involved in thiol/disulfide exchange, a hypothetical protein, and a di-hydroxy-acid dehydratase. A comparative genomic analysis revealed that the och operon is a characteristic genetic determinant of the Leptospirillum genus that is not present in other acidophiles. Conclusions Altogether, these results suggest that the och operon plays a protective role against chromate and hydrogen peroxide and is an important mechanism required to face polyextremophilic conditions in acid environments.

Background: Acidophilic microorganisms like Leptospirillum sp. CF-1 thrive in environments with extremely low pH and high concentrations of dissolved heavy metals that can induce the generation of reactive oxygen species (ROS). Several hypothetical genes and proteins from Leptospirillum sp. CF-1 are known to be up-regulated under oxidative stress conditions. Results: In the present work, the function of hypothetical gene ABH19_09590 from Leptospirillum sp. CF-1 was studied. Heterologous expression of this gene in Escherichia coli led to an increase in the ability to grow under oxidant conditions with 5 mM K2CrO4 or 5 mM H2O2. Similarly, a significant reduction in ROS production in E. coli transformed with a plasmid carrying ABH19_09590 was observed after exposure to these oxidative stress elicitors for 30 min, compared to a strain complemented with the empty vector. A co-transcriptional study using RT-PCR showed that ABH19_09590 is contained in an operon, here named the "och" operon, that also contains ABH19_09585, ABH19_09595 and ABH19_09600 genes. The expression of the och operon was significantly up-regulated in Leptospirillum sp. CF-1 exposed to 5 mM K2CrO4 for 15 and 30 min. Genes of this operon potentially encode a NADH:ubiquinone oxidoreductase, a CXXC motif-containing protein likely involved in thiol/disulfide exchange, a hypothetical protein, and a di-hydroxy-acid dehydratase. A comparative genomic analysis revealed that the och operon is a characteristic genetic determinant of the Leptospirillum genus that is not present in other acidophiles. Conclusions: Altogether, these results suggest that the och operon plays a protective role against chromate and hydrogen peroxide and is an important mechanism required to face polyextremophilic conditions in acid environments. [ABSTRACT FROM AUTHOR]

Acidophiles play a vital catalytic role in the formation of acid mine drainage (AMD). In this study, acidophilic communities in a stream, two lakes and five ponds in Nanshan Mine, Anhui Province, China were comparatively investigated. Among the three types of AMDs, the stream which originated from a rock wall had the strongest acidity (pH 2.30–2.42), while the ponds (pH 2.35–2.60) formed in mine dump and the two large-scale AMD lakes (pH 2.66–3.05) were less acidic. The chemolithotrophic iron-oxidizer Leptospirillum dominated at the stream starting point with the lowest pH and highest Fe content. However, with streamflow, pH increased and Fe concentration decreased dramatically, and the dominance of Leptospirillum was replaced by the less acid-resistant iron-oxidizer Ferrovum. In contrast to the stream, prokaryotic communities in the ponds were mostly dominated by chemoheterotrophic Acidiphilium because of the abundant organic matter from surrounding mine waste. Both chemolithotrophic Ferrovum and chemoheterotrophic Acidiphilium were abundant in the AMD lakes, which may form a symbiotic relationship around the carbon cycle. Statistical analyses indicated that pH was the predominant environmental factor influencing the prokaryotic communities. As for the eukaryotic communities, the microeukaryotes in the stream were mostly uncultured groups. Both photoautotrophic algae Chrysonebula and chemoheterotrophic fungi were abundant in the ponds, while the two AMD lakes were only dominated by photoautotrophic algae Coccomyxa and Ochromonas. [ABSTRACT FROM AUTHOR]

The exact role that cytochrome 579 plays in the aerobic iron respiratory chain of Leptospirillum ferriphilum is unclear. This paper presents genomic, structural, and kinetic data on the cytochrome 579 purified from cell-free extracts of L. ferriphilum cultured on soluble iron. Electrospray mass spectrometry of electrophoretically homogeneous cytochrome 579 yielded two principal peaks at 16,015 and 16,141 Daltons. N-terminal amino acid sequencing of the purified protein yielded data that were used to determine the following: there are seven homologs of cytochrome 579; each homolog possesses the CXXCH heme-binding motif found in c-type cytochromes; each of the seven sequenced strains of L. ferriphilum expresses only two of the seven homologs of the cytochrome; and each homolog contains an N-terminal signal peptide that directs the mature protein to an extra-cytoplasmic location. Static light scattering and macroion mobility measurements on native cytochrome 579 yielded masses of 125 and 135 kDaltons, respectively. The reduced alkaline pyridine hemochromogen spectrum of the purified cytochrome had an alpha absorbance maximum at 567 nm, a property not exhibited by any known heme group. The iron-dependent reduction and oxidation of the octameric cytochrome exhibited positively cooperative kinetic behavior with apparent Hill coefficients of 5.0 and 3.7, respectively, when the purified protein was mixed with mM concentrations of soluble iron. Consequently, the extrapolated rates of reduction at sub-mM iron concentrations were far too slow for cytochrome 579 to be the initial iron oxidase in the aerobic respiratory chain of L. ferriphilum. Rather, these observations support the hypothesis that the acid-stable cytochrome 579 is a periplasmic conduit of electrons from initial iron oxidation in the outer membrane of this Gram-negative bacterium to a terminal oxidase in the plasma membrane.

