Bioleaching review part B:

This review describes the historical development and current state of metals leaching and sulfide mineral biooxidation by the minerals industries. During the past 20 years commercial processes employing microorganisms for mineral recovery have progressed from rather uncontrolled copper dump leaching to mineral oxidation and leaching in designed bioheaps for oxidation of refractory gold ores and for copper recovery. Also during this period of time, stirred tank bioleaching has been commercialized for cobalt recovery and for biooxidation of refractory gold ores. Chalcopyrite bioleaching in stirred tanks is on the verge of commercialization. Commercial applications of biohydrometallurgy have advanced due to favorable process economics and, in some cases, reduced environmental problems compared to conventional metal recovery processes such as smelting. Process development has included recognition of the importance of aeration of bioheaps, and improvements in stirred tank reactor design and operation. Concurrently, knowledge of the key microorganisms involved in these processes has advanced, aided by advances in molecular biology to characterize microbial populations.     

Two-stage process of bacterial-chemical oxidation of refractory pyrite-arsenopyrite gold-bearing concentrate

A technology for tank biooxidation of refractory gold-bearing concentrate under variable temperature conditions has been improved: the temperature of the first of two stages was changed from 30°C to 34–36°C. Gold in this concentrate is mainly associated with sulfide minerals: arsenopyrite and pyrite, which underlies a low gold recovery (16.68%) as a result of cyanidation. To resolve the problem, an association of mesophilic acidophilic chemolithotrophic microorganisms and moderately thermophilic bacteria of the Sulfobacillus genus were used for the concentrate oxidation. The composition of the used microbial association was studied; it was shown that it depends upon temperature: at 42°C, the population of the mesophilic thiobacteria decreased, whereas that of thermophilic sulfobacilli enhanced as compared to 36°C. The accepted scheme of the process ensures a high extent of gold recovery (94.6%) within a short space of time for biooxidation (96 h).     

Molecular genetics of Thiobacillus ferrooxidans.

Thiobacillus ferrooxidans is a gram-negative, highly acidophilic (pH 1.5 to 2.0), autotrophic bacterium that obtains its energy through the oxidation of ferrous iron or reduced inorganic sulfur compounds. It is usually dominant in the mixed bacterial populations that are used industrially for the extraction of metals such as copper and uranium from their ores. More recently, these bacterial consortia have been used for the biooxidation of refractory gold-bearing arsenopyrite ores prior to the recovery of gold by cyanidation. The commercial use of T. ferrooxidans has led to an increasing interest in the genetics and molecular biology of the bacterium. Initial investigations were aimed at determining whether the unique physiology and specialized habitat of T. ferrooxidans had been accompanied by a high degree of genetic drift from other gram-negative bacteria. Early genetic studies were comparative in nature and concerned the isolation of genes such as nifHDK, glnA, and recA, which are widespread among bacteria. From a molecular biology viewpoint, T. ferrooxidans appears to be a typical member of the proteobacteria. In most instances, cloned gene promoters and protein products have been functional in Escherichia coli. Although T. ferrooxidans has proved difficult to transform with DNA, research on indigenous plasmids and the isolation of the T. ferrooxidans merA gene have resulted in the development of a low-efficiency electroporation system for one strain of T. ferrooxidans. The most recent studies have focused on the molecular genetics of the pathways associated with nitrogen metabolism, carbon dioxide fixation, and components of the energy-producing mechanisms.     

A genomic island provides <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis> ATCC 53993 additional copper resistance: a possible competitive advantage

