Characterization and identification of an iron-oxidizing,Leptospirillum-like bacterium,present in the high sulfate leaching solution of a commercial bioleaching plant

Most copper bioleaching plants operate with a high concentration of sulfate salts, caused by the continuous addition of sulfuric acid and the recycling of the leaching solution. Since the bacteria involved in bioleaching have been generally isolated at low sulfate concentrations, the bacterial population present in the high-sulfate (150 gl(-1)) leaching solution, employed in a copper production plant, was investigated. The iron-oxidizing bacteria able to grow in the leaching solution were enriched by several batch cultivations and, after serial dilution, an abundant bacterial strain was isolated. This strain, called LA, exhibited a relatively constant rate of iron-oxidation in media containing sulfate ions at concentrations ranging from 10 to 150 gl(-1). Culture collection strains of Leptospirillum ferrooxidans and Acidithiobacillus ferrooxidans showed limited abilities to grow at sulfate ion concentrations higher than 70 gl(-1). In spite of its tolerance to high sulfate concentrations, strain LA was as sensitive to NaCl as A. ferrooxidans. Comparative sequence analysis of the 16S rRNA gene of strain LA indicated that it is phylogenetically related to strains described as Leptospirillum ferrooxidans. Bacterial community DNA restriction patterns of 16S rRNA genes suggested that strain LA was a minor component of the bacterial population present in leaching solution, but is abundant in ore leached with this solution.     

Microbially-influenced corrosion: damage to prostheses, delight for bacteria

In natural and man-made environments, microbial communities thrive as biofilms on living (e.g. tissue) and inanimate (e.g. plastic, metal, wood, mineral) surfaces. Biofilms are found in a wide range of aqueous habitats, including physiological fluids. Numerous types of microorganisms are able to colonize catheters, implants, prosthetics, and other medical devices manufactured from different metallic and non-metallic materials dwelling within a human body. The development of biofilm is facilitated by the production of extracellular polymeric substances (EPS). Biofilms formed on surfaces of metallic materials may alter interfacial electrochemical processes, which can lead to increased corrosion of the colonized substratum. Deterioration of metallic materials in the presence of a biofilm is termed biocorrosion or microbially-influenced corrosion (MIC). In the field of biomaterials, "biocorrosion" is commonly used when describing the effect of host tissue on the corrosion of implant metals and alloys. Therefore, to avoid confusion, we will here use the term MIC as a reference to biofilm-influenced corrosion. It is important to realise that although most metals are prone to microbial colonization, i.e. to biofouling, this does not imply that they are susceptible to MIC. For example, a metal such as titanium, accumulates biofilm, however, it still demonstrates excellent resistance against MIC. Corrosion is, by definition, an electrochemical process, therefore, electrochemical techniques are frequently employed to determine and measure the rate of abiotic, as well as biologically driven corrosion reactions. This communication addresses the use of electrochemical techniques for monitoring (i) biofilm formation on and (ii) MIC of implant metals and alloys.     

Isolation and characterization of a novel Acidithiobacillus ferrivorans strain from the Chilean Altiplano: attachment and biofilm formation on pyrite at low temperature

Microorganisms are used to aid the extraction of valuable metals from low-grade sulfide ores in mines worldwide, but relatively little is known about this process in cold environments. This study comprises a preliminary analysis of the bacterial diversity of the polyextremophilic acid River Aroma located in the Chilean Altiplano, and revealed that Betaproteobacteria was the most dominant bacterial group (Gallionella-like and Thiobacillus-like). Taxa characteristic of leaching environments, such Acidithiobacillus and Leptospirillum, were detected at low abundances. Also, bacteria not associated with extremely acidic, metal-rich environments were found. After enrichment in iron- and sulfur-oxidizing media, we isolated and identified a novel psychrotolerant Acidithiobacillus ferrivorans strain ACH. This strain can grow using ferrous iron, sulfur, thiosulfate, tetrathionate and pyrite, as energy sources. Optimal growth was observed in the presence of pyrite, where cultures reached a cell number of 6.5·10⁷ cells mL⁻¹. Planktonic cells grown with pyrite showed the presence of extracellular polymeric substances (10 °C and 28 °C), and a high density of cells attached to pyrite grains were observed at 10 °C by electron microscopy. The attachment of cells to pyrite coupons and the presence of capsular polysaccharides were visualized by using epifluorescence microscopy, through nucleic acid and lectin staining with Syto^®9 and TRITC-Con A, respectively. Interestingly, we observed high cell adhesion including the formation of microcolonies within 21 days of incubation at 4 °C, which was correlated with a clear induction of capsular polysaccharides production. Our data suggests that attachment to pyrite is not temperature-dependent in At. ferrivorans ACH. The results of this study highlight the potential of this novel psychrotolerant strain in oxidation and attachment to minerals under low-temperature conditions.     

