Bioleaching Of Metal Sulfides Research Articles

Insights into the effect of ethyl xanthate on the adhesion and biofilm formation by Acidianus manzaensis on chalcopyrite

Adhesion and biofilm formation play important roles in the bio-dissolution of metal sulfides, which can initiate dissolution and enhance bioleaching. Ethyl xanthate, a commonly used flotation reagent, remained in flotation concentrates of metal sulfides and may have an impact on the bioleaching of metal sulfides. Therefore, it is important to analyze the effect of ethyl xanthate on microbial initial adhesion and biofilm formation. The results showed that the adhesion percentage of Acidianus manzaensis on chalcopyrite decreased with increasing ethyl xanthate concentration after adsorption equilibrium, due to the toxicity of ethyl xanthate to A. manzaensis. However, a low concentration (0.01 mg/L) of ethyl xanthate had little effect on the growth of A. manzaensis. The hydrophobicity of chalcopyrite increased after the addition of ethyl xanthate, leading to an increase in total interaction energy and adhesion force. The presence of ethyl xanthate inhibited the early growth of biofilm and delayed the formation of microcolonies on chalcopyrite. While biofilm coverage was larger with the addition of ethyl xanthate than that of the control when biofilm was mature. Furthermore, in the presence of ethyl xanthate, more and larger microcolonies were observed on the chalcopyrite surface when biofilm was matured, resulting in an increased corrosion degree and more jarosite produced on the chalcopyrite surface. This study suggested that a low concentration (0.01 mg/L) of ethyl xanthate could promote the dissolution of chalcopyrite, which could be significant for bioleaching of flotation reagent-bearing flotation concentrates.

Biofilm formation and development during the pyrite bioleaching of moderately thermophilic microorganisms

Bioleaching is a process that uses microorganisms to recover metals from intractable metal sulfide ores. Metal sulfide bioleaching is an interfacial process, and biofilm growth is crucial during the early phases. To study the interactions between microbes and minerals during the bioleaching of moderately thermophilic microorganisms, the formation and development of biofilms on pyrite coupons incubated in shake flasks for 40 days were evaluated. The biofilm biomass progressively increased as bioleaching proceeded. The most extensive biofilm was formed on day 30, when bacteria were attached to the mineral surface or submerged in extracellular polymeric substances. Confocal laser scanning microscopy indicated that the biofilm biomass of pyrite on day 40 was 6.55, 5.28, 3.17, and 1.70 times higher than that on days 5, 10, 20, and 30, respectively. Extracellular protein content exhibited an increasing trend throughout the process, whereas extracellular polysaccharides played a significant role during the early stages. Live/dead staining showed the highest mortality rate of 94% when cells were in contact with pyrite in the early stage, and the percentage of live bacteria increased in the later period. In the bioleaching environment, mature biofilms appeared to form relatively quickly after day 20. Real-time quantitative fluorescent quantitative analysis indicated that biofilm formation played an important role in defense against hostile external environments. In this study, biofilm formation and the associated RNA transcript numbers of moderately thermophilic microorganisms during pyrite bioleaching were investigated, contributing to the understanding of cell-mineral interactions.

In situ detection of Cu2+, Fe3+ and Fe2+ ions at the microbe-mineral interface during bioleaching of chalcopyrite by moderate thermophiles

Bioleaching of metal sulfides represents an interfacial process in which the spatial relationship of metal ions with microbial cells and extracellular polymeric substances (EPS) is complex. The dissolution of chalcopyrite, cell attachment, and biofilm formation patterns were assessed during bioleaching with mixed cultures at 45 °C. The analysis of confocal laser scanning microscopy (CLSM) indicated that EPS were laid flat on the surface of chalcopyrite coupons, forming monolayer biofilms, with significant amounts of Fe3+, Fe2+ and Cu2+ enriched at the cell-chalcopyrite interface. In addition, the metal ions from mineral surfaces were extracted by ultrasonication. The results showed that the mineral surface is enriched with large amounts of Fe3+ (12.07 mg/g) and Cu2+ (10.82 mg/g) during the final stage of bioleaching, and more Cu2+ ions and iron maters from bio-oxidizing maybe were enclosed in the EPS space. It was inferred that the EPS with jarosite on the surface of chalcopyrite gradually acted as a weak diffusion barrier for Cu2+, and Fe3+ ions transference during bioleaching. These results can provide important evidence to reveal the hypothesis of ''metal ions enrichment on the mineral surface'' and benefit a further understanding of the bioleaching mechanism.

