Temperature Effects on Bacterial Leaching of Sulfide Minerals in Shake Flask Experiments

The microbiological leaching of a sulfide ore sample was investigated in shake flask experiments. The ore sample contained pyrite, pyrrhotite, pentlandite, sphalerite, and chalcopyrite as the main sulfide minerals. The tests were performed at eight different temperatures in the range of 4 to 37°C. The primary data were used for rate constant calculations, based on kinetic equations underlying two simplified models of leaching, i.e., a shrinking particle model and a shrinking core model. The rate constants thus derived were further used for the calculation of activation energy values for some of the sulfide minerals present in the ore sample. The chalcopyrite leaching rates were strongly influenced by the interaction of temperature, pH, and redox potential. Sphalerite leaching could be explained with the shrinking particle model. The data on pyrrhotite leaching displayed good fit with the shrinking core model. Pyrite leaching was found to agree with the shrinking particle model. Activation energies calculated from the rate of constants suggested that the rate-limiting steps were different for the sulfide minerals examined; they could be attributed to a chemical or biochemical reaction rather than to diffusion control.     

Alterations in surfaces and textures of minerals during the bacterial leaching of a complex sulfide ore

Abstract

The purpose of the work was to characterize changes in surface textures of minerals during the biological leaching of a complex sulfide ore. The ore contained pyrrhotite (Fe_I__xS), pyrite (FeS₂), sphalerite (ZnS), pentlandite [(Ni,Fe,Co)₉S₈], and chalcopyrite (CuFeS₂). Several mixed cultures were initially screened using the ore material as the sole substrate. Shake flask leaching experiments showed no major differences among test cultures, which were all derived by enrichment techniques using environmental samples collected from a mine site. Leached pyrrhotite surfaces were invariably surrounded by a dark rim of elemental S. A reaction zone was also associated with leached sphalerite grains. Chemical analyses of leach solutions indicated that the relative ranking of biological leaching of the sulfide minerals was Zn > Ni > Co > Cu. Microscopic observations were in keeping with this rankin     

Mineral Products of Pyrrhotite Oxidation by Thiobacillus ferrooxidans

The biological leaching of pyrrhotite (Fe_1-xS) by Thiobacillus ferrooxidans was studied to characterize the oxidation process and to identify the mineral weathering products. The process was biphasic in that an initial phase of acid consumption and decrease in redox potential was followed by an acid-producing phase and an increase in redox potential. Elemental S was one of the first products of pyrrhotite degradation detected by X-ray diffraction. Pyrrhotite oxidation also yielded K-jarosite [KFe₃(SO₄)₂(OH)₆], goethite (α-FeOOH), and schwertmannite [Fe₈O₈(OH)₆SO₄] as solid-phase products. Pyrrhotite was mostly depleted after 14 days, whereas impurities in the form of pyrite (cubic FeS₂) and marcasite (orthorhombic FeS₂) accumulated in the leach residue.     

Microbiological Oxidation of Ferrous Iron at Low Temperatures

Acidophilic iron-oxidizing bacteria were enriched from mine water samples with ferrous sulfate as the substrate at incubation temperatures in the range of 4 to 46°C. After several subcultures at each test temperature except 46°C, which was prohibitive to growth, the rates of iron oxidation were determined in batch cultures. The results yielded linear rates in a semilogarithmic scale. The rate constants of iron oxidation by growing cultures were fitted into the Arrhenius equation, which displayed linearity in the 4 to 28°C range and yielded an activation energy value of 83 ± 3 kJ/mol.     

Kinetics of Sulfur Oxidation at Suboptimal Temperatures

Chemolithoautotrophic bacteria were enriched from mine water at incubation temperatures ranging from 4 to 46°C, using elemental sulfur as a substrate in acid mineral salts media. Thiobacillus-type bacteria were successfully enriched for at all test temperatures except 46°C. Changes in pH (−dpH/dt) were used to estimate the rate constants for the enrichment cultures. The rate constants yielded a linear Arrhenius plot, an activation energy of 65 kJ/mol, and a temperature coefficient (Q₁₀) of 2.1 for the 4 to 37°C temperature interval.     