The exact role that cytochrome 579 plays in the aerobic iron respiratory chain of Leptospirillum ferriphilum is unclear. This paper presents genomic, structural, and kinetic data on the cytochrome 579 purified from cell-free extracts of L. ferriphilum cultured on soluble iron. Electrospray mass spectrometry of electrophoretically homogeneous cytochrome 579 yielded two principal peaks at 16,015 and 16,141 Daltons. N-terminal amino acid sequencing of the purified protein yielded data that were used to determine the following: there are seven homologs of cytochrome 579; each homolog possesses the CXXCH heme-binding motif found in c -type cytochromes; each of the seven sequenced strains of L. ferriphilum expresses only two of the seven homologs of the cytochrome; and each homolog contains an N-terminal signal peptide that directs the mature protein to an extra-cytoplasmic location. Static light scattering and macroion mobility measurements on native cytochrome 579 yielded masses of 125 and 135 kDaltons, respectively. The reduced alkaline pyridine hemochromogen spectrum of the purified cytochrome had an alpha absorbance maximum at 567 nm, a property not exhibited by any known heme group. The iron-dependent reduction and oxidation of the octameric cytochrome exhibited positively cooperative kinetic behavior with apparent Hill coefficients of 5.0 and 3.7, respectively, when the purified protein was mixed with mM concentrations of soluble iron. Consequently, the extrapolated rates of reduction at sub-mM iron concentrations were far too slow for cytochrome 579 to be the initial iron oxidase in the aerobic respiratory chain of L. ferriphilum. Rather, these observations support the hypothesis that the acid-stable cytochrome 579 is a periplasmic conduit of electrons from initial iron oxidation in the outer membrane of this Gram-negative bacterium to a terminal oxidase in the plasma membrane. [ABSTRACT FROM AUTHOR]

Aquatic environments of volcanic origin provide an exceptional opportunity to study the adaptations of microorganisms to early planet life conditions. Here, we characterized the prokaryotic communities and physicochemical properties of seepage sites at the bottom of the Poas Volcano crater and the Agrio River, two geologically related extremely acidic environments located in Costa Rica. Both locations hold a low pH (1.79–2.20) and have high sulfate and iron concentrations (Fe = 47–206 mg/L, SO42− = 1170–2460 mg/L), but significant differences in their temperature (90.0–95.0 ºC in the seepages at Poas Volcano, 19.1–26.6 ºC in Agrio River) and in the elemental sulfur content. Based on the analysis of 16S rRNA gene sequences, we determined that Sulfobacillus spp. represented more than half of the sequences in Poas Volcano seepage sites, while Agrio River was dominated by Leptospirillum and members of the archaeal order Thermoplasmatales. Both environments share some chemical characteristics and part of their microbiota, however, the temperature and the reduced sulfur are likely the main distinguishing features, ultimately shaping their microbial communities. Our data suggest that in the Poas Volcano–Agrio River system there is a common metabolism but with specialization of species that adapt to the physicochemical conditions of each environment. [ABSTRACT FROM AUTHOR]

Extreme acidophilic bacteria like Leptospirillum sp. require an efficient enzyme system to counteract strong oxygen stress conditions in their natural habitat. The genome of Leptospirillum sp. CF-1 encodes the thioredoxin-fold protein TFP2, which exhibits a high structural similarity to the thioredoxin domain of E. coli CnoX. CnoX from Escherichia coli is a chaperedoxin that protects protein substrates from oxidative stress conditions using its holdase function and a subsequent transfer to foldase chaperones for refolding. Recombinantly produced and purified Leptospirillum sp. TFP2 possesses both thioredoxin and chaperone holdase activities in vitro. It can be reduced by thioredoxin reductase (TrxR). The tfp2 gene co-locates with genes for the chaperone foldase GroES/EL on the chromosome. The " tfp2 cluster" ( ctpA-groES-groEL-hyp-tfp2-recN ) was found between 1.9 and 8.8-fold transcriptionally up-regulated in response to 1 mM hydrogen peroxide (H 2 O 2 ). Leptospirillum sp. tfp2 heterologously expressed in E. coli wild type and cnoX mutant strains lead to an increased tolerance of these E. coli strains to H 2 O 2 and significantly reduced intracellular protein aggregates. Finally, a proteomic analysis of protein aggregates produced in E. coli upon exposition to oxidative stress with 4 mM H 2 O 2 , showed that Leptospirillum sp. tfp2 expression caused a significant decrease in the aggregation of 124 proteins belonging to fifteen different metabolic categories. These included several known substrates of DnaK and GroEL/ES. These findings demonstrate that Leptospirillum sp. TFP2 is a chaperedoxin-like protein, acting as a key player in the control of cellular proteostasis under highly oxidative conditions that prevail in extreme acidic environments.

A multitude of microbes are involved in the solubilisation of minerals and metals as this approach offers numerous advantages over traditional methods. This strategy is preferred as it is eco-friendly and economical, thus overcoming the drawbacks of the traditional approach of pyrometallurgy. Many different types of bacteria are employed in the process of Bioleaching, which are collectively grouped under chemolithotrophs, as they derive their energy from inorganic compounds. Bioleaching is the mobilization of metal cations from insoluble ores by microorganisms. All chemolithotropic bacteria are extremophiles since they have the ability to survive in extreme conditions. They carry out the process of Bioleaching through three mechanisms: Indirect, contact/ direct and cooperative bioleaching. This review gives a sneak peek into the different strains of chemolithotrophs which are used in bioleaching, and some recent work in the field. It also gives an insight into the general process and mechanism of Bioleaching, the study of which will pave way for developing better and efficient industrial bioleaching operations.

