Dissimilatory Oxidation and Reduction of Elemental Sulfur in Thermophilic Archaea

The oxidation and reduction of elemental sulfur and reduced inorganic sulfur species are some of the most important energy-yielding reactions for microorganisms living in volcanic hot springs, solfataras, and submarine hydrothermal vents, including both heterotrophic, mixotrophic, and chemolithoautotrophic, carbon dioxide-fixing species. Elemental sulfur is the electron donor in aerobic archaea like Acidianus and Sulfolobus. It is oxidized via sulfite and thiosulfate in a pathway involving both soluble and membrane-bound enzymes. This pathway was recently found to be coupled to the aerobic respiratory chain, eliciting a link between sulfur oxidation and oxygen reduction at the level of the respiratory heme copper oxidase. In contrast, elemental sulfur is the electron acceptor in a short electron transport chain consisting of a membrane-bound hydrogenase and a sulfur reductase in (facultatively) anaerobic chemolithotrophic archaea Acidianus and Pyrodictium species. It is also the electron acceptor in organoheterotrophic anaerobic species like Pyrococcus and Thermococcus, however, an electron transport chain has not been described as yet. The current knowledge on the composition and properties of the aerobic and anaerobic pathways of dissimilatory elemental sulfur metabolism in thermophilic archaea is summarized in this contribution.     

Evidence of cell surface iron speciation of acidophilic iron-oxidizing microorganisms in indirect bioleaching process

While indirect model has been widely accepted in bioleaching, but the evidence of cell surface iron speciation has not been reported. In the present work the iron speciation on the cell surfaces of four typically acidophilic iron-oxidizing microorganism (mesophilic Acidithiobacillus ferrooxidans ATCC 23270, moderately thermophilic Leptospirillum ferriphilum YSK and Sulfobacillus thermosulfidooxidans St, and extremely thermophilic Acidianus manzaensis YN25) grown on different energy substrates (chalcopyrite, pyrite, ferrous sulfate and elemental sulfur (S⁰)) were studied in situ firstly by using synchrotron-based micro- X-ray fluorescence analysis and X-ray absorption near-edge structure spectroscopy. Results showed that the cells grown on iron-containing substrates had apparently higher surface iron content than the cells grown on S⁰. Both ferrous iron and ferric iron were detected on the cell surface of all tested AIOMs, and the Fe(II)/Fe(III) ratios of the same microorganism were affected by different energy substrates. The iron distribution and bonding state of single cell of A. manzaensis were then studied in situ by scanning transmission soft X-ray microscopy based on dual-energy contrast analysis and stack analysis. Results showed that the iron species distributed evenly on the cell surface and bonded with amino, carboxyl and hydroxyl groups.     

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.     

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.     

Thermoacidophilic microbial community oxidizing the gold-bearing flotation concentrate of a pyrite-arsenopyrite ore

An aboriginal community of thermophilic acidophilic chemolithotrophic microorganisms (ACM) was isolated from a sample of pyrite gold-bearing flotation concentrate at 45–47°C and pH 1.8–2.0. Compared to an experimental thermoacidophilic microbial consortium formed in the course of cultivation in parallel bioreactors, it had lower rates of iron leaching and oxidation, while its rate of sulfur oxidation was higher. A new thermophilic acidophilic microbial community was obtained by mutual enrichment with the microorganisms from the experimental and aboriginal communities during the oxidation of sulfide ore flotation concentrate at 47°C. The dominant bacteria of this new ACM community were Acidithiobacillus caldus (the most active sulfur oxidize) and Sulfobacillus thermotolerans (active oxidizer of both iron and sulfur), while iron-oxidizing archaea of the family Ferroplasmaceae and heterotrophic bacteria Alicyclobacillus tolerans were the minor components. The new ACM community showed promise for leaching/oxidation of sulfides from flotation concentrate at high pulp density (S : L = 1 : 4).     

