Method for Extracting Platinum Group Metals

Abstract

The present disclosure relates to a method for extracting platinum group metals (PGMs) from a material, the method comprising surface oxidizing the material, sonicating the surface oxidized material of step a) in the presence of an acid, and biolyzing the sonicated material in the presence of cyanide formed from cyanide-producing microorganisms in the presence of a cyanide precursor to form a leachate containing PGMs.

Description

Technical Field

[0001] The present invention generally relates to a method for extracting platinum group metals (PGMs) from materials further comprising Cu, Zn, Fe, Ti, or combinations thereof.

Background Art

[0002] Among the precious metals, platinum group metals [PGMs; platinum (Pt), palladium (Pd), and rhodium (Rh)] are widely used in the manufacture of cosmic materials, catalysts, hydrogen fuel cells, and chemical and biomedical industries. Automotive catalytic converters (ACCs) are major consumers in the world's PGM production. PGMs are widely used in ACCs due to their high stability, activity, and selectivity. They act as active catalysts to convert toxic exhaust gases into less harmful products. Since the mid-1970s, ACCs have increasingly been used to convert carbon monoxide and hydrocarbons into carbon dioxide and water (H 2 O). Binary (oxidation) catalytic converters have been replaced by three-way (oxidation-reduction) catalytic converters because they can purify nitrous oxide, which is one of the important greenhouse gases. The catalytic converter has a honeycomb monolith structure and has a refractory oxide support made of a stainless steel container or cordierite (Mg 2 ) incorporated into a cerianite (CeO 2 Al 4 Si 5 O 18 ). In most cases, the inner surface of the monolith structure is coated with 90% γ-Al 2 O 3 and a mixture of metal oxide additives such as cerium (Ce), zirconium (Zr), nickel (Ni), and iron (Fe). And these catalysts containing PGM and other metals are fixed on the coated surface in the form of reduced metals to detoxify the exhaust gas. The metal composition of ACCs varies significantly depending on the age, origin, and manufacturer. For example, in ACCs, the platinum (Pt) content is 300 - 1000 μg g -1within the range, and the contents of palladium (Pd) and rhodium (Rh) are respectively 200 to 800 μg / g -1 and 50 to 100 μg / g -1 respectively. However, the total concentration of PGM in ACC is always less than 0.1%.

[0003] Autocatalysts contain very small amounts (0.1 to 0.3 wt% of the monolith) of two or more precious metals. Approximately 50% of the produced platinum, 80% of the rhodium, and 80% of the palladium, which are the major amounts of the world's PGM production, are used in ACC. Due to high demand and prices, and decreasing PGM concentrations in natural ores, it has become necessary to extract PGM from secondary resources. In fact, these secondary resources contain PGM at higher concentrations than natural ores. Recovering PGM from used automotive catalysts (SAC) not only protects natural primary ores to meet future demand but also supports sustainable development. It also minimizes waste disposal, suppresses power consumption, and reduces environmental pollution. For example, to obtain 1 kg of platinum from primary ore, 150 Mg (tons) of ore must be processed, generating 400 Mg of waste, while the same amount of platinum can be recovered by recycling 2 Mg of SAC. Recovering these metals from SAC is more economical and environmentally friendly than mining from ore. Conventional techniques such as hydrometallurgy and pyrometallurgy can obtain these metals with high recovery rates, but the use of additional solvents, generation of harmful liquid waste and exhaust gases, high operating costs, and energy requirements are the main constraints.

[0004] It is desirable to overcome or improve at least one of the above problems.

Summary of the Invention

Means for Solving the Problems

[0005] The present invention provides a method for extracting platinum group metals (PGM) from a material, the method comprising a) surface oxidizing the above material, b) subjecting the surface-oxidized material of step a) to ultrasonic treatment in the presence of an acid, and c) performing bioleaching of the ultrasonicated material of step b) in the presence of cyanide formed from cyanide-producing microorganisms in the presence of a cyanide precursor to form a leachate containing PGM.

[0006] In some embodiments, the surface oxidation is performed at about 700 °C to about 900 °C, preferably about 850 °C.

[0007] In some embodiments, the surface oxidized material of step a) is sonicated at a frequency of about 30 kHz to about 80 kHz, preferably about 37 kHz to about 80 kHz.