Leaching Soluble Salts Increases Population Densities of Tylenchulus semipenetrans

The effect of salinity on population densities of Tylenchulus semipenetrans was measured on 3-month-old salt-tolerant Rangpur lime growing on either loamy sand, sand, or organic mix and on 4-month-old salt-sensitive Sweet lime in organic mix. Salinity treatments were initiated by watering daily with 25 mol/m³ NaCl + 3.3 mol/m³ CaCl₂ for 3 days and every other day with 50 mol/m³ NaC1 + 6.6 mol/m³ CaC1₂ for one week, with no salt (NS) treatments as controls. Salinity was discontinued in one treatment (DS) by leaching with tap water prior to inoculation with nematodes, whereas the continuous salinity (CS) treatment remained unchanged. Overall, in Rangpur lime organic soil supported the highest population densities of T. semipenetrans, followed by loamy sand and sand. The DS treatment resulted in the highest (P ≤ 0.05) mean population densities of T. semipenetrans in the three soil types. Similarly, the DS treatment in Sweet lime resulted in the highest (P ≤ 0.05) nematode populations. The DS treatment predisposed citrus to nematode infection through accumulated salt stress, whereas leaching soluble salt in soil solution offered nematodes a suitable nonosmotic habitat. Nematode females under the DS treatment also had the highest (P ≤ 0.05) fecundity.     

Rates of sulfide mineral oxidation by acidophilic chemolithotrophic microbial communities from various sources

A correlation was observed between the rate of oxidation of pure sulfide minerals (pyrite, pyrrhotite, and arsenopyrite) by communities of acidophilic chemolithotrophic microorganisms (ACM) and the mineral substrate where these communities were formed. The ACM community formed during continuous oxidation of the pyrite-arsenopyrite ore concentrate (Kyuchus deposit) exhibited the highest rate of pyrite oxidation. The highest rate of pyrrhotite oxidation was observed for the ACM community developed during semicontinuous oxidation of the pyrrhotite-containing pyrite-arsenopyrite ore concentrate (Olympiadinskoe deposit), by the communities isolated from the pyrrhotite concentrate, and ore of the Shanuch deposit. In the case of arsenopyrite oxidation, the ACM community isolated during oxidation of the Olympiadinskoe ore concentrate grew without a lag phase. Other communities commenced arsenopyrite oxidation at various rates only after a two-day lag phase. The similarity of the mineralogical characteristics of pure sulfide minerals with those of the minerals in the substrates where the ACM communities developed may affect the rates of oxidation.     

Immobilised Histidine Tagged β 2-Adrenoceptor Oriented by a Diazonium Salt Reaction and Its Application in Exploring Drug-Protein Interaction Using Ephedrine and Pseudoephedrine as Probes

A new oriented method using a diazonium salt reaction was developed for linking β ₂-adrenoceptor (β ₂-AR) on the surface of macroporous silica gel. Stationary phase containing the immobilised receptor was used to investigate the interaction between β ₂-AR and ephedrine plus pseudoephedrine by zonal elution. The isotherms of the two drugs best fit the Langmuir model. Only one type of binding site was found for ephedrine and pseudoephedrine targeting β ₂-AR. At 37 °C, the association constants during the binding were (5.94±0.05)×10³/M for ephedrine and (3.80±0.02) ×10³/M for pseudoephedrine, with the binding sites of (8.92±0.06) ×10⁻⁴ M. Thermodynamic studies showed that the binding of the two compounds to β ₂-AR was a spontaneous reaction with exothermal processes. The ΔG^θ, ΔH^θ and ΔS^θ for the interaction between ephedrine and β ₂-AR were −(22.33±0.04) kJ/mol, −(6.51±0.69) kJ/mol and 50.94±0.31 J/mol·K, respectively. For the binding of pseudoephedrine to the receptor, these values were −(21.17±0.02) kJ/mol, −(7.48±0.56) kJ/mol and 44.13±0.01 J/mol·K. Electrostatic interaction proved to be the driving force during the binding of the two drugs to β ₂-AR. The proposed immobilised method will have great potential for attaching protein to solid substrates and realizing the interactions between proteins and drugs.     

