Microbial Ecology of an Extreme Acidic Environment, the Tinto River

The Tinto River (Huelva, southwestern Spain) is an extreme environment with a rather constant acidic pH along the entire river and a high concentration of heavy metals. The extreme conditions of the Tinto ecosystem are generated by the metabolic activity of chemolithotrophic microorganisms thriving in the rich complex sulfides of the Iberian Pyrite Belt. Molecular ecology techniques were used to analyze the diversity of this microbial community. The community's composition was studied by denaturing gradient gel electrophoresis (DGGE) using 16S rRNA and by 16S rRNA gene amplification. A good correlation between the two approaches was found. Comparative sequence analysis of DGGE bands showed the presence of organisms related to Leptospirillum spp., Acidithiobacillus ferrooxidans, Acidiphilium spp., “Ferrimicrobium acidiphilum,” Ferroplasma acidiphilum, and Thermoplasma acidophilum. The different phylogenetic groups were quantified by fluorescent in situ hybridization with a set of rRNA-targeted oligonucleotide probes. More than 80% of the cells were affiliated with the domain Bacteria, with only a minor fraction corresponding to Archaea. Members of Leptospirillum ferrooxidans, Acidithiobacillus ferrooxidans, and Acidiphilium spp., all related to the iron cycle, accounted for most of the prokaryotic microorganisms detected. Different isolates of these microorganisms were obtained from the Tinto ecosystem, and their physiological properties were determined. Given the physicochemical characteristics of the habitat and the physiological properties and relative concentrations of the different prokaryotes found in the river, a model for the Tinto ecosystem based on the iron cycle is suggested.     

Kinetic study of ferrous sulphate oxidation of Acidithiobacillus ferrooxidans in the presence of heavy metal ions

Acidophilic microorganisms such as Acidithiobacillus ferrooxidans have the capability to carry out processes of bioleaching, biosorption and bioprecipitation of heavy metal ions, which have important environmental applications. At. ferrooxidans derives the energy for their metabolism from ferrous iron oxidation, process, which can be affected by the presence of heavy metals in the medium. Moreover, organic matter produces an inhibitory effect over the ferrous iron oxidation of At. ferrooxidans. In this work, heterotrophic bacterium Acidiphilium sp. was added when the medium is supplemented with organic matter to reduce this negative effect. The purpose of this work is the kinetic study of ferrous sulphate oxidation by At. ferrooxidans in the presence of different concentrations of several heavy metal ions (Cr(III), Cu(II), Cd(II), Zn(II) and Ni(II)) and compare this kinetic behaviour with a mixed culture with Acidiphilium sp.The obtained results show a non-competitive inhibition of heavy metals over bacterial oxidation of ferrous sulphate. In accordance with this kind of inhibition, a kinetic equation has been proposed to predict the behaviour of At. ferrooxidans in the presence of heavy metals in the range of concentrations studied.     

Toxicity of flotation reagents to moderately thermophilic bioleaching microorganisms

The toxicity of 15 flotation reagents (including xanthates, carbamates, thiophosphates, a mercaptobenzthiazole and a frothing reagent) used for concentrating sulfide minerals to five species of mineral-oxidising, moderately thermophilic and acidophilic microorganisms was assessed. The acidophiles tested included four bacteria (a Leptospirillum isolate, Acidimicrobium ferrooxidans, Acidithiobacillus caldus and a Sulfobacillusisolate) and one archaeon (a Ferroplasma isolate). There was wide variation both in terms of the relative toxicities of the different flotation reagents and the sensitivities of the microorganisms tested. In general, the dithiophosphates and the mercaptobenzothiol were the most toxic, while the Leptospirillum and Ferroplasma isolates were the most sensitive of the acidophilic microorganisms. The significance of these findings, in view of the expanding application of ore concentrates bioprocessing, is discussed.     

