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 degrees 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.     

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。     

Microbial communities in a porphyry copper tailings impoundment and their impact on the geochemical dynamics of the mine waste

The distribution and diversity of acidophilic bacteria of a tailings impoundment at the La Andina copper mine, Chile, was examined. The tailings have low sulfide (1.7% pyrite equivalent) and carbonate (1.4% calcite equivalent) contents and are stratified into three distinct zones: a surface (0-70-80 cm) 'oxidation zone' characterized by low-pH (2.5-4), a 'neutralization zone' (70-80 to 300-400 cm) and an unaltered 'primary zone' below 400 cm. A combined cultivation-dependent and biomolecular approach (terminal restriction enzyme fragment length polymorphism and 16S rRNA clone library analysis) was used to characterize the indigenous prokaryotic communities in the mine tailings. Total cell counts showed that the microbial biomass was greatest in the top 125 cm of the tailings. The largest numbers of bacteria (10(9) g(-1) dry weight of tailings) were found at the oxidation front (the junction between the oxidation and neutralization zones), where sulfide minerals and oxygen were both present. The dominant iron-/sulfur-oxidizing bacteria identified at the oxidation front included bacteria of the genus Leptospirillum (detected by molecular methods), and Gram-positive iron-oxidizing acidophiles related to Sulfobacillus (identified both by molecular and cultivation methods). Acidithiobacillus ferrooxidans was also detected, albeit in relatively small numbers. Heterotrophic acidophiles related to Acidobacterium capsulatum were found by molecular methods, while another Acidobacterium-like bacterium and an Acidiphilium sp. were isolated from oxidation zone samples. A conceptual model was developed, based on microbiological and geochemical data derived from the tailings, to account for the biogeochemical evolution of the Piuquenes tailings impoundment.     

Leptospirillum-like bacteria and their role in pyrite oxidation

Two strains of Leptospirillum-like bacteria isolated from dumps of Alaverdi and Akhtala sulfide ore deposits in Armenia were studied. The optimum and maximum temperatures for the growth of both strains were 37 and 40 degrees C, respectively. The pH optimum was 2.0-2.3. Bacterial growth and ferrous iron oxidation were inhibited by yeast extract. The pyrite-leaching activity of the Leptospirillum-like bacteria under mesophilic conditions was close to that of Acidithiobacillus ferroxidans and exceeded by 2.0-2.7 times the activity of these moderately thermophilic bacteria at 37 degrees C. The leaching of pyrite by Leptospirillum-like bacteria increased in the presence of sulfur-oxidizing bacteria, particularly, in their association with a thermotolerant sulfur-oxidizing bacterium.     

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.     

Leptospirillum-Like Bacteria and Evaluation of Their Role in Pyrite Oxidation

Two strains of Leptospirillum-like bacteria isolated from dumps of Alaverdi and Akhtala sulfide ore deposits in Armenia were studied. The optimum and maximum temperatures for the growth of both strains were 37 and 40°C, respectively. The pH optimum was 2.0–2.3. Bacterial growth and ferrous iron oxidation were inhibited by yeast extract. The pyrite-leaching activity of the Leptospirillum-like bacteria under mesophilic conditions was close to that of Acidithiobacillus ferrooxidans and exceeded by 2.0–2.7 times the activity of these moderately thermophilic bacteria at 37°C. The leaching of pyrite by Leptospirillum-like bacteria increased in the presence of sulfur-oxidizing bacteria, particularly, in their association with a thermotolerant sulfur-oxidizing bacterium.     