[0008] In some embodiments, the surface oxidized material of step a) is sonicated at an output of about 30 W to about 120 W.

[0009] In some embodiments, the surface oxidized material of step a) is sonicated for a duration of about 10 minutes to about 150 minutes, preferably about 70 minutes to about 80 minutes.

[0010] In some embodiments, the surface oxidized material of step a) is sonicated at a temperature of about 30 °C to about 80 °C, preferably about 70 °C.

[0011] In some embodiments, the acid is selected from nitric acid, hydrochloric acid, sulfuric acid, or combinations thereof.

[0012] In some embodiments, the concentration of the acid is about 2 M to about 16 M, preferably about 8 M to about 9 M.

[0013] In some embodiments, the surface oxidized material of step a) is sonicated at a frequency of about 37 kHz, a temperature of about 70 °C, an output of about 100 W, and a duration of about 80 minutes, and the acid is about 8 M to about 8.5 M nitric acid.

[0014] In some embodiments, the method further includes, after step b), reducing the ultrasonicated material of step b) in the presence of a reducing agent.

[0015] According to some embodiments, the reducing agent is selected from formic acid, ascorbic acid, glycolic acid, malonic acid, or combinations thereof.

[0016] According to some embodiments, the concentration of the reducing agent is from about 1% v / v to about 15% v / v, preferably from about 5% v / v to about 10% v / v.

[0017] According to some embodiments, the ultrasonic-treated material in step b) is reduced for a duration of from about 30 minutes to about 120 minutes, preferably from about 60 minutes to about 90 minutes.

[0018] According to some embodiments, the ultrasonic-treated material in step b) is reduced at a temperature of from about 50°C to about 90°C, preferably from about 50°C to about 80°C.

[0019] According to some embodiments, the ultrasonic-treated material in step b) is reduced at a concentration of the reducing agent of about 5 vol%, for a duration of about 90 minutes, and at a temperature of about 80°C.

[0020] According to some embodiments, the concentration of the cyanide precursor is from about 0.5 g / L to about 20 g / L, preferably about 10 g / L.

[0021] According to some embodiments, the ultrasonic-treated material in step b) is bioleached at a pulp density of from about 0.1% w / v to about 12% w / v, or about 0.5% w / v.

[0022] According to some embodiments, the ultrasonic-treated material in step b) is bioleached at a pH of from about 7 to about 11, preferably at a pH of from about 9 to about 10.5.

[0023] According to some embodiments, the ultrasonic-treated material in step b) is bioleached at a temperature of from about 22°C to 38°C, preferably at about 30°C.

[0024] Depending on the embodiment, the ultrasonically treated material in step b) is bioremediated at a cyanide precursor concentration of about 10 g / L, a pulp density of about 0.5% w / v, a pH of about 9 to about 10.5, and a temperature of about 30 °C.

[0025] Depending on the embodiment, the ultrasonically treated material in step b) is bioremediated under aerobic conditions in the presence of the cyanide-producing microorganism characterized by the HCN synthase operon (hcnA, hcnB, and hcnC) in the genome and the cyanide precursor.

[0026] Depending on the embodiment, the bioremediation step i) pre-incubating the cyanide-producing microorganism and the cyanide precursor under aerobic conditions to produce the cyanide, and ii) mixing the cyanide-producing microorganism and the cyanide precursor in step i) with the ultrasonically treated material in step b), The cyanide-producing microorganism is characterized by the HCN synthase operon (hcnA, hcnB, and hcnC) in the genome.

[0027] Depending on the embodiment, the bioremediation step i) pre-incubating the cyanide-producing microorganism and the cyanide precursor under aerobic conditions to produce the cyanide, and ii) isolating the cyanide from the cyanide-producing microorganism to form a cell-free medium, and mixing the cell-free medium with the ultrasonically treated material in step b), The cyanide-producing microorganism is characterized by the HCN synthase operon (hcnA, hcnB, and hcnC) in the genome.

[0028] Depending on the embodiment, the aerobic conditions are O 2 The saturation percentage is about 30%.

[0029] Depending on the embodiment, the cyanide-producing microorganism is selected from Chromobacterium violaceum, Pseudomonas fluorescens, Bacillus megaterium, or a combination thereof.

