Evaluation of Bacterial Community Structure and Its Influence on Sulfide Oxidation in a Bio-Leaching Environment

The mechanism of sulfide oxidation by adhering bacteria (direct oxidation mechanism) and by ferric ion in the aqueous phase was studied by quantitative assessment of bacterial activity on the sulfide surface. To probe for the principal bacterial species on the surface and in the supernatant, a library of DNA genes encoding portions of bacterial 16S rRNA was constructed. The PCR-amplified DNA from the bacterial populations was cloned employing PROMEGA's pGEM-T Easy Vector system. The clone frequency indicated that iron-oxidizing bacteria were dominant in the liquid phase, while Acidithiobacillus ferroixdans, which is both sulfur and iron oxidizer was the most prevalent on the sulfide surface. Samples of crystalline pyrite were exposed to the bacterial consortium to evaluate surface alterations caused by bacteria. Chemical (abiotic) oxidation experiments with ferric ion as the oxidant were carried out in parallel with the biological oxidation tests. Changes in the surface topography were monitored by atomic force microscopy (AFM) while changes in surface chemistry were examined by Raman spectroscopy. Bacterial attachment resulted in a 53% increase in the specific surface area in comparison to a 13% increase caused by chemical (ferric ion) oxidation. The oxidation rate was assessed by evaluating the iron release. After corrections for surface area changes, the specific abiotic (oxidation by Fe^{3 +}) and biotic oxidation rates with adhering bacteria were nearly the same (2.6 × 10^{? 9} mol O₂/s/m² versus 3.3 × 10^{? 9} mol O₂/s/m²) at pH = 2 and a temperature of 25°C. The equality of rates implies that the availability of ferric ion as the oxidant is rate limiting.     

Reactive oxygen species generated in the presence of fine pyrite particles and its implication in thermophilic mineral bioleaching

In the tank bioleaching process, maximising solid loading and mineral availability, the latter through decreasing particle size, are key to maximising metal extraction. In this study, the effect of particle size distribution on bioleaching performance and microbial growth was studied through applying knowledge based on medical geology research to understand the adverse effects of suspended fine pyrite particles. Small-scale leaching studies, using pyrite concentrate fractions (106–75, 75–25, ?25 μm fines), were used to confirm decreasing performance with decreasing particle size (D ₅₀ <40 μm). Under equivalent experimental conditions, the generation of the reactive oxygen species (ROS), hydrogen peroxide and hydroxyl radicals from pyrite was illustrated. ROS generation measured from the different pyrite fractions was found to increase with increasing pyrite surface area loading (1.79–74.01 m² L^?1) and Fe²⁺ concentration (0.1–2.8 g?L^?1) in solution. The highest concentration of ROS was measured from the finest fraction of pyrite (0.85 mM) and from the largest concentration of Fe²⁺ (0.78 mM). No ROS was detected from solutions containing only Fe³⁺ under the same conditions tested. The potential of ROS to inhibit microbial performance under bioleaching conditions was demonstrated. Pyrite-free Sulfolobus metallicus cultures challenged with hydrogen peroxide (0.5–2.5 mM) showed significant decrease in both cell growth and Fe²⁺ oxidation rates within the concentration range 1.5–2.5 mM. In combination, the results from this study suggest that conditions of large pyrite surface area loading, coupled with high concentrations of dissolved Fe²⁺, can lead to the generation of ROS, resulting in oxidative stress of the microorganisms.     

Effect of Aluminum on the Production of Siderophore by Rhizobium sp. (Cicer arietinum)

Rhizobium sp. strain BICC 651 in the presence of 100 μM Al³⁺ produced a threefold higher level of siderophore than in the control culture under iron limitation during the stationary phase. Al³⁺ in increasing concentrations resulted in decreased growth, and the effect was alleviated by the addition of iron. Siderophore production decreased gradually in Al³⁺-treated culture as well as in the control with the addition of increasing concentrations of Fe³⁺, and at 50 μM Fe³⁺ the level of siderophore was practically undetectable. The siderophore binds Fe³⁺ and also Al³⁺. The outer membrane protein profiles of the bacteria grown in the presence or absence of Al³⁺ were indistinguishable. Received: 15 November 1999 / Accepted: 21 December 1999     

Adaptation of Thiobacillus ferrooxidans to nickel ion and bacterial oxidation of nickel sulfide

Summary The Ni²⁺ resistance of Thiobacillus ferrooxidans was enhanced by repeated culturing in medium containing Ni²⁺ and gradually increasing the Ni²⁺ concentration. The extraction of nickel sulfide was enhanced by the adapted strain following the direct leaching mechanism of the microorganism.     