A simplified in vivo approach for evaluating the bioabsorbable behavior of candidate stent materials

Metal stents are commonly used to revascularize occluded arteries. A bioabsorbable metal stent that harmlessly erodes away over time may minimize the normal chronic risks associated with permanent implants. However, there is no simple, low-cost method of introducing candidate materials into the arterial environment. Here, we developed a novel experimental model where a biomaterial wire is implanted into a rat artery lumen (simulating bioabsorbable stent blood contact) or artery wall (simulating bioabsorbable stent matrix contact). We use this model to clarify the corrosion mechanism of iron (≥99.5 wt %), which is a candidate bioabsorbable stent material due to its biocompatibility and mechanical strength. We found that iron wire encapsulation within the arterial wall extracellular matrix resulted in substantial biocorrosion by 22 days, with a voluminous corrosion product retained within the vessel wall at 9 months. In contrast, the blood-contacting luminal implant experienced minimal biocorrosion at 9 months. The importance of arterial blood versus arterial wall contact for regulating biocorrosion was confirmed with magnesium wires. We found that magnesium was highly corroded when placed in the arterial wall but was not corroded when exposed to blood in the arterial lumen for 3 weeks. The results demonstrate the capability of the vascular implantation model to conduct rapid in vivo assessments of vascular biomaterial corrosion behavior and to predict long-term biocorrosion behavior from material analyses. The results also highlight the critical role of the arterial environment (blood vs. matrix contact) in directing the corrosion behavior of biodegradable metals.     

Micro-Raman analysis and AFM imaging of Acidithiobacillus ferrooxidans biofilm grown on uranium ore

Acidithiobacillus ferrooxidans biofilm grown on uranium ore substrate was analyzed by a micro-Raman spectrometer and an atomic force microscope (AFM). The bacterium employed for this study, A. ferrooxidans BM1, was isolated from a uranium mine (Jaduguda, India). Micro-Raman analysis revealed the different constituents of molecular fragments present in microbial cells and in secreted extracellular polymeric substances (EPSs). AFM images clearly revealed bacterial cells surrounded by EPS. From Raman spectral data, the composition of EPS from A. ferrooxidans BM1 appeared to be similar to that of EPS secreted in a different Pseudomonas bacterium.     

Insights into the pathways of iron- and sulfur-oxidation,and biofilm formation from the chemolithotrophic acidophile Acidithiobacillus ferrivorans CF27

The iron-oxidizing acidithiobacilli cluster into at least four groups, three of which (Acidithiobacillus ferrooxidans, Acidithiobacillus ferridurans and Acidithiobacillus ferrivorans) have been designated as separate species. While these have many physiological traits in common, they differ in some phenotypic characteristics including motility, and pH and temperature minima. In contrast to At. ferrooxidans and At. ferridurans, all At. ferrivorans strains analysed to date possess the iro gene (encoding an iron oxidase) and, with the exception of strain CF27, the rusB gene encoding an iso-rusticyanin whose exact function is uncertain. Strain CF27 differs from other acidithiobacilli by its marked propensity to form macroscopic biofilms in liquid media. To identify the genetic determinants responsible for the oxidation of ferrous iron and sulfur and for the formation of extracellular polymeric substances, the genome of At. ferrivorans CF27 strain was sequenced and comparative genomic studies carried out with other Acidithiobacillus spp.. Genetic disparities were detected that indicate possible differences in ferrous iron and reduced inorganic sulfur compounds oxidation pathways among iron-oxidizing acidithiobacilli. In addition, strain CF27 is the only sequenced Acidithiobacillus spp. to possess genes involved in the biosynthesis of fucose, a sugar known to confer high thickening and flocculating properties to extracellular polymeric substances.     