[0030] Depending on the embodiment, the step of pre-incubating the microorganism includes incubating the microorganism at a pH of about 7.5 and then incubating the microorganism at a pH of about 9.

[0031] Depending on the embodiment, the ultrasonicated material in step b) undergoes bioleaching in the presence of the microorganism and H 2 O 2 and the concentration of H 2 O 2 is about 0.02% v / v to about 0.16% v / v, preferably about 0.08% v / v.

[0032] Depending on the embodiment, the ultrasonicated material in step b) undergoes bioleaching in the presence of a ROS scavenger and / or a dispersant.

[0033] Depending on the embodiment, the ROS scavenger is added at least 10 hours after mixing the reduced material with the microorganism.

[0034] Depending on the embodiment, the concentration of the ROS scavenger is about 0.2 g / L to about 2 g / L, preferably about 0.6 g / L.

[0035] Depending on the embodiment, the concentration of the dispersant is about 0.2 g / L to about 1 g / L, preferably about 0.4 g / L.

[0036] Depending on the embodiment, the pH of the cell-free medium is about 10.5.

[0037] In some embodiments, the above material is a used catalytic converter or a used automotive catalyst.

[0038] In some embodiments, the above material further comprises Cu, Zn, Fe, Ti, or a combination thereof.

[0039] In some embodiments, the above method is characterized by a volume of at least 1 L.

[0040] In some embodiments, the above method further comprises a step of bioreducing the leachate of step c) to form nanoparticles.

[0041] In some embodiments, the leachate of step c) is bioreduced using Cupriavidus metallidurans.

[0042] In some embodiments, Cupriavidus metallidurans is pre-incubated.

[0043] In some embodiments, the leachate of step c) is bioreduced at a pH of about 4 to about 8, preferably about 6.

[0044] In some embodiments, the above nanoparticles are characterized by an average particle size of about 10 nm to about 80 nm.

[0045] In some embodiments, the above nanoparticles are characterized by a hydrodynamic diameter of about 80 nm to about 110 nm.

[0046] In some embodiments, the above nanoparticles are characterized by a polydispersity index of about 0.2 to about 0.3.

[0047] The present invention also provides a method for extracting platinum group metals (PGMs) from a material, the method comprising Bioleaching the above material in the presence of cyanide formed from cyanide-producing microorganisms in the presence of a cyanide precursor, and further in the presence of a ROS scavenger and / or a dispersant to form a leachate containing PGM.

[0048] The present invention also provides a material containing a platinum group metal (PGM), and the above method comprises sonicating the above material in the presence of an acid, wherein at least the surface of the above material is oxidized.

[0049] The present invention also provides a method for pretreating a material containing a platinum group metal (PGM), and the above method comprises reducing the above material in the presence of a reducing agent, wherein at least the surface of the above material is oxidized.

[0050] In some embodiments, the above method further comprises sonicating the above material in the presence of an acid before the above reducing step.

[0051] Hereinafter, as non-limiting examples, embodiments of the present invention will be described with reference to the drawings.

Brief Description of the Drawings

[0052]

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Mode for Carrying Out the Invention

[0053] The inventors believe that bio-metallurgical processes are generally more environmentally friendly, sustainable, closer to natural biogeochemical cycles, and thus reduce the demand for natural resources such as ores, energy, and landfill sites.

[0054] The term bioleaching is defined as "the mobilization of metal ions from insoluble materials by biological oxidation and complexation processes". In bioleaching, microorganisms produce secondary metabolites that dissolve the target metal and leave behind the unwanted complex matrix. Certain heterotrophic microorganisms (bacteria and fungi) have been reported to be able to recover precious metals from waste and secondary resources. These microorganisms produce cyanide in an aqueous medium by oxidatively decarboxylating glycine and form soluble metal-cyanide complexes of each metal ion. Bioleaching is generally considered an environmentally friendly metal recovery process. Due to the unique capabilities of microorganisms in microbial metabolism and mineral transformation, bioleaching has become a simple and efficient treatment technology. Also, a biological approach is considered to be economical for treating small-scale deposits, low-purity ores, secondary waste, and mixed ores. However, its potential use for the recovery of precious metals from secondary resources has not been well documented in the literature.