Role of Ferrous Ions in Synthetic Cobaltous Sulfide Leaching of Thiobacillus ferrooxidans

Microbiological leaching of synthetic cobaltous sulfide (CoS) was investigated with a pure strain of Thiobacillus ferroxidans. The strain could not grow on CoS-salts medium in the absence of ferrous ions (Fe²⁺). However, in CoS-salts medium supplemented with 18 mM Fe²⁺, the strain utilized both Fe²⁺ and the sulfur moiety in CoS for growth, resulting in an enhanced solubilization of Co²⁺. Cell growth on sulfur-salts medium was strongly inhibited by Co²⁺, and this inhibition was completely protected by Fe²⁺. Cobalt-resistant cells, obtained by subculturing the strain in medium supplemented with both Fe²⁺ and Co²⁺, brought a marked decrease in the amount of Fe²⁺ absolutely required for cell growth on CoS-salts medium. As one mechanism of protection by Fe²⁺, it is proposed that the strain utilizes one part of Fe²⁺ externally added to CoS-salts medium to synthesize the cobalt-resistant system. Since a similar protective effect by Fe²⁺ was also observed for cell inhibition by stannous, nickel, zinc, silver, and mercuric ions, a new role of Fe²⁺ in bacterial leaching in T. ferrooxidans is proposed.     

Production of Microbial Cells from Hydrocarbons

Hydrocarbon-assimilating yeasts and bacteria were isolated from soil and sewage. The optimal conditions of cell yield from liquid paraffine by a Torulopsis yeast and a Pseudomonas strain were studied. A Torulopsis yeast gave, in optimal condition, 70 percent cell yield on a weight conversion basis from light oil fraction. In a strain of Pseudomonas the additions of amino acids, Fe^{+ +}, Mg^{+ +} and Ca^{+ +} ions were effective for cell production. This strain showed, in optimal condition, 80 percent cell yield (wt%) from kerosene.     

Pyrite oxidation by Thiobacillus ferrooxidans with special reference to the sulphur moiety of the mineral

Available cultures of Thiobacillus ferrooxidans were found to be contaminated with bacteria very similar to Thiobacillus acidophilus. The experiments described were performed with a homogeneous culture of Thiobacillus ferrooxidans.Pyrite (FeS₂) was oxidized by Thiobacillus ferrooxidans grown on iron (Fe²⁺), elemental sulphur (S^o) or FeS₂.Evidence for the direct utilization of the sulphur moiety of pyrite by Thiobacillus ferrooxidans was derived from the following observations: a. Known inhibitors of Fe²⁺ and S^o oxidation, NaN₃ and NEM, respectively, partially abolished FeS₂ oxidation. b. A b-type cytochrome was detectable in FeS₂-and S^o-grown cells but not in Fe²⁺-grown cells. c. FeS₂ and S^o reduced b-type cytochromes in whole cells grown on S^o. d. CO₂ fixation at pH 4.0 per mole of oxygen consumed was the highest with S^o, lowest with Fe²⁺ and medium with FeS₂ as substrate. e. Bacterial Fe²⁺ oxidation was found to be negligible at pH 5.0 whereas both FeS₂ and S^o oxidation was still appreciable above this pH. f. Separation of pyrite and bacteria by means of a dialysis bag caused a pronounced drop of the oxidation rate which was similar to the reduction of pyrite oxidation by NEM; indirect oxidation of the sulphur moiety by Fe³⁺ was not affected by separation of pyrite and bacteria.Bacterial oxidation and utilization of the sulphur moiety of pyrite were relatively more important with increasing pH.     

Modelling of Fe2 + oxidation by Thiobacillus ferrooxidans

Summary The kinetics of oxidation of aqueous acidic ferrous sulphate by Thiobacillus ferrooxidans has been studied in a batch reactor. The contribution of cell wall envelopes to the oxidation rate has been shown to be negligible. A model which accounts for the oxidation of Fe^{2 +}, death of bacteria due to Fe^{3 +} poisoning, existence of an optimal pH and precipitation of Fe^{3 +} has been proposed. The model is able to predict the concentration of Fe^{2 +} and pH quite satisfactorily. The predictions of Fe^{3 +} are not so accurate because of simplifying assumptions made about its precipitation. Offprint requests to: R. Kumar     

Sugar-specific Attachment of Pseudomonas syringae pv. glycinea to Isolated Single Leaf Cells of Resistant Soybean Cultivars