Leaching of Pyrite by Acidophilic Heterotrophic Iron-Oxidizing Bacteria in Pure and Mixed Cultures

Seven strains of heterotrophic iron-oxidizing acidophilic bacteria were examined to determine their abilities to promote oxidative dissolution of pyrite (FeS₂) when they were grown in pure cultures and in mixed cultures with sulfur-oxidizing Thiobacillus spp. Only one of the isolates (strain T-24) oxidized pyrite when it was grown in pyrite-basal salts medium. However, when pyrite-containing cultures were supplemented with 0.02% (wt/vol) yeast extract, most of the isolates oxidized pyrite, and one (strain T-24) promoted rates of mineral dissolution similar to the rates observed with the iron-oxidizing autotroph Thiobacillus ferrooxidans. Pyrite oxidation by another isolate (strain T-21) occurred in cultures containing between 0.005 and 0.05% (wt/vol) yeast extract but was completely inhibited in cultures containing 0.5% yeast extract. Ferrous iron was also needed for mineral dissolution by the iron-oxidizing heterotrophs, indicating that these organisms oxidize pyrite via the “indirect” mechanism. Mixed cultures of three isolates (strains T-21, T-23, and T-24) and the sulfur-oxidizing autotroph Thiobacillus thiooxidans promoted pyrite dissolution; since neither strains T-21 and T-23 nor T. thiooxidans could oxidize this mineral in yeast extract-free media, this was a novel example of bacterial synergism. Mixed cultures of strains T-21 and T-23 and the sulfur-oxidizing mixotroph Thiobacillus acidophilus also oxidized pyrite but to a lesser extent than did mixed cultures containing T. thiooxidans. Pyrite leaching by strain T-23 grown in an organic compound-rich medium and incubated either shaken or unshaken was also assessed. The potential environmental significance of iron-oxidizing heterotrophs in accelerating pyrite oxidation is discussed.     

Significance of microbial communities and interactions in safeguarding reactive mine tailings by ecological engineering

Pyritic mine tailings (mineral waste generated by metal mining) pose significant risk to the environment as point sources of acidic, metal-rich effluents (acid mine drainage [AMD]). While the accelerated oxidative dissolution of pyrite and other sulfide minerals in tailings by acidophilic chemolithotrophic prokaryotes has been widely reported, other acidophiles (heterotrophic bacteria that catalyze the dissimilatory reduction of iron and sulfur) can reverse the reactions involved in AMD genesis, and these have been implicated in the "natural attenuation" of mine waters. We have investigated whether by manipulating microbial communities in tailings (inoculating with iron- and sulfur-reducing acidophilic bacteria and phototrophic acidophilic microalgae) it is possible to mitigate the impact of the acid-generating and metal-mobilizing chemolithotrophic prokaryotes that are indigenous to tailing deposits. Sixty tailings mesocosms were set up, using five different microbial inoculation variants, and analyzed at regular intervals for changes in physicochemical and microbiological parameters for up to 1 year. Differences between treatment protocols were most apparent between tailings that had been inoculated with acidophilic algae in addition to aerobic and anaerobic heterotrophic bacteria and those that had been inoculated with only pyrite-oxidizing chemolithotrophs; these differences included higher pH values, lower redox potentials, and smaller concentrations of soluble copper and zinc. The results suggest that empirical ecological engineering of tailing lagoons to promote the growth and activities of iron- and sulfate-reducing bacteria could minimize their risk of AMD production and that the heterotrophic populations could be sustained by facilitating the growth of microalgae to provide continuous inputs of organic carbon.     

Prevention of formation of acid drainage from high-sulfur coal refuse by inhibition of iron- and sulfur-oxidizing microorganisms. II. Inhibition in "run of mine" refuse under simulated field conditions

The combination of sodium lauryl sulfate and benzoic acid effectively inhibits iron- and sulfur-oxidizing bacteria in coal refuse and prevents the conversion of iron pyrite to sulfate, ferric iron, and sulfuric acid, thereby significantly reducing the formation of acidic drainage from coal refuse. The inhibitors were effective in a concentration of 1.1 mg/kg refuse, and data indicate that the SLS was in excess of the concentration required. The treatment was compatible with the use of lime for neutralization of acid present prior to inhibition of its formation.     