Crystal structure studies on sulfur oxygenase reductase from Acidianus tengchongensis

Sulfur oxygenase reductase (SOR) simultaneously catalyzes oxidation and reduction of elemental sulfur to produce sulfite, thiosulfate, and sulfide in the presence of molecular oxygen. In this study, crystal structures of wild type and mutants of SOR from Acidianus tengchongensis (SOR-AT) in two different crystal forms were determined and it was observed that 24 identical SOR monomers form a hollow sphere. Within the icosatetramer sphere, the tetramer and trimer channels were proposed as the paths for the substrate and products, respectively. Moreover, a comparison of SOR-AT with SOR-AA (SOR from Acidianus ambivalens) structures showed that significant differences existed at the active site. Firstly, Cys31 is not persulfurated in SOR-AT structures. Secondly, the iron atom is five-coordinated rather than six-coordinated, since one of the water molecules ligated to the iron atom in the SOR-AA structure is lost. Consequently, the binding sites of substrates and a hypothetical catalytic process of SOR were proposed.     

Dependence of effectiveness of leaching of metallic sulphides on enzymes involved in inorganic sulphur metabolism in Thiobacillus ferrooxidans

Summary The leaching activity of five batches of Thiobacillus ferrooxidans, strain F26-77, cultivated under various conditions, towards elemental sulphur, ferrous ions, pyrite, covellite, chalcopyrite and sphalerite was studied. The activities of sulphite oxidase, thiosulphate oxidase and rhodanese were determined in crude, cell-free bacterial extracts. The effectiveness of leaching was directly correlated with the enzymic activity of the cultures. The results suggest that the activities of the enzymes metabolizing sulphur and its inorganic compounds in Thiobacillus ferrooxidans, or bacterial leaching activity on sulphur and sulphides, rather than the rate of oxidation of ferrous ions, should be taken as the criterion of usefulness for the leaching of sulphide minerals.     

Progress in bioleaching: part B: applications of microbial processes by the minerals industries

This review presents developments and applications in bioleaching and mineral biooxidation since publication of a previous mini review in 2003 (Olson et al. Appl Microbiol Biotechnol 63:249–257, 2003). There have been discoveries of newly identified acidophilic microorganisms that have unique characteristics for effective bioleaching of sulfidic ores and concentrates. Progress has been made in understanding and developing bioleaching of copper from primary copper sulfide minerals, chalcopyrite, covellite, and enargite. These developments point to low oxidation–reduction potential in concert with thermophilic bacteria and archaea as a potential key to the leaching of these minerals. On the commercial front, heap bioleaching of nickel has been commissioned, and the mineral biooxidation pretreatment of sulfidic-refractory gold concentrates is increasingly used on a global scale to enhance precious metal recovery. New and larger stirred-tank reactors have been constructed since the 2003 review article. One biooxidation–heap process for pretreatment of sulfidic-refractory gold ores was also commercialized. A novel reductive approach to bioleaching nickel laterite minerals has been proposed.     

Biooxidation of Gold-, Silver,and Antimony-Bearing Highly Refractory Polymetallic Sulfide Concentrates,and its Comparison with Abiotic Pretreatment Techniques

Effectiveness of different pure and mixed cultures of three moderately thermophilic, extremely acidophilic bacterial strains (Acidimicrobium ferrooxidans ICP, Sulfobacillus sibiricus N1, Acidithiobacillus caldus KU) were investigated for biooxidation of highly refractory polymetallic gold ore concentrates. Despite of its complex mineralogy and the presence of a mixture of potentially inhibitory metals and metalloids, the concentrate was readily dissolved in defined mixed cultures including both iron and sulfur oxidizers, releasing as much as 80% of soluble Fe and 61% of soluble As. Factors to affect microbial mineral dissolution efficiencies (i.e. microbial As(III) oxidation ability, formation of secondary mineral precipitation (e.g. jarosite, elemental sulfur, scorodite, anglesite), and microbial population dynamics during biooxidation) were studied, based on which roles of individual microbes and their synergistic interactions during biooxidation were discussed. Applying the biooxidation pretreatment using the most efficient mixed cultures containing all three strains significantly improved the recovery of both Au (from 1.1% to 86%) and Ag (from 3.2% to 87%). Finally, this study provides one of the very few available comparisons of the effectiveness of different pretreatment techniques for refractory gold ore concentrates: Compared with other abiotic pretreatment approaches (roasting, pressure oxidation, and alkali dissolution), biooxidation was shown to be one of the most effective options in terms of the recovery of Au and Ag.     

