Advanced Materials Research Vol. 825

Abstract: Acidophilic iron-oxidizing microorganisms are important in both environmental and biotechnological applications. These microorganisms are known to accelerate the dissolution of sulfur minerals such as pyrite (FeS₂), leading to the acid mine drainage generation , a serious pollution problem, that makes these microorganisms essential to the commercial processing of minerals and sulfur. In order to answer this question, diversity of native acidophilic bacteria isolated from acid mine drainage of Peru was evaluated. The samples were collected from Yanacocha mining (3000 m.a.s.l.) located in the North of Cajamarca region, Yanamina mining (4440 m.a.s.l.) located in the middle of Huancavelica region; finally, SPCC mining (2000 m.a.s.l.) located in the South of Moquegua region. We isolated 11 strains from which three were identified as Acidithiobacillus ferrooxidans, two as At. ferrivorans, two as At. ferridurans, three as Leptospirillum ferrooxidans and one as Acidiphilium sp. by comparative sequencing of PCR-amplified 16S rRNA genes. Phylogenetic analysis of the 16S rRNA genes revealed that some of the strains isolated are closely related to other already known, but there are some with similarities lower than < 95 percent. Our results provide the first study on the diversity of iron-oxidizing bacteria isolated from acid mine drainage of Peru.

Abstract: Abstract. Pan de Azúcar abandoned mine is located in Jujuy province, Argentina. The impact of that pollution was evaluated determining the density and the composition of the vegetation around the tailings. Also soil and plants samples were collected and their metal contents were determined. Deyeuxia breviaristata was the main native plant in the most polluted soils and its biomass had the highest concentration of zinc and lead. Acid mine drainage (AMD) samples were collected and their toxic effects were analyzed by ecotoxicology assays employed Lactuca sativa L. seeds; CI50₁₂₀ of 0.03 % v/v was determined. From mineral and AMD samples some iron- and sulphur-oxidizing microorganisms were isolated and also characterised by molecular techniques. AMD potential was evaluated for different tailing samples inoculating native microorganisms; deeper samples produced drainage with pH close to 1 and released iron above of 1000 ppm. AMD can reach Cincel River which is the main tributary of Laguna Pozuelos (just 25 km from the mine). Laguna Pozuelos is a Natural Monument, Biosphere Reserve and Ramsar site.

Abstract: Nanostructure forms of semiconductor materials are of great interest. Among these compounds, copper sulfide as a variable stoichiometric composition attracts considerable attention. In the present study, copper sulfide nanoparticles were synthesized biologically from a chalcopyrite concentratemainly containing chalcopyrite (46%) and pyrite (23%). Firstly, the copper contents of the concentrate were bioleached using thermophile bacteria, then the grown Fusarium oxysporum was added to the prepared solution and the biosynthesized nanoparticles collected and their characteristics compared with the product derived from the pure copper sulfate solution. The characterization was performed by UV spectrometry, Fourier Transform Infrared Spectroscopy (FTIR), Energy Dispersive X-ray Spectroscopy (EDS), Thermogravimetery (TG), Differential Scanning Calorimetery (DSC), Mass Spectrometery (MS), and Transmission Electron Microscopy (TEM). Finally, it wasproved that the produced nanoparticles had a covellite composition and their size was about 5-40 nm.

Abstract: In the bioleaching of mineral sulphides under the catalytic action of At. ferrooxidans, ferrous ion oxidation and sulfides/sulfur solubilization uses oxygen as the final electron acceptor. Also, under anaerobic conditions, At. ferrooxidans can alternatively catalize the oxidation of sulfur or reduced inorganic sulfur compounds (RISC) using ferric iron as electron acceptor [1]. The formation of Fe (II) from pyrite and covellite in the ferric anaerobic bioleaching with A. ferrooxidans, has been studied and well documented [2,3]. The requirements of ferric iron as electron acceptor for the anaerobic growth of At. ferrooxidans on elemental sulfur has been demonstrated and a linear relationship was obtained between the concentration of ferrous iron accumulated in the cultures and the increase in cell density [4]. It has been suggested a possible role in the solubilization of metals from sulfide ores involving the participation of the enzyme sulfur (sulfide): Fe (III) oxidoreductase [5]. Bacterial growth of At. ferrooxidans has also been reported in the oxidative anaerobic respiration using hydrogen as electron donor and ferric iron as electron acceptor [6]. Anaerobic reduction of ferric iron and ferrous iron production from pyrite by At. ferrooxidans has been demonstrated [2], however there are no reports about bacterial growth using this mineral. In this work, we studied the anaerobic bioleaching of pyrite with the aim to determine if At. ferrooxidans is capable to anaerobic growth on pyrite using ferric iron as electron acceptor.