Phase-contrast and scanning electron microscopy showed races of P. synngae pv. glycinea uniformly distributed over and attached to the whole surface of isolated single leaf cells of resistant soybean cultivars, as early as 30 to 180 min after inoculation. On the contrary, attachment in the compatible interaction did not occur within 10—15 h. In a later period, compatibility was characterized by the formation of adherent bacterial clusters. Early attachment of races 1 and 6 to cv. Harosoy and that of race 5 to cv. Flambeau leaf cells, each representing incompatible interaction, could be inhibited by L-rhamnose and D-glucose, respectively. Furthermore, the lack of Mn²⁺ and Fe²⁺ and heat-treatment of plant cells also affected the early attachment in incompatible combinations and resulted in cluster formation, suggesting incompatibility rather than compatibility to be the active phenomenon. Pre-inoculation of cells with an incompatible race induced changes that caused compatible bacteria also to distributively attach to plant cell surface indicating that a transfer of information or surface alterations occur upon attachment in incompatible interaction.     

Rate Equations and Kinetic Parameters of the Reactions Involved in Pyrite Oxidation by Thiobacillus ferrooxidans

Rate equations and kinetic parameters were obtained for various reactions involved in the bacterial oxidation of pyrite. The rate constants were 3.5 μM Fe²⁺ per min per FeS₂ percent pulp density for the spontaneous pyrite dissolution, 10 μM Fe²⁺ per min per mM Fe³⁺ for the indirect leaching with Fe³⁺, 90 μM O₂ per min per mg of wet cells per ml for the Thiobacillus ferrooxidans oxidation of washed pyrite, and 250 μM O₂ per min per mg of wet cells per ml for the T. ferrooxidans oxidation of unwashed pyrite. The K_m values for pyrite concentration were similar and were 1.9, 2.5, and 2.75% pulp density for indirect leaching, washed pyrite oxidation by T. ferrooxidans, and unwashed pyrite oxidation by T. ferrooxidans, respectively. The last reaction was competitively inhibited by increasing concentrations of cells, with a K_i value of 0.13 mg of wet cells per ml. T. ferrooxidans cells also increased the rate of Fe²⁺ production from Fe³⁺ plus pyrite.     

Process for biological oxidation and control of dissolved iron in bioleach liquors

Iron has a central role in bioleaching and biooxidation processes. Fe²⁺ produced in the dissolution of sulfidic minerals is re-oxidized to Fe³⁺ mostly by biological action in acid bioleaching processes. To control the concentration of iron in solution, it is important to precipitate the excess as part of the process circuit. In this study, a bioprocess was developed based on a fluidized-bed reactor (FBR) for Fe²⁺ oxidation coupled with a gravity settler for precipitative removal of ferric iron. Biological iron oxidation and partial removal of iron by precipitation from a barren heap leaching solution was optimized in relation to the performance and retention time (τ_FBR) of the FBR. The biofilm in the FBR was dominated by Leptospirillum ferriphilum and “Ferromicrobium acidiphilum.” The FBR was operated at pH 2.0 ± 0.2 and at 37 °C. The feed was a barren leach solution following metal recovery, with all iron in the ferrous form. 98–99% of the Fe²⁺ in the barren heap leaching solution was oxidized in the FBR at loading rates below 10 g Fe²⁺/L h (τ_FBR of 1 h). The optimal performance with the oxidation rate of 8.2 g Fe²⁺/L h was achieved at τ_FBR of 1 h. Below the τ_FBR of 1 h the oxygen mass transfer from air to liquid limited the iron oxidation rate. The precipitation of ferric iron ranged from 5% to 40%. The concurrent Fe²⁺ oxidation and partial precipitative iron removal was maximized at τ_FBR of 1.5 h, with Fe²⁺ oxidation rate of 5.1 g Fe²⁺/L h and Fe³⁺ precipitation rate of 25 mg Fe³⁺/L h, which corresponded to 37% iron removal. The precipitates had good settling properties as indicated by the sludge volume indices of 3–15 mL/g but this step needs additional characterization of the properties of the solids and optimization to maximize the precipitation and to manage sludge disposal.     

Biological Oxidation of Sulfide Raw Material Using a Culture of Thiobacillus ferrooxidans under Various Conditions of Leaching

Major parameters of the first stage of leaching of a copper–zinc sulfide product (raw material) by a culture of Thiobacillus ferrooxidans have been studied, including the effects of solid-phase concentration, Fe²⁺ and Fe³⁺ ions, pH, and the intensity of mixing. The first stage of leaching of the sulfide raw material is optimum under the following conditions: pH of the original leaching solution equal to 1.6; Fe³⁺ concentration of the order of 10 g/l; and vigorous mixing of the suspension at solid-phase concentrations of 30–35%. A theoretical substantiation of the observed dependences is proposed.     