Comparative Leaching of Chalcopyrite by Selected Acidophilic Bacteria and Archaea

A systematic study of the bioleaching of chalcopyrite (CuFeS 2 ) was conducted using axenic cultures of 11 species of acidophilic Bacteria and Archaea to obtain a direct comparison of the microbial chalcopyrite leaching capabilities of the different cultures and to determine the factors that affect Cu release. The characteristics of chalcopyrite leaching by the moderate thermophile Sulfobacillus thermosulfidooxidans , the mesophile Acidithiobacillus ferrooxidans , and the thermophile Acidianus brierleyi were used to elucidate the leaching process. Moderately thermophilic cultures of Sulfobacillus acidophilus, Acidimicrobium ferrooxidans , and Acidithiobacillus caldus were used to study the effects of different metabolic capabilities and relate those to leaching efficiency. The greatest rate of Cu solubilization from chalcopyrite was achieved at high temperatures (up to 70°C) at redox potentials below +550 mV (Ag/AgCl). The enhanced Cu solubilization observed at high temperatures resulted from accelerated chemical reaction rates, rather than from the rates at which individual acidophiles generated the mineral leaching reactants such as Fe 3+ .     

嗜酸硫氧化细菌消解元素硫的研究进展

浸矿酸性环境下,金属硫化矿在Fe3+作用下,经过硫代硫酸盐途径或多聚硫化氢途径而分解的过程中导致大量元素硫的累积,进而可能在金属硫化矿表面形成疏水元素硫层,阻碍金属离子的进一步浸出。酸性环境下,惰性元素硫的消解必须借助嗜酸硫氧化细菌来实现。该消解过程包括嗜酸硫氧化细菌对元素硫的吸附、转运以及氧化转化等过程。本文对近年来嗜酸硫氧化细菌消解元素硫过程的相关研究进行了全面评述,认为有关嗜酸硫氧化细菌消解元素硫的分子机制的清晰阐述还有待人们通过对消解过程的各个环节的分子机制进行大量研究来实现。     

Enumeration and Characterization of Acidophilic Microorganisms Isolated from a Pilot Plant Stirred-Tank Bioleaching Operation

Microorganisms were enumerated and isolated on selective solid media from a pilot-scale stirred-tank bioleaching operation in which a polymetallic sulfide concentrate was subjected to biologically accelerated oxidation at 45°C. Four distinct prokaryotes were isolated: three bacteria (an Acidithiobacillus caldus-like organism, a thermophilic Leptospirillum sp., and a Sulfobacillus sp.) and one archaeon (a Ferroplasma-like isolate). The relative numbers of these prokaryotes changed in the three reactors sampled, and the Ferroplasma isolate became increasingly dominant as mineral oxidation progressed, eventually accounting for >99% of plate isolates in the third of three in-line reactors. The identities of the isolates were confirmed by analyses of their 16S rRNA genes, and some key physiological traits (e.g., oxidation of iron and/or sulfur and autotrophy or heterotrophy) were examined. More detailed studies were carried out with the Leptospirillum and Ferroplasma isolates. The data presented here represent the first quantitative study of the microorganisms in a metal leaching situation and confirm that mixed cultures of iron- and sulfur-oxidizing prokaryotic acidophiles catalyze the accelerated dissolution of sulfidic minerals in industrial tank bioleaching operations. The results show that indigenous acidophilic microbial populations change as mineral dissolution becomes more extensive.     

Potential Role of Thiobacillus caldus in Arsenopyrite Bioleaching

We investigated the potential role of the three strains of Thiobacillus caldus (KU, BC13, and C-SH12) in arsenopyrite leaching in combination with a moderately thermophilic iron oxidizer, Sulfobacillus thermosulfidooxidans. Pure cultures of T. caldus and S. thermosulfidooxidans were used as well as defined mixed cultures. By measuring released iron, tetrathionate, and sulfur concentrations, we found that the presence of T. caldus KU and BC13 in the defined mixed culture lowered the concentration of sulfur, and levels of tetrathionate were comparable to or lower than those in the presence of S. thermosulfidooxidans. This suggests that T. caldus grows on the sulfur compounds that build up during leaching, increasing the arsenopyrite-leaching efficiency. This result was similar to leaching arsenopyrite with a pure culture of S. thermosulfidooxidans in the presence of yeast extract. Therefore, three possible roles of T. caldus in the leaching environment can be hypothesized: to remove the buildup of solid sulfur that can cause an inhibitory layer on the surface of the mineral, to aid heterotrophic and mixotrophic growth by the release of organic chemicals, and to solubilize solid sulfur by the production of surface-active agents. The results showed that T. caldus KU was the most efficient at leaching arsenopyrite under the conditions tested, followed by BC13, and finally C-SH12.     