Extreme zinc tolerance in acidophilic microorganisms from the bacterial and archaeal domains

Zinc can occur in extremely high concentrations in acidic, heavy metal polluted environments inhabited by acidophilic prokaryotes. Although these organisms are able to thrive in such severely contaminated ecosystems their resistance mechanisms have not been well studied. Bioinformatic analysis of a range of acidophilic bacterial and archaeal genomes identified homologues of several known zinc homeostasis systems. These included primary and secondary transporters, such as the primary heavy metal exporter ZntA and Nramp super-family secondary importer MntH. Three acidophilic model microorganisms, the archaeon ??Ferroplasma acidarmanus??, the Gram negative bacterium Acidithiobacillus caldus, and the Gram positive bacterium Acidimicrobium ferrooxidans, were selected for detailed analyses. Zinc speciation modeling of the growth media demonstrated that a large fraction of the free metal ion is complexed, potentially affecting its toxicity. Indeed, many of the putative zinc homeostasis genes were constitutively expressed and with the exception of ??F. acidarmanus?? ZntA, they were not up-regulated in the presence of excess zinc. Proteomic analysis revealed that zinc played a role in oxidative stress in At. caldus and Am. ferrooxidans. Furthermore, ??F. acidarmanus?? kept a constant level of intracellular zinc over all conditions tested whereas the intracellular levels increased with increasing zinc exposure in the remaining organisms.     

“Bioshrouding”—a novel approach for securing reactive mineral tailings

A novel technique (“bioshrouding”) for safeguarding highly reactive sulfidic mineral tailings deposits is proposed. In this, freshly milled wastes are colonised with ferric iron-reducing heterotrophic acidophilic bacteria that form biofilms on reactive mineral surfaces, thereby preventing or minimising colonisation by iron sulfide-oxidising chemolithotrophs such as Acidithiobacillus ferrooxidans and Leptospirillum spp. Data from initial experiments showed that dissolution of pyrite could be reduced by between 57 and 75% by “bioshrouding” the mineral with three different species of heterotrophic acidophiles (Acidiphilium, Acidocella and Acidobacterium spp.), under conditions that were conducive to microbial oxidative dissolution of the iron sulfide.     

Attachment of Acidithiobacillus ferrooxidans and Leptospirillum ferriphilum cultured under varying conditions to pyrite, chalcopyrite, low-grade ore and quartz in a packed column reactor

The attachment of Acidithiobacillus ferrooxidans and Leptospirillum ferriphilum spp. grown on ferrous medium or adapted to a pyrite mineral concentrate to four mineral substrata, namely, chalcopyrite and pyrite concentrates, a low-grade chalcopyrite ore (0.5 wt%) and quartzite, was investigated. The quartzite represented a typical gangue mineral and served as a control. The attachment studies were carried out in a novel particle-coated column reactor. The saturated reactor containing glass beads, which were coated with fine mineral concentrates, provided a quantifiable surface area of mineral concentrate and maintained good fluid flow. A. ferrooxidans and Leptospirillum spp. had similar attachment characteristics. Enhanced attachment efficiency occurred with bacteria grown on sulphide minerals relative to those grown on ferrous sulphate in an ore-free environment. Selective attachment to sulphide minerals relative to gangue materials occurred, with mineral adapted cultures attaching to the minerals more efficiently than ferrous grown cultures. Mineral-adapted cultures showed highest levels of attachment to pyrite (74% and 79% attachment for A. ferrooxidans and L. ferriphilum, respectively). This was followed by attachment of mineral-adapted cultures to chalcopyrite (63% and 58% for A. ferrooxidans and L. ferriphilum, respectively). A. ferrooxidans and L. ferriphilum exhibited lower levels of attachment to low-grade ore and quartz relative to the sulphide minerals.     