Diversity and functional analysis of bacterial communities associated with natural hydrocarbon seeps in acidic soils at Rainbow Springs, Yellowstone National Park

In this paper we describe the bacterial communities associated with natural hydrocarbon seeps in nonthermal soils at Rainbow Springs, Yellowstone National Park. Soil chemical analysis revealed high sulfate concentrations and low pH values (pH 2.8 to 3.8), which are characteristic of acid-sulfate geothermal activity. The hydrocarbon composition of the seep soils consisted almost entirely of saturated, acyclic alkanes (e.g., n-alkanes with chain lengths of C15 to C30, as well as branched alkanes, predominately pristane and phytane). Bacterial populations present in the seep soils were phylogenetically characterized by 16S rRNA gene clone library analysis. The majority of the sequences recovered (>75%) were related to sequences of heterotrophic acidophilic bacteria, including Acidisphaera spp. and Acidiphilium spp. of the alpha-Proteobacteria. Clones related to the iron- and sulfur-oxidizing chemolithotroph Acidithiobacillus spp. were also recovered from one of the seep soils. Hydrocarbon-amended soil-sand mixtures were established to examine [14C]hexadecane mineralization and corresponding changes in the bacterial populations using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. Approximately 50% of the [14C]hexadecane added was recovered as 14CO2 during an 80-day incubation, and this was accompanied by detection of heterotrophic acidophile-related sequences as dominant DGGE bands. An alkane-degrading isolate was cultivated, whose 16S rRNA gene sequence was identical to the sequence of a dominant DGGE band in the soil-sand mixture, as well as the clone sequence recovered most frequently from the original soil. This and the presence of an alkB gene homolog in this isolate confirmed the alkane degradation capability of one population indigenous to acidic hydrocarbon seep soils.     

三株氧化硫硫杆菌的分离与鉴定

从煤堆废水中分离得到3株嗜温嗜酸硫氧化细菌.这3株菌株为革兰氏阴性、菌体大小0.4～0.7 μm×1～2 μm、短杆状运动细菌,其最适生长温度为 30 ℃和最适生长pH 2.0～2.5.它们能够利用元素硫,硫代硫酸钠和连四硫酸钾为能源进行自养生长,不能利用有机物质以及硫酸亚铁、黄铁矿和黄铜矿等无机物质作为能源生长.细菌的形态、生理生化特性研究以及基于16S rRNA序列同源性构建的系统发育树结果表明,这3株细菌初步鉴定为氧化硫硫杆菌.氧化硫硫杆菌能够通过产酸有效促进黄铜矿的浸出速率和浸出率.     

Biodiversity and geochemistry of an extremely acidic, low-temperature subterranean environment sustained by chemolithotrophy

The geochemical dynamics and composition of microbial communities within a low-temperature (≈ 8.5°C), long-abandoned (> 90 years) underground pyrite mine (Cae Coch, located in north Wales) were investigated. Surface water percolating through fractures in the residual pyrite ore body that forms the roof of the mine becomes extremely acidic and iron-enriched due to microbially accelerated oxidative dissolution of the sulfide mineral. Water droplets on the mine roof were found to host a very limited diversity of exclusively autotrophic microorganisms, dominated by the recently described psychrotolerant iron/sulfur-oxidizing acidophile Acidithiobacillus ferrivorans, and smaller numbers of iron-oxidizing Leptospirillum ferrooxidans. In contrast, flowing water within the mine chamber was colonized with vast macroscopic microbial growths, in the form of acid streamers and microbial stalactites, where the dominant microorganisms were Betaproteobacteria (autotrophic iron oxidizers such as 'Ferrovum myxofaciens' and a bacterium related to Gallionella ferruginea). An isolated pool within the mine showed some similarity (although greater biodiversity) to the roof droplets, and was the only site where archaea were relatively abundant. Bacteria not previously associated with extremely acidic, metal-rich environments (a Sphingomonas sp. and Ralstonia pickettii) were found within the abandoned mine. Data supported the hypothesis that the Cae Coch ecosystem is underpinned by acidophilic, mostly autotrophic, bacteria that use ferrous iron present in the pyrite ore body as their source of energy, with a limited role for sulfur-based autotrophy. Results of this study highlight the importance of novel bacterial species (At. ferrivorans and acidophilic iron-oxidizing Betaproteobacteria) in mediating mineral oxidation and redox transformations of iron in acidic, low-temperature environments.     