[0055] Unfortunately, the potential of bio-metallurgical processes for the recovery of PGMs has been scarcely investigated. Despite the advantages over conventional metal recovery technologies, the bio-recovery of PGMs from SAC is particularly difficult mainly in the following aspects: (i) the complexity of SAC, (ii) the toxicity imposed by the metals interfering with the microorganisms, (iii) the low yield of the lixiviant, (iv) the competition of metal-cyanide complexes between the target metal and non-target metals, (v) the low metal recovery rate at high pulp density, (vi) the low metal recovery rate in the presence of metal oxides, and (vii) the slow leaching rate. To overcome at least some of these problems, the present invention provides an effective, sustainable, and environmentally friendly process by bio-extraction of PGMs from SAC.

[0056] Furthermore, the synthesis of nanoparticles (NPs) has recently received significant research interest. Due to the unique crystalline, catalytic, optical, and surface-related physicochemical properties of these nanoparticles, applications in numerous fields such as biomedicine, pharmaceuticals, optics, biosensors, fuel cells, semiconductors, optical engineering, electronics, petrochemistry, and catalytic reactions have been found. Several methods are used to synthesize nanoparticles, such as chemical methods, physical methods, and biological methods. In the early days of nanoparticle synthesis, physicochemical methods were used. In physical methods, metal atoms are generated from bulk metals using high temperatures or laser ablation, and then the atoms are combined to form nanoparticles. In chemical methods, wet chemistry is used to generate metal atoms containing two elements: a reducing agent and a metal ion source. An additional capping agent may be used to control the shape, particle size, and dispersibility of the nanoparticles. The use of toxic substances in physicochemical synthesis approaches has led to an interest in the "green synthesis" of environmentally friendly materials. For this reason, the biosynthesis of nanoparticles, classified as microbial synthesis and plant synthesis, has been introduced. In the biosynthesis of nanoparticles, proteins and / or peptides are used as green reducing agents to reduce the environmental footprint. Also, biosynthesized nanoparticles are biocompatible, contain no chemicals, are sustainable, environmentally friendly, and cost-effective. However, the limitations associated with the biological synthesis of nanoparticles include challenges in controlling shape and particle size, stability, and aggregation. Although stable nanoparticles with different shapes and particle sizes can be obtained by chemical methods, the biomedical applications of these nanoparticles are limited due to concerns about biosafety.

[0057] Biosorption is an enzyme-assisted metal precipitation process in which metal ions are reduced from a high valence state to a zero valence state. Bacteria produce nanoparticles extracellularly or intracellularly through a reduction reaction mechanism that converts metal ions into nanoparticles using an intracellular signaling pathway containing bacterial enzymes. In the leachate, biosorption by microorganisms causes conversions that play an important role in metal recovery. It must be recognized that in all studies on the biosynthesis of Pt and Pd nanoparticles, only aqueous solutions containing PGM-chloride complexes or model synthetic solutions, i.e., metal salts as metal ion sources, have been used. Model synthetic solutions contain only one or two target metals and are much simpler compared to actual waste leachates. In model synthetic solutions, the harmful effects of metal toxicity on the function of microorganisms are low, and thus these solutions can be easily treated. Conversely, in actual waste leachates, the composition of metal ions is very complex, containing several metals at high concentrations, which has an adverse effect on the normal function of microorganisms and limits the efficiency of the process. Spent automotive catalysts (SACs) contain many heavy metals that reduce biosorption efficiency. The presence of these metals is toxic to viable cells. Among these metals, certain metals cause metal-cyanide complexation using cyanide. As a result, the presence of metal ions of these undesirable metals makes the treatment of actual waste leachates more difficult. The inventors are not aware of any studies dealing with the biosorption of Pt and Pd from SAC leachates.

[0058] Therefore, it is desirable to (1) develop and optimize an efficient pretreatment technique to remove interfering elements (copper, zinc, iron, and titanium) from SAC before bioleaching, (2) optimize the biomobilization of PGM using hydrogen cyanide-forming (HCN) bacteria and optimize the leaching rate to enhance bioleaching efficiency, (3) study the microorganism-metal interaction and minimize the harmful effects of metal toxicity and oxidative stress in bacteria during bioleaching to increase the PGM extraction rate at high pulp density, and (4) explore the bio-reduction reaction mechanism for the bacterial-mediated bio-recovery of Pt and Pd and the green synthesis of Pt and Pd nanoparticles from the SAC leachate, as well as the effects of various process parameters on the bio-reduction efficiency and particle size and shape of the synthesized nanoparticles.