Abstract: Biofilm development of F. acidiphilum BRGM4 on polycarbonate filters floating on liquid medium and pyrite surfaces were studied by confocal laser scanning microscopy (CLSM) and atomic force microscopy (AFM) combined with epifluorescence microscopy (EFM). Results show that F. acidiphilum biofilms were heterogeneously distributed, and varied among different growth conditions, such as inorganic phosphate (P_i) starvation and glucose supplementation. Biofilm and planktonic cells showed significant morphological differences. Capsular EPS were observed in both biofilm and planktonic cells. Cells showed preferential attachment to the cracks/defects of pyrite surfaces.

Abstract: A functional luxIR-type Quorum Sensing (QS) system is present in Acidithiobacillus ferrooxidans. However, cell-cell communication among various acidophilic chemolithoautotrophs growing on pyrite has not been studied in detail. These aspects are the scope of this study with emphasis on the effects exerted by the N-acyl-homoserine lactone (AHL) type signaling molecules which are produced by Acidithiobacillus ferrooxidans. Their effects on attachment and leaching efficiency by other leaching bacteria, such as Acidithiobacillus ferrivorans, Acidiferrobacter spp. SPIII/3 and Leptospirillum ferrooxidans in pure and mixed cultures growing on pyrite is shown.

Abstract: Bioleaching is the extraction of metals, such as copper or gold, from sulfidic ores by microorganisms. Their energy for growth is obtained by oxidation of ferrous iron and/or reduced inorganic sulfur compounds. Bacterial attachment to ores increase leaching activities through the formation of a special microenvironment between the bacterium and the metal sulfide surface, filled by extracellular polymeric substances (EPS). Recently, a high-throughput proteomic comparison from biofilm cells attached to pyrite (FeS₂) and planktonic cells of the mesophilic iron and/or sulfur oxidizer Acidithiobacillus ferrooxidans ATCC 23270 was done. Several proteins were found to be up-regulated in biofilm cells. Among them, membrane and outer membrane proteins probably involved in osmotic regulation, polysaccharide biosynthesis and protein secretion, as well as proteins probably involved in cofactor metabolism were present. In order to extend our knowledge of the genus Acidithiobacilli, we started a high-throughput proteomic analysis of the sulfur oxidizer Acidithiobacillus caldus ATCC 51756 by comparing cells grown with an insoluble energy substrate such as elemental sulfur (S°) against cells grown on a soluble energy substrate, such as thiosulfate. The results revealed several differences in proteins related to sulfur metabolism, potential EPS biosynthesis pathways as well as membrane and transport functions. In both microorganisms several conserved hypothetical proteins were found. Some of them were also found to be induced in sessile cells, suggesting their potential involvement in biofilm formation. This study will provide new insights into the biology of Acidithiobacilli and will probably help assigning functions to poorly characterized and unknown proteins. Keywords: Biofilm, Proteomics, Acidithiobacilli

