From Laboratory towards Industrial Operation: Biomarkers for Acidophilic Metabolic Activity in Bioleaching Systems.

In the actual mining scenario, copper bioleaching, mainly raw mined material known as run-of-mine (ROM) copper bioleaching, is the best alternative for the treatment of marginal resources that are not currently considered part of the profitable reserves because of the cost associated with leading technologies in copper extraction. It is foreseen that bioleaching will play a complementary role in either concentration-as it does in Minera Escondida Ltd. (MEL)-or chloride main leaching plants. In that way, it will be possible to maximize mines with installed solvent-extraction and electrowinning capacities that have not been operative since the depletion of their oxide ores. One of the main obstacles for widening bioleaching technology applications is the lack of knowledge about the key events and the attributes of the technology's critical events at the industrial level and mainly in ROM copper bioleaching industrial operations. It is relevant to assess the bed environment where the bacteria-mineral interaction occurs to learn about the limiting factors determining the leaching rate. Thus, due to inability to accurately determine in-situ key variables, their indirect assessment was evaluated by quantifying microbial metabolic-associated responses. Several candidate marker genes were selected to represent the predominant components of the microbial community inhabiting the industrial heap and the metabolisms involved in microbial responses to changes in the heap environment that affect the process performance. The microbial community's predominant components were Acidithiobacillus ferrooxidans, At. thiooxidans, Leptospirillum ferriphilum, and Sulfobacillus sp. Oxygen reduction, CO2 and N2 fixation/uptake, iron and sulfur oxidation, and response to osmotic stress were the metabolisms selected regarding research results previously reported in the system. After that, qPCR primers for each candidate gene were designed and validated. The expression profile of the selected genes vs. environmental key variables in pure cultures, column-leaching tests, and the industrial bioleaching heap was defined. We presented the results obtained from the industrial validation of the marker genes selected for assessing CO2 and N2 availability, osmotic stress response, as well as ferrous iron and sulfur oxidation activity in the bioleaching heap process of MEL. We demonstrated that molecular markers are useful for assessing limiting factors like nutrients and air supply, and the impact of the quality of recycled solutions. We also learned about the attributes of variables like CO2, ammonium, and sulfate levels that affect the industrial ROM-scale operation.

Microbially Influenced Corrosion of Stainless Steel by Acidithiobacillus ferrooxidans Supplemented with Pyrite: Importance of Thiosulfate.

Microbially influenced corrosion (MIC) results in significant damage to metallic materials in many industries. Anaerobic sulfate-reducing bacteria (SRB) have been well studied for their involvement in these processes. Highly corrosive environments are also found in pulp and paper processing, where chloride and thiosulfate lead to the corrosion of stainless steels. Acidithiobacillus ferrooxidans is a critically important chemolithotrophic acidophile exploited in metal biomining operations, and there is interest in using A. ferrooxidans cells for emerging processes such as electronic waste recycling. We explored conditions under which A. ferrooxidans could enable the corrosion of stainless steel. Acidic medium with iron, chloride, low sulfate, and pyrite supplementation created an environment where unstable thiosulfate was continuously generated. When combined with the chloride, acid, and iron, the thiosulfate enabled substantial corrosion of stainless steel (SS304) coupons (mass loss, 5.4 ± 1.1 mg/cm2 over 13 days), which is an order of magnitude higher than what has been reported for SRB. There results were verified in an abiotic flow reactor, and the importance of mixing was also demonstrated. Overall, these results indicate that A. ferrooxidans and related pyrite-oxidizing bacteria could produce aggressive MIC conditions in certain environmental milieus.IMPORTANCE MIC of industrial equipment, gas pipelines, and military material leads to billions of dollars in damage annually. Thus, there is a clear need to better understand MIC processes and chemistries as efforts are made to ameliorate these effects. Additionally, A. ferrooxidans is a valuable acidophile with high metal tolerance which can continuously generate ferric iron, making it critical to copper and other biomining operations as well as a potential biocatalyst for electronic waste recycling. New MIC mechanisms may expand the utility of these cells in future metal resource recovery operations.

Effect of particle size on uranium bioleaching in column reactors from a low-grade uranium ore.