This comprehensive study summarizes kinetic data from experiments performed over a wide temperature range that overlaps the physiological operating temperature windows for most mesophilic Fe-oxidizers. Three acidophilic autotrophic bacterial strains Acidithiobacillus (At.) ferrivorans, At. ferrooxidans and Leptospirillum (L.) ferriphilum were investigated in this work in order to compare their growth and iron oxidation kinetics over a temperature range of 5–45 °C. On-line gas analysis was applied to determine the O 2 and CO 2 consumption rates during the culture incubation. Despite the genotypic differences these three strains share many common physiological traits. At t = 30 °C, the specific growth rate (μ) at all three species reached 0.11 h−1 which is equivalent to generation time 6.5 h. Maximum ferrous‑iron oxidation rate per unit of biomass was achieved with L. ferriphilum at t = 40 °C and approached 10 mmol Fe2+ (C-mmol) h−1. The temperature of incubation had a profound effect on the specific rates, while the kinetic parameter Ks / Ki at the Acidithiobacilli strains remained unaffected. Cultures with Leptospirillum incubated at temperatures >35 °C were capable of iron oxidation at redox potentials close to +950 mV and kept constant specific rates until Fe2+ depletion. The parameter Ks/Ki at L. ferriphilum was two order of magnitude lower than for the Acidithiobacilli. However, this feature disappears at temperatures below 30 °C, where Ks / Ki has a value comparable to Acidithiobacilli , which suggests that L. ferriphillum uses different Fe oxidation systems depending on temperature conditions. Strong correlation was observed between ferrous iron oxidation, and carbon dioxide fixation in the exponentially growing cultures with the yield coefficient (Y SX) approaching 14 μmol C fixed per mmol Fe2+ oxidized. The kinetic parameters differed significantly between bacterial strains under "extreme" temperatures (t < 10 °C; t > 35 °C). All three bacterial strains were capable of oxidizing iron over the entire range (5–45 °C) of incubation temperatures. Neither At. ferrivorans nor At. ferrooxidans showed exponential growth at the temperature 40 °C. Bacterial iron oxidation not associated with growth was observed during incubation at 5 °C. At. ferrivorans , was the only species that can grow exponentially at a temperature of 5 °C. Moreover, it differed from the other species by greater sensitivity to low pH which seems to be more pronounced at temperatures t < 10 °C, with minimum of pH 1.9 for growth. • Bacteria oxidized Fe2+ to Fe3+ in the temperature range of 5–45 °C. • Significant differences in growth and Fe2+ oxidation rate at t < 10 °C, t > 30 °C. • A. ferrivorans showed exponential growth at 5 °C, pH > 1.9, sensitivity to low pH. • Bacterial Fe2+ oxidation not associated with cell growth at suboptimal temperatures. • L. ferriphilum manifest different Fe oxidation systems depending on the temperature. [ABSTRACT FROM AUTHOR]

A multitude of microbes are involved in the solubilisation of minerals and metals as this approach offers numerous advantages over traditional methods. This strategy is preferred as it is ecofriendly and economical, thus overcoming the drawbacks of the traditional approach of pyrometallurgy. Many different types of bacteria are employed in the process of Bioleaching, which are collectively grouped under chemolithotrophs, as they derive their energy from inorganic compounds. Bioleaching is the mobilization of metal cations from insoluble ores by microorganisms. All chemolithotropic bacteria are extremophiles since they have the ability to survive in extreme conditions. They carry out the process of Bioleaching through three mechanisms: Indirect, contact/direct and cooperative bioleaching. This review gives a sneak peek into the different strains of chemolithotrophs which are used in bioleaching, and some recent work in the field. It also gives an insight into the general process and mechanism of Bioleaching, the study of which will pave way for developing better and efficient industrial bioleaching operations. [ABSTRACT FROM AUTHOR]

Leptospirillum ferriphilum plays a major role in acidic, metal-rich environments, where it represents one of the most prevalent iron oxidizers. These milieus include acid rock and mine drainage as well as biomining operations. Despite its perceived importance, no complete genome sequence of the type strain of this model species is available, limiting the possibilities to investigate the strategies and adaptations that Leptospirillum ferriphilum DSM 14647T (here referred to as Leptospirillum ferriphilumT) applies to survive and compete in its niche. This study presents a complete, circular genome of Leptospirillum ferriphilumT obtained by PacBio singlemolecule real-time (SMRT) long-read sequencing for use as a high-quality reference. Analysis of the functionally annotated genome, mRNA transcripts, and protein concentrations revealed a previously undiscovered nitrogenase cluster for atmospheric nitrogen fixation and elucidated metabolic systems taking part in energy conservation, carbon fixation, pH homeostasis, heavy metal tolerance, the oxidative stress response, chemotaxis and motility, quorum sensing, and biofilm formation. Additionally, mRNA transcript counts and protein concentrations were compared between cells grown in continuous culture using ferrous iron as the substrate and those grown in bioleaching cultures containing chalcopyrite (CuFeS2). Adaptations of Leptospirillum ferriphilumT to growth on chalcopyrite included the possibly enhanced production of reducing power, reduced carbon dioxide fixation, as well as elevated levels of RNA transcripts and proteins involved in heavy metal resistance, with special emphasis on copper efflux systems. Finally, the expression and translation of genes responsible for chemotaxis and motility were enhanced. [ABSTRACT FROM AUTHOR]

