Isolation and characterization of Ferroplasma thermophilum sp. nov., a novel extremely acidophilic, moderately thermophilic archaeon and its role in bioleaching of chalcopyrite

Aims:  To isolate Ferroplasma thermophilum L1^T from a low pH environment and to understand its role in bioleaching of chalcopyrite.
Methods and Results:  Using serial dilution method, a moderately thermophilic and acidophilic ferrous iron-oxidizing archaeon, named L1^T, was isolated from a chalcopyrite-leaching bioreactor. The morphological, biochemical and physiological characteristics of strain L1^T and its role in bioleaching of chalcopyrite were studied. Strain L1^T was a nonmotile coccus that lacked cell wall. Strain L1^T had a temperature optimum of 45°C and the optimum pH for growth was 1·0. Strain L1^T was capable of chemomixotrophic growth on ferrous iron and yeast extract. Results of fatty acid analysis, DNA–DNA hybridization, G+C content, and analysis based on 16S rRNA gene sequence indicated that strain L1^T should be grouped in the genus Ferroplasma , and represented a new species, Ferroplasma thermophilum . Ferroplasma thermophilum in combination with Acidithiobacillus caldus and Leptospirillum ferriphilum could improve the copper dissolution in bioleaching of chalcopyrite.
Conclusions:  A novel extremely acidophilic, moderately thermophilic archaeon isolated from a bioleaching reactor has been identified as F. thermophilum that played an important role in bioleaching of chalcopyrite at low pH.
Significance and Impact of the Study:  This study contributes to understand the characteristics of F. thermophilum L1^T and its role in bioleaching of sulfide ores.     

Leaching of nonferrous metals from copper converter slag with application of acidophilic microorganisms

The leaching process of copper and zinc from copper converter slag with ferric iron in sulfuric acid solutions obtained using the association of acidophilic chemolithotrophic microorganisms was investigated. The best parameters of chemical leaching (temperature 70°C, an initial concentration of ferric iron in the leaching solution of 10.1 g/L, and a solid phase content in the pulp of 10%) were selected. Carrying out the process under these parameters resulted in the recovery of 89.4% of copper and 39.3% of zinc into the solution. The possibility of the bioregeneration of ferric iron in the solution obtained after the chemical leaching of slag by iron-oxidizing acidophilic chemolithotrophic microorganisms without inhibiting their activity was demonstrated.     

铜蓝精矿的生物浸出

采用两种嗜酸硫杆菌(嗜酸氧化亚铁硫杆菌和喜温硫杆菌)对铜蓝进行生物浸出,实验在有或没有4 g/L硫酸亚铁pH 2．0、150转/分、35℃的三角瓶中进行。实验结果表明:用两种菌混合浸出的铜几乎等于嗜酸氧化亚铁硫杆菌单独浸出的铜;另外,亚铁的加入能提高铜的浸出。     

Chemical leaching of copper-zinc concentrate with ferric iron biosolution

The process of leaching of copper-zinc concentrate with a solution containing biogenic iron, which is a product of the metabolism of iron-oxidizing microorganisms, was studied. The dependence of leaching rate of metals on temperature and pH was determined. It was shown that up to 98% of zinc and 70% of iron could be removed from the concentrate, while up to 7 and 4 g/L of zinc and copper, respectively, were accumulated in the liquid phase, which was sufficient for metal recovery. It was established that a copper concentrate with copper content up to 16% and only 0.5% of zinc could be obtained after chemical leaching for 340 min at 80°C.     

Bioleaching of copper- and zinc-bearing ore using consortia of indigenous iron-oxidizing bacteria

Indigenous iron-oxidizing bacteria were isolated on modified selective 9KFe²⁺ medium from Baiyin copper mine stope, China. Three distinct acidophilic bacteria were isolated and identified by analyzing the sequences of 16S rRNA gene. Based on published sequences of 16S rRNA gene in the GenBank, a phylogenetic tree was constructed. The sequence of isolate WG101 showed 99% homology with Acidithiobacillus ferrooxidans strain AS2. Isolate WG102 exhibited 98% similarity with Leptospirillum ferriphilum strain YSK. Similarly, isolate WG103 showed 98% similarity with Leptospirillum ferrooxidans strain L15. Furthermore, the biotechnological potential of these isolates in consortia form was evaluated to recover copper and zinc from their ore. Under optimized conditions, 77.68 ± 3.55% of copper and 70.58 ± 3.77% of zinc were dissolved. During the bioleaching process, analytical study of pH and oxidation–reduction potential fluctuations were monitored that reflected efficient activity of the bacterial consortia. The FTIR analysis confirmed the variation in bands after treatment with consortia. The impact of consortia on iron speciation within bioleached ore was analyzed using Mössbauer spectroscopy and clear changes in iron speciation was reported. The use of indigenous bacterial consortia is more efficient compared to pure inoculum. This study provided the basic essential conditions for further upscaling bioleaching application for metal extraction.