Leaching of copper ore of the Udokanskoe deposit at low temperatures by an association of acidophilic chemolithotrophic microorganisms

Pure cultures of indigenous microorganisms Acidithiobacillus ferrooxidans strain TFUd, Leptospirillum ferrooxidans strain LUd, and Sulfobacillus thermotolerans strain SUd have been isolated from the oxidation zone of sulfide copper ore of the Udokanskoe deposit. Regimes of bacterial-chemical leaching of ore have been studied over a temperature range from −10 to +20°C. Effects of pH, temperature, and the presence of microorganisms on the extraction of copper have been shown. Bacterial leaching has been detected only at positive values of temperature, and has been much more active at +20 than at +4°C. The process of leaching was more active when the ore contained more hydrophilic and oxidized minerals. The possibility of copper ore leaching of the Udokanskoe deposit using sulfuric acid with pH 0.4 at negative values of temperature and applying acidophilic chemolithotrophic microorganisms at positive values of temperature and low pH values was shown.     

Life in blue: Copper resistance mechanisms of bacteria and Archaea used in industrial biomining of minerals

Industrial biomining processes to extract copper, gold and other metals involve the use of extremophiles such as the acidophilic Acidithiobacillus ferrooxidans (Bacteria), and the thermoacidophilic Sulfolobus metallicus (Archaea). Together with other extremophiles these microorganisms subsist in habitats where they are exposed to copper concentrations higher than 100 mM. Herein we review the current knowledge on the Cu-resistance mechanisms found in these microorganisms. Recent information suggests that biomining extremophiles respond to extremely high Cu concentrations by using simultaneously all or most of the following key elements: 1) a wide repertoire of Cu-resistance determinants; 2) duplication of some of these Cu-resistance determinants; 3) existence of novel Cu chaperones; 4) a polyP-based Cu-resistance system, and 5) an oxidative stress defense system. Further insight of the biomining community members and their individual response to copper is highly relevant, since this could provide key information to the mining industry. In turn, this information could be used to select the more fit members of the bioleaching community to attain more efficient industrial biomining processes.     

Bioleaching of chalcopyrite by pure and mixed cultures of Acidithiobacillus spp. and Leptospirillum ferriphilum

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.     

A review of biotechnology processes applied for manganese recovery from wastes

The global consumption of manganese is rising due to its growing industrial requirement while the natural reserves of manganese are diminishing at an alarming rate. Consequently, recovery of manganese from metal containing wastes has become highly crucial. Bioleaching of metal from wastes using microbes provides an adequate advantage over the traditional method of recovery. A molecular level understanding of microbial catalyzed manganese recovery is essential for the exploitation of novel microorganisms for similar applications. In current scenario, the application of bioleaching concentrates on cost effective and eco-friendly recovery of precious metals from mining and industrial wastes. This review encompasses the modern improvements in biomining, highlights the comprehensive factors that emphasize the selection of manganese recovery technique, shed insights into spectacular progress in developing molecular based technologies and also identifies the applicability of different models in metal bioremediation which will not only aid in pollution abatement but also in the prevention of occupational health disorder.     

Acdidophilic thermophilic archaebacteria: Potential application for metals recovery

Abstract The acidophilic thermophilic archaebacteria Sulfolobus and Acidianus have the potential for applid use in the recovery of metal values from ores through the process of baterial leaching. These microbes readily adapt to the conditions of low pH and high concentrations of metals required for bacterial leaching. In addition, these archaebacteria can exist at high temperatures which can occur during the oxidation of metal sulfides in bioleaching reactors. The acidophilic of copper and molybdenum from chalcopyrite and molybdenite minerals, respectively. The microbes can also enhance the recovery of gold by oxidation of pyrite which occludes gold preventing recovery by standard metallurgical procedures. The ability of this group of microbes to facilitate metals recovery is yet to be developed on a commercial scale.     