Biofilm dynamics and EPS production of a thermoacidophilic bioleaching archaeon

Bioleaching of metal sulfides represents an interfacial process where biofilm formation is important in the initial steps of this process. In technical applications of bioleaching, such as reactor leaching in the temperature range of 50 up to 90 °C and also in (self-heating) heaps, thermophilic archaea play an important role and often are the leaching organisms of choice. Nevertheless, to date there is little information available on the interactions between thermoacidophilic archaea and their natural mineral substrates such as pyrite. Especially for extracellular polymeric substances (EPS) of archaea and their biofilms in bioleaching environments information is rather limited. The present work focused on investigations of biofilm dynamics and EPS production of the thermoacidophilic archaeon Acidianus sp. DSM 29099 under bioleaching conditions. The results show that biofilms are dispersed non-homogeneously on pyrite. Large parts of the pyrite surfaces remain free of cells. Cell detachment from pyrite results in microbial “footprints” which, based on lectin binding assays, consist of mannose, glucose and fucose containing compounds. A monolayer biofilm develops on pyrite after 2–4 days of incubation. In addition, the pyrite surface is covered with a layer of organic compounds. EPS analysis indicates the presence of proteins, polysaccharides and uronic acids, the composition of which varies according to substrate and lifestyle (i.e. planktonic, biofilm cells). This report provides insight into EPS and biofilm characteristics of thermophilic archaea and improves understanding of the mineral-microbial-biofilm interfacial interactions in extreme environments. Moreover, the results on interaction dynamics of archaeal microbial consortia will facilitate the understanding of thermophilic bioleaching.

Adhesion to Mineral Surfaces by Cells of Leptospirillum, Acidithiobacillus and Sulfobacillus from Armenian Sulfide Ores

Bioleaching of metal sulfides is an interfacial process where adhesion and subsequent biofilm formation are considered to be crucial for this process. In this study, adhesion and biofilm formation by several acidophiles (Acidithiobacillus, Leptospirillum and Sulfobacillus) isolated from different biotopes with sulfide ores in Armenia were studied. Results showed that: (1) these bacteria adhere to pyrite surfaces to various extents. A correlation between pyrite biooxidation and adhesion of S. thermosulfidooxidans 6, L. ferriphilum CC, L. ferrooxidans ZC on pyrite surfaces is shown. It is supposed that bioleaching of pyrite by S. thermosulfidooxidans 6, L. ferriphilum CC, L. ferrooxidans ZC occurs by means of indirect leaching: by ferric iron of bacterial origin; (2) cells of At. ferrooxidans 61, L. ferrooxidans ZC and St. thermosulfidooxidans 6 form a monolayer biofilm on pyrite surfaces. The coverage of pyrite surfaces varies among these species. The order of the biofilm coverage is: L. ferrooxidans ZC ≥ At. ferrooxidans 61 > St. thermosulfidooxidans 6; (3) the extracellular polymeric substances (EPS) analysis indicates that the tested strains produce EPS, if grown either on soluble ferrous iron or solid pyrite. EPS are mainly composed of proteins and carbohydrates. Cells excrete higher amounts of capsular EPS than of colloidal EPS. In addition, cells grown on pyrite produce more EPS than ones grown on ferrous iron.

Application of β-glucuronidase (GusA) as an effective reporter for extremely acidophilic Acidithiobacillus ferrooxidans.

Acidithiobacillus ferrooxidans is a model organism for investigating metal sulfide bioleaching. The regulatory mechanism of gene expression by metabolizing various substrates is critical for understanding its role in bioleaching processes. However, no reporter has been successfully employed to study gene expression in A. ferrooxidans to date. In this study, a sensitive and robust reporter system based on β-glucuronidase (GusA) was described for feasible application in A. ferrooxidans. A set of vectors, which contained the transcriptional and translational fusions of gusA, were constructed and employed to analyze promoter activity and efficiency of translation initiation in A. ferrooxidans. Ptac and P2811 were screened out from ten tested promoters and could be used as strong promoters for gene overexpression in A. ferrooxidans. Among the four translational fusions of gusA with different start codons, ATG was most active, followed by TTG and GTG, while CTG showed the least activity. The transcriptional inhibition effect of an IclR-like transcription factor was also observed on its own encoding gene AFE_1668 as well as its neighboring AFE_1667. In addition, the specific chromogenic reaction of GusA could be detected and visualized by colonies of A. ferrooxidans containing gusA expression plasmids. Generally, the established GusA reporter system would be applied not only for quantitative analysis of promoter strength and its transcriptional regulation but also for qualitative colony screening in A. ferrooxidans in the future.