Cross-correlating analyses of mineral-associated microorganisms in an unsaturated packed bed flow-through column test; cell number,activity and EPS

In heap bioleaching and waste-rock dumps, complex microbial communities exist in the flowing and interstitial liquid phases and mineral surface-associated biofilms, often embedded in extracellular polymeric substances (EPS). Microbial activity in the interstitial phase and mineral ore surface facilitates mineral degradation, resulting in either metal recovery or acidic, metal -bearing drainage from sulfidic waste-rock. Determining microbial presence and activity through microorganisms leaving the heap or dump has severe limitations. Hence, increasingly the ore-bed is sampled to quantify and characterise this. Here, methods for cell detachment and quantification, microbial activity measurement on the mineral surface and evaluation of EPS, quantitatively and biochemically, were refined and validated to assess microbial presence, using mineral coated beads in continuous flow-through columns. Number of wash steps required were assessed over increasing colonisation times over 30 days. Microbial cells colonising the mineral surface, pre- and post-washing were visualised by scanning electron microscopy (SEM) and their activity quantified by isothermal microcalorimetry (IMC). Using IMC, detachment and enumeration of detached cells, we demonstrated that 6–8 washes provided a reliable estimation of mineral-associated microorganisms, with less than 10% of cells or microbial activity associated with the surface following treatment. This allowed consolidated refinement of the protocol using traditional detachment method, SEM and IMC to provide correlative data. Extraction of EPS in a complete flow-through system is reported for the first time and the biochemical composition was similar to those reported under batch bioleaching conditions.     

An evaluation into the potential of biological processing for the removal of metals from sewage sludges

The use of sewage sludge in agricultural land as a means of sludge disposal and recycling has been shown to be economical and suitable because of the presence of nutrients such as nitrogen and phosphorus. However, municipal sludges often contain high quantities of toxic metals and other compounds that must be removed for its safe use in agricultural soils. The biological leaching of metals from sewage sludges has been shown to be a promising technique for metal detoxifying in such complex matrix. The process efficiency is dependent on several physico-chemical parameters, such as total solids concentration, metal forms, pH-ORP, and temperature. Scale-up of the process has not yet been defined and is still pursuing the correct operational design. Current research involving the bioleaching of metals from sewage sludge and its application to land, which affects soil physical properties, are presented and discussed.     

Metal transformation as a strategy for bacterial detoxification of heavy metals

Microorganisms can modify the chemical and physical characters of metals leading to an alteration in their speciation, mobility, and toxicity. Aqueous heavy metals solutions (Hg, Cd, Pb, Ag, Cu, and Zn) were treated with the volatile metabolic products (VMPs) of Escherichia coli Z3 for 24 h using aerobic bioreactor. The effect of the metals treated with VMPs in comparison to the untreated metals on the growth of E. coli S1 and Staphylococcus aureus S2 (local isolates) was examined. Moreover, the toxic properties of the treated and untreated metals were monitored using minimum inhibitory concentration assay. A marked reduction of the treated metals toxicity was recorded in comparison to the untreated metals. Scanning electron microscopy and energy dispersive X‐ray analysis revealed the formation of metal particles in the treated metal solutions. In addition to heavy metals at variable ratios, these particles consisted of carbon, oxygen, sulfur, nitrogen elements. The inhibition of metal toxicity was attributed to the existence of ammonia, hydrogen sulfide, and carbon dioxide in the VMPs of E. coli Z3 culture that might responsible for the transformation of soluble metal ions into metal complexes. This study clarified the capability of E. coli Z3 for indirect detoxification of heavy metals via the immobilization of metal ions into biologically unavailable species.     