Pure-Culture Growth of Fermentative Bacteria, Facilitated by H2 Removal: Bioenergetics and H2 Production

We used an H₂-purging culture vessel to replace an H₂-consuming syntrophic partner, allowing the growth of pure cultures of Syntrophothermus lipocalidus on butyrate and Aminobacterium colombiense on alanine. By decoupling the syntrophic association, it was possible to manipulate and monitor the single organism's growth environment and determine the change in Gibbs free energy yield (ΔG) in response to changes in the concentrations of reactants and products, the purging rate, and the temperature. In each of these situations, H₂ production changed such that ΔG remained nearly constant for each organism (−11.1 ± 1.4 kJ mol butyrate⁻¹ for S. lipocalidus and −58.2 ± 1.0 kJ mol alanine⁻¹ for A. colombiense). The cellular maintenance energy, determined from the ΔG value and the hydrogen production rate at the point where the cell number was constant, was 4.6 × 10⁻¹³ kJ cell⁻¹ day⁻¹ for S. lipocalidus at 55°C and 6.2 × 10⁻¹³ kJ cell⁻¹ day⁻¹ for A. colombiense at 37°C. S. lipocalidus, in particular, seems adapted to thrive under conditions of low energy availability.     

Nutrient effect on the biological leaching of a black-schist ore

The purpose of the study was to examine the influence of inorganic N (NH(4), NO(3)) and phosphate on the biological oxidation of a sulfidic black-schist ore which contained pyrrhotite as the main iron sulfide. Iron was initially solubilized as Fe from the ore and subsequently oxidized to Fe in shake flask experiments. Under these experimental conditions, iron dissolution from pyrrhotite was mainly a chemical reaction, with some enhancement by bacteria, whereas the subsequent Fe oxidation was bacterially mediated, with negligible contribution from chemical oxidation. Phosphate amendment did not enhance Fe oxidation. Chemical analysis of leach solutions with no exogenous phosphate revealed that phosphate was solubilized from the black-schist ore. Ammonium amendment (6 mM) enhanced Fe oxidation, whereas the addition of nitrate (6 and 12 mM) had a negative effect. An increase in the temperature from 30 to 35 degrees C slightly enhanced Fe oxidation, but the effect was statistically not significant. The precipitation of potassium jarosite was indicative of Fe oxidation and was absent in nitrate-inhibited cultures because of the lack of Fe oxidation. The black-schist ore also contained phlogopite, which was altered to vermiculite in iron-oxidizing cultures.     

Kinetics of Phosphomevalonate Kinase from Saccharomyces cerevisiae

The mevalonate-based isoprenoid biosynthetic pathway is responsible for producing cholesterol in humans and is used commercially to produce drugs, chemicals, and fuels. Heterologous expression of this pathway in Escherichia coli has enabled high-level production of the antimalarial drug artemisinin and the proposed biofuel bisabolane. Understanding the kinetics of the enzymes in the biosynthetic pathway is critical to optimize the pathway for high flux. We have characterized the kinetic parameters of phosphomevalonate kinase (PMK, EC 2.7.4.2) from Saccharomyces cerevisiae, a previously unstudied enzyme. An E. coli codon-optimized version of the S. cerevisiae gene was cloned into pET-52b+, then the C-terminal 6X His-tagged protein was expressed in E. coli BL21(DE3) and purified on a Ni²⁺ column. The K_M of the ATP binding site was determined to be 98.3 µM at 30°C, the optimal growth temperature for S. cerevisiae, and 74.3 µM at 37°C, the optimal growth temperature for E. coli. The K_M of the mevalonate-5-phosphate binding site was determined to be 885 µM at 30°C and 880 µM at 37°C. The V_max was determined to be 4.51 µmol/min/mg enzyme at 30°C and 5.33 µmol/min/mg enzyme at 37°C. PMK is Mg²⁺ dependent, with maximal activity achieved at concentrations of 10 mM or greater. Maximum activity was observed at pH = 7.2. PMK was not found to be substrate inhibited, nor feedback inhibited by FPP at concentrations up to 10 µM FPP.     