Energy storage is currently one of the most significant problems associated with mass-scale usage of renewable (i.e. wind and solar) power sources. The use of hydrogen as an energy storage medium is very promising, but is hampered by the lack of commercially available hydrogen-to-electricity (H2e) converters. Here we are presenting the first commercially viable, biologically based technology for H2e conversion named the BioGenerator. It is a microbial fuel cell based on electron consumption resulting from the respiration of chemolithoautotrophic microorganisms. The results obtained during the scale-up study of the BioGenerator showed a maximum specific current of 1.35 A/cm 2 , maximum power density of 1800 W/m 2 and stable electricity generation over a period spanning longer than four years. The largest unit studied so far has a volume of 600 L and a power output of 0.3 kW. [ABSTRACT FROM AUTHOR]

Recently, extraction of metals from different resources using a simple, efficient, and low-cost technique-known as bioleaching-has been widely considered, and has turned out to be an important global technology. Leptospirillum ferrooxidans and Acidithiobacillus (Thiobacillus) ferrooxidans are ubiquitous bacteria in the biomining industry. To date, the effects of commercial flotation reagents on the biooxidation activities of these bacteria have not been thoroughly studied. This investigation, by using various systematic measurement methods, studied the effects of various collectors and frothers (collectors: potassium amylxanthate, potassium isobutyl-xanthate, sodium ethylxanthate, potassium isopropylxanthate, and dithiophosphate; and frothers: pine oil and methyl isobutyl carbinol) on L. ferrooxidans and A. ferrooxidans activities. In general, results indicate that in the presence of these collectors and frothers, L. ferrooxidans is less sensitive than T. ferrooxidans. In addition, the inhibition effect of collectors on both bacteria is recommended in the following order: for the collectors, potassium isobutyl-xanthate > dithiophosphate > sodium ethylxanthate > potassium isobutyl-xanthate > potassium amylxanthate; and for the frothers, methyl isobutyl carbinol > pine oil. These results can be used for the optimization of biometallurgical processes or in the early stage of a process design for selection of flotation reagents. [ABSTRACT FROM AUTHOR]

Monitoring of the microbial community in bioleaching processes is essential in order to control process parameters and enhance the leaching efficiency. Suitable methods are, however, limited as they are usually not adapted to bioleaching samples and often no taxon-specific assays are available in the literature for these types of consortia. Therefore, our study focused on the development of novel quantitative real-time PCR (qPCR) assays for the quantification of Acidithiobacillus caldus, Leptospirillum ferriphilum, Sulfobacillus thermosulfidooxidans and Sulfobacillus benefaciens and comparison of the results with data from other common molecular monitoring methods in order to evaluate their accuracy and specificity. Stirred tank bioreactors for the leaching of copper concentrate, housing a consortium of acidophilic, moderately-thermophilic bacteria, relevant in several bioleaching operations, served as a model system. The microbial community analysis via qPCR allowed a precise monitoring of the evolution of total biomass as well as abundance of specific species. Data achieved by the standard fingerprinting methods, terminal restriction fragment length polymorphism (T-RFLP) and capillary electrophoresis single strand conformation polymorphism (CE-SSCP) on the same samples followed the same trend as qPCR data. The main added value of qPCR was, however, to provide quantitative data for each species whereas only relative abundance could be deduced from T-RFLP and CE-SSCP profiles. Additional value was obtained by applying two further quantitative methods which do not require nucleic acid extraction, total cell counting after SYBR Green staining and metal sulfide oxidation activity measurements via microcalorimetry. Overall, these complementary methods allow for an efficient quantitative microbial community monitoring in various bioleaching operations.

Monitoring of the microbial community in bioleaching processes is essential in order to control process parameters and enhance the leaching efficiency. Suitable methods are, however, limited as they are usually not adapted to bioleaching samples and often no taxon-specific assays are available in the literature for these types of consortia. Therefore, our study focused on the development of novel quantitative real-time PCR (qPCR) assays for the quantification of Acidithiobacillus caldus, Leptospirillum ferriphilum, Sulfobacillus thermosulfidooxidans, and Sulfobacillus benefaciens and comparison of the results with data from other common molecular monitoring methods in order to evaluate their accuracy and specificity. Stirred tank bioreactors for the leaching of copper concentrate, housing a consortium of acidophilic, moderately thermophilic bacteria, relevant in several bioleaching operations, served as a model system. The microbial community analysis via qPCR allowed a precise monitoring of the evolution of total biomass as well as abundance of specific species. Data achieved by the standard fingerprinting methods, terminal restriction fragment length polymorphism (T-RFLP) and capillary electrophoresis single strand conformation polymorphism (CE-SSCP) on the same samples followed the same trend as qPCR data. The main added value of qPCR was, however, to provide quantitative data for each species whereas only relative abundance could be deduced from T-RFLP and CE-SSCP profiles. Additional value was obtained by applying two further quantitative methods which do not require nucleic acid extraction, total cell counting after SYBR Green staining and metal sulfide oxidation activity measurements via microcalorimetry. Overall, these complementary methods allow for an efficient quantitative microbial community monitoring in various bioleaching operations. [ABSTRACT FROM AUTHOR]