Influence of Sulfobacillus thermosulfidooxidans on Initial Attachment and Pyrite Leaching by Thermoacidophilic Archaeon Acidianus sp. DSM 29099

At the industrial scale, bioleaching of metal sulfides includes two main technologies, tank leaching and heap leaching. Fluctuations in temperature caused by the exothermic reactions in a heap have a pronounced effect on the growth of microbes and composition of mixed microbial populations. Currently, little is known on the influence of pre-colonized mesophiles or moderate thermophiles on the attachment and bioleaching efficiency by thermophiles. The objective of this study was to investigate the interspecies interactions of the moderate thermophile Sulfobacillus thermosulfidooxidans DSM 9293T and the thermophile Acidianus sp. DSM 29099 during initial attachment to and dissolution of pyrite. Our results showed that: (1) Acidianus sp. DSM 29099 interacted with S. thermosulfidooxidansT during initial attachment in mixed cultures. In particular, cell attachment was improved in mixed cultures compared to pure cultures alone; however, no improvement of pyrite leaching in mixed cultures compared with pure cultures was observed; (2) active or inactivated cells of S. thermosulfidooxidansT on pyrite inhibited or showed no influence on the initial attachment of Acidianus sp. DSM 29099, respectively, but both promoted its leaching efficiency; (3) S. thermosulfidooxidansT exudates did not enhance the initial attachment of Acidianus sp. DSM 29099 to pyrite, but greatly facilitated its pyrite dissolution efficiency. Our study provides insights into cell-cell interactions between moderate thermophiles and thermophiles and is helpful for understanding of the microbial interactions in a heap leaching environment.

Sulfide Oxidation by a Noncanonical Pathway in Red Blood Cells Generates Thiosulfate and Polysulfides

A cardioprotectant at low concentrations, H2S is a toxin at high concentrations and inhibits cytochrome c oxidase. A conundrum in H2S homeostasis is its fate in red blood cells (RBCs), which produce H2S but lack the canonical mitochondrial sulfide oxidation pathway for its clearance. The sheer abundance of RBCs in circulation enhances the metabolic significance of their clearance strategy for H2S, necessary to avoid systemic toxicity. In this study, we demonstrate that H2S generation by RBCs is catalyzed by mercaptopyruvate sulfurtransferase. Furthermore, we have discovered the locus of sulfide oxidation in RBCs and describe a new role for an old protein, hemoglobin, which in the ferric or methemoglobin state binds H2S and oxidizes it to a mixture of thiosulfate and hydropolysulfides. Our study reveals a previously undescribed route for the biogenesis of hydropolysulfides, which are increasingly considered important for H2S-based signaling, but their origin in mammalian cells is unknown. An NADPH/flavoprotein oxidoreductase system restores polysulfide-carrying hemoglobin derivatives to ferrous hemoglobin, thus completing the methemoglobin-dependent sulfide oxidation cycle. Methemoglobin-dependent sulfide oxidation in mammals is complex and has similarities to chemistry reported for the dissolution of iron oxides in sulfidic waters and during bioleaching of metal sulfides. The catalytic oxidation of H2S by hemoglobin explains how RBCs maintain low steady-state H2S levels in circulation, and suggests that additional hemeproteins might be involved in sulfide homeostasis in other tissues.