Making sticky cells: effect of galactose and ferrous iron on the attachment of Leptospirillum ferrooxidans to mineral surfaces

The purpose of this study was to compare the efficacy of galactose and high initial ferrous iron concentrations as inducers for extracellular polymeric substances (EPS) production in planktonic cells of Leptospirillum ferrooxidans and to study cell attachment to a mineral surface in comparison to cells not exposed to such substances. L. ferrooxidans was successfully adapted to grow in a modified 9K medium at different concentrations of galactose (0.15, 0.25, 0.35%) and also at different initial ferrous iron concentrations (18, 27, 36 g/L), which are higher than 9K medium (9 g/L). The experiments were done in shake flasks using ferrous iron as energy source. A comparison of growth kinetics showed a decreasing of maximum specific growth rate of L. ferrooxidans with increasing concentrations of galactose and initial ferrous iron. The EPS content increased and the EPS chemical composition (relative abundance of carbohydrates, proteins and ferric iron) changed with increasing concentrations of galactose and initial ferrous iron. Results revealed that the increase of the bacterial adhesion rather depended on the chemical composition, i.e. relative abundance of the constituents of the EPS, than on the total amount of EPS. The EPS induced by galactose seemed to be “stickier” than the one induced by ferrous iron. Based on the results of this study it is proposed that galactose might enhance biooxidation processes which needs to be tested in future studies.     

Biofilm formation and interspecies interactions in mixed cultures of thermo-acidophilic archaea Acidianus spp. and Sulfolobus metallicus

The understanding of biofilm formation by bioleaching microorganisms is of great importance for influencing mineral dissolution rates and to prevent acid mine drainage (AMD). Thermo-acidophilic archaea such as Acidianus, Sulfolobus and Metallosphaera are of special interest due to their ability to perform leaching at high temperatures, thereby enhancing leaching rates. In this work, leaching experiments and visualization by microscopy of cell attachment and biofilm formation patterns of the crenarchaeotes Sulfolobus metallicus DSM 6482^T and the Acidianus isolates DSM 29038 and DSM 29099 in pure and mixed cultures on sulfur or pyrite were studied. Confocal laser scanning microscopy (CLSM) combined with fluorescent dyes as well as fluorescently labeled lectins were used to visualize different components (e.g. DNA, proteins or glycoconjugates) of the aforementioned species. The data indicate that cell attachment and the subsequently formed biofilms were species- and substrate-dependent. Pyrite leaching experiments coupled with pre-colonization and further inoculation with a second species suggest that both species may negatively influence each other during pyrite leaching with respect to initial attachment and pyrite dissolution rates. In addition, the investigation of binary biofilms on pyrite showed that both species were heterogeneously distributed on pyrite surfaces in the form of individual cells or microcolonies. Physical contact between the two species seems to occur, as revealed by specific lectins able to specifically bind single species within mixed cultures.     

The amyloid paradox: amyloid-beta-metal complexes can be neurotoxic and neuroprotective

Senile plaques in the brains of people with Alzheimer disease (AD) are primarily composed of the amyloid-beta (Abeta) peptide and contain substantially elevated levels of iron, copper and zinc. These metals bind to Abeta and have been reported to increase the toxicity of Abeta to cultured neurones. Other reports have demonstrated that Abeta can reduce the neurotoxicity of metal ions, suggesting that the interaction can, under some circumstances, be protective. To investigate these apparently conflicting results, human Abeta1-42 was co-injected with iron, copper or zinc (at the concentrations found in plaques) into rat cerebral cortex, and the resulting numbers of dying neurones were compared. It was found that Abeta complexed with either iron or zinc was more toxic than Abeta alone. In contrast, Abeta-copper complexes were not neurotoxic. Surprisingly, we observed that when iron or copper were combined with Abeta, the neurotoxicity of these metals was substantially reduced, suggesting that Abeta may help to limit the toxicity of redox-active metal ions, thereby assisting the antioxidant defence of the brain. Thus paradoxical effects occur when Abeta complexes with metal ions, where Abeta-metal complexes are capable of being neurotoxic and neuroprotective.     