Mine tailings are a serious threat to the environment and public health. Remediation of these residues can be carried out effectively by the activation of specific microbial processes. This article presents detailed information about temporal changes in bacterial community composition during the remediation of a section of porphyry copper tailings deposited on the Bahía de Ite shoreline (Peru). An experimental remediation cell was flooded and transformed into a wetland in order to prevent oxidation processes, immobilizing metals. Initially, the top oxidation zone of the tailings deposit displayed a low pH (3.1) and high concentrations of metals, sulfate, and chloride, in a sandy grain size geological matrix. This habitat was dominated by sulfur- and iron-oxidizing bacteria, such as Leptospirillum spp., Acidithiobacillus spp., and Sulfobacillus spp., in a microbial community which structure resembled acid mine drainage environments. After wetland implementation, the cell was water-saturated, the acidity was consumed and metals dropped to a fraction of their initial respective concentrations. Bacterial communities analyzed by massive sequencing showed time-dependent changes both in composition and cell numbers. The final remediation stage was characterized by the highest bacterial diversity and evenness. Aside from classical sulfate reducers from the phyla δ-Proteobacteria and Firmicutes, community structure comprised taxa derived from very diverse habitats. The community was also characterized by an elevated proportion of rare phyla and unaffiliated sequences. Numerical ecology analysis confirmed that the temporal population evolution was driven by pH, redox, and K. Results of this study demonstrated the usefulness of a detailed follow-up of the remediation process, not only for the elucidation of the communities gradually switching from autotrophic, oxidizing to heterotrophic and reducing living conditions, but also for the long term management of the remediation wetlands. [ABSTRACT FROM AUTHOR]

Leptospirillum sp. YQP-1, a member of iron-oxidizing bacteria was isolated from volcanic lake in northeast China. Here, we report the draft genome sequence of the strain YQP-1 with a total genome size of 3,103,789 bp from 85 scaffolds (104 contigs) with 58.64% G + C content. The genome sequence can be accessed at DDBJ/EMBL/GenBank under the accession no. LIEB00000000.

Mesophilic bacteria (Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans, and Leptospirillum ferrooxidans) and moderate thermophiles (Acidithiobacillus caldus and Sulfobacillus thermosulfidooxidans) were used in bioleaching experiments with five select sphalerite samples, which varied in the Zn- and Fe-content. Each sample was leached with and without additional Fe2+ and S0 under mesophilic and moderately thermophilic conditions. A sphalerite sample with a high Fe-content (8.95% Fe) reached a higher yield of zinc bioleaching than those with less iron (0.20–2.04% Fe). The maximum Zn recovery in the mesophilic tests was 82%, obtained with the sample of the highest Fe-content. In the moderately thermophilic experiments, the highest Zn recovery was 32%. The mesophiles increased the oxidation reduction potential due to the oxidation of Fe2+. The formation of jarosite-type precipitates and sulfur on sphalerite surfaces in the mesophilic tests and the formation of sulfur in the moderate thermophilic tests were indicated by analysis with powder X-ray diffraction and scanning electron microscopy equipped with energy-dispersive X-ray spectroscopy. [Display omitted] • Select sphalerite samples were leached with Fe- and S-oxidizing acidophiles. • Mesophiles (35 °C) and moderate thermophiles (45 °C) were used in the study. • The Zn- and Fe-content and crystallographic parameters varied in the samples. • The highest yield of Zn bioleaching was with the sample of the highest Fe-content. • Additional Fe and S enhanced the bioleaching more at 35 °C than at 45 °C. [ABSTRACT FROM AUTHOR]

Fe-S clusters are versatile and essential cofactors that participate in multiple and fundamental biological processes. In Escherichia coli , the biogenesis of these cofactors requires either the housekeeping Isc pathway, or the stress-induced Suf pathway which plays a general role under conditions of oxidative stress or iron limitation. In the present work, the Fe-S cluster assembly Isc and Suf systems of acidophilic Bacteria and Archaea, which thrive in highly oxidative environments, were studied. This analysis revealed that acidophilic microorganisms have a complete set of genes encoding for a single system (either Suf or Isc). In acidophilic Proteobacteria and Nitrospirae, a complete set of isc genes ( iscRSUAX-hscBA-fdx ), but not genes coding for the Suf system, was detected. The activity of the Isc system was studied in Leptospirillum sp. CF-1 (Nitrospirae). RT-PCR experiments showed that eight candidate genes were co-transcribed and conform the isc operon in this strain. Additionally, RT-qPCR assays showed that the expression of the iscS gene was significantly up-regulated in cells exposed to oxidative stress imposed by 260 mM Fe 2 (SO 4 ) 3 for 1 h or iron starvation for 3 h. The activity of cysteine desulfurase (IscS) in CF-1 cell extracts was also up-regulated under such conditions. Thus, the Isc system from Leptospirillum sp. CF-1 seems to play an active role in stressful environments. These results contribute to a better understanding of the distribution and role of Fe-S cluster protein biogenesis systems in organisms that thrive in extreme environmental conditions.