There is great interest in understanding how extremophilic biomining bacteria adapt to exceptionally high copper concentrations in their environment. Acidithiobacillus ferrooxidans ATCC 53993 genome possesses the same copper resistance determinants as strain ATCC 23270. However, the former strain contains in its genome a 160-kb genomic island (GI), which is absent in ATCC 23270. This GI contains, amongst other genes, several genes coding for an additional putative copper ATPase and a Cus system. A. ferrooxidans ATCC 53993 showed a much higher resistance to CuSO₄ (>100 mM) than that of strain ATCC 23270 (<25 mM). When a similar number of bacteria from each strain were mixed and allowed to grow in the absence of copper, their respective final numbers remained approximately equal. However, in the presence of copper, there was a clear overgrowth of strain ATCC 53993 compared to ATCC 23270. This behavior is most likely explained by the presence of the additional copper-resistance genes in the GI of strain ATCC 53993. As determined by qRT-PCR, it was demonstrated that these genes are upregulated when A. ferrooxidans ATCC 53993 is grown in the presence of copper and were shown to be functional when expressed in copper-sensitive Escherichia coli mutants. Thus, the reason for resistance to copper of two strains of the same acidophilic microorganism could be determined by slight differences in their genomes, which may not only lead to changes in their capacities to adapt to their environment, but may also help to select the more fit microorganisms for industrial biomining operations.     

Bacterial Oxidation of Refractory Sulfide Ores for Gold Recovery

Abstract

The microbiological leaching of refractory sulfide ores (pyrite, arsenopyrite) for recovery of gold is reviewed in this article. The underlying physiological, biochemical, and genetic fundamentals of the bacteria involved (Thiobacillus and Sulfolobus spp.) are complex and have yet to be elucidated in depth. The chemistry of acid and biological leaching of pyrite and arsenopyrite minerals is also complex, and many of the individual reactions are not known in detail. Bacterial leaching is discussed in relation to chemical speciation at acid pH values. Attempts to develop models for a better understanding of bioleaching processes are summarized. The importance of pH, redox potential, temperature, sulfur balance, and toxic metals is evaluated for optimizing conditions for bacterial activity. Gold is finely disseminated in refractory sulfide ores, thereby decreasing Au recoveries upon conventional cyanidation for gold dissolution. In the bioleaching process, bacteria remove the sulfide minerals by oxidative dissolution and thus expose Au to extraction with cyanide solution. Stirred tank reactors appear most suited for this biological leaching process. The overall oxidation of the sulfides is an important variable for gold recovery. Pilot- and commercial-scale bioleaching processes for gold-containing pyrite and arsenopyrite ores are reviewed. This application of mineral biotechnology competes favorably with pressure leaching and roasting processes, both of which are problematic and energy-intensive alternatives for pretreatment of auriferous pyrite/arsenopyrite ores.     

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.     

Isolation of Arsenic Resistant and Arsenopyrite Oxidizing Acidithiobacillus Species from pH Neutral Colombian Mine Effluents

Abstract

Inactive mines provide a great source of bacterial diversity for studying acidophilic communities and their biotechnological applications, but prospecting of these anthropogenic environments in Colombia has been limited. Conventional microbiological methods were used to isolate acidophilic bacterial strains from effluents emanating from the Colombian gold mine ‘El Zancudo’ (Titiribí, Antioquia). Despite the drainage waters having circumneutral pH, all of the isolated strains were phylogenetically related to the extreme acidophile Acidithiobacillus genus. However, based upon 16S rRNA gene sequences the mesophilic sulfur-oxidizing indigenous strains could not be assigned to a species. Pure cultures were selected by screening in medium with soluble inorganic arsenic (III) and their mineral-oxidative activity was evaluated at 30?°C in Erlenmeyer flasks with arsenopyrite ore under rotary shaking conditions. The indigenous strains were able to catalyze arsenopyrite oxidation in a mixed culture with a pulp density of 10%, maintaining their growth in the presence of >80?mM leached arsenic. This research provides information regarding the isolation of arsenic resistant bacterial communities from neutral effluents from El Zancudo mine and the possibility of the isolated strains to be useful in the biooxidation pretreatment of refractory gold-bearing arsenopyrite ores and concentrates.     