Microbiological leaching of sulfide minerals with different percolation regimes

Summary The microbiological leaching of Fe, Al, Zn, Cu, Ni and Co from sulfide ore material was evaluated with four percolation regimes involving trickle and flood leaching. Continuous circulation of the leach solution associated with flood leaching resulted in the highest rates of leaching of Ni (44% recovery), Zn (25%), Co (18%), and Cu (8%) over a period of about half a year. Iron and aluminum recoveries remained low because of their precipitation. Bacterial counts increased from 3.2×10⁶ to 4.8×10⁷ iron-oxidizers and from 6.6×10⁶ to 1.8×10⁷ glucose-oxidizers per ml leach solution. Microscopic counts reached a maximum of 4.9×10⁸ cells per ml. Neither microscopic nor viable counts reflected the time course and the progress of the leaching. However, both the microscopic and viable counts were highest with the continuous flooding technique which also yielded the fastest rates of metal solubilization.     

Acetate Threshold Concentrations Suggest Varying Energy Requirements during Anaerobic Respiration by Anaeromyxobacter dehalogenans

Acetate threshold concentrations were determined under chlororespiring and Fe(III)-reducing conditions for Anaeromyxobacter dehalogenans strain 2CP-C. The acetate threshold concentrations measured were 69 ± 4, 19 ± 8, and <1 nM for chlororespiration, amorphous Fe(III) reduction, and Fe(III) citrate reduction, respectively. Residual ΔG values of −75.4 kJ/mol of electrons for chlororespiration and −41.5 kJ/mol of electrons for amorphous Fe(III) reduction were calculated at the acetate threshold concentration. By comparing threshold concentrations for different metabolisms in a single organism, this study provides insight into the metabolic use of energy under different growth conditions.     

Effects of Abiotic Factors on Acetylene Reduction by Cyanobacteria Epiphytic on Moss at a Subantarctic Island

Acetylene reduction (AR) rates by cyanobacteria epiphytic on a moss at Marion Island (46°54′ S, 37°45′ E) increased from −5°C to a maximum at 25 to 27°C. Q₁₀ values between 0 and 25°C were between 2.3 and 2.9, depending on photosynthetic photon flux density. AR rates declined sharply at temperatures above the optimum and were lower at 35°C than at 0°C. Photosynthetic photon flux density at low levels markedly influenced AR, and half of the maximum rate occurred at 84 μmol m⁻² s⁻¹, saturation occurring at ca. 1,000 μmol m⁻² s⁻¹. Higher photosynthetic photon flux density levels decreased AR rates. AR increased up to the highest sample moisture content investigated (3,405%), and the pH optimum was between 5.9 and 6.2. The addition of P, Co, and Mo, individually or together, depressed AR.     

Rational Design,Synthesis, and Biological Evaluation of Third Generation α-Noscapine Analogues as Potent Tubulin Binding Anti-Cancer Agents

Systematic screening based on structural similarity of drugs such as colchicine and podophyllotoxin led to identification of noscapine, a microtubule-targeted agent that attenuates the dynamic instability of microtubules without affecting the total polymer mass of microtubules. We report a new generation of noscapine derivatives as potential tubulin binding anti-cancer agents. Molecular modeling experiments of these derivatives 5a, 6a-j yielded better docking score (-7.252 to -5.402 kCal/mol) than the parent compound, noscapine (-5.505 kCal/mol) and its existing derivatives (-5.563 to -6.412 kCal/mol). Free energy (ΔG _bind) calculations based on the linear interaction energy (LIE) empirical equation utilizing Surface Generalized Born (SGB) continuum solvent model predicted the tubulin-binding affinities for the derivatives 5a, 6a-j (ranging from -4.923 to -6.189 kCal/mol). Compound 6f showed highest binding affinity to tubulin (-6.189 kCal/mol). The experimental evaluation of these compounds corroborated with theoretical studies. N-(3-brormobenzyl) noscapine (6f) binds tubulin with highest binding affinity (K_D, 38 ± 4.0 µM), which is ~ 4.0 times higher than that of the parent compound, noscapine (K_D, 144 ± 1.0 µM) and is also more potent than that of the first generation clinical candidate EM011, 9-bromonoscapine (K_D, 54 ± 9.1 µM). All these compounds exhibited substantial cytotoxicity toward cancer cells, with IC₅₀ values ranging from 6.7 µM to 72.9 µM; compound 6f showed prominent anti-cancer efficacy with IC₅₀ values ranging from 6.7 µM to 26.9 µM in cancer cells of different tissues of origin. These compounds perturbed DNA synthesis, delayed the cell cycle progression at G2/M phase, and induced apoptotic cell death in cancer cells. Collectively, the study reported here identified potent, third generation noscapinoids as new anti-cancer agents.     