Abstract: The ability of Acidithiobacillus ferrooxidans to get its energy from the oxidation of ferrous iron and the inhibitory effect of high ferric iron concentrations on its growth behaviour has been extensively studied. Furthermore it is known that A. ferrooxidans exudes organic substances called extracellular polymeric substances (EPS), which could play a role in its protection against adverse environmental conditions. In this context, the aim of this work was to study the production of EPS during adaptation of A. ferrooxidans to high ferric ion concentrations. The experiments were performed in shake flasks of 250 mL at 30 °C, 200 rpm and at an initial pH of 1.8. In order to establish the natural tolerance of the strain, its growth behaviour was evaluated at high ferric iron concentrations by adding consecutively the equivalent of 9 g/L of ferrous iron each time it was depleted in the broth. Cell growth stopped once ferric iron concentration increased up to 38 g/L. The adaptation consisted in eight sub-cultures run in parallel at initial concentrations of ferrous iron of 18, 27 and 36 g/L. The EPS was quantified as micro volumes using confocal laser scanning microscopy (CLSM), labelling the cells with propidium iodide and EPS carbohydrates with wheat germ agglutinin (WGA). During the adaptation procedure it was observed an increase in the ferric ion volumetric productivity of subcultures run with 27 and 36 g/L, as a result of cell adaptation. The amount of EPS exuded by cells was higher along with those experimental conditions having higher ferric iron concentrations. It was not detected EPS on cells grown on 9 g/L of ferrous iron. This study found that the adapted strain showed higher production of EPS at high ferric ion concentrations and higher ferric ion tolerance than non-adapted ones.

Abstract: The presence of extracellular polymeric substances (EPS) is important in the building of biofilms on mineral surfaces, increasing the bioleaching activity, as well as protecting the cells from adverse environmental conditions. The objective of this work was to study the effect of galactose in EPS production by Acidithiobacillus ferrooxidans. The experiences were performed in shake flask of 250 mL at 30 °C, 200 rpm and at an initial pH of 1.8. In order to establish the natural tolerance of the strain, its growth behaviour was evaluated at high ferric iron concentrations by adding consecutively the equivalent of 9 g/L of ferrous iron each time it was depleted in the broth. Cell growth stopped once ferric iron concentration increased up to 38 g/L. In order to determine the optimal conditions for EPS production, experiments were run in a chemostat of 0.5 L, operated at a constant dilution rate of 0.03 h^-1. Different steady states were obtained varying feeding concentrations of galactose (0.15%; 0.25% and 0.35%) and carbon dioxide (180 ppm and 360 ppm). , Cells grown in the chemostat at optimum operation conditions were used as inoculum to determine oxidative capacity of the microorganisms overproducing EPS. The EPS was quantified using confocal laser scanning microscopy (CLSM), labelling the cells with propidium iodide and EPS carbohydrates with wheat germ agglutinin (WGA). The higher volume production of EPS was observed in cells grown using 360 ppm of CO₂ and 0.35% of galactose. Also it was observed a size increment of cells, compared to cells grown in culture medium having 9 g/L of ferrous iron where presence of EPS was no detected. The results revealed that EPS overproducing A. ferrooxidans showed a tolerance to ferric iron concentration almost 9.5 g/L higher than the natural tolerance of cells grown in absence of galactose. Presence of galactose in culture medium stimulated the EPS production.

Abstract: Extracellular polymeric substances (EPS) play an important role in the attachment of bacteria to sulphide minerals, biofilm formation and efficiency of the bioleaching process. Previous studies have suggested a potential connection between galactose and EPS formation. In this context, the influence of exogenous galactose on EPS formation during the bioleaching of pyrite was studied. In order to fully adapt the microorganism to bioleaching conditions it was performed a total of five consecutive sub cultures, one every fifteen days, taking for each one inocula from previous culture in shake flasks with 200 ml of fermentation medium at 30°C, 200 rpm, 40 gL^-1 mineral and an initial pH of 1,8. Assays were performed in a medium supplemented with exogenous galactose (0.25% w/v) and without exogenous galactose (control), both with an initial concentration of ferric sulphate in the first three sub cultures of 5 gL^-1, decreasing in the last two sub cultures to 2.5 gL^-1. Samples of three cultures in both conditions were analyzed using confocal laser scanning microscopy (CLSM) labelling the cells with propidium iodide and EPS carbohydrates with Wheat Germ Agglutinin (WGA). Samples obtained on the last day of the fifth culture showed that the EPS layer on the particle surface was 5.00 μm³/μm² in the case of the control condition and 6.10 μm³/μm² when bioleaching was carried out in the presence of exogenous galactose. Also it was observed that in the fifth sub culture the volumetric productivity of total iron in the control experiment was 0.0065 gL^-1.h^-1 compared with 0.0076 gL^-1.h^-1 obtained in presence of galactose. The results reveal that the presence of galactose in the bioleaching solution stimulates EPS's formation and apparently also favour the pyrite bioleaching process.