Biogeochemical phenomena induced by bacteria within sulfidic mine tailings

Summary Mill tailings resulting from mining and metallurgical processes are usually disposed of into open-air impoundments, where they become subjected to chemical or microbial leaching. At the surface of the tailings, where oxic conditions prevail, acidophilic bacteria, such as thiobacilli, can oxidize sulfidic minerals (e.g. pyrite and pyrrhotite) and generate acidic metal-rich leachates as by-products of their metabolism. This, combined with chemical oxidation, leads to acid mine drainage (AMD). Biomineralization, whereby a proportion of the metal leachate is precipitated, can also occur in the oxidized tailings, often as a result of a close metal-bacteria interaction. Iron-rich precipitates are usually found on bacterial cell walls, and are thought to serve as nucleation sites for further mineralization within the tailings impoundments. As depth increases in mine tailings, oxygen depletion and the presence of water-saturated pores usually lead to anoxic conditions. Under such redox and chemical conditions, populations of sulfate-reducing bacteria (SRBs) can colonize the tailings. As a result of their metabolic activity, sulfate is reduced to hydrogen sulfide, which in turn can react with dissolved metals to form metal sulfide precipitates. Microbial sulfate reduction also generates alkalinity, although chemical dissolution of carbonate and oxide minerals probably also play an important role in the generation of alkaline conditions in mine tailings.     

Microbial leaching of metals from sulfide minerals

Microorganisms are important in metal recovery from ores, particularly sulfide ores. Copper, zinc, gold, etc. can be recovered from sulfide ores by microbial leaching. Mineral solubilization is achieved both by 'direct (contact) leaching' by bacteria and by 'indirect leaching' by ferric iron (Fe(3+)) that is regenerated from ferrous iron (Fe(2+)) by bacterial oxidation. Thiobacillus ferrooxidans is the most studied organism in microbial leaching, but other iron- or sulfide/sulfur-oxidizing bacteria as well as archaea are potential microbial agents for metal leaching at high temperature or low pH environment. Oxidation of iron or sulfur can be selectively controlled leading to solubilization of desired metals leaving undesired metals (e.g., Fe) behind. Microbial contribution is obvious even in electrochemistry of galvanic interactions between minerals.     

Quantitative Microbial Community Analysis of Three Different Sulfidic Mine Tailing Dumps Generating Acid Mine Drainage

The microbial communities of three different sulfidic and acidic mine waste tailing dumps located in Botswana, Germany, and Sweden were quantitatively analyzed using quantitative real-time PCR (Q-PCR), fluorescence in situ hybridization (FISH), catalyzed reporter deposition-FISH (CARD-FISH), Sybr green II direct counting, and the most probable number (MPN) cultivation technique. Depth profiles of cell numbers showed that the compositions of the microbial communities are greatly different at the three sites and also strongly varied between zones of oxidized and unoxidized tailings. Maximum cell numbers of up to 10⁹ cells g⁻¹ dry weight were determined in the pyrite or pyrrhotite oxidation zones, whereas cell numbers in unoxidized tailings were significantly lower. Bacteria dominated over Archaea and Eukarya at all tailing sites. The acidophilic Fe(II)- and/or sulfur-oxidizing Acidithiobacillus spp. dominated over the acidophilic Fe(II)-oxidizing Leptospirillum spp. among the Bacteria at two sites. The two genera were equally abundant at the third site. The acidophilic Fe(II)- and sulfur-oxidizing Sulfobacillus spp. were generally less abundant. The acidophilic Fe(III)-reducing Acidiphilium spp. could be found at only one site. The neutrophilic Fe(III)-reducing Geobacteraceae as well as the dsrA gene of sulfate reducers were quantifiable at all three sites. FISH analysis provided reliable data only for tailing zones with high microbial activity, whereas CARD-FISH, Q-PCR, Sybr green II staining, and MPN were suitable methods for a quantitative microbial community analysis of tailings in general.     