This study evaluated the effectiveness of ore particle size on column bioleaching from low-grade uranium ore using an indigenous Acidithiobacillus ferrooxidans, isolated from local uranium ore. The uranium content was 0.033% by weight and ore particle size was crushed to <50 mm, <30 mm, and <15 mm. The additive content of sulfuric acid 5 g/L, Fe3+ dosage of 5.0 g/L, spray strength of 2.57 L/(h·m2) and temperature of 25 °C were controlled. After 150 days of leaching, acid consumption amounted to 2.73 g H2SO4 per kg ore, the obtained maximum uranium extraction was 64.85% with the ore particle size of <15 mm. The results showed that a smaller particle size ore had a higher uranium extraction and that an economic uranium extraction can be obtained by correctly controlling the ore granularity.

Valorization of Phosphorus Secondary Raw Materials by Acidithiobacillus ferrooxidans.

This paper presents the possibility of producing phosphorus fertilizers through Acidithiobacillus ferrooxidans utilization in secondary raw materials solubilization. Phosphorus was obtained from the bones of poultry and fish as well as from Morocco phosphorite. Four doses of poultry bones and fish bones were used in the experiment (2, 4, 10 and 20 g/L) and two doses (2 and 4 g/L) of phosphorite were also used. The experimenters measured the final pH, which increased in proportion to the increase in the number of poultry bone doses, whereas in the case of fish bones it decreased in proportion to the increase in the number of fish bone doses. Only in the case of phosphorite, where 10 g/L were used, there was a slight increase in pH during solubilization observed. The highest phosphorus concentration of 1.9% (expressed as P2O5) was found for the solubilization performed on fish bones with the highest dose (20 g/L). The formulation obtained in this study meets the necessary requirements for use as a bio-fertilizer because of the relatively low content of P2O5 and the low content of toxic elements. The results confirm the utilization of Acidithiobacillus ferrooxidans in the biosolubilization of phosphorus renewable raw materials that can alleviate the problem of the world's depleting phosphorite deposits.

Effect of metal sulfide pulp density on gene expression of electron transporters in Acidithiobacillus sp. FJ2.

In Acidithiobacillus ferrooxidans, one of the most important bioleaching bacterial species, the proteins encoded by the rus operon are involved in the electron transfer from Fe2+ to O2. To obtain further knowledge about the mechanism(s) involved in the adaptive responses of the bacteria to growth on the different uranium ore pulp densities, we analyzed the expression of the four genes from the rus operon by real-time PCR, when Acidithiobacillus sp. FJ2 was grown in the presence of different uranium concentrations. The uranium bioleaching results showed the inhibitory effects of the metal pulp densities on the oxidation activity of the bacteria which can affect Eh, pH, Fe oxidation and uranium extractions. Gene expression analysis indicated that Acidithiobacillus sp. FJ2 tries to survive in the stress with increasing in the expression levels of cyc2, cyc1, rus and coxB, but the metal toxicity has a negative effect on the gene expression in different pulp densities. These results indicated that Acidithiobacillus sp. FJ2 could leach the uranium even in high pulp density (50%) by modulation in rus operon gene responses.

Realgar transforming solution displays anticancer potential against human hepatocellular carcinoma HepG2 cells by inducing ROS.

Realgar (As4S4), as a mineral drug containing arsenic compound, has been employed in clinical therapy of cancer for its good therapeutic reputation in Chinese traditional medicine. However, large dose of realgar and long period of treatment are necessary for achieving the effective blood medicine concentration due to its low bioavailability resulted from poor solubility. In this study, we obtained realgar transforming solution (RTS) using intrinsic biotransformation in microorganism, and investigated underlying mechanisms of RTS for HepG2 cells. Our results demonstrated that an effective biotransformation of realgar method by A. ferrooxidans was established, in which realgar was biologically converted into an aqueous solution, and RTS had a strong activity inducing apoptosis and interrupting G2/M progression in HepG2 cells via upregulation of cellular ROS. Importantly, RTS inhibited the cellular antioxidant defense system leading to abundant ROS accumulation, and activated cell cycle arrest and mitochondrial pathway of apoptosis mediated by activating p53 due to cellular uncontrolled ROS. Collectively, our findings suggest that RTS is a potential candidate for therapy of human hepatocellular carcinoma.