Biofilm formation, communication and interactions of leaching bacteria during colonization of pyrite and sulfur surfaces

Bioleaching of metal sulfides is an interfacial process where biofilm formation is considered to be important in the initial steps of this process. Among the factors regulating biofilm formation, molecular cell-to-cell communication such as quorum sensing is involved. A functional LuxIR-type I quorum sensing system is present in Acidithiobacillus ferrooxidans. However, cell-to-cell communication among different species of acidophilic mineral-oxidizing bacteria has not been studied in detail. These aspects were the scope of this study with emphasis on the effects exerted by the external addition of mixtures of synthetic N-acyl-homoserine-lactones on pure and binary cultures. Results revealed that some mixtures had inhibitory effects on pyrite leaching. Some of them correlated with changes in biofilm formation patterns on pyrite coupons. We also provide evidence that A. thiooxidans and Acidiferrobacter spp. produce N-acyl-homoserine-lactones. In addition, the observation that A. thiooxidans cells attached more readily to pyrite pre-colonized by living iron-oxidizing acidophiles than to heat-inactivated or biofilm-free pyrite grains suggests that other interactions also occur. Our experiments show that pre-cultivation conditions influence A. ferrooxidans attachment to pre-colonized pyrite surfaces. The understanding of cell-to-cell communication may consequently be used to develop attempts to influence biomining/bioremediation processes.

Identification and characterization of an ETHE1-like sulfur dioxygenase in extremely acidophilic Acidithiobacillus spp.

Elemental sulfur (S(0)) oxidation in Acidithiobacillus spp. is an important process in metal sulfide bioleaching. However, the gene that encodes the sulfur dioxygenase (SDO) for S(0) oxidation has remained unclarified in Acidithiobacillus spp. By BLASTP with the eukaryotic mitochondrial sulfur dioxygenases (ETHE1s), the putative sdo genes (AFE_0269 and ACAL_0790) were recovered from the genomes of Acidithiobacillus ferrooxidans ATCC 23270 and Acidithiobacillus caldus MTH-04. The purified recombinant proteins of AFE_0269 and ACAL_0790 exhibited remarkable SDO activity at optimal mildly alkaline pH by using the GSH-dependent in vitro assay. Then, a sdo knockout mutant and a sdo overexpression strain of A. ferrooxidans ATCC 23270 were constructed and characterized. By overexpressing sdo in A. ferrooxidans ATCC 23270, a significantly increased transcriptional level of sdo (91-fold) and a 2.5-fold increase in SDO activity were observed when S(0) was used as sole energy source. The sdo knockout mutant of A. ferrooxidans displayed a slightly reduced growth capacity in S(0)-medium compared with the wild type but still maintained high S(0)-oxidizing activity, suggesting that there is at least one other S(0)-oxidizing enzyme besides SDO in A. ferrooxidans ATCC 23270 cells. In addition, no obvious changes in transcriptional levels of selected genes related to sulfur oxidation was observed in response to the sdo overexpression or knockout in A. ferrooxidans when cultivated in S(0)-medium. All the results might suggest that SDO is involved in sulfide detoxification rather than bioenergetic S(0) oxidation in chemolithotrophic bacteria.

Effect of pyrite, elemental sulfur and ferrous ions on EPS production by metal sulfide bioleaching microbes

Extracellular polymeric substances (EPS) produced by acidophilic bioleaching microorganisms play an important role in the production of acid mine drainage and metal sulfide bioleaching. EPS mediate the contact between microbial cells and growth substrates, having a pivotal role in organic film formation and bacterium-substratum interactions. The production and chemical composition of EPS produced by seven bioleaching strains grown with different substrates were studied. Analysis of the EPS extracted from these strains indicated that the EPS consisted of carbohydrates, proteins and galacturonic acid. The contents of EPS, carbohydrates, proteins and galacturonic acid of EPS were largely related to the kind of strain used and culture condition. The results show that EPS productions of microbes grown with pyrite were significantly higher than those of microbes grown with sulfur or FeSO4·7H2O. The highest EPS production of the seven acidiphilic strains was (159.43±3.93) mg/g, which was produced by Leptospirillum ferriphilum CBCBSUCSU208015 when cultivated with pyrite.