Microbiological oxidation of synthetic chalcocite and covellite by Thiobacillus ferrooxidans.

The microbiological oxidation of synthetic chalcocite and covellite has been investigated using an adapted strain of Thiobacillus ferrooxidans. Biodegradation of chalcocite was found to be 90 to 100% and that of covellite 45 to 60%. Optimum conditions for the oxidation of chalcocite were: pH, 1.7 to 2.3; temperature, 35 C; and ferric iron concentration in the range of 0.004 to 0.01 M. For covellite, the optimum conditions were: pH 2.3; temperature, 35 C; and ferric iron concentration in the range of 0.004 to 0.02 M. The energies of activation were determined to be 16.3 kcal (ca. 6.8 X 10(4) J) per mol and 11.7 kcal (ca. 4.8 X 10(4) J) per mol for chalcocite and covellite, respectively.     

Microbiological Leaching of Metallic Sulfides

The percentage of chalcopyrite leached in percolators by Thiobacillus ferrooxidans was dependent on the surface area of the ore but not on the amount. Typical examples of ore leaching, which demonstrate the role of the bacteria, are presented. In stationary fermentations, changes in KH(2)PO(4) concentration above or below 0.1% decreased copper leaching as did reduction in the MgSO(4).7H(2)O and increase in the (NH(4))(2)SO(4) concentration. Bacterial leaching of chalcopyrite was more effective than nonbiological leaching with ferric sulfate; ferric sulfate appeared to retard biological leaching, but this effect was likely caused by formation of an insoluble copper-iron complex. Ferrous sulfate and sodium chloride singly accentuated both bacterial and nonbiological leaching of chalcocite but jointly depressed bacterial action. Sodium chloride appeared to block bacterial iron oxidation without interfering with sulfide oxidation. Bacterial leaching of millerite, bornite, and chalcocite was greatest at pH 2.5. The economics of leaching a number of British Columbia ore bodies was discussed.     

Intermediary sulfur compounds in pyrite oxidation: implications for bioleaching and biodepyritization of coal

Accumulation of elemental sulfur during pyrite oxidation lowers the efficiency of coal desulfurization and bioleaching. In the case of pyrite bioleaching by Leptospirillum ferrooxidans, an iron(II)-ion-oxidizing organism without sulfur-oxidizing capacity, from the pyritic sulfur moiety about 10% elemental sulfur, 2% pentathionate, and 1% tetrathionate accumulated by a recently described cyclic pyrite oxidation mechanism. In the case of pure cultures of Thiobacillus ferrooxidans and mixed cultures of L. ferrooxidans and T. thiooxidans, pyrite was nearly completely oxidized to sulfate because of the capacity of these cultures to oxidize both iron(II) ions and sulfur compounds. Pyrite oxidation in acidic solutions, mediated chemically by iron(III) ion, resulted in an accumulation of similar amounts of sulfur compounds as obtained with L. ferrooxidans. Changes of pH to values below 2 or in the iron ion concentration are not decisive for diverting the flux of sulfur compounds. The literature on pyrite bioleaching is in agreement with the findings indicating that the chemistry of direct and indirect pyrite leaching is identical. Received: 20 April 1998 / Received revision: 27 August 1998 / Accepted: 3 September 1998     

Influence of the surface speciation on biofilm attachment to chalcopyrite by Acidithiobacillus thiooxidans

Surfaces of massive chalcopyrite (CuFeS₂) electrodes were modified by applying variable oxidation potential pulses under growth media in order to induce the formation of different secondary phases (e.g., copper-rich polysulfides, S _n ^2?; elemental sulfur, S⁰; and covellite, CuS). The evolution of reactivity (oxidation capacity) of the resulting chalcopyrite surfaces considers a transition from passive or inactive (containing CuS and S _n ^2?) to active (containing increasing amounts of S⁰) phases. Modified surfaces were incubated with cells of sulfur-oxidizing bacteria (Acidithiobacillus thiooxidans) for 24 h in a specific culture medium (pH 2). Abiotic control experiments were also performed to compare chemical and biological oxidation. After incubation, the density of cells attached to chalcopyrite surfaces, the structure of the formed biofilm, and their exopolysaccharides and nucleic acids were analyzed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy coupled to dispersive X-ray analysis (SEM-EDS). Additionally, CuS and S _n ^2?/S⁰ speciation, as well as secondary phase evolution, was carried out on biooxidized and abiotic chalcopyrite surfaces using Raman spectroscopy and SEM-EDS. Our results indicate that oxidized chalcopyrite surfaces initially containing inactive S _n ^2? and S _n ^2?/CuS phases were less colonized by A. thiooxidans as compared with surfaces containing active phases (mainly S⁰). Furthermore, it was observed that cells were partially covered by CuS and S⁰ phases during biooxidation, especially at highly oxidized chalcopyrite surfaces, suggesting the innocuous effect of CuS phases during A. thiooxidans performance. These results may contribute to understanding the effect of the concomitant formation of refractory secondary phases (as CuS and inactive S _n ^2?) during the biooxidation of chalcopyrite by sulfur-oxidizing microorganisms in bioleaching systems.     