     

Bioleaching of copper from chalcopyrite ore by fungi

Microorganisms have been geologically active in mineral formation, mineral diagenesis and sedimentation via direct action of their enzymes or indirectly through chemical action of their metabolic products. This property of microorganisms is being harnessed during the recent years for extraction of metals from their ores, especially from low-grade ores. In the present study bioleaching of copper from its low-grade chalcopyrite ore using 26 isolates of acidophilic fungi is reported. Most of these fungal strains belonged to the genera Aspergillus, Penicillium and Rhizopus. The leaching experiments were conducted in Czepek Dox minimal medium containing 1% (100 mesh) ore with shaking at room temperature for 20 days. Out of these, 4 isolates exhibited significant bioleaching activities. Maximum leaching of copper (78 mg/L) was observed with Aspergillus flavus (DSF-8) and Aspergillus niger (DOF-1). Nutritional and environmental conditions for optimum bioleaching were standardized. Present study indicates the usefulness of acidophilic fungi in bioleaching of copper from its low-grade ores.     

Isolation of an extremely acidophilic and highly efficient strain Acidithiobacillus sp. for chalcopyrite bioleaching

An extremely acidophilic sulfur-oxidizing bacterium was isolated from an industrial-scale bioheap of the Zijinshan copper mine and was named ZJJN. A tuft of flagella and a layer of thick capsule outside the cell envelope were clearly observed under transmission electron microscopy (TEM), which might be closely related to the extremely acid-proof capacity of ZJJN cells in the bioleaching system; 16S ribosomal RNA (rRNA) phylogeny showed that the isolated strain was highly homologous to the genera of Acidithiobacillus. The optimum temperature of ZJJN was determined at 30?°C and pH at 1.0. It was capable of growth at even pH 0. Strain ZJJN can utilize reduced sulfur as an energy source but not with organics or ferrous ion. Strain ZJJN was sensitive to all antibiotics with different concentrations; when it showed a certain resistance to different concentrations of Cu²⁺. In the mixed strains of ZJJN and A. ferrooxidans system (initial pH 1.0), the copper-leaching efficiency was up to 60.1?%, which was far higher than other systems. Scanning electron microscopy (SEM) analysis showed that less jarosite precipitation was produced in the most efficient system. The extremely acidophilic strain ZJJN would be of great potential in the application of chalcopyrite bioleaching.     

Selective removal of transition metals from acidic mine waters by novel consortia of acidophilic sulfidogenic bacteria

Two continuous‐flow bench‐scale bioreactor systems populated by mixed communities of acidophilic sulfate‐reducing bacteria were constructed and tested for their abilities to promote the selective precipitation of transition metals (as sulfides) present in synthetic mine waters, using glycerol as electron donor. The objective with the first system (selective precipitation of copper from acidic mine water containing a variety of soluble metals) was achieved by maintaining a bioreactor pH of ～2.2–2.5. The second system was fed with acidic (pH 2.5) synthetic mine water containing 3 mM of both zinc and ferrous iron, and varying concentrations (0.5–30 mM) of aluminium. Selective precipitation of zinc sulfide was possible by operating the bioreactor at pH 4.0 and supplementing the synthetic mine water with 4 mM glycerol. Analysis of the microbial populations in the bioreactors showed that they changed with varying operational parameters, and novel acidophilic bacteria (including one sulfidogen) were isolated from the bioreactors. The acidophilic sulfidogenic bioreactors provided ‘proof of principle’ that segregation of metals present in mine waters is possible using simple online systems within which controlled pH conditions are maintained. The modular units are versatile and robust, and involve minimum engineering complexity.     

