Culture-dependent hunt and characterization of iron-oxidizing bacteria in Baiyin Copper Mine, China, and their application in metals extraction.

The exploration of microbial diversity in extremely acidic habitats has provided a vital base for the progression of minerals biotechnology. Three indigenous iron-oxidizing acidophilic bacterial strains were isolated through serial dilution of enriched bacterial culture from Baiyin Copper Mine Stope, China. The morphological, biochemical, physiological, and phylogenetic characteristics of isolates were investigated. These isolates were motile, Gram-negative, and curved shape with pleomorphism except isolate WG101 that was a straight rod. The optimum growth pH and temperature for all isolates were 1.5 and 30 °C, respectively, and showed extreme acidophilic nature. All the isolates showed obligate chemoautotrophic nature and used ferrous iron and pyrite as an energy source, however, isolates WG102 and WG103 were unable to use sulfur, while isolate WG101 could use elemental sulfur and reduced inorganic sodium thiosulfate as an energy source. The phylogenetic analyses based on 16S rRNA sequences revealed that the isolates WG101, WG102, and WG103 were homologous with Acidithiobacillus ferrooxidans strain AS2 (99%), Leptospirillum ferriphilum strain YSK (98%), and Leptospirillum ferrooxidans strain L15 (98%), respectively. These bacterial isolates showed efficient copper and zinc dissolution from the ore. The metals dissolution rate of At. ferrooxidans strain WG101 was 54.5 ± 4.33% (copper) and 49.6 ± 5% (zinc). The metals recovery rate of L. ferriphilum strain WG102 was 45.7 ± 3.5% (copper) and 40.5 ± 2.5% (zinc). The recovery rate of copper and zinc was 49.6 ± 4% and 46.5 ± 3% respectively in the case of L. ferrooxidans strain WG103. The findings of this study are consistent with the notion that the indigenous bacteria are more efficient in minerals dissolution.

Generation of acid mine drainage around the Karaerik copper mine (Espiye, Giresun, NE Turkey): implications from the bacterial population in the Acisu effluent.

The Karaerik Cu mine is a worked-out deposit with large volumes of tailings and slags which were left around the mine site without any protection. Natural feeding of these material and run-off water from the mineralised zones into the Acisu effluent causes a serious environmental degradation and creation of acid mine drainage (AMD) along its entire length. This research aims at modelling the formation of AMD with a specific attempt on the characterisation of the bacterial population in association with AMD and their role on its occurrence. Based on 16SrRNA analyses of the clones obtained from a composite water sample, the bacterial community was determined to consist of Acidithiobacillus ferrivorans, Ferrovum myxofaciens, Leptospirillum ferrooxidans and Acidithiobacillus ferrooxidans as iron-oxidising bacteria, Acidocella facilis, Acidocella aluminiidurans, Acidiphilium cryptum and Acidiphilium multivorum as iron-reducing bacteria, and Acidithiobacillus ferrivorans, Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans and Acidiphilium cryptum as sulphur-oxidising bacteria. This association of bacteria with varying roles was interpreted as evidence of a concomitant occurrence of sulphur and iron cycles during the generation of AMD along the Acisu effluent draining the Karaerik mine.

Model-based evaluation of ferrous iron oxidation by acidophilic bacteria in chemostat and biofilm airlift reactors.

This article presents a model-based evaluation of ferrous iron oxidation in chemostat and biofilm airlift reactors inoculated with a mixed culture of Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans bacteria. The competition between the two types of bacteria in the chemostat and in the biofilm airlift reactors together with the distribution of both bacteria along the biofilm thickness at different time sections has been studied. The bacterial distribution profiles along the biofilm in the airlift reactor at different time scales show that in the beginning A. ferrooxidans bacteria are dominant, but when the reactor operates for a long time the desirable L. ferrooxidans species outcompete A. ferrooxidans as a result of the low Fe(2+) and high Fe(3+) concentrations. The results obtained from the simulation were compared with the experimental data of continuously operated internal loop airlift biofilm reactor. The model results are in good agreement with the experimental results.

Bio/phyremediation potential of Leptospirillum ferrooxidans and Ricinus communis on metal contaminated mine sludge.

The heavy metal pollution is a serious environmental pollution around the globe and threatens the ecosystem. The physicochemical traits (pH, Electrical conductivity, hardness, NPK, Al, Fe, Cd, Cr, Pb, Mg, and Mn) of soil sample collected from the polluted site were analyzed and found that the most of the metal contents were beyond the acceptable limits of national standards. The metals such as Mn (1859.37 ± 11.25 mg kg-1), Cd (24.86 ± 1.85 mg kg-1), Zn (795.64 ± 9.24 mg kg-1), Pb (318.62 ± 5.85 mg kg-1), Cr (186.84 ± 6.84 mg kg-1), and Al (105.84 ± 5.42 mg kg-1) were crossing the permissible limits. The pre-isolated L. ferrooxidans showed considerable metal tolerance to metals such as Al, Cd, Cr, Pb, Mg, and Mn at up to the concentration of 750 µg mL-1 and also have remediation potential on polluted soil in a short duration of treatment. The greenhouse study demonstrated that the bio/phytoremediation potential of metal tolerant L. ferrooxidans and R. communis under various remediation (A, B, and C) groups. Surprisingly, remediation group C demonstrated greater phytoextraction potential than the other remediation groups (A and B). These results strongly suggest that coexistence of L. ferrooxidans and R. communis had a significant positive effect on phytoextraction on metal-contaminated soil.

Bioleaching of Chalcopyrite Waste Rock in the Presence of the Copper Solvent Extractant LIX984N.