Chemolithoautotrophic acidophilic bacteria, which belong to the genus Leptospirillum, can only grow with Fe(II) as electron donor and oxygen as an electron acceptor. Members of this genus play an important role in bioleaching sulfide ores. We used nearly complete genome sequences of Leptospirillum ferrooxidans (group I), Leptospirillum rubarum, Leptospirillum '5-way CG' (group II) and Leptospirillum ferrodiazotrophum (group III) to identify cytochromes that are likely involved in electron transfer chain(s). The results show the presence of genes encoding a number of c-type cytochromes (18-20 genes were identified in each species), as well as bd and cbb3 oxidases. Genes encoding cbb3 oxidase are clustered, with predicted genes involved in cbb3 maturation proteins. Duplication of cbb3 encoding genes (ccoNO) was detected in all four genomes. Interestingly, these micro-organisms also contain genes that potentially encode bc1 and b6f-like complexes organized into two putative operon structures. To date, the Leptospirillum genus includes the only organisms reported to have genes coding for two different bc complexes. This study provides detailed insights into the components of electron transfer chains of Leptospirillum spp., revealing their conservation among leptospirilla groups and suggesting that there may be a single common pathway for electron transport between Fe(II) and oxygen. Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Construction and analysis of the 16S rDNA clone libraries was used to investigate the species composition of two thermotolerant communities of acidophilic chemolithotrophic microorganisms (ACM) isolated from the pulp of laboratory reactors used for oxidation of different gold-containing ore concentrates. The first community was formed during oxidation of the pyrite-arsenopyrite ore concentrate from the Kyuchus deposit. The clones of the bacterial component of this community belonged to the genera Sulfobacillus (32 clones) and Leptospirillum (33 clones). The Sulfobacillus clones belonged to three groups: Sb. thermosulfidooxidans, Sb. benefaciens, and Sb. thermotolerans. All Leptospirillum clones were closely related to L. ferriphilum. All clones of the archaeal component belonged to Ferroplasma acidiphilum. The microorganisms of this community were used as inoculum for biooxidation of a different mineral concentrate, the pyrrhotite-containing pyrite-arsenopyrite ore concentrate from the Olympiadinskoe deposit, and the structure of the community formed in the process was investigated. The clones of the bacterial component of the second community also belonged to the genera Sulfobacillus (14 clones) and Leptospirillum (48 clones). The Sulfobacillus clones belonged to the species Sb. thermosulfidooxidans (13 clones) and Sb. thermotolerans (1 clone). All Leptospirillum clones were closely related to L. ferriphilum. All clones of the archaeal component belonged to Ferroplasma acidiphilum. During the adaptation of the community to a new oxidized mineral substrate, both the composition and the ratio of the constituent microbial species changed. [ABSTRACT FROM AUTHOR]

The species composition of the microbial association involved in industrial tank biooxidation of the concentrate of refractory pyrrhotite-containing pyrite-arsenopyrite gold-arsenic ore of the Olympiadinskoe deposit at 39°C was studied by cultural and molecular biological techniques. Pure microbial cultures were isolated, their physiological characteristics were investigated, and their taxonomic position was determined by 16S rRNA gene sequencing. The library of 16S rRNA gene clones obtained from the total DNA isolated from the biomass of the pulp of industrial reactors was analyzed. The diversity of microorganisms revealed by cultural techniques in the association of acidophilic chemolithotrophs ( Acidithiobacillus ferrooxidans, Leptospirillum ferriphilum, Sulfobacillus thermosulfidooxidans, Ferroplasma acidiphilum, Alicyclobacillus tolerans, and Acidiphilium cryptum) was higher than the diversity of the 16S rDNA clone library ( At. ferrooxidans, L. ferriphilum, and F. acidiphilum). The combination of microbiological and molecular biological techniques for the investigation of the biodiversity in natural and anthropogenic microbial communities promotes detection of new phylogenetic microbial groups in these communities. [ABSTRACT FROM AUTHOR]

Bacterial communities of moderately acidic waste piles of sulfide nickel ore and of the nickel-leaching enrichments obtained from them are analyzed. The structure of bacterial communities was determined by molecular biological techniques. The PCR profiles of bacterial communities were obtained with the primers to a variable 433 bp site of the eubacterial 16S rRNA gene. The differences in community compositions were determined by comparison of their DGGE profiles. Sequencing of the DNA fragments was then carried out and the results were compared with the GenBank gene sequences. Analysis of the 16S rRNA gene sequences revealed few bacterial genera in the moderately acidic waste piles of sulfide nickel ore, with predomination of Acidithiobacillus sp. and Leptospirillum sp. A number of the bacteria revealed belonged to the species never obtained in pure culture. Molecular biological analysis showed the presence of the same groups of bacteria in enriched cultures obtained by inoculating the liquid medium containing ground ore with the waste pile samples (5 : 1). The geochemical activity of these bacteria was confirmed by their capacity for leaching nickel from the sulfide ore in enriched cultures, resulting in nickel solubilization. Thus, new information was obtained concerning the structure composition of the bacterial communities of sulfide ore waste piles: the dominant forms were determined, their leaching activity was confirmed, and the activity of thiobacilli from the waste, which have not been isolated in pure cultures, was confirmed in liquid medium in the presence of ore. [ABSTRACT FROM AUTHOR]

Abstract: An empirical technique for the rapid assessment of active biomass of acidophilic microorganisms in pure cultures and in leach liquors from mineral bio-processing operations is described. The method is based on the measurement of ATP concentrations in test solutions, using commercially-available kits and a portable luminometer. The ATP contents of five Gram-negative biomining bacteria mineral-degrading acidophiles assayed varied from 0.87×10−20 to 18×10−20 mol per cell in actively-growing cultures. Depletion of ferrous iron in cultures of a strain of Leptospirillum ferriphilum resulted in rapid mortality of the iron-oxidizing bacterium (denoted by decreasing numbers of plate isolates) and corresponding changes in ATP concentrations in culture aliquots. The technique was applied successfully to mineral leachates from stirred tank biomining operations, where it was shown to determine populations of active microorganisms in liquors where most of the cells present were not viable. The method is highly sensitive (detecting as few as 103 acidophilesml−1 of test solution) and would therefore have generic application in monitoring microbial populations in other types of biomining operations, such as heap bioreactors. [Copyright &y& Elsevier]