[0059] The present invention provides a method for extracting platinum group metals (PGM) from a material, the method comprising: a) surface oxidizing the material; b) sonicating the surface-oxidized material of step a) in the presence of an acid; and c) performing bioleaching of the sonicated material of step b) in the presence of cyanide formed from cyanide-producing microorganisms in the presence of a cyanide precursor to form a leachate containing PGM.

[0060] The material can be a catalytic converter or a used automotive catalyst. Generally, such materials contain metals such as Pt, Pd, Rh, Cu, Zn, Fe, Ti, Al, Ba, Ce, Zr, Ni, Ca, and / or Mg, and in other examples, may contain V, Mn, Nb, Cd, Sn, Sb, and / or Hf. These metals can be present in various amounts depending on the manufacturer.

[0061] Surface oxidation has been found to reduce the dissolution of PGM from the above materials in step b). Also, the ultrasonic treatment step (step b)) substantially removes metals that may interfere with the extraction of PGM. These metals may include Cu, Zn, Fe, and Ti. Therefore, the PGM recovery rate is improved.

[0062] The present invention also provides a method for extracting platinum group metals (PGM) from a material, the material further comprising Cu, Zn, Fe, Ti, or a combination thereof, the method comprising: a) surface oxidizing the material; b) ultrasonically treating the surface-oxidized material of step a) in the presence of an acid; and c) performing bioleaching of the ultrasonically treated material of step b) in the presence of cyanide formed from cyanide-producing microorganisms in the presence of a cyanide precursor to form a leachate containing PGM.

[0063] In some embodiments, the surface oxidation is carried out at about 700 °C to about 900 °C, preferably about 850 °C. In some embodiments, the temperature is about 750 °C to about 900 °C, about 800 °C to about 900 °C, or about 800 °C to about 850 °C. The surface oxidation can be carried out for about 2 hours to about 10 hours, preferably about 4 hours. In some embodiments, the duration is about 3 hours to about 10 hours, about 4 hours to about 10 hours, or about 4 hours to about 8 hours.

[0064] In some embodiments, the surface-oxidized material of step a) is ultrasonically treated at a frequency of about 30 kHz to about 80 kHz, preferably about 37 kHz to about 80 kHz.

[0065] Depending on the embodiment, the surface-oxidized material in step a) is ultrasonically treated at an output of about 30% to about 100%, preferably about 70% to about 80%. For example, the output of the ultrasonic treatment can vary from about 30% to about 100% when the frequency is 37 kHz. When the frequency is 37 kHz, the output of 100% ultrasonic treatment is about 120 W. For example, the output of the ultrasonic treatment can vary from about 30% to about 100% when the frequency is 80 kHz. When the frequency is 80 kHz, the output of 100% ultrasonic treatment is about 100 W. Therefore, the output can be from about 30 W to about 120 W.

[0066] Depending on the embodiment, the surface-oxidized material in step a) is ultrasonically treated for a duration of about 10 minutes to about 150 minutes, preferably about 70 minutes to about 80 minutes. Depending on the embodiment, the above duration is about 20 minutes to about 150 minutes, about 30 minutes to about 150 minutes, about 40 minutes to about 150 minutes, about 50 minutes to about 150 minutes, about 60 minutes to about 150 minutes, or about 70 minutes to about 150 minutes.

[0067] Depending on the embodiment, the surface-oxidized material in step a) is ultrasonically treated at a temperature of about 30°C to about 80°C, preferably about 70°C.

[0068] Depending on the embodiment, the above acid is selected from nitric acid, hydrochloric acid, sulfuric acid, or a combination thereof.

[0069] Depending on the embodiment, the concentration of the above acid is about 2 M to about 16 M, preferably about 8 M to about 9 M. Depending on the embodiment, the above concentration is about 4 M to about 16 M, about 6 M to about 16 M, about 6 M to about 14 M, about 6 M to about 12 M, about 6 M to about 10 M, or about 8 M to about 10 M.