Selection of a community of acidochemolithotrophic microorganisms with a high oxidation rate of pyrrhotite-containing sulphide ore flotation concentrate

A community of acidochemolithotrophic microorganisms with a high oxidation rate of pyrrhotite-containing sulphide ore flotation concentrate was selected. The Acidithiobacillus caldus OP-1 and Ferroplasma acidiphilum OP-2 cultures were identified to be dominating members. The presence of the Acidithiobacillus ferrooxidans OP-3, Leptospirillum ferriphilum OP-4, and Sulfobacillus thermosulfidooxidans OP-5 cultures in the community’s composition was also mentioned. The analysis results of solid residues of the process showed a greater elemental sulfur oxidation level and gold recovery when the initial pH value in tank I was maintained at a level of 1.8–2.0 (90.5%) rather than 1.6–1.8 (86.3%).     

Evaluation of the Biotechnological Potential of Pure and Mixture of Indigenous Iron-Oxidizing Bacteria to Dissolve Trace Metals from Cu Bearing Ore

Abstract

This study aimed to investigate the ability of pure and consortia of indigenous iron-oxidizing bacteria to enhance the dissolution of trace metals from Cu and Zn-bearing ore. Three bacterial strains Acidithiobacillus ferrooxidans strain WG101, Leptospirillum ferriphilum strain WG102, Leptospirillum ferrooxidans strain WG103 isolated from Baiyin copper mine, China were used in this study. The biotechnological potential of these indigenous isolates was evaluated both in pure and in consortia to extract cobalt, chromium, and lead from the copper and zinc bearing ore. The sulfur and iron-oxidizing bacterial isolate Acidithiobacillus ferrooxidans strain WG101 exhibited efficient dissolution compared to sole iron-oxidizing Leptospirillum ferriphilum strain WG102, and Leptospirillum ferrooxidans strain WG103. Initial medium pH, pulp density, and temperature were studied as influential parameters in bioleaching carried out by bacterial consortia. The achieved optimum conditions were; initial pH of 1.5, 10% of pulp density, and temperature 30?°C with 68.7?±?3.9% cobalt, 56.6?±?3.9% chromium, and 36?±?3.7% lead recovery. Analytical study of oxidation-reduction potential and pH fluctuation were observed during this whole process that shows the metal dissolution efficiency of bacterial consortia. Alterations in spectral bands of processed residues were reported through FTIR analysis compared with control ore sample. Mössbauer spectroscopy analysis showed the influence of bacterial consortia on iron speciation in bioleached samples. The findings confirm that the indigenous acidophilic iron-oxidizing bacterial strains are highly effective in the dissolution of trace elements present in ore samples. This study not only supports the notion that indigenous bacterial strains are highly effectual in metal dissolution but provides the basic vital conditions to upscale the bioleaching technique for metals dissolution.     

Gene expression modulation by chalcopyrite and bornite in Acidithiobacillus ferrooxidans

Acidithiobacillus ferrooxidans is a mesophilic, acidophilic, chemolithoautotrophic bacterium that obtains energy from the oxidation of ferrous iron (Fe²⁺), elemental sulfur and reduced sulfur compounds. The industrial interest in A. ferrooxidans resides in its capacity to oxidize insoluble metal sulfides into soluble metal sulfates, thus allowing the recovery of the desired metals from low-grade sulfide ores. In the present work, RNA arbitrarily primed PCR (RAP-PCR) was performed to identify cDNAs differentially expressed in A. ferrooxidans cells grown in the presence of Fe²⁺ and cells maintained for 24 h in the presence of the copper sulfides bornite and chalcopyrite. Eighteen cDNAs corresponding to genes with known function were identified, and their relative expression was further characterized by real-time quantitative PCR. Bornite had a mild effect on the expression of the 18 genes analyzed. None of these genes was down-regulated and among the few genes up-regulated, it is worth mentioning lepA and def-2 that are involved in protein synthesis. Chalcopyrite presented the most significant changes. Five genes related to protein processing were down-regulated, and another 5 genes related to the transport system were up-regulated. The up- and down-regulation of these genes in the presence of bornite and chalcopyrite could be due to alterations in the ideal pH, presence of copper ions in solution and nutrient limitation. The results suggest that gene expression modulation might be important for the A. ferrooxidans early response to copper sulfides.     