Abstract: The microbial consortium in continuous-flow, stirred tank processes used to treat gold-bearing arsenopyrite concentrates in South Africa has become adapted to high concentrations of arsenic over several years. The dominant microorganisms, Acidithiobacillus caldus and Leptospirillum ferriphilum, were found to contain two sets of arsenic-resistance genes. One set was present in all isolates of a species irrespective of whether they were highly arsenic resistant or not, while a second, transposon-located set was present in only those strains that had been adapted to high concentrations of arsenic. The arsenopyrite treatment tanks from Tamboraque (near Lima, Peru) have not been inoculated with an arsenic-adapted consortium from South Africa. Isolation of a Leptospirillum ferrooxidans from the Tamboraque consortium allowed us to determine whether these microorganisms had acquired similar arsenic-resistance mechanisms as contained on the transposons in the highly arsenic resistant South African cultures. Several isolates of both L. ferriphilum and L. ferrooxidans from Europe as well as a “Leptospirillum ferrodiazotrophum” were also screened to detect whether they contained similar arsenic-resistance transposons even though they had not been selected for enhanced arsenic resistance. Transposons containing arsenic-resistance genes that were identical or closely related to those from South Africa were found in both L. ferrooxidans and L. ferriphilum isolates from South America and Europe. The widespread occurrence of arsenic-resistance transposons suggests that it should be possible to select for highly arsenic resistant biomining microorganisms from many different sources and therefore, unnecessary to use pre-adapted arsenic resistant consortia. [Copyright &y& Elsevier]

Abstract: Members of the Leptospirillum genus have recently been found to be not only the most representative prokaryotes in the Río Tinto, a natural extreme acidic habitat, but also in other acidic environments (AMD), as well as in biohydrometallurgical operations. The main objective of this work was to better understand and control industrial biohydrometallurgical processes by studying the role of chemolithoautotrophic bacteria of this genus in the Tinto ecosystem. Different strains of Leptospirillum were isolated from the Tinto basin and physiologically and genetically characterized by Pulsed Field Gel Electrophoresis (PFGE) and DNA microarrays. Certain metabolic capabilities, such as iron oxidation, pyrite leaching and nitrogen fixation, were determined for each strain. Complementary molecular ecology techniques (fluorescence in situ hybridization (FISH and CARD-FISH) and 16S rRNA gene cloning) were used to study the microbial diversity and the distribution of leptospirilli along the iron gradient of the Tinto ecosystem. [Copyright &y& Elsevier]

The article explores the geochemical and microbiological dynamics of a polymetallic sulfide ore from the Talvivaara mine in Finland in simulated-heap leaching. Mineral oxidation was fast in shake flask cultures of acidophilic microorganisms in which 68% of zinc and 80% of both manganese and nickel were leached within 6 weeks. Leptospirillum ferriphilum and Acidimicrobium ferroxidans were among the dominated microorganisms in shake flasks. Acidithiobacillus ferrooxidans were the dominant microorganisms in the early phase of the study.

Abstract: Bioleaching is an economical method for the recovery of metals that requires low investment and operation costs. Furthermore, it is generally more environmentally friendly than many physicochemical metal extraction processes. The bioleaching of chalcopyrite in shake flasks was investigated with pure and mixed cultures of Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans, Acidithiobacillus caldus, and Leptospirillum ferriphilum. The mixed cultures containing both iron- and sulfur-oxidizing bacteria were more efficient than the pure culture alone. The presence of sulfur-oxidizing bacteria positively increased the dissolution rate and the percentage recovery of copper from chalcopyrite. Mixed cultures consisting of moderately thermophilic L. ferriphilum and A. caldus leached chalcopyrite more effectively than mesophilic A. ferrooxidans pure and mixed cultures. The decrease of the chalcopyrite dissolution rate in leaching systems containing A. ferrooxidans after 12–16 days coincided with the formation of jarosite precipitation as a passivation layer on the mineral surface during bioleaching. Low pH significantly reduces jarosite formation in pure and mixed cultures of L. ferriphilum and A. caldus. [Copyright &y& Elsevier]

Abstract: Two strains of Leptospirillum ferrooxidans were isolated from samples taken from La Silvita polymetallic sulphide mine, Neuquen, Argentina. Isolates were identified by 16S rRNA gene restriction enzyme analysis. The specific growth rate (μ) at different temperatures and pH values were examined. The potential of two isolates to assist the leaching process using a mineral ore from the same mine was evaluated. The tests were carried out in shake flask at 1% (w/v) pulp density. The main sulphides found in the mineralogical composition of La Silvita ore were: sphalerite, galena, pyrite and chalcopyrite. Zinc and copper recoveries with a native isolate were 100% in 20 days and 12 days, respectively. Iron solubilization was 42% after 33 days of operation. These values were greater than those obtained using a laboratory reference strain, L. ferrooxidans DSMZ 2705, or abiotic conditions. [Copyright &y& Elsevier]

Abstract: Bioleaching of low-grade complex sulfide ores from La Silvita and La Resbalosa (Patagonia Argentina) were carried out in a reverse flow airlift reactor by a native strain of Leptospirillum ferrooxidans (Lf-LS04). The reactor was filled with iron free 9K medium pH 1.8, with mineral particle size of –74 μm, pulp density of 1% and a superficial air velocity of 0.01m s− 1. Zinc recoveries from the two sulfide ores – mainly in the case of La Silvita ore – were higher than those obtained previously with a collection strain of A. ferrooxidans. By applying the shrinking core-product layer diffusion model to sphalerite leaching, diffusion was found to be rate limiting due to the formation of an insoluble product layer (elemental sulfur, anglesite and ferric iron precipitates) as well as to the presence of an unreacted layer of gangue minerals (quartz, feldspar). Even in this case, the indigenous strain of L. ferrooxidans was shown to be highly efficient for processing both complex sulfides in the absence of sulfur-oxidizing bacteria. [Copyright &y& Elsevier]