Thermal Inactivation of Bacillus anthracis Spores in Cow's Milk

Decimal reduction time (time to inactivate 90% of the population) (D) values of Bacillus anthracis spores in milk ranged from 3.4 to 16.7 h at 72°C and from 1.6 to 3.3 s at 112°C. The calculated increase of temperature needed to reduce the D value by 90% varied from 8.7 to 11.0°C, and the Arrhenius activation energies ranged from 227.4 to 291.3 kJ/mol. Six-log-unit viability reductions were achieved at 120°C for 16 s. These results suggest that a thermal process similar to commercial ultrahigh-temperature pasteurization could inactivate B. anthracis spores in milk.     

Technique for Simultaneous Determination of [35S]Sulfide and [14C]Carbon Dioxide in Anaerobic Aqueous Samples

A technique for the simultaneous determination of [³⁵S]sulfide and [¹⁴C]carbon dioxide produced in anaerobic aqueous samples dual-labeled with [³⁵S]sulfate and a ¹⁴C-organic substrate is described. The method involves the passive distillation of sulfide and carbon dioxide from an acidified water sample and their subsequent separation by selective chemical absorption. The recovery of sulfide was 93% for amounts ranging from 0.35 to 50 μmol; recovery of carbon dioxide was 99% in amounts up to 20 μmol. Within these delineated ranges of total sulfide and carbon dioxide, 1 nmol of [³⁵S]sulfide and 7.5 nmol of [¹⁴C]carbon dioxide were separated and quantified. Correction factors were formulated for low levels of radioisotopic cross-contamination by sulfide, carbon dioxide, and volatile organic acids. The overall standard error of the method was ±4% for sulfide and ±6% for carbon dioxide.     

Archaeal diversity and geochemical energy yields in a geothermal well on Vulcano Island, Italy

The archaeal diversity in a shallow geothermal well on Vulcano Island, Italy was characterized using culture‐independent 16S rDNA sequence analysis. Environmental DNA was extracted from 56 °C well water, and the 16S ribosomal RNA gene was amplified with archaea‐specific primers. Restriction fragment length polymorphism (RFLP) analysis of ～250 clones revealed 35 unique patterns, which were sequenced and analyzed. These yielded 17 operational taxonomic units, of which 13, 3, and 1 were unique cren‐, eury‐, and korarchaeotal sequences, respectively. The majority of the crenarchaeotal phylotypes formed a novel, deeply‐branching clade that includes sequences from other hydrothermal environments, but no cultured representatives. Three phylotypes represent novel lineages in the Thermoproteales and two phylotypes represent a novel genus of Euryarchaeota. One euryarchaeotal phylotype was nearly identical (99%) to Palaeococcus helgesonii, an aerotolerant, hyperthermophilic fermenter previously isolated from the same well. To place this diverse archaeal community in the geochemical framework of this ecosystem, we calculate values of Gibbs free energy of 145 organic and inorganic redox reactions at in situ conditions. Energy yields ranged from 0 to 125 kJ per mole of electrons transferred. The most exergonic organic reactions were organic carbon oxidation with O₂ (>100 kJ/mol e^?), followed by oxidation with (61–93 kJ/mol e^?), Fe(III) (43–60 kJ/mol e^?), and S⁰/ (6–27 kJ/mol e^?) as terminal electron acceptors. Overall, energy yields from inorganic reactions were similar to those of the organic reactions considered, but were less systematic with respect to terminal electron acceptor. The oxidation of methane coupled with Fe(III) reduction yielded the most energy (123 kJ/mol e^?). However, the most exergonic inorganic reactions were predominantly O₂, , or reduction. Reduction of , S⁰, CO₂, and CO yielded significantly less energy (0–18 kJ/mol e^?). Metabolisms of the cultured organisms identified in the Pozzo Istmo archaeal clone library were exergonic. However, most of the archaeal diversity remains uncultured and energetic calculations reveal an extensive suite of potential lithotrophic and heterotrophic metabolisms that could be exploited by these novel organisms.     