Use of Walnut Shell Powder to Inhibit Expression of Fe(2+)-Oxidizing Genes of Acidithiobacillus Ferrooxidans.

Acidithiobacillus ferrooxidans is a Gram-negative bacterium that obtains energy by oxidizing Fe(2+) or reduced sulfur compounds. This bacterium contributes to the formation of acid mine drainage (AMD). This study determined whether walnut shell powder inhibits the growth of A. ferrooxidans. First, the effects of walnut shell powder on Fe(2+) oxidization and H⁺ production were evaluated. Second, the chemical constituents of walnut shell were isolated to determine the active ingredient(s). Third, the expression of Fe(2+)-oxidizing genes and rus operon genes was investigated using real-time polymerase chain reaction. Finally, growth curves were plotted, and a bioleaching experiment was performed to confirm the active ingredient(s) in walnut shells. The results indicated that both walnut shell powder and the phenolic fraction exert high inhibitory effects on Fe(2+) oxidation and H⁺ production by A. ferrooxidans cultured in standard 9K medium. The phenolic components exert their inhibitory effects by down-regulating the expression of Fe(2+)-oxidizing genes and rus operon genes, which significantly decreased the growth of A. ferrooxidans. This study revealed walnut shell powder to be a promising substance for controlling AMD.

Effect of calcium oxide on the efficiency of ferrous ion oxidation and total iron precipitation during ferrous ion oxidation in simulated acid mine drainage treatment with inoculation of Acidithiobacillus ferrooxidans.

Calcium oxide was added into ferrous ion oxidation system in the presence of Acidithiobacillus ferrooxidans at concentrations of 0-4.00 g/L. The pH, ferrous ion oxidation efficiency, total iron precipitation efficiency, and phase of the solid minerals harvested from different treatments were investigated during the ferrous ion oxidation process. In control check (CK) system, pH of the solution decreased from 2.81 to 2.25 when ferrous ions achieved complete oxidation after 72 h of Acidithiobacillus ferrooxidans incubation without the addition of calcium oxide, and total iron precipitation efficiency reached 20.2%. Efficiency of ferrous ion oxidation and total iron precipitation was significantly improved when the amount of calcium oxide added was ≤1.33 g/L, and the minerals harvested from systems were mainly a mixture of jarosite and schwertmannite. For example, the ferrous ion oxidation efficiency reached 100% at 60 h and total iron precipitation efficiency was increased to 32.1% at 72 h when 1.33 g/L of calcium oxide was added. However, ferrous ion oxidation and total iron precipitation for jarosite and schwertmannite formation were inhibited if the amount of calcium oxide added was above 2.67 g/L, and large amounts of calcium sulfate dihydrate were generated in systems.

Comparative proteomics of Acidithiobacillus ferrooxidans grown in the presence and absence of uranium.

Acidithiobacillus ferrooxidans is an acidophile that thrives in metal-contaminated environments and tolerates high levels of uranium. To gain a better understanding of the processes involved in U(VI) resistance, comparative proteomics was used. The proteome of A. ferrooxidans was grown in the presence and absence of 0.5 mM U(VI); expression of 17 proteins was upregulated and one was downregulated. Most proteins with increased expression are part of the general stress response or are involved in reactive oxygen species detoxification. Four novel proteins showed increased expression in the presence of U(VI) and may contribute to U(VI) resistance via thiol homoeostasis and U(VI) binding.

Global transcriptional responses of Acidithiobacillus ferrooxidans Wenelen under different sulfide minerals.

In order to provide new information about the adaptation of Acidithiobacillus ferrooxidans during the bioleaching process, the current analysis presents the first report of the global transcriptional response of the native copper mine strain Wenelen (DSM 16786) oxidized under different sulfide minerals. Microarrays were used to measure the response of At. ferrooxidans Wenelen to shifts from iron supplemented liquid cultures (reference state) to the addition of solid substrates enriched in pyrite or chalcopyrite. Genes encoding for energy metabolism showed a similar transcriptional profile for the two sulfide minerals. Interestingly, four operons related to sulfur metabolism were over-expressed during growth on a reduced sulfur source. Genes associated with metal tolerance (RND and ATPases type P) were up-regulated in the presence of pyrite or chalcopyrite. These results suggest that At. ferrooxidans Wenelen presents an efficient transcriptional system developed to respond to environmental conditions, namely the ability to withstand high copper concentrations.