A dual-specific Glu-tRNA and Asp-tRNA amidotransferase is involved in decoding glutamine and asparagine codons in Acidithiobacillus ferrooxidans

The gatC, gatA and gatB genes encoding the three subunits of glutamyl-tRNA^Gln amidotransferase from Acidithiobacillus ferrooxidans, an acidophilic bacterium used in bioleaching of minerals, have been cloned and expressed in Escherichia coli. As in Bacillus subtilis the three gat genes are organized in an operon-like structure in A. ferrooxidans. The heterologously overexpressed enzyme converts Glu-tRNA^Gln to Gln-tRNA^Gln and Asp-tRNA^Asn to Asn-tRNA^Asn. Biochemical analysis revealed that neither glutaminyl-tRNA synthetase nor asparaginyl-tRNA synthetase is present in A. ferrooxidans, but that glutamyl-tRNA synthetase and aspartyl-tRNA synthetase enzymes are present in the organism. These data suggest that the transamidation pathway is responsible for the formation of Gln-tRNA and Asn-tRNA in A. ferrooxidans.     

Analysis of the microbial community in moderately acidic drainage from the Yanahara pyrite mine in Japan

Acid rock drainage (ARD) originating from the Yasumi-ishi tunnel near the main tunnel of the Yanahara mine in Japan was characterized to be moderately acidic (pH 4.1) and contained iron at a low concentration (51?mg/L). The composition of the microbial community was determined by sequence analysis of 16S rRNA genes using PCR and denaturing gradient gel electrophoresis. The analysis of the obtained sequences showed their similarity to clones recently detected in other moderately acidic mine drainages. Uncultured bacteria related to Ferrovum- and Gallionella-like clones were dominant in the microbial community. Analyses using specific primers for acidophilic iron- or sulfur-oxidizing bacteria, Acidithiobacillus ferrooxidans, Leptospirillum spp., Acidithiobacillus caldus, Acidithiobacillus thiooxidans, and Sulfobacillus spp. revealed the absence of these bacteria in the microbial community in ARD from the Yasumi-ishi tunnel. Clones affiliated with a member of the order Thermoplasmatales were detected as the dominant archaea in the ARD microbial population.     

Investigation of energy gene expressions and community structures of free and attached acidophilic bacteria in chalcopyrite bioleaching

In order to better understand the bioleaching mechanism, expression of genes involved in energy conservation and community structure of free and attached acidophilic bacteria in chalcopyrite bioleaching were investigated. Using quantitative real-time PCR, we studied the expression of genes involved in energy conservation in free and attached Acidithiobacillus ferrooxidans during bioleaching of chalcopyrite. Sulfur oxidation genes of attached A. ferrooxidans were up-regulated while ferrous iron oxidation genes were down-regulated compared with free A. ferrooxidans in the solution. The up-regulation may be induced by elemental sulfur on the mineral surface. This conclusion was supported by the results of HPLC analysis. Sulfur-oxidizing Acidithiobacillus thiooxidans and ferrous-oxidizing Leptospirillum ferrooxidans were the members of the mixed culture in chalcopyrite bioleaching. Study of the community structure of free and attached bacteria showed that A. thiooxidans dominated the attached bacteria while L. ferrooxidans dominated the free bacteria. With respect to available energy sources during bioleaching of chalcopyrite, sulfur-oxidizers tend to be on the mineral surfaces whereas ferrous iron-oxidizers tend to be suspended in the aqueous phase. Taken together, these results indicate that the main role of attached acidophilic bacteria was to oxidize elemental sulfur and dissolution of chalcopyrite involved chiefly an indirect bioleaching mechanism.