Bacteria and Acidic Drainage from Coal Refuse: Inhibition by Sodium Lauryl Sulfate and Sodium Benzoate

The application of an aqueous solution of sodium lauryl sulfate and sodium benzoate to the surface of high-sulfur coal refuse resulted in the inhibition of iron-and sulfur-oxidizing chemoautotrophic bacteria and in the decrease of acidic drainage from the refuse, suggesting that acid drainage can be abated in the field by inhibiting iron- and sulfur-oxidizing bacteria.     

Influence of coal source and treatment upon indigenous microbial communities

Summary The leaching of six Eastern coals was investigated using experimental coal columns subjected to simulated leaching events. Measurements of CO₂ assimilation and specific enrichment cultures indicated that the microbial communities of all leachates were dominated by iron- and sulfur-oxidizing chemoautotrophic bacteria. Comparison of CO₂ assimilation rates in leachates and core samples of leached coal indicated that most chemoautotrophs remained within coal columns during leaching. Mean numbers of chemoautotrophic bacteria in leachate samples were correlated with concentrations of dissolved iron and sulfate. Leachates from unwashed, run-of-mine coals contained more chemoautotrophs and more iron and sulfate than did leachates from washed, final product coals. After several leachings, the ratio of sulfur oxidizers to iron oxidizers tended to increase. These data suggest that the chemoautotrophic community of final product coals may be pyritelimited. Aerobic heterotrophs constituted a minor component of the microbial community in leachates from the six coals and their abundance and metabolic activity were apparently not influenced by the beneficiation history of the coal. Changes in rates of acetate metabolism may have been related to microbial succession within the heterotrophic community of coal columns. In all leachates, rates of tritiated methylthymidine assimilation were correlated with rates of acetate incorporation but not with CO₂ assimilation, even though autotrophs dominated the microflora. Thus, thymidine assimilation rates appear to reflect activities or growth of mainly heterotrophic microorganisms in leachate.     

嗜酸藻类研究进展

酸性矿山废水(acid mine drainage,AMD)是硫化矿物暴露于地表,与水、大气及微生物相互作用发生氧化性溶解而形成的,其pH通常在3.5以下,并含有高浓度的金属离子,危害十分严重。因为嗜酸原核生物可通过氧化硫化矿物获得能量,从而加速酸性矿山废水的形成过程,所以一直以来都是嗜酸微生物研究的焦点。但事实上,真核生物在酸性生态系统中往往发挥着更重要的作用。尤其是光合藻类,它们在很多酸性环境都是主要的初级生产者,对酸性矿山废水的演化和修复起着关键的推动作用。本文从嗜酸藻类的常见种类和生态分布、适应极端环境的生理机制以及它们在污染治理和工业生产中的应用3个方面,综述了该领域近30年来的研究进展,并以此为基础提出了将来需要着力加强的5个研究方向。     

Iron and carbon metabolism by a mineral-oxidizing Alicyclobacillus-like bacterium

A novel iron-oxidizing, moderately thermophilic, acidophilic bacterium (strain “GSM”) was isolated from mineral spoil taken from a gold mine in Montana. Biomolecular analysis showed that it was most closely related to Alicyclobacillus tolerans, although the two bacteria differed in some key respects, including the absence (in strain GSM) of ϖ-alicyclic fatty acids and in their chromosomal base compositions. Isolate GSM was able to grow in oxygen-free media using ferric iron as terminal electron acceptor confirming that it was a facultative anaerobe, a trait not previously described in Alicyclobacillus spp.. The acidophile used both organic and inorganic sources of energy and carbon, although growth and iron oxidation by isolate GSM was uncoupled in media that contained both fructose and ferrous iron. Fructose utilization suppressed iron oxidation, and oxidation of ferrous iron occurred only when fructose was depleted. In contrast, fructose catabolism was suppressed when bacteria were harvested while actively oxidizing iron, suggesting that both ferrous iron- and fructose-oxidation are inducible in this acidophile. Isolate GSM accelerated the oxidative dissolution of pyrite in liquid media either free of, or amended with, organic carbon, although redox potentials were significantly different in these media. The potential of this isolate for commercial mineral processing is discussed.