Effect of Cell Lysis (CLs) Products on Acidophilic Chemolithotrophic Microorganisms and the Role of Acidocella Species

Acidophilic chemolithotrophic microorganisms (CMs) are widely used for bioleaching of mineral resources. However, the growth of bacteria and their leaching activity are often inhibited (restricted) by organic components, e.g. lysates and exudates. The aims of this study were to examine the extent of cell lysis (CLs) inhibition on acidophilic microorganisms and to identify microorganisms that can utilize CLs products and eliminate their inhibition effect on acidophilic microorganisms. Specifically, it was revealed that Acidithiobacillus caldus was severely inhibited at 5% CLs products, whereas A. ferrooxidans and Leptospirillum ferriphilum are severely inhibited at 20%. It has been found that strains RBA and RBB of heterotrophic bacteria, isolated from anaerobic sludge, can biodegrade CLs products and when co-cultured with A. ferrooxidans, they can alleviate the toxic effect of CLs products under low pH (2–3). It has been shown that besides CLs, isolated strains can grow on glucose, glycerol, yeast extract, citric acid, and tryptone soya broth with an optimum temperature of 35°C and a pH of 3. The strains showed the ability to reduce ferric ions to ferrous ions when glycerol was used as a substrate after 2 days under both aerobic and anaerobic conditions. On the basis of morphophysiological and molecular biological studies, the isolated strains RBA and RBB were identified as Acidocella spp.     

Bioregeneration of the pregnant leach solutions obtained during the leaching of nonferrous metals from slag waste by acidophilic microorganisms

The bioregeneration of the solutions obtained after the leaching of copper and zinc from slag waste by sulfuric acid solutions containing ferric iron was examined. For bioregeneration, associations of mesophilic and moderately thermqophilic acidophilic chemolithotrophic microorganisms were made. It has been shown that the complete oxidation of iron ions in solutions is possible at a dilution of the pregnant leach solution with a nutrient medium. It has been found that the maximal rate of oxidation of iron ions is observed at the use of a mesophilic association of microorganisms at a threefold dilution of the pregnant leach solution with a nutrient medium. The application of bioregeneration during the production of nonferrous metals from both copper converter slag and its waste would make it possible to approach the technology of their processing using the closed cycle of workflows.     

Iron uptake by the microaerophilic anaerobe Bifidobacterium bifidum var. pennsylvanicus

A system was designed to investigate ferrous iron transport into Bifidobacterium bifidum var. pennsylvanicus. It involved the incubation of the organisms with labeled ferrous iron in the Norris medium at pH 5, in which the bacteria had grown. Iron uptakes were similar under aerobic and anaerobic conditions. Ferrous but not ferric iron was taken up by the organisms. Iron uptake showed saturation kinetics and a marked temperature dependence. 2,4-Dinitrophenol and thenoltrifluoroacetate but not azide or trypsin treatment inhibited iron uptake. Zinc inhibited iron uptake competitively. Iron uptake from used medium was much greater than that from fresh medium at the same pH. It is concluded that ferrous iron uptake by the microorganisms is a carrier-mediated active phenomenon, inhibited by zinc, which may involve a substance elaborated into the medium by the organism.     

Bioleaching of pyrite by acidophilic thermophile Acidianus brierleyi

The kinetics of bioleaching of pyrite (FeS(2)) by the acidophilic thermophilic bacterium Acidianus brierleyi was studied in a well-mixed batch reactor. Experiments were done at 65 degrees C and pH 1.5 on adsorption of A. brierleyi onto pyrite particles, liquid-phase oxidation of ferrous iron by A. brierleyi, and microbial leaching of pyrite. The adsorption of A. brierleyi was a fast process; equilibrium was attained within the first 30 min of exposure to pyrite. The adsorption equilibrium data were well correlated with the Langmuir isotherm. The oxidation of ferrous iron was markedly accelerated in the presence of A. brierleyi, and the growth yield on ferrous iron was determined. The bioleaching of pyrite by A. brierleyi was found to take place with a direct attack by adsorbed cells on the surface of pyrite, the chemical leaching of pyrite by ferric iron being insignificant. Rate data collected under a wide variety of operating variables were analyzed to determine kinetic and stoichiometric parameters for the microbial pyrite leaching. The specific growth rate on pyrite for A. brierleyi was about four times that for the mesophilic bacterium, Thiobacillus ferrooxidans, whereas the growth yields on pyrite for the two microbes were approximately equal to one another in magnitude. A comparison of A. brierleyi with T. ferrooxidans for pyrite leachability demonstrated the thermophile to be much more effective. (c) 1995 John Wiley & Sons, Inc.     