Progress in bioleaching: fundamentals and mechanisms of bacterial metal sulfide oxidation—part A

Bioleaching of metal sulfides is performed by a diverse group of microorganisms. The dissolution chemistry of metal sulfides follows two pathways, which are determined by the mineralogy and the acid solubility of the metal sulfides: the thiosulfate and the polysulfide pathways. Bacterial cells can effect this metal sulfide dissolution via iron(II) ion and sulfur compound oxidation. Thereby, iron(III) ions and protons, the metal sulfide-attacking agents, are available. Cells can be active either in planktonic state or in forming biofilms on the mineral surface; however, the latter is much more efficient in terms of bioleaching kinetics. In the case of Acidithiobacillus ferrooxidans, bacterial exopolymers contain iron(III) ions, each complexed by two uronic acid residues. The resulting positive charge allows an electrostatic attachment to the negatively charged pyrite. Thus, the first function of complexed iron(III) ions is the mediation of cell attachment, while their second function is oxidative dissolution of the metal sulfide, similar to the role of free iron(III) ions in non-contact leaching. In both cases, the electrons extracted from the metal sulfide reduce molecular oxygen via a redox chain forming a supercomplex spanning the periplasmic space and connecting both outer and inner membranes. In this review, we summarize some recent discoveries relevant to leaching bacteria which contribute to a better understanding of these fascinating microorganisms. These include surface science, biochemistry of iron and sulfur metabolism, anaerobic metabolism, and biofilm formation. The study of microbial interactions among multispecies leaching consortia, including cell-to-cell communication mechanisms, must be considered in order to reveal more insights into the biology of bioleaching microorganisms and their potential biotechnological use.

Some quantitative data on bacterial attachment to pyrite

Bacterial attachment and biofilm formation are important for microbial bioleaching of metal sulfides, however, many details of the role played by bacteria are still unknown. Attachment as the first step in biofilm formation is critical. Our investigations are focused on these processes to control bacterial bioleaching processes. In moderately thermophilic pure cultures of Acidithiobacillus caldus and Leptospirillum ferriphilum, only the latter one showed significant attachment and biofilm formation to metal sulfides. In pure cultures of the mesophilic strains Acidithiobacillus spp., Leptospirillum ferrooxidans, Ferrimicrobium acidiphilum and a novel undescribed leaching organism designated SPIII/3, attachment was significant by L. ferrooxidans, At. thiooxidans and SPIII/3. In general, interaction of different species in mixed cultures resulted in increased attachment, increased production of extracellular polymeric substances (EPS) and leaching. Bacteria like Leptospirillum spp. enhanced the attachment of other species in mixed biofilms. Another finding is that large areas of the surface of minerals remain uncolonized, whereas at some places bacteria attach in clusters. Based on these findings we conclude that attachment and thereby bacterial leaching can be improved by the production of EPS by several strains especially in combination with Leptospirillum spp.

Kinetics of ferrous iron oxidation by Sulfobacillus thermosulfidooxidans

The biological oxidation of ferrous iron is an important sub-process in the bioleaching of metal sulfides and other bioprocesses such as the removal of H 2S from gases, the desulfurization of coal and the treatment of acid mine drainage (AMD). As a consequence, many Fe(II) oxidation kinetics studies have mostly been carried out with mesophilic microorganisms, but only a few with moderately thermophilic microorganisms. In this work, the ferrous iron oxidation kinetics in the presence of Sulfobacillus thermosulfidooxidans (DSMZ 9293) was studied. The experiments were carried out in batch mode (2L STR) and the effect of the initial ferrous iron concentration (2–20 g L −1) on both the substrate consumption and bacterial growth rate was assessed. The Monod equation was applied to describe the growth kinetics of this microorganism and values of μ max and K s of 0.242 h −1 and 0.396 g L −1, respectively, were achieved. Due to the higher temperature oxidation, potential benefits on leaching kinetics are forecasted.

Comparative study of interaction between pyrite and cysteine by thermogravimetric and electrochemical techniques

Adsorption mechanism of l-cysteine on pyrite was investigated by thermogravimetric and electrochemical techniques. TG curves provided the direct evidence for chemisorption of cysteine on pyrite surface. Once cysteine adsorbed to pyrite surface, E corr (corrosion potential) sharply lowered whereas I corr (corrosion current) increased rapidly. Pyrite became more susceptible to be oxidized even at lower potential as cysteine was added. However, the mechanism for pyrite oxidation does not fundamentally change, although cysteine can obviously accelerate oxidation rate of pyrite. These findings have important implications for understanding the mechanism of bacterial adhesion to pyrite and even metal sulfide bioleaching.