Structural changes ofNoetia ponderosa red blood cell membranes during cell volume regulation in reduced salinities: A freeze fracture study

Summary Changes in molluscan blood cell membrane structure coincided with changes in membrane amino acid permeability during cell volume regulation. Blood cells were freeze fractured after the free amino acid permeability of their membranes had been altered by modifying the extracellular Ca²⁺ and intracellular ATP levels and the membrane particles examined for changes in size, number/area and distribution. Test substances that altered the divalent cation or ATP levels also altered membrane particle densities, but not size or distribution, of freeze fractured blood cells. Those test substances (Ca²⁺-free seawater, DNP, low temperature) that inhibited volume regulation and the FAA efflux caused decreased membrane particle density, while those test substances (Co²⁺, Mn²⁺) that potentiated volume regulation and the FAA efflux increased the number of membrane particles/unit area. These changes in membrane particle density appear to result from the changes in surface area due to the treatment effects on cell volume, so that the number of membrane particles per cell remained constant. Therefore, altered membrane FAA permeability is associated with altered membrane particle density, but the effect of this structural alteration on membrane permeability is not clear.Abbreviations FAA free amino acid - DMSO dimethylsulfoxide - DNP dinitrophenol - ASW artificial seawater     

Regulation of Fe3+-oxide Formation Among Fe2+-oxidizing Bacteria

Helical stalks (resembling Gallionella ferruginea, Mariprofundus ferrooxydans) and filamentous sheaths (resembling Leptothrix ochracea) of Fe²⁺-oxidizing bacteria (FeOB) are mineralized by hydrous ferric oxides (HFO). To perform both inter-species and inter-site size comparisons of HFO particles on stalks and sheaths we measured HFO particles in samples of natural bacteriogenic iron oxides (BIOS) from 3 contrasting field sites: the Loihi Seamount (southern Hawaii); Äspö Hard Rock Laboratory (eastern Sweden); and Chalk River Laboratories (northern Canada) representing seafloor saline, underground brackish, and surface freshwater aqueous conditions. Ambient temperatures were in the psychrophilic range and pHs measured for Loihi, CRL, and Äspö were 5.6, 6.9 and 7.4, respectively. Dissolved Fe was lowest for CRL (0.2 mg · L^?1) followed by Äspö (1.5 mg · L^?1), then Loihi (4.5–14.9 mg · L^?1). L. ochraceasheaths appear to have surface properties that restrict HFO particle growth in comparison to G.ferruginea-M.ferrooxydans stalks in the same environment, which we attribute to interfacial surface energy (γ). An inverse relationship between particle size and stalk/sheath length due to restrictions in reactive surface area was also observed, which may provide insight into FeOB survival strategies to alleviate oxidative stress arising from Fe³⁺ production.     

Antimicrobial activity of Fe‐loaded chitosan nanoparticles

To increase the antimicrobial activities of chitosan, chitosan nanoparticles loaded with Fe²⁺ or Fe³⁺ were prepared by surfactant‐assisted chitosan chelating Fe²⁺, Fe³⁺ and ionic gelation chitosan. Their chelating rates were determined by spectrophotometry. The particle sizes and zeta potentials of chitosan nanoparticles loaded with Fe²⁺ or Fe³⁺ were measured by size and zeta potential analysis. The nanoparticles antimicrobial activities were evaluated by different concentration against Escherichia coli, Staphylococcus aureus, Candida albicans in vitro. Results showed that the mean diameter of chitosan nanoparticles loaded with Fe²⁺ or Fe³⁺ were 206.4 and 195.2 nm, respectively. Their zeta potentials were +28.82 and +28.26 mV, respectively. The chelating rate of chitosan nanoparticles loaded with Fe²⁺ was greatly higher than with Fe³⁺. Their antimicrobial activity was showed greatly higher at lower concentrations compared to chitosan, and the antibacterial effect of chitosan nanoparticles loaded with Fe²⁺ or Fe³⁺ was preliminary observed.     

The role of iron uptake in pathogenicity and symbiosis in <Emphasis Type="Italic">Photorhabdus luminescens</Emphasis> TT01

Background  

Photorhabdus are Gram negative bacteria that are pathogenic to insect larvae whilst also having a mutualistic interaction with nematodes from the family Heterorhabditis. Iron is an essential nutrient and bacteria have different mechanisms for obtaining both the ferrous (Fe²⁺) and ferric (Fe³⁺) forms of this metal from their environments. In this study we were interested in analyzing the role of Fe³⁺ and Fe²⁺ iron uptake systems in the ability of Photorhabdus to interact with its invertebrate hosts.     