Microbial communities and geochemical dynamics in an extremely acidic, metal-rich stream at an abandoned sulfide mine (Huelva, Spain) underpinned by two functional primary production systems

An extremely acidic (pH 2.5-2.75) metal-rich stream draining an abandoned mine in the Iberian Pyrite Belt, Spain, was ramified with stratified macroscopic gelatinous microbial growths ('acid streamers' or 'mats'). Microbial communities of streamer/mat growths sampled at different depths, as well as those present in the stream water itself, were analysed using a combined biomolecular and cultivation-based approach. The oxygen-depleted mine water was dominated by the chemolithotrophic facultative anaerobe Acidithiobacillus ferrooxidans, while the streamer communities were found to be highly heterogeneous and very different to superficially similar growths reported in other extremely acidic environments. Microalgae accounted for a significant proportion of surface streamer biomass, while subsurface layers were dominated by heterotrophic acidophilic bacteria (Acidobacteriacae and Acidiphilium spp.). Sulfidogenic bacteria were isolated from the lowest depth streamer growths, where there was also evidence for selective biomineralization of copper sulfide. Archaeal clones (exclusively Euryarchaeota) were recovered from streamer samples, as well as the mine stream water. Both sunlight and reduced inorganic chemicals (predominantly ferrous iron) served as energy sources for primary producers in this ecosystem, promoting complex microbial interactions involving transfer of electron donors and acceptors and of organic carbon, between microorganisms in the stream water and the gelatinous streamer growths. Microbial transformations were shown to impact the biogeochemical cycling of iron and sulfur in the acidic stream, severely restricting the net oxidation of ferrous iron even when the initially anoxic waters were oxygenated by indigenous acidophilic algae. A model accounting for the biogeochemistry of iron and sulfur in the mine waters is described, and the significance of the acidophilic communities in regulating the geochemistry of acidic, metal-rich waters is described.     

嗜酸异养菌对自养菌Acidithiobacillus ferrooxidans金属离子抗性和生物浸出的影响

【目的】了解嗜酸异养菌在诸如酸性矿坑水(AMD)和生物浸出体系等极端酸性环境中对浸矿微生物产生的影响。【方法】研究由嗜酸异养菌Acidiphilium acidophilum和自养菌Acidithiobacillus ferrooxidans经长期驯化后形成的共培养体系分别在Cd2+、Cu2+、Ni2+和Mg2+胁迫下的稳定性;并将此共培养体系应用于黄铁矿和低品位黄铜矿的生物浸出实验。【结果】在上述4种金属离子分别存在的条件下,异养菌Aph.acidophilum均能促进At.ferrooxidans对亚铁的氧化,提高其对能源利用的效率。共培养体系中的异养菌Aph.acidophilum使At.ferrooxidans对Cu2+的最大耐受浓度(MTC)由2.0 g/L提高到5.0 g/L,而且共培养的细胞数量与2.0 g/L Cu2+条件下生长的At.ferrooxidans纯培养相似。另外,共培养中的At.ferrooxidans对Mg2+的MTC也由12.0 g/L提高到17.0 g/L。生物浸出实验中嗜酸异养菌Aph.acidophilum促进了At.ferrooxidans对黄铁矿样品的浸出,浸出率较其纯培养提高了22.7%;但在含铁量较低的低品位黄铜矿浸出体系中共培养和At.ferrooxidans纯培养的浸出率均低于33%。在加入2.0 g/L Fe2+的低品位黄铜矿浸出体系中,共培养和At.ferrooxidans纯培养的浸出率均得到提高,分别达到52.22%和41.27%。【结论】以上结果表明,Aph.acidophilum与At.ferrooxidans共培养在一定的环境胁迫下仍能保持其稳定性并完成各自的生态功能,并且嗜酸异养菌Aph.acidophilum适合在含铁量较高的浸出体系中与铁氧化细菌共同作用来提高生物浸出的效率。     

Characteristics of a moderately thermophilic and acidophilic iron-oxidizing Thiobacillus

Summary Thiobacillus TH1 is an acidophilic chemolithotrophic heterotroph growing at temperatures up to about 50°C on media containing ferrous iron or pyrite when supplemented with yeast extract or glutathione. Virtually no carbon dioxide fixation occurred during growth on iron with yeast extract. Its DNA contains 48 mol % guanine + cytosine. The organism effects the thermophilic leaching of metals from pyrite, chalcopyrite, CuS, and copper concentrates. Oxidation of soluble ferrous iron at pH 1.6 was competitively inhibited by ferric iron and had a K_m of 7.3 mM FeSO₄.     

Bioleaching of arsenic from medicinal realgar by pure and mixed cultures

The aim of this paper is to determine the efficiency of bioleaching of arsenic in realgar, a Chinese mineral drug, using pure cultures of Acidithiobacillus ferrooxidans or Acidithiobacillus thiooxidans and a mixed culture of A. ferrooxidans and A. thiooxidans. The experiments were carried out in shaker flasks, at 150 rpm, 30 °C at a culture pH of 1.80. To investigate the mechanism of the bioleaching in realgar, media with and without ferrous iron were chosen for the experiments. The results showed that the leaching rate of arsenic in realgar after 20 days was higher (43%) in A. ferrooxidans cultures with ferrous iron compared to cultures without ferrous iron (10%), and the leaching rate of A. thiooxidans cultures only increased from 21% to 23% in the presence of ferrous iron. The leaching rate of arsenic in mixed culture with ferrous iron was greatly enhanced from 16% to 56%, indicating that bioleaching in mixed culture is preferable for the dissolution of realgar.     