Thermophilic microorganisms in biomining

Biomining is an applied biotechnology for mineral processing and metal extraction from ores and concentrates. This alternative technology for recovering metals involves the hydrometallurgical processes known as bioleaching and biooxidation where the metal is directly solubilized or released from the matrix for further solubilization, respectively. Several commercial applications of biomining can be found around the world to recover mainly copper and gold but also other metals; most of them are operating at temperatures below 40–50 °C using mesophilic and moderate thermophilic microorganisms. Although biomining offers an economically viable and cleaner option, its share of the world´s production of metals has not grown as much as it was expected, mainly considering that due to environmental restrictions in many countries smelting and roasting technologies are being eliminated. The slow rate of biomining processes is for sure the main reason of their poor implementation. In this scenario the use of thermophiles could be advantageous because higher operational temperature would increase the rate of the process and in addition it would eliminate the energy input for cooling the system (bioleaching reactions are exothermic causing a serious temperature increase in bioreactors and inside heaps that adversely affects most of the mesophilic microorganisms) and it would decrease the passivation of mineral surfaces. In the last few years many thermophilic bacteria and archaea have been isolated, characterized, and even used for extracting metals. This paper reviews the current status of biomining using thermophiles, describes the main characteristics of thermophilic biominers and discusses the future for this biotechnology.     

Metabolic reconstruction of sulfur assimilation in the extremophile <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis> based on genome analysis

Background  

Acidithiobacillus ferrooxidansis a gamma-proteobacterium that lives at pH2 and obtains energy by the oxidation of sulfur and iron. It is used in the biomining industry for the recovery of metals and is one of the causative agents of acid mine drainage. Effective tools for the study of its genetics and physiology are not in widespread use and, despite considerable effort, an understanding of its unusual physiology remains at a rudimentary level. Nearly complete genome sequences ofA. ferrooxidansare available from two public sources and we have exploited this information to reconstruct aspects of its sulfur metabolism.     

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.     

Characteristics and adaptability of iron- and sulfur-oxidizing microorganisms used for the recovery of metals from minerals and their concentrates

Microorganisms are used in large-scale heap or tank aeration processes for the commercial extraction of a variety of metals from their ores or concentrates. These include copper, cobalt, gold and, in the past, uranium. The metal solubilization processes are considered to be largely chemical with the microorganisms providing the chemicals and the space (exopolysaccharide layer) where the mineral dissolution reactions occur. Temperatures at which these processes are carried out can vary from ambient to 80°C and the types of organisms present depends to a large extent on the process temperature used. Irrespective of the operation temperature, biomining microbes have several characteristics in common. One shared characteristic is their ability to produce the ferric iron and sulfuric acid required to degrade the mineral and facilitate metal recovery. Other characteristics are their ability to grow autotrophically, their acid-tolerance and their inherent metal resistance or ability to acquire metal resistance. Although the microorganisms that drive the process have the above properties in common, biomining microbes usually occur in consortia in which cross-feeding may occur such that a combination of microbes including some with heterotrophic tendencies may contribute to the efficiency of the process. The remarkable adaptability of these organisms is assisted by several of the processes being continuous-flow systems that enable the continual selection of microorganisms that are more efficient at mineral degradation. Adaptability is also assisted by the processes being open and non-sterile thereby permitting new organisms to enter. This openness allows for the possibility of new genes that improve cell fitness to be selected from the horizontal gene pool. Characteristics that biomining microorganisms have in common and examples of their remarkable adaptability are described.     

The extremophile Acidithiobacillus ferrooxidans possesses a c-di-GMP signalling pathway that could play a significant role during bioleaching of minerals

Aims: The primary goal of this study was to characterize the existence of a functional c‐di‐GMP pathway in the bioleaching bacterium Acidithiobacillus ferrooxidans. Methods and Results: A bioinformatic search revealed that the genome sequence of At. ferrooxidans ATCC 23270 codes for several proteins involved in the c‐di‐GMP pathway, including diguanylate cyclases (DGC), phosphodiesterases and PilZ effector proteins. Overexpression in Escherichia coli demonstrated that four At. ferrooxidans genes code for proteins containing GGDEF/EAL domains with functional DGC activity. MS/MS analysis allowed the identification of c‐di‐GMP in nucleotide preparations obtained from At. ferrooxidans cells. In addition, c‐di‐GMP levels in cells grown on the surface of solid energetic substrates such as sulfur prills or pyrite were higher than those measured in ferrous iron planktonic cells. Conclusions: At. ferrooxidans possesses a functional c‐di‐GMP pathway that could play a key role in At. ferrooxidans biofilm formation during bioleaching processes. Significance and Impact of the Study: This is the first global study about the c‐di‐GMP pathway in an acidophilic bacterium of great interest for the biomining industry. It opens a new way to explore the regulation of biofilm formation by biomining micro‐organisms during the bioleaching process.     