Commercial bioleaching of copper and the biooxidation of gold is a cost-effective and environmentally friendly process for metal recovery. A partial genome sequence of the acidophilic, bioleaching bacterium Acidithiobacillus ferrooxidans is available from two public sources. This information has been used to build preliminary models that describe how this microorganism confronts unusually high iron loads in the extremely acidic conditions (pH 2) found in natural environments and in bioleaching operations. A. ferrooxidans contains candidate genes for iron uptake, sensing, storage, and regulation of iron homeostasis. Predicted proteins exhibit significant amino acid similarity with known proteins from neutrophilic organisms, including conservation of functional motifs, permitting their identification by bioinformatics tools and allowing the recognition of common themes in iron transport across distantly related species. However, significant differences in amino acid sequence were detected in pertinent domains that suggest ways in which the periplasmic and outer membrane proteins of A. ferrooxidans maintain structural integrity and relevant protein-protein contacts at low pH. Unexpectedly, the microorganism also contains candidate genes, organized in operon-like structures that potentially encode at least 11 siderophore systems for the uptake of Fe(III), although it does not exhibit genes that could encode the biosynthesis of the siderophores themselves. The presence of multiple Fe(III) uptake systems suggests that A. ferrooxidans can inhabit aerobic environments where iron is scarce and where siderophore producers are present. It may also help to explain why it cannot tolerate high Fe(III) concentrations in bioleaching operations where it is out-competed by Leptospirillum species. [ABSTRACT FROM AUTHOR]

Acidic, metal-rich waters generated by the microbially accelerated dissolution of pyrite and other sulfide minerals, are frequently encountered in derelict mine sites, including many that have been long-abandoned. While these waters are major causes of environmental pollution and are toxic to the majority of prokaryotic and eukaryotic organisms, some life forms (mostly bacteria and archaea) thrive within them. “Acidophiles” comprise a surprisingly wide diversity (in terms of both physiology and phylogeny) of microorganisms. This article reviews current knowledge of the distribution and biodiversity of this group of extremophiles. [Copyright &y& Elsevier]

Reddish filaments in two fragments of unusual iron oxide bearing stalactites, "the Rusticles" from Lechuguilla Cave, New Mexico, are found only within the central canals of the Rusticles. The curved, helical, and/or vibrioidal filaments vary from 1 to 6μm in outer diameter and 10 to > 50μm in length. SEM and TEM show the filaments have 0.5-μm diameter central tubes, with goethite crystals radiating outwardly along their lengths. The diameter of the central tubes is consistent with the diameter of many ironoxidizing filamentous bacteria. Although most iron oxide depositing bacteria do not deposit well-crystallized radiating goethite, we propose thick hydrous iron oxide was slowly crystallized from amorphous material to goethite, in place, over a relatively long period of time. From the gross morphology and the particular setting, we suggest this represents an occurrence of fossilized, acidophilic iron-oxidizing bacteria. [ABSTRACT FROM AUTHOR]

Spermidine was found to be the predominant polyamine in all the acidophilic as well as neutrophilic strains of chemolithotrophic iron- and sulphur-oxidizing bacteria and associated organotrophic cultures inhabiting a copper mining ecosystem. Adaptation of neutrophilic Thiobacillus thioparus strain to pH 5.0 was found to increase the spermidine content of the cells 2.3 times while the biomass yield decreased by 50%. However, exogenous supplementation of spermidine could restore the biomass yield to levels obtainable at pH 7.0. These observations indicate that spermidine plays a significant role in adaptive behaviour of microorganisms in natural ecosystems, especially under acid conditions. [ABSTRACT FROM AUTHOR]

Ultraviolet-A (UVA) radiation is one of the most stressful oxidative agents that bacteria have to face in natural environments. The effect of this radiation on growth, survival, biofilm formation, and bioleaching activity of two Leptospirillum ferrooxidans strains was studied, comparing the collection strain DSM2705 with FAM-RA2, a strain recently isolated from Famatina (La Rioja, Argentina), at 3869 m above sea level (ma.s.l.). We demonstrated that the isolate FAM-RA2 was more UVA resistant than the collection strain DSM2705 in kinetic growth and biofilm formation assays. However, both strains presented a similar behavior in their bioleaching capacity. Higher levels of intracellular reactive oxygen species (ROS) after UVA exposure were detected in DSM2705 strain, suggesting the presence of stronger defensive mechanisms against oxidative stress in FAM-RA2 strain. Assays of biofilm formation showed that UVA radiation induced cell adhesion and extracellular polymeric substance (EPS) production in the collection strain DSM2705, but not in the FAM-RA2 strain. The ability of UVA radiation to increase biofilm formation in DSM2705 led us to suppose that this agent could promote its bioleaching activity; however, under these studied conditions, this stress agent did not enhance metal sulfide dissolution. This study provides first insights into the effects of UVA radiation on the physiological features of acidophilic microorganisms. • The effect of UVA on two Leptospirillum ferrooxidans strains was studied. • The recently isolated strain is more resistant to UVA than the collection strain. • Both strains presented a similar detrimental bioleaching capacity under UVA exposure. • UVA induced biofilm formation in the collection strain. • UVA is an important stress factor in high altitude mining sites. [ABSTRACT FROM AUTHOR]