Microbial iron management mechanisms in extremely acidic environments: comparative genomics evidence for diversity and versatility

Background  

Iron is an essential nutrient but can be toxic at high intracellular concentrations and organisms have evolved tightly regulated mechanisms for iron uptake and homeostasis. Information on iron management mechanisms is available for organisms living at circumneutral pH. However, very little is known about how acidophilic bacteria, especially those used for industrial copper bioleaching, cope with environmental iron loads that can be 10¹⁸ times the concentration found in pH neutral environments. This study was motivated by the need to fill this lacuna in knowledge. An understanding of how microorganisms thrive in acidic ecosystems with high iron loads requires a comprehensive investigation of the strategies to acquire iron and to coordinate this acquisition with utilization, storage and oxidation of iron through metal responsive regulation. In silico prediction of iron management genes and Fur regulation was carried out for three Acidithiobacilli: Acidithiobacillus ferrooxidans (iron and sulfur oxidizer) A. thiooxidans and A. caldus (sulfur oxidizers) that can live between pH 1 and pH 5 and for three strict iron oxidizers of the Leptospirillum genus that live at pH 1 or below.     

Genomics, metagenomics and proteomics in biomining microorganisms

The use of acidophilic, chemolithotrophic microorganisms capable of oxidizing iron and sulfur in industrial processes to recover metals from minerals containing copper, gold and uranium is a well established biotechnology with distinctive advantages over traditional mining. A consortium of different microorganisms participates in the oxidative reactions resulting in the extraction of dissolved metal values from ores. Considerable effort has been spent in the last years to understand the biochemistry of iron and sulfur compounds oxidation, bacteria-mineral interactions (chemotaxis, quorum sensing, adhesion, biofilm formation) and several adaptive responses allowing the microorganisms to survive in a bioleaching environment. All of these are considered key phenomena for understanding the process of biomining. The use of genomics, metagenomics and high throughput proteomics to study the global regulatory responses that the biomining community uses to adapt to their changing environment is just beginning to emerge in the last years. These powerful approaches are reviewed here since they offer the possibility of exciting new findings that will allow analyzing the community as a microbial system, determining the extent to which each of the individual participants contributes to the process, how they evolve in time to keep the conglomerate healthy and therefore efficient during the entire process of bioleaching.     

Genomic insights into the iron uptake mechanisms of the biomining microorganism Acidithiobacillus ferrooxidans

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.     

不同能源条件下中度嗜热嗜酸细菌多样性分析

采用黄铁矿、黄铜矿、硫酸亚铁和硫粉混合物作为主要能源物质在50°C条件下分别培养中度嗜热细菌混合物,研究其细菌多样性。提取细菌基因组总DNA,采用PCR结合限制性酶切片段多态性分析(RFLP)方法进行细菌16SrRNA基因的系统发育分析,比较不同能源条件下富集培养的混合细菌群落构成的差异。从3个培养物中共获得阳性克隆303个并进行RFLP分析,对29种不同酶切谱型的克隆插入序列进行测定和系统发育分析。大部分序列与已报道的浸矿微生物16SrRNA序列相似性较高(89.1%~99.7%),归属于硫化叶菌属的耐温氧化硫化杆菌(Sulfobacillus thermotolerans)和热氧化硫化杆菌(Sulfobacillus thermosulfidooxidans),嗜酸硫杆菌属的喜温硫杆菌(Acidithiobacillus caldus),钩端螺旋菌属的嗜铁钩端螺旋菌(Leptospirillumferriphilum)以及unculturedforest soil bacterium、uncultured proteobacterium。其中Acidithiobacillus caldus,Sulfobacillus thermotolerans,Leptospirillumferriphilum3种细菌为三类能源物质培养物中的优势细菌类群。L.ferriphilum在黄铁矿培养体系(53.8%)和硫酸亚铁和硫粉为能源的培养体系中(45.9%)中丰度最高;在以黄铜矿为能源物质的培养体系中,S.thermotolerans的比例大幅上升(70.1%)。关键词:中度嗜热细菌;生物多样性;生物浸矿;喜温硫杆菌(Acidithiobacillus caldus);耐温氧化硫化杆菌(Sulfobacillus thermotolerans);嗜铁钩端螺旋菌(Leptospirillumferriphilum)