[0070] Depending on the embodiment, the surface-oxidized material in step a) is ultrasonically treated at a frequency of about 37 kHz, a temperature of about 70°C, an output of about 80% (about 96 W to about 100 W), and a duration of about 80 minutes, and the above acid is nitric acid with a concentration of about 8 M to about 8.5 M.

[0071] Depending on the embodiment, the above method further includes, after step b), a step of reducing the ultrasonically treated material of step b) in the presence of a reducing agent.

[0072] Depending on the embodiment, the above reducing agent is selected from formic acid, ascorbic acid, glycolic acid, malonic acid, or a combination thereof. Depending on the embodiment, the above reducing agent is selected from formic acid, ascorbic acid, or a combination thereof.

[0073] Depending on the embodiment, the concentration of the above reducing agent is about 1% v / v to about 15% v / v, preferably about 5% v / v to about 10% v / v. Depending on the embodiment, the above concentration is about 2% v / v to about 15% v / v, about 3% v / v to about 15% v / v, about 4% v / v to about 15% v / v, or about 5% v / v to about 15% v / v.

[0074] Depending on the embodiment, the ultrasonically treated material of step b) is reduced for a duration of about 30 minutes to about 120 minutes, preferably about 60 minutes to about 90 minutes. Depending on the embodiment, the above duration is about 40 minutes to about 120 minutes, about 50 minutes to about 120 minutes, about 60 minutes to about 120 minutes, or about 60 minutes to about 100 minutes.

[0075] Depending on the embodiment, the ultrasonically treated material of step b) is reduced at a temperature of about 50°C to about 90°C, preferably about 50°C to about 80°C.

[0076] Depending on the embodiment, the ultrasonically treated material of step b) is reduced at a concentration of the reducing agent of about 5 vol%, for a duration of about 90 minutes, and at a temperature of about 80°C.

[0077] In the presence of cyanide-producing microorganisms, the cyanide precursor is converted to cyanide. Then, cyanide is used to leach PGM from the above material. The above cyanide precursor can be, for example, glycine.

[0078] Depending on the embodiment, the concentration of the cyanide precursor is about 0.5 g / L to about 20 g / L, preferably about 10 g / L. Depending on the embodiment, the concentration is about 1 g / L to about 20 g / L, about 2 g / L to about 20 g / L, about 3 g / L to about 20 g / L, about 4 g / L to about 20 g / L, about 5 g / L to about 20 g / L, about 6 g / L to about 20 g / L, about 7 g / L to about 20 g / L, about 8 g / L to about 20 g / L, about 9 g / L to about 20 g / L, or about 10 g / L to about 20 g / L.

[0079] Depending on the embodiment, the ultrasonically treated material in step b) is subjected to bioleaching at a pulp density of about 0.1% w / v to about 12% w / v, or about 0.5% w / v. Pulp density refers to the ratio of the solid component to the liquid component, or the mass of the used automotive catalyst (SAC) or material in a unit volume of the reagent. In other embodiments, the pulp density is about 0.1% w / v to about 11% w / v, about 0.1% w / v to about 10% w / v, about 0.1% w / v to about 9% w / v, about 0.1% w / v to about 8% w / v, about 0.1% w / v to about 7% w / v, about 0.1% w / v to about 6% w / v, about 0.1% w / v to about 5% w / v, about 0.1% w / v to about 4% w / v, about 0.1% w / v to about 3% w / v, about 0.1% w / v to about 2% w / v, about 0.1% w / v to about 1% w / v, about 0.1% w / v to about 0.8% w / v, or about 0.1% w / v to about 0.5% w / v.

[0080] Depending on the embodiment, the ultrasonically treated material in step b) is subjected to bioleaching at a pH of about 7 to about 11, preferably at a pH of about 9 to about 10.5.

[0081] Depending on the embodiment, the ultrasonically treated material in step b) is subjected to bioleaching at a temperature of about 22°C to 38°C, preferably about 30°C.

[0082] Depending on the embodiment, the ultrasonically treated material in step b) is subjected to bioleaching at a concentration of the cyanide precursor of about 10 g / L, a pulp density of about 0.5% w / v, a pH of about 9 to about 10.5, and a temperature of about 30°C.