Effect of neutralized solid waste generated in lime neutralization on the ferrous ion bio-oxidation process during acid mine drainage treatment.

Bio-oxidation of ferrous ions prior to lime neutralization exhibits great potential for acid mine drainage (AMD) treatment, while slow ferrous ion bio-oxidation or total iron precipitation is a bottleneck in this process. In this study, neutralized solid waste (NSW) harvested in an AMD lime neutralization procedure was added as a crystal seed in AMD for iron oxyhydroxysulfate bio-synthesis. The effect of this waste on ferrous ion oxidation efficiency, total iron precipitation efficiency, and iron oxyhydroxysulfate minerals yield during ferrous ion bio-oxidation by Acidithiobacillus ferrooxidans was investigated. Ferrous ion oxidation efficiency was greatly improved by adding NSW. After 72 h incubation, total iron precipitation efficiency in treatment with 24 g/L of NSW was 1.74-1.03 times higher than in treatment with 0-12 g/L of NSW. Compared with the conventional treatment system without added NSW, the iron oxyhydroxysulfate minerals yield was increased by approximately 21.2-80.9% when 3-24 g/L of NSW were added. Aside from NSW, jarosite and schwertmannite were the main precipitates during ferrous ion bio-oxidation with NSW addition. NSW can thus serve as the crystal seed for iron oxyhydroxysulfate mineral bio-synthesis in AMD, and improve ferrous ion oxidation and total iron precipitation efficiency significantly.

Enhanced dewaterability of anaerobically digested sewage sludge using Acidithiobacillus ferrooxidans culture as sludge conditioner.

Role of Acidithiobacillus ferrooxidans culture in bioacidification and dewaterability of anaerobically digested sewage sludge (ADS) was investigated. A. ferrooxidans culture grown in 9K medium along with Fe(2+) produced iron flocculant containing, secondary iron minerals and biopolymeric substances as confirmed by FT-IR, XRD, and SEM-EDX. Bioacidification of ADS was performed using 10% (v/v) A. ferrooxidans culture, isolated cells and cell-free culture filtrate; and dewaterability was assessed using the capillary suction time (CST) and specific resistance to filtration (SRF). Isolated bacterial cells significantly (P<0.05) reduced the sludge dewaterability when supplemented with Fe(2+) while the whole culture and cell-free filtrate rapidly acidified the sludge without Fe(2+) and showed significant reduction of CST (71.3-73.5%) and SRF (84-88%). Results clearly indicated that the culture and filtrate of the A. ferrooxidans facilitated rapid sludge dewaterability while the cells supplemented with Fe(2+) also enhanced dewaterability but required 2-4 days.

Novel integration strategy for enhancing chalcopyrite bioleaching by Acidithiobacillus sp. in a 7-L fermenter.

An integrated strategy (additional energy substrate-three stage pH control-fed batch) was firstly proposed for efficiently improving chalcopyrite bioleaching by Acidithiobacillus sp. in a 7-L fermenter. The strain adaptive-growing phase was greatly shortened from 8days into 4days with the supplement of additional 2g/L Fe(2+)+2g/L S(0). Jarosite passivation was effectively weakened basing on higher biomass via the three-stage pH-stat control (pH 1.3-1.0-0.7). The mineral substrate inhibition was attenuated by fed-batch fermentation. With the integrated strategy, the biochemical reaction was promoted and achieved a better balance. Meanwhile, the domination course of A. thiooxidans in the microbial community was shortened from 14days to 8days. As the results of integrated strategy, the final copper ion and productivity reached 89.1mg/L and 2.23mg/(Ld), respectively, which was improved by 52.8% compared to the uncontrolled batch bioleaching. The integrated strategy could be further exploited for industrial chalcopyrite bioleaching.

Radionuclide release from simulated waste material after biogeochemical leaching of uraniferous mineral samples.