Heap bioleaching, the solubilization of metal ions from metal sulfides by microbial oxidation, is often combined with solvent extraction (SX) and electrowinning to recover, e.g., copper from low-grade ores. After extraction, the leaching solution is recycled, but the entrained organic solvents may be toxic to the microorganisms. Here Acidithiobacillus ferrooxidans, Leptospirillum ferrooxidans, and Sulfobacillus thermosulfidooxidans were selected to perform bioleaching of chalcopyrite waste rock in the presence of the SX reagent (2.5% v/v LIX984N in kerosene). Possibly inhibitory effects have been evaluated by copper extraction, bacterial activity, number of actively Fe(II)-oxidizing cells, and biofilm formation. Microcalorimetry, most probable number determination, and atomic force microscopy combined with epifluorescence microscopy were applied. The results show that 100 and 300 mg/L SX reagent could hardly inhibit At. ferrooxidans from oxidizing Fe2+, but they seriously interfered with the biofilm formation and the oxidization of sulfur, thereby hindering bioleaching. L. ferrooxidans was sensitive to 50 mg/L SX reagent, which inhibited its bioleaching completely. Sb. thermosulfidooxidans showed different metabolic preferences, if the concentration of the SX reagent differed. With 10 mg/L LIX984N Sb. thermosulfidooxidans preferred to oxidize Fe2+ and extracted the same amount of copper as the assay without LIX984N. With 50 mg/L extractant the bioleaching stopped, since Sb. thermosulfidooxidans preferred to oxidize reduced inorganic sulfur compounds.

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.

Stoichiometric model and flux balance analysis for a mixed culture of Leptospirillum ferriphilum and Ferroplasma acidiphilum.

The oxidation process of sulfide minerals in natural environments is achieved by microbial communities from the Archaea and Bacteria domains. A metabolic reconstruction of two dominant species, Leptospirillum ferriphilum and Ferroplasma acidiphilum, which are always found together as a mixed culture in this natural environments, was made. The metabolic model, composed of 152 internal reactions and 29 transport reactions, describes the main interactions between these species, assuming that both use ferrous iron as energy source, and F. acidiphilum takes advantage of the organic compounds secreted by L. ferriphilum for chemomixotrophic growth. A first metabolic model for a mixed culture used in bacterial leaching is proposed in this article, which pretends to represent the characteristics of the mixed culture in a simplified manner. It was evaluated with experimental data through flux balance analysis (FBA) using as objective function the maximization of biomass. The growth yields on ferrous iron obtained for each microorganism are consistent with experimental data, and the flux distribution obtained allows understanding of the metabolic capabilities of both microorganisms growing together in a bioleaching process. The model was used to simulate the growth of F. acidiphilum on different substrates, to determine in silico which compounds maximize cell growth, and which are essential. Knockout simulations were carried out for L. ferriphilum and F. acidiphilum metabolic models, predicting key enzymes of central metabolism. The results of this analysis are consistent with experimental data from literature, showing a robust behavior of the metabolic model.

Quantifying adhesion of acidophilic bioleaching bacteria to silica and pyrite by atomic force microscopy with a bacterial probe.

The adhesion of acidophilic bacteria to mineral surfaces is an important phenomenon in bioleaching processes. In this study, functionalized colloidal probes covered by bioleaching bacterial cells (Acidithiobacillus thiooxidans and Leptospirillum ferrooxidans) were developed and used to sense specific adhesion forces to a silica surface and a pyrite surface in various solutions. Experimentally, recorded retraction curves of A. thiooxidans revealed sawtooth features that were in good agreement with the wormlike chain model, while that of L. ferrooxidans exhibited stair-step separation. The magnitudes of adhesion forces and snap-off distances were strongly influenced by the ionic strength and pH. Macroscopic surface properties including hydrophobicity and surface potential for bacterial cells and substrata were measured by a sessile drop method and microelectrophoresis. The ATR-FTIR spectra indicated the presence of different types of biopolymers on two strains of bacteria.

In situ Spectroscopy on Intact Leptospirillum ferrooxidans Reveals that Reduced Cytochrome 579 is an Obligatory Intermediate in the Aerobic Iron Respiratory Chain.

Electron transfer reactions among colored cytochromes in intact bacterial cells were monitored using an integrating cavity absorption meter that permitted the acquisition of accurate absorbance data in suspensions of cells that scatter light. The aerobic iron respiratory chain of Leptospirillum ferrooxidans was dominated by the redox status of an abundant cellular cytochrome that had an absorbance peak at 579 nm in the reduced state. Intracellular cytochrome 579 was reduced within the time that it took to mix a suspension of the bacteria with soluble ferrous iron at pH 1.7. Steady state turnover experiments were conducted where the initial concentrations of ferrous iron were less than or equal to that of the oxygen concentration. Under these conditions, the initial absorbance spectrum of the bacterium observed under air-oxidized conditions was always regenerated from that of the bacterium observed in the presence of Fe(II). The kinetics of aerobic respiration on soluble iron by intact L. ferrooxidans conformed to the Michaelis-Menten formalism, where the reduced intracellular cytochrome 579 represented the Michaelis complex whose subsequent oxidation appeared to be the rate-limiting step in the overall aerobic respiratory process. The velocity of formation of ferric iron at any time point was directly proportional to the concentration of the reduced cytochrome 579. Further, the integral over time of the concentration of the reduced cytochrome was directly proportional to the total concentration of ferrous iron in each reaction mixture. These kinetic data obtained using whole cells were consistent with the hypothesis that reduced cytochrome 579 is an obligatory steady state intermediate in the iron respiratory chain of this bacterium. The capability of conducting visible spectroscopy in suspensions of intact cells comprises a powerful post-reductionist means to study cellular respiration in situ under physiological conditions for the organism.