Biogeochemical Properties of Bacteriogenic Iron Oxides

Bacteriogenic iron oxides (BIOS) are composite materials that consist of intact and partly degraded remains of bacterial cells intermixed with variable amounts of poorly ordered hydrous ferric oxide (HFO) minerals. They form in response to chemical or bacterial oxidation of Fe²⁺, which gives rise to Fe³⁺. Once formed, Fe³⁺ tends to undergo hydrolysis to precipitate in association with bacterial cells. In acidic systems where the chemical oxidation of Fe²⁺ is slow, bacteria are capable of accelerating the reaction by several orders of magnitude. At circumneutral pH, the chemical oxidation of Fe²⁺ is fast. This requires Fe²⁺ oxidizing bacteria to exploit steep redox gradients where low pO₂ slows the abiotic reaction enough to allow the bacteria to compete kinetically. Because of their reactive surface properties, BIOS behave as potent sorbents of dissolved metal ions. Strong enrichments of Al, Cu, Cr, Mn, Sr, and Zn in the solid versus aqueous phase (log 10 K_d values range from 1.9 to 4.2) are common; however, the metal sorption properties of BIOS are not additive owing to surface chemical interactions between the constituent HFO and bacteria. These interactions have been investigated using acid-base tritrations, which show that the concentration of high pK_a sites is reduced in BIOS compared to HFO. At the same time, hydroxylamine insoluble material (i.e., residual bacterial fraction) is enriched in low pK_a sites relative to both BIOS and HFO. These differences indicate that low pK_a or acidic sites associated with bacteria in BIOS interact specifically with high pK_a or basic sites on intermixed HFO.     

A conserved haem redox and trafficking pathway for cofactor attachment

A pathway for cytochrome c maturation (Ccm) in bacteria, archaea and eukaryotes (mitochondria) requires the genes encoding eight membrane proteins (CcmABCDEFGH). The CcmABCDE proteins are proposed to traffic haem to the cytochrome c synthetase (CcmF/H) for covalent attachment to cytochrome c by unknown mechanisms. For the first time, we purify pathway complexes with trapped haem to elucidate the molecular mechanisms of haem binding, trafficking and redox control. We discovered an early step in trafficking that involves oxidation of haem (to Fe³⁺), yet the final attachment requires reduced haem (Fe²⁺). Surprisingly, CcmF is a cytochrome b with a haem never before realized, and in vitro, CcmF functions as a quinol:haem oxidoreductase. Thus, this ancient pathway has conserved and orchestrated mechanisms for trafficking, storing and reducing haem, which assure its use for cytochrome c synthesis even in limiting haem (iron) environments and reducing haem in oxidizing environments.     

Influence of Trace Elements Mixture on Bacterial Diversity and Fermentation Characteristics of Liquid Diet Fermented with Probiotics under Air-Tight Condition

Cu²⁺, Zn²⁺, Fe²⁺ and I⁻ are often supplemented to the diet of suckling and early weaning piglets, but little information is available regarding the effects of different Cu²⁺, Zn²⁺, Fe²⁺ and I⁻ mixtures on bacteria growth, diversity and fermentation characteristics of fermented liquid diet for piglets. Pyrosequencing was performed to investigate the effect of Cu²⁺, Zn²⁺, Fe²⁺ and I⁻ mixtures on the diversity, growth and fermentation characteristics of bacteria in the liquid diet fermented with Bacillus subtilis and Enterococcus faecalis under air-tight condition. Results showed that the mixtures of Cu²⁺, Zn²⁺, Fe²⁺ and I⁻ at different concentrations promoted Bacillus growth, increased bacterial diversity and lactic acid production and lowered pH to about 5. The importance of Cu²⁺, Zn²⁺, Fe²⁺ and I⁻ is different for Bacillus growth with the order Zn²⁺> Fe²⁺>Cu²⁺> I⁻ in a 21-d fermentation and Cu²⁺>I⁻>Fe²⁺>Zn²⁺ in a 42-d fermentation. Cu²⁺, Zn²⁺, Fe²⁺ and I⁻ is recommended at a level of 150, 60, 150 and 0.6 mg/kg respectively for the production of fermented liquid diet with Bacillus subtilis. The findings improve our understanding of the influence of trace elements on liquid diet fermentation with probiotics and support the proper use of trace elements in the production of fermented liquid diet for piglets.