Inhibition of bactericidal and bacteriolytic activities of poly-D-lysine and lysozyme by chitotriose and ferric iron.

In a previous report from this laboratory (N. J. Laible and G. R. Germaine, Infect. Immun. 48:720-728, 1985), evidence was presented to suggest that the bactericidal actions of both reduced (i.e., muramidase-inactive) human placental lysozyme and the synthetic cationic homopolymer poly-D-lysine involved the activation of a bacterial endogenous activity that was inhibitable by N,N',N"-triacetylchitotriose (chitotriose). In the present investigation however, we found that the bactericidal and bacteriolytic action of poly-D-lysine could be prevented only by some commercially available chitotriose preparations and not by others. Analysis by physical and chemical methods failed to distinguish protective chitotriose (CTa) and nonprotective chitotriose (CTi) preparations. CTi and CTa preparations displayed equal capacities to competitively inhibit binding of [3H]chitotriose by immobilized lysozyme and were indistinguishable in their abilities to block the lytic activity of lysozyme against Micrococcus lysodeikticus cells. Elemental analysis revealed significantly higher levels of phosphorus, calcium, iron, sodium, manganese, and copper in CTa. Removal of metals from CTa by chelate chromatography completely abolished the poly-D-lysine-protective capacity. Of the metals detected, only ferric iron (5 to 10 microM) mimicked the protective action of CTa. A Fe(III) concentration of 50 microM was required to inhibit lysozyme (5 micrograms/ml). Both Fe(III) and CTa (but not CTi) quantitatively blocked the labeling of poly-D-lysine by fluorescamine, suggesting that the primary amino groups of the lysine residues participate in iron binding. Thus, it appears that the poly-D-lysine-protective capacity of certain chitotriose preparations was due not to the chitotriose itself but to contaminating metal ions which interact directly with the polycationic agent. In contrast, Fe(III) cannot account for inhibition of either the bactericidal or bacteriolytic activity of lysozyme by chitotriose.     

Bioleaching of pyritic coal wastes: bioprospecting and efficiency of selected consortia

Pyrite-bearing coal wastes are responsible of the formation of acid mine drainage (AMD), and their management to mitigate environmental impacts is a challenge to the coal mine industry in Europe and worldwide. The European CEReS project sought to develop a generic co-processing strategy to reuse and recycle coal wastes, based on removal of AMD generating potential through bioleaching. Chemolitoautotrophic iron- and sulfur-oxidizing microbial consortia were enriched from a Polish coal waste at 30 °C and 48 °C, but not 42 °C. Pyrite leaching yield, determined from bioleaching tests in 2-L stirred bioreactors, was best with the 48 °C endogenous consortium (80%), then the 42 °C exogenous BRGM-KCC consortium (71%), and finally the 30 °C endogenous consortium (50%). 16S rRNA gene-targeted metagenomics from five surface locations on the dump waste revealed a microbial community adapted to the site context, composed of iron- and/or sulfur-oxidizing genera thriving in low pH and metal rich environments and involved in AMD generation. All together, the results confirmed the predisposition of the pyritic coal waste to bioleaching and the potential of endogenous microorganisms for efficient bioleaching at 48 °C. The good leaching yields open the perspective to optimize further and scale-up the bioleaching process.     

Effects of different compounds of metals and of their mixtures on the growth and survival of Thiobacillus ferrooxidans

The iron oxidation by Thiobacillus ferrooxidans was studied in the presence of different concentrations of metals as sulphates, chlorides, nitrates, and in some other compounds. The bacteriological oxidation of ferro-ions develops in all solutions in nearly the same way, with the only difference that, as the ion concentration increases, the beginning of measurable oxidation is postponed, i. e., with ion concentration increasing, the lag-phase expands. When salts are mixed in different proportions, the toxic concentration of such a mixture corresponds, in a defined way, to the total of salts, i. e., of their ions.     