C-di-GMP Pathway in Biomining Bacteria

Acidithiobacillus ferrooxidans, A. thiooxidans, and A. caldus are acidophilic Gram-negative -proteobacteria involved in the bioleaching of metal sulfides. Bacterial attachment to mineral surface and biofilm development play a pivotal role in this process. Therefore, the understanding of biofilm formation has relevance to the design of biological strategies to improve the efficiency of bioleaching processes. For this reason, our laboratory is focused on the characterization of the molecular mechanisms involved in biofilm formation in biomining bacteria. In many bacteria, the intracellular level of c-di-GMP molecules regulates the transition from the motile planktonic state to sessile community-based behaviors, such as biofilm development. Thus, we recently started the study of c-di-GMP pathway in biomining bacteria. C-di-GMP molecules are synthesized by diguanylate cyclases (DGCs) and degraded by phosphodiesterases (PDEs). So far, two kinds of effectors have been identified, including three protein families (pilZ, PleD and FleQ) and a conserved RNA domain (GEMM) which acts as a riboswitch. We previously reported the existence of different molecular players involved in c-di-GMP pathway in A. ferrooxidans ATCC 23270. Here, we expanded our work to other Acidithioibacillus species: A. thiooxidans ATCC 19377 and A. caldus ATCC 51756. In both, we identified several putative-ORFs encoding DGC, PDE and effector proteins. By using total RNA extracted from A. ferrooxidans and A. caldus cells in RT-PCR and qPCR experiments, we demonstrated that these genes are expressed. In addition, we characterized the presence of c-di-GMP in A. ferrooxidans ATCC 23270 and A. caldus ATCC 51756 cell extracts. Taken together, these results strongly suggest that A. ferrooxidans, A. caldus and A. thiooxidans possess functional c-di-GMP pathways. As it occurs in other Gram-negative bacteria, this pathway should be involved in the regulation of the planktonic/biofilm switch. In the future, we have to integrate this new biological dimension to improve the biological understanding of bioleaching.

C-di-GMP Pathway in <i>Acidithiobacillus ferrooxidans</i>: Analysis of Putative Diguanylate Cyclases (DGCs) and Phosphodiesterases (PDEs) Bifunctional Proteins

Acidithiobacillus ferrooxidans, an acidophilic, chemolithotrophic, γ-proteobacterium, is involved in the bioleaching of metal sulfides. For this process, bacterial attachment to mineral surface and biofilm development play a pivotal role. Generally, biofilm formation and production of exopolysaccharides is regulated by the second messenger cyclic diguanylic acid (c-di-GMP) whose cellular level depends on the synthesis and degradation activities of diguanylate cyclase (DGCs, with GGDEF domain) and phosphodiesterase (PDE, with EAL or HD-GYP domains), respectively. The analysis of the genomic sequence of A. ferrooxidans ATCC 23270 allowed us to identify 5 putative orfs encoding DGC and/or PDE-like proteins. Four of them encode for bifunctional putative proteins with GGDEF and EAL domains and are named AFE_0053, AFE_1360, AFE_1373 and AFE_1379. The fifth one named AFE_1852 has an EAL domain. The putative proteins also include PAS and GAF domains involved in signal transduction. These features suggest an involvement in signalling transduction through the metabolism of c-di-GMP. The amino acid sequences of these putative proteins were aligned with known DGCs and PDEs. Alignments indicate that AFE_1360 and AFE_1373 share more consensus sequences with active PDEs, whereas AFE_0053 and AFE_1379 do with active DGCs. On the other hand, in AFE_1852 some conserved residues of known active PDEs are changed. RT-PCR-experiments revealed that the genes that encode for these putative DGCs and/or PDEs are expressed by growth on two different substrates. These preliminary results suggest that A. ferrooxidans possesses a c-di-GMP pathway that should be involved in biofilm formation, as it occurs in many bacteria.