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.     

Occurrences at mineral-bacteria interface during oxidation of arsenopyrite by Thiobacillus ferrooxidans

The combination of an improved bacterial desorption method, scanning electron microscopy (SEM), diffuse reflectance and transmission infrared Fourier transform spectroscopy, and a desorption-leaching device like high-pressure liquid chromatography (HPLC) was used to analyze bacterial populations (adhering and free bacteria) and surface-oxidized phases (ferric arsenates and elemental sulfur) during the arsenopyrite biooxidation by Thiobacillus ferrooxidans. The bacterial distribution, the physicochemical composition of the leachate, the evolution of corrosion patterns, and the nature and amount of the surface-oxidized chemical species characterized different behavior for each step of arsenopyrite bioleaching. The first step is characterized by a slow but strong adhesion of bacteria to mineral surfaces, the appearance of a surface phase of elemental sulfur, the weak solubilization of Fe(II), As(III), and As(V), and the presence of the first corrosion patterns, which follow the fragility zones and the crystallographic orientation of mineral grains. After this short step, growth of the unattached bacteria begins, while ferrous ions in solution are oxidized by them. Ferric ions produced by the bacteria can oxidize the sulfide directly and are regenerated by Fe(II) bacterial oxidation. At this time, a bioleaching cycle takes place and a coarse surface phase of ferric arsenate (FeAsO(4) . xH(2)O where x approximately 2) and deep ovoid pores appear. At the end of the bioleaching cycle, the high concentration of Fe(III) and As(V) in solution promotes the precipitation of a second phase of amorphous ferric arsenate (FeAsO(4) . xH(2)O where x approximately 4) in the leachate. Then the biooxidation process ceases: The bacteria adhering to the mineral sufaces are coated by the ferric arsenates and the concentration of Fe(III) on the leachate is found to have decreased greatly. Both oxidation mechanisms (direct and indirect oxidation) have been stopped. (c) 1995 John Wiley & Sons, Inc.     

An Immunological Strategy To Monitor In Situ the Phosphate Starvation State in Thiobacillus ferrooxidans

Thiobacillus ferrooxidans is one of the chemolithoautotrophic bacteria important in industrial biomining operations. During the process of ore bioleaching, the microorganisms are subjected to several stressing conditions, including the lack of some essential nutrients, which can affect the rates and yields of bioleaching. When T. ferrooxidans is starved for phosphate, the cells respond by inducing the synthesis of several proteins, some of which are outer membrane proteins of high molecular weight (70,000 to 80,000). These proteins were considered to be potential markers of the phosphate starvation state of these microorganisms. We developed a single-cell immunofluorescence assay that allowed monitoring of the phosphate starvation condition of this biomining microorganism by measuring the increased expression of the surface proteins. In the presence of low levels of arsenate (2 mM), the growth of phosphate-starved T. ferrooxidans cells was greatly inhibited compared to that of control nonstarved cells. Therefore, the determination of the phosphorus nutritional state is particularly relevant when arsenic compounds are solubilized during the bioleaching of different ores.     

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.     

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.

     

Resistance determinants of a highly arsenic-resistant strain of Leptospirillum ferriphilum isolated from a commercial biooxidation tank

Two sets of arsenic resistance genes were isolated from the highly arsenic-resistant Leptospirillum ferriphilum Fairview strain. One set is located on a transposon, TnLfArs, and is related to the previously identified TnAtcArs from Acidithiobacillus caldus isolated from the same arsenopyrite biooxidation tank as L. ferriphilum. TnLfArs conferred resistance to arsenite and arsenate and was transpositionally active in Escherichia coli. TnLfArs and TnAtcArs were sufficiently different for them not to have been transferred from one type of bacterium to the other in the biooxidation tank. The second set of arsenic resistance genes conferred very low levels of resistance in E. coli and appeared to be poorly expressed in both L. ferriphilum and E. coli.