Biofouling and Biocorrosion in Industrial Water Systems

Corrosion associated with microorganisms has been recognized for over 50 years and yet the study of microbiologically influenced corrosion (MIC) is relatively new. MIC can occur in diverse environments and is not limited to aqueous corrosion under submerged conditions, but also takes place in humid atmospheres.

Biofouling of industrial water systems is the phenomenon whereby surfaces in contact with water are colonized by microorganisms, which are ubiquitous in our environment. However, the economic implications of biofouling in industrial water systems are much greater than many people realize. In a survey conducted by the National Association of Corrosion Engineers of the United States ten years ago, it was found that many corrosion engineers did not accept the role of bacteria in corrosion, and many of them that did, could not recognize and mitigate the problem.

Biofouling can be described in terms of its effects on processes and products such as material degradation (bio-corossion), product contamination, mechanical blockages, and impedance of heat transfer. Microorganisms distinguish themselves from other industrial water contaminants by their ability to utilize available nutrient sources, reproduce, and generate intra- and extracellular organic and inorganic substances in water. A sound understanding of the molecular and physiological activities of the microorganisms involved is necessary before strategies for the long term control of biofouling can be format. Traditional water treatment strategies however, have largely failed to address those factors that promote biofouling activities and lead to biocorrosion.

Some of the major developments in recent years have been a redefinition of biofilm architecture and the realization that MIC of metals can be best understood as biomineralization.     

Biofouling and biocorrosion in industrial water systems

Corrosion associated with microorganisms has been recognized for over 50 years and yet the study of microbiologically influenced corrosion (MIC) is relatively new. MIC can occur in diverse environments and is not limited to aqueous corrosion under submerged conditions, but also takes place in humid atmospheres. Biofouling of industrial water systems is the phenomenon whereby surfaces in contact with water are colonized by microorganisms, which are ubiquitous in our environment. However, the economic implications of biofouling in industrial water systems are much greater than many people realize. In a survey conducted by the National Association of Corrosion Engineers of the United States ten years ago, it was found that many corrosion engineer did not accept the role of bacteria in corrosion, and many of then that did, could not recognize and mitigate the problem. Biofouling can be described in terms of its effects on processes and products such as material degradation (bio-corossion), product contamination, mechanical blockages, and impedance of heat transfer. Microorganisms distinguish themselves from other industrial water contaminants by their ability to utilize available nutrient sources, reproduce, and generate intra- and extracellular organic and inorganic substances in water. A sound understanding of the molecular and physiological activities of the microorganisms involved is necessary before strategies for the long term control of biofouling can be format. Traditional water treatment strategies however, have largely failed to address those factors that promote biofouling activities and lead to biocorrosion. Some of the major developments in recent years have been a redefinition of biofilm architecture and the realization that MIC of metals can be best understood as biomineralization.     

T cell cross-reactivity to heavy metals: identical cryptic peptides may be presented from protein exposed to different metals

The mechanisms by which metals induce activation of T cells and thus produce allergic and/or autoimmune reactions are still obscure, and the same is true for the mechanisms that underly T cell cross-reactivity to different heavy metal ions. In the present study, we investigated induction by metals of T cell reactions to cryptic peptides of bovine RNase A. Murine CD4⁺ T cell hybridomas specific for cryptic RNase peptides presented from Au(III)-treated RNase were used as detection probes. We showed that in vitro treatment of RNase with Pd(II), Pd(IV), Ni(IV), and partially Pt(IV), but not Au(I), Ni(II), or Pt(II), induced presentation of the same cryptic peptides as those presented from Au(III)-treated RNase. That the former heavy metal ions, but not the latter, were able to alter the antigenicity of RNase was reflected by their ability to induce conformational changes of RNase, as detected by circular dichroism spectroscopy. Furthermore, upon immunization against RNase pretreated with these metals, CD4⁺ T cell hybridomas specific for unidentified cryptic peptides were obtained. In conclusion, “metal-specific” T cell reactions may be directed against cryptic peptides, and metal cross-reactivity in allergic individuals might be due to metal-induced presentation of overlapping, but not identical, panels of cryptic peptides.