Bioleaching review part A:

Bioleaching of metal sulfides is caused by astonishingly diverse groups of bacteria. Today, at least 11 putative prokaryotic divisions can be related to this phenomenon. In contrast, the dissolution (bio)chemistry of metal sulfides follows only two pathways, which are determined by the acid-solubility of the sulfides: the thiosulfate and the polysulfide pathway. The bacterial cell can effect this sulfide dissolution by "contact" and "non-contact" mechanisms. The non-contact mechanism assumes that the bacteria oxidize only dissolved iron(II) ions to iron(III) ions. The latter can then attack metal sulfides and be reduced to iron(II) ions. The contact mechanism requires attachment of bacteria to the sulfide surface. The primary mechanism for attachment to pyrite is electrostatic in nature. In the case of Acidithiobacillus ferrooxidans, bacterial exopolymers contain iron(III) ions, each complexed by two uronic acid residues. The resulting positive charge allows attachment to the negatively charged pyrite. Thus, the first function of complexed iron(III) ions in the contact mechanism is mediation of cell attachment, while their second function is oxidative dissolution of the metal sulfide, similar to the role of free iron(III) ions in the non-contact mechanism. In both cases, the electrons extracted from the metal sulfide reduce molecular oxygen via a complex redox chain located below the outer membrane, the periplasmic space, and the cytoplasmic membrane of leaching bacteria. The dominance of either At. ferrooxidans or Leptospirillum ferrooxidans in mesophilic leaching habitats is highly likely to result from differences in their biochemical iron(II) oxidation pathways, especially the involvement of rusticyanin.

Study of Bacterial Attachment During the Bioleaching of Pyrite, Chalcopyrite, and Sphalerite

In this study the degree of bacterial attachment of the main microorganisms involved in the bioleaching of metal sulfides and their influence on the dissolution rate and final metal extractions were determined. Three different mineral sulfides were bioleached: chalcopyrite (CuFeS 2 ), sphalerite (ZnS) and pyrite (FeS 2 ). A mixed culture of mesophilic bacteria ( Acidithiobacillus and Leptospirillum spp.) was tested at 35°C and thermophilic bacteria ( Sulfolobus spp.) were tested at 68°C. The results confirmed that a relationship exists between attachment and mineral dissolution rates. The bioleaching process can be divided into three stages. An initial stage with extensive bacterial attachment to the pyritic phase, which is of major importance in order to obtain high dissolution rates (since attached cells liberate Fe 2+ by contact bioleaching and oxidize Fe 2+ to Fe 3+ avoiding iron accumulation at the surface). In a second stage the bacterial attachment diminishes due to the saturation of the surface by the attached cells. This limitation, together with the presence of Fe 2+ in solution, produces an increase in the concentration of free cells, which use Fe 2+ as an energy source. Finally, in a third stage, a balance between free and attached cells is reached, giving rise to a cooperative mechanism. In this case, the attached cells attack the pyrite phase of the mineral generating Fe 2+ . This in turn is oxidized by the free cells in solution, regenerating the oxidizing agent (Fe 3+ ) for the indirect bioleaching of the mineral.

(Bio)chemistry of bacterial leaching—direct vs. indirect bioleaching

Bioleaching of metal sulfides is effected by bacteria, like Thiobacillus ferrooxidans, Leptospirillum ferrooxidans, Sulfolobus/Acidianus, etc., via the (re)generation of iron(III) ions and sulfuric acid. According to the new integral model for bioleaching presented here, metal sulfides are degraded by a chemical attack of iron(III) ions and/or protons on the crystal lattice. The primary iron(III) ions are supplied by the bacterial extracellular polymeric substances, where they are complexed to glucuronic acid residues. The mechanism and chemistry of the degradation is determined by the mineral structure. The disulfides pyrite (FeS 2), molybdenite (MoS 2), and tungstenite (WS 2) are degraded via the main intermediate thiosulfate. Exclusively iron(III) ions are the oxidizing agents for the dissolution. Thiosulfate is, consequently, degraded in a cyclic process to sulfate, with elemental sulfur being a side product. This explains, why only iron(II) ion-oxidizing bacteria are able to oxidize these metal sulfides. The metal sulfides galena (PbS), sphalerite (ZnS), chalcopyrite (CuFeS 2), hauerite (MnS 2), orpiment (As 2S 3), and realgar (As 4S 4) are degradable by iron(III) ion and proton attack. Consequently, the main intermediates are polysulfides and elemental sulfur (thiosulfate is only a by-product of further degradation steps). The dissolution proceeds via a H 2S* +-radical and polysulfides to elemental sulfur. Thus, these metal sulfides are degradable by all bacteria able to oxidize sulfur compounds (like T. thiooxidans, etc.). The kinetics of these processes are dependent on the concentration of the iron(III) ions and, in the latter case, on the solubility product of the metal sulfide.