[0083] The bioleaching process can be carried out in a single step by mixing the cyanide-producing microorganism and the cyanide precursor with the ultrasonicated material. In this regard, cyanide is produced in situ. Depending on the embodiment, the ultrasonicated material of step b) is subjected to bioleaching under aerobic conditions in the presence of a cyanide-producing microorganism and a cyanide precursor characterized by the HCN synthase operon (hcnA, hcnB, and hcnC) in the genome.

[0084] The bioleaching process may also be carried out as a two-step process by first pre-incubating the cyanide-producing microorganism and the cyanide precursor separately under aerobic conditions to produce cyanide, and then mixing the cyanide-producing microorganism and the cyanide precursor with the ultrasonicated material of step b). The cyanide-producing microorganism can be pre-incubated under aerobic conditions to achieve the mid-logarithmic growth phase of the microorganism, after which the cyanide precursor can be mixed. In addition, once the cyanide production reaches its maximum, the pH can be adjusted to achieve the optimal cyanide concentration. This has the advantage of improving bioleaching efficiency since the toxicity of the metal can potentially cause loss of bacterial activity.

[0085] Therefore, depending on the embodiment, the bioleaching process comprises i) pre-incubating a cyanide-producing microorganism and a cyanide precursor under aerobic conditions to produce cyanide, and ii) mixing the cyanide-producing microorganism and the cyanide precursor of step i) with the ultrasonicated material of step b).

[0086] Alternatively, the bioleaching process can be carried out as spent media leaching. In spent media leaching, first, cyanide-producing microorganisms and cyanide precursors are pre-incubated under aerobic conditions to produce cyanide, and then the cyanide is isolated from the cyanide-producing microorganisms to obtain a spent medium (cell-free medium). Next, the spent medium is mixed with the above materials. Since the toxicity of the leached metal may cause the loss of bacterial activity, this has the advantage of improving bioleaching efficiency. The absence of cells ensures a higher concentration of DO that can be reliably utilized for PGM-cyanide complexation. There is no consumption of cyanide by cells, and as a result, the PGM recovery rate can be increased. Also, the leaching can be carried out under alkaline conditions to minimize the loss of cyanide leaching agent and increase the PGM recovery rate.

[0087] Accordingly, in some embodiments, the bioleaching process comprises i) pre-incubating cyanide-producing microorganisms and cyanide precursors under aerobic conditions to produce cyanide, and ii) isolating cyanide from the cyanide-producing microorganisms to form a cell-free medium and mixing the cell-free medium with the sonicated material of step b).

[0088] Since the toxicity of the metal may cause the loss of bacterial activity, this has the advantage of improving bioleaching efficiency.

[0089] In some embodiments, step i) comprises pre-incubating cyanide-producing microorganisms under aerobic conditions to achieve the mid-logarithmic growth phase of the microorganisms, and then mixing the pre-incubated cyanide-producing microorganisms with cyanide precursors. In some embodiments, step i) further comprises adjusting the pH to increase the cyanide concentration.

[0090] Cyanide-producing microorganisms can be characterized by the HCN synthase operon (hcnA, hcnB, and hcnC) in their genome.

[0091] Depending on the embodiment, the aerobic condition is that the O 2 saturation percentage is about 30%.

[0092] Depending on the embodiment, the microorganism is selected from Chromobacterium violaceum, Pseudomonas fluorescens, Bacillus megaterium, or a combination thereof.

[0093] Depending on the embodiment, the pre-incubation step is carried out at a pH of about 7 to about 11.

[0094] Depending on the embodiment, the step of pre-incubating the cyanide-producing microorganism includes incubating the cyanide-producing microorganism at a pH of about 7.5 and then incubating the microorganism at a pH of about 9. The above pH may be maintained using a base such as sodium hydroxide or an acid such as hydrochloric acid.

[0095] Depending on the embodiment, the pH of the cell-free medium is about 10.5.

[0096] Depending on the embodiment, the ultrasonicated material in step b) is subjected to bioleaching in the presence of the above microorganism and H 2 O 2 and the concentration of H 2 O 2 is about 0.02% v / v to about 0.16% v / v, preferably about 0.08% v / v.

[0097] Depending on the embodiment, the ultrasonicated material in step b) is subjected to bioleaching in the presence of a ROS scavenger and / or a dispersant. Depending on the embodiment, the ultrasonicated material in step b) is subjected to bioleaching in the presence of glutathione and polyvinylpyrrolidone (PVP).