万座嗜酸两面菌硫代谢相关膜蛋白基因的筛选

活性巯基在浸矿微生物硫代谢的过程中起着重要的作用,半胱氨酸残基作为蛋白质中活性巯基的提供者,为筛选硫代谢相关蛋白质基因提供了依据。本研究以极端嗜酸热古菌万座嗜酸两面菌Acidianus manzaensis为研究对象,基于其全基因组注释信息,筛选出编码富半胱氨酸残基的潜在硫代谢相关膜蛋白基因,并通过RT-qPCR实验对筛选出来的基因进行表达水平验证,同时利用生物信息学方法对其进一步分析。研究表明,与在亚铁中生长的细胞相比,单质硫培养下的细菌中与能量代谢相关的β-葡糖苷酶,与电子传递相关的ATP合成酶、NADH-辅酶Q氧化还原酶基因均表达上调,说明硫代谢途径可能与能量代谢和电子传递有着重要的联系。此外,还有三个假定蛋白基因表达上调,这三个假定膜蛋白中,ARM75161.1、ARM75436.1中的半胱氨酸都位于保守区域,且均有一个半胱氨酸残基暴露于膜外,而ARM75580.1中的半光氨酸不位于保守区域。其中ARM75436.1具有CXXXC结构域,且该结构域中半胱氨酸残基处于同一个β-折叠中。这些假定蛋白可能参与A. manzaensis中硫代谢途径。     

Sequence-specific bacterial growth inhibition by peptide nucleic acid targeted to the mRNA binding site of 16S rRNA

To compare oxidative dissolution rates of chalcopyrite by different consortia of moderately thermophilic acidophiles, various defined mixed cultures of three bacteria Acidithiobacillus caldus s2, Leptospirillum ferriphilum YSK, and Sulfobacillus sp. LN and one archaeon Ferroplasma thermophilum L1 were studied in batch shake flask cultures incubated at 45 °C. Chalcopyrite dissolution was determined by measuring variations of soluble copper, ferric iron, and pH. Microbial population dynamics involved in bioleaching process were monitored using real-time quantitative polymerase chain reaction (PCR) technology. The complex consortia containing both chemoautotrophic (L. ferriphilum and At. caldus) and chemomixotrophic (Sulfobacillus LN and F. thermophilum) moderate thermophiles were found to be the most efficient in all of those tested. Mutualistic interactions between physiologically distinct moderately thermophilic acidophiles, involving transformations of iron and sulfur and transfer of organic compound, were considered to play a critical role in promoting chalcopyrite dissolution. The real-time PCR assay was reliable to analyze population dynamics of moderate thermophiles in bioleaching systems, and the analysis results were consistent with physiological characteristics of these strains.     

Corrosion and Electrochemical Oxidation of a Pyrite by Thiobacillus ferrooxidans

The oxidation of a pure pyrite by Thiobacillus ferrooxidans is not really a constant phenomenon; it must be considered to be more like a succession of different steps which need characterization. Electrochemical studies using a combination of a platinum electrode and a specific pyrite electrode (packed-ground-pyrite electrode) revealed four steps in the bioleaching process. Each step can be identified by the electrochemical behavior (redox potentials) of pyrite, which in turn can be related to chemical (leachate content), bacterial (growth), and physical (corrosion patterns) parameters of the leaching process. A comparison of the oxidation rates of iron and sulfur indicated the nonstoichiometric bacterial oxidation of a pure pyrite in which superficial phenomena, aqueous oxidation, and deep crystal dissolution are successively involved.