Biogeochemical mineral dissolution is a promising method for the released of metals in low-grade host mineralization that contain sulphidic minerals. The application of biogeochemical mineral dissolution to engineered leach heap piles in the Elliot Lake region may be considered as a promising passive technology for the economic recovery of low grade Uranium-bearing ores. In the current investigation, the decrease of radiological activity of uraniferous mineral material after biogeochemical mineral dissolution is quantified by gamma spectroscopy and compared to the results from digestion/ICP-MS analysis of the ore materials to determine if gamma spectroscopy is a simple, viable alternative quantification method for heavy nuclides. The potential release of Uranium (U) and Radium-226 ((226)Ra) to the aqueous environment from samples that have been treated to represent various stages of leaching and passive closure processes are assessed. Dissolution of U from the solid phase has occurred during biogeochemical mineral dissolution in the presence of Acidithiobacillus ferrooxidans, with gamma spectroscopy indicating an 84% decrease in Uranium-235 ((235)U) content, a value in accordance with the data obtained by dissolution chemistry. Gamma spectroscopy data indicate that only 30% of the (226)Ra was removed during the biogeochemical mineral dissolution. Chemical inhibition and passivation treatments of waste materials following the biogeochemical mineral dissolution offer greater protection against residual U and (226)Ra leaching. Pacified samples resist the release of (226)Ra contained in the mineral phase and may offer more protection to the aqueous environment for the long term, compared to untreated or inhibited residues, and should be taken into account for future decommissioning.

Reversal effect of arsenic sensitivity in human leukemia cell line K562 and K562/ADM using realgar transforming solution.

The success of arsenic trioxide (ATO) in treatment of acute promyelocytic leukemia (APL) attracts a great deal of attention to researchers to explore its activity of anti-leukemia. However, ATO has unavailable effect on chronic myeloid leukemia (CML), especially multidrug resistant (MDR)-CML, unless using high concentration. Realgar (As(4)S(4)) has been employed in Chinese traditional medicine for 1500 years. Research evidences confirmed realgar has similar effect on treating with APL as ATO, but the problem of large dose and long period in the CML/MDR-CML treatment still exist. By using a microbial leaching process with Acidithiobacillus ferrooxidans, we obtained realgar transforming solution (RTS) which showed significantly higher extent in inhibiting CML cell line K562 and MDR-CML cell line K562/ADM, and then trigger apoptosis. Both K562 and K562/ADM showed arsenic-dose-dependent effect on RTS. Interestingly, the overexpression of MDR1 mRNA and P-glucoprotein (P-gp) in K562/ADM cells were down-regulated by RTS, where there are no obvious effects on ATO and realgar and arsenic can be subsequently accumulated in K562/ADM cells efficiently. The intracellular accumulation of arsenic in K562/ADM cells treated with RTS for 4 h was 2-fold and 16-folds higher than those treated with realgar or ATO. Meanwhile, Western blot analysis of AQP9, the main transporter of arsenic, was increased by RTS treatment particularly in K562/ADM. Thus, these results suggested that the effect from a certain arsenical or a variety of arsenicals in RTS might be a promising candidate both for treating CML/MDR-CML alone and as combinations with currently used anti-CML/MDR-CML drug, although arsenical forms in RTS are undefined.

Microbial processing of apatite rich low grade Indian uranium ore in bioreactor.

Bioreactor leaching using enriched culture of Acidithiobacillus ferrooxidans and Leptosprillium ferrooxidans was investigated for the apatite rich Indian (Narwapahar) uranium ore. Bioreactor leaching of Narwapahar ore of <45 µm size at pH 2.0 and 10% (w/v) PD using 10% (v/v) inoculum of the bacterium at 35 °C (A. ferrooxidans) and 40 °C (L. ferrooxidans), solubilised 57% and 63% uranium in 5 days, respectively; the E(SCE) values being 561 and 588 mV. Leaching kinetics improved so much so that ~83% uranium was recovered in just 10h with 10% inoculum of A. ferrooxidans containing biogenic Fe(3+); at 20% PD uranium recovery rose to 87%. Role of temperature (25-40 °C) was noticed with 90.3% uranium bioleaching in 10h at 40 °C with L. ferrooxidans as against 77% leaching with A. ferrooxidans at pH 2.0, 40 °C and 20% (w/v) PD.

Supplementation of inorganic phosphate enhancing the removal efficiency of tannery sludge-borne Cr through bioleaching.