Thermoacidophilic microbial community oxidizing the gold-bearing flotation concentrate of a pyrite-arsenopyrite ore

An aboriginal community of thermophilic acidophilic chemolithotrophic microorganisms (ACM) was isolated from a sample of pyrite gold-bearing flotation concentrate at 45–47°C and pH 1.8–2.0. Compared to an experimental thermoacidophilic microbial consortium formed in the course of cultivation in parallel bioreactors, it had lower rates of iron leaching and oxidation, while its rate of sulfur oxidation was higher. A new thermophilic acidophilic microbial community was obtained by mutual enrichment with the microorganisms from the experimental and aboriginal communities during the oxidation of sulfide ore flotation concentrate at 47°C. The dominant bacteria of this new ACM community were Acidithiobacillus caldus (the most active sulfur oxidize) and Sulfobacillus thermotolerans (active oxidizer of both iron and sulfur), while iron-oxidizing archaea of the family Ferroplasmaceae and heterotrophic bacteria Alicyclobacillus tolerans were the minor components. The new ACM community showed promise for leaching/oxidation of sulfides from flotation concentrate at high pulp density (S : L = 1 : 4).     

Benzoate Fermentation by the Anaerobic Bacterium Syntrophus aciditrophicus in the Absence of Hydrogen-Using Microorganisms

The anaerobic bacterium Syntrophus aciditrophicus metabolized benzoate in pure culture in the absence of hydrogen-utilizing partners or terminal electron acceptors. The pure culture of S. aciditrophicus produced approximately 0.5 mol of cyclohexane carboxylate and 1.5 mol of acetate per mol of benzoate, while a coculture of S. aciditrophicus with the hydrogen-using methanogen Methanospirillum hungatei produced 3 mol of acetate and 0.75 mol of methane per mol of benzoate. The growth yield of the S. aciditrophicus pure culture was 6.9 g (dry weight) per mol of benzoate metabolized, whereas the growth yield of the S. aciditrophicus-M. hungatei coculture was 11.8 g (dry weight) per mol of benzoate. Cyclohexane carboxylate was metabolized by S. aciditrophicus only in a coculture with a hydrogen user and was not metabolized by S. aciditrophicus pure cultures. Cyclohex-1-ene carboxylate was incompletely degraded by S. aciditrophicus pure cultures until a free energy change (ΔG′) of −9.2 kJ/mol was reached (−4.7 kJ/mol for the hydrogen-producing reaction). Cyclohex-1-ene carboxylate, pimelate, and glutarate transiently accumulated at micromolar levels during growth of an S. aciditrophicus pure culture with benzoate. High hydrogen (10.1 kPa) and acetate (60 mM) levels inhibited benzoate metabolism by S. aciditrophicus pure cultures. These results suggest that benzoate fermentation by S. aciditrophicus in the absence of hydrogen users proceeds via a dismutation reaction in which the reducing equivalents produced during oxidation of one benzoate molecule to acetate and carbon dioxide are used to reduce another benzoate molecule to cyclohexane carboxylate, which is not metabolized further. Benzoate fermentation to acetate, CO₂, and cyclohexane carboxylate is thermodynamically favorable and can proceed at free energy values more positive than −20 kJ/mol, the postulated minimum free energy value for substrate metabolism.