Four inorganic mineral nutrients including NH4+, K+, Mg2+ and soluble inorganic phosphate (Pi) were investigated to reveal the potential limiting nutrients for tannery sludge bioleaching process driven by Acidithiobacillus species, and the feasibility of supplementing the limiting nutrients to accelerate tannery sludge bioleaching was studied in the present study. It was found that the concentration of Pi was lower than 3.5 mg/L throughout the whole bioleaching process, which is the most probable restricting nutrient for tannery sludge bioleaching. Further experiments revealed that the deficiency of Pi could seriously influence the growth of Acidithiobacillus thiooxidans and lower its oxidization capacity for S0, and the limiting concentration of Pi for the growth of A. thiooxidans was 6 mg/L. The low concentration of soluble Pi in sludge matrix was resulted from the extremely strong sorbing/binding capacity of tannery sludge for phosphate. The supplementation of more than 1.6 g/L KH2PO4 into tannery sludge bioleaching system could effectively stimulate the growth of Acidithiobacillus species, enhance Cr removal rate and further shorten tannery sludge bioleaching period from 10 days to 7 days. Therefore, inorganic phosphate supplementation is an effective and feasible method to accelerate tannery sludge bioleaching process, and the optimum dosage of KH2PO4 was 1.6 g/L for tannery sludge with 5.1% of total solids.

Column bioleaching of uranium embedded in granite porphyry by a mesophilic acidophilic consortium.

A mesophilic acidophilic consortium was enriched from acid mine drainage samples collected from several uranium mines in China. The performance of the consortium in column bioleaching of low-grade uranium embedded in granite porphyry was investigated. The influences of several chemical parameters on uranium extraction in column reactor were also investigated. A uranium recovery of 96.82% was achieved in 97 days column leaching process including 33 days acid pre-leaching stage and 64 days bioleaching stage. It was reflected that indirect leaching mechanism took precedence over direct. Furthermore, the bacterial community structure was analyzed by using Amplified Ribosomal DNA Restriction Analysis. The results showed that microorganisms on the residual surface were more diverse than that in the solution. Acidithiobacillus ferrooxidans was the dominant species in the solution and Leptospirillum ferriphilum on the residual surface.

Metal recovery from spent refinery catalysts by means of biotechnological strategies.

A bioleaching study aimed at recovering metals from hazardous spent hydroprocessing catalysts was carried out. The exhaust catalyst was rich in nickel (4.5 mg/g), vanadium (9.4 mg/g) and molybdenum (4.4 mg/g). Involved microorganisms were iron/sulphur oxidizing bacteria. Investigated factors were elemental sulphur addition, ferrous iron addition and actions contrasting a possible metal toxicity (either adding powdered activated charcoal or simulating a cross current process by means of periodical filtration). Ferrous iron resulted to be essential for metal extraction: nickel and vanadium extraction yields were 83% and 90%, respectively, while about 50% with no iron. The observed values for molybdenum extraction yields were not as high as Ni and V ones (the highest values were around 30-40%). The investigated actions aimed at contrasting a possible metal toxicity resulted not to be effective; in contrast, sequential filtration of the liquor leach had a significant negative effect on metals extraction. Nickel and vanadium dissolution kinetics resulted to be significantly faster than molybdenum dissolution ones. Furthermore, a simple first order kinetic model was successfully fitted to experimental data. All the observed results supported the important role of the indirect mechanism in bioleaching of LC-Finer catalysts.

Bioleaching kinetics and multivariate analysis of spent petroleum catalyst dissolution using two acidophiles.

Bioleaching studies were conducted to evaluate the recovery of metal values from waste petroleum catalyst using two different acidophilic microorganisms, Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans. Various leaching parameters such as contact time, pH, oxidant concentration, pulp densities, particle size, and temperature were studied in detail. Activation energy was evaluated from Arrhenius equation and values for Ni, V and Mo were calculated in case of both the acidophiles. In both cases, the dissolution kinetics of Mo was lower than those of V and Ni. The lower dissolution kinetics may have been due to the formation of a sulfur product layer, refractoriness of MoS(2) or both. Multivariate statistical data were presented to interpret the leaching data in the present case. The significance of the leaching parameters was derived through principle component analysis and multi linear regression analyses for both iron and sulfur oxidizing bacteria.