[0098] Depending on the embodiment, glutathione is added at least 10 hours after the reduced material is mixed with the microorganism. Glutathione acts as an antioxidant or ROS scavenger to minimize oxidative stress in the presence of high concentrations of metals. Other antioxidants such as nicotinamide adenine dinucleotide phosphate (NADPH), vitamin A, vitamin C, β-carotene, ubiquinone, flavonoids, fullerenes, and alginates may also be used.

[0099] Depending on the embodiment, the concentration of the antioxidant (such as glutathione) is about 0.2 g / L to about 2 g / L, preferably about 0.6 g / L.

[0100] A dispersant may be added to minimize bacteria-metal interactions. For example, a non-ionic dispersant such as polyvinylpyrrolidone (PVP) may be used. Also, other dispersants such as poly(ethylene oxide) (PEO), poly(vinyl alcohol-co-vinyl acetate) (PVAL), carboxymethyl cellulose (CMC), polyethylene glycol octylphenyl ether, cetyltrimethylammonium bromide (CTAB), ethylene glycol, poly(ethylene glycol) (PEG), sodium dodecylbenzenesulfonate (SDBS), sodium dodecyl sulfate (SDS), sodium polyacrylate, and sodium lignosulfonate (SLS) may be used. Depending on the embodiment, the concentration of the dispersant is about 0.2 g / L to about 1 g / L, preferably about 0.4 g / L.

[0101] The material from which PGM (such as Pt, Pd, Rh) is extracted can be a used catalyst. Catalysts usually gradually lose their catalytic activity due to structural changes, poisoning, overheating, or the deposition of foreign materials such as coke. A catalyst is considered "used" when it no longer exhibits the required activity or specificity demanded by the user.

[0102] Depending on the embodiment, the above material is a used catalytic converter or a used automotive catalyst. A catalytic converter is an exhaust gas control device that converts toxic gases and pollutants in the exhaust gas from an internal combustion engine into less toxic pollutants by catalyzing redox reactions. Catalytic converters are typically used with internal combustion engines fueled by gasoline or diesel, including lean burn engines, and in some cases kerosene heaters and stoves. Catalytic converters generally consist of a multi-component material containing rhodium, platinum and palladium, ceria (CeO 2 ), γ-alumina (Al 2 O 3 ), and other metal oxides. Typically, it consists of a cordierite (2Mg·2Al 2 O 3 ·5SiO 2 ) ceramic monolith and has strong porous walls surrounding an array of parallel channels. Cordierite is used because it can withstand the high temperature of the exhaust gas and the high coefficient of thermal expansion encountered when the engine is first started (typically, the exhaust gas temperature can reach several hundred degrees in less than a minute). Also, a metal monolith can be used. To achieve a large surface area for the catalytic reaction, the inner surface of the monolith is covered with a thin film (30 - 50 μm) of a very porous material known as a washcoat. Generally, the washcoat consists of alumina (70 - 85%) and oxides that act as structural promoters (stabilizers such as BaO for maintaining surface area) and chemical promoters (such as CeO 2 ). This system serves as a support for PGM (Pt, Pd, and Rh). When unburned fuel passes through the catalytic converter, the catalytic converter can be used. Since the catalytic converter can get very hot during use, unburned fuel can ignite inside the converter, and as a result, the ceramic catalyst can be completely or partially melted. Also, other fluids such as antifreeze or oil can enter the vehicle's exhaust system. This generates thick soot and carbon, covering and clogging the air passages of the converter's ceramic honeycomb catalyst.

[0103] According to some embodiments, the above method is characterized by a volume of at least 1 L.

[0104] According to some embodiments, the above method further includes a step of bioreducing the leachate of step c) to form nanoparticles.

[0105] According to some embodiments, the leachate of step c) is bioreduced using Cupriavidus metallidurans. Cupriavidus metallidurans is a gram-negative bacterium that does not form spores and may be adapted to withstand heavy metal stress.

[0106] According to some embodiments, C. metallidurans is pre-incubated.

[0107] According to some embodiments, the leachate of step c) is bioreduced at a pH of about 4 to about 8, preferably about 6.

[0108] According to some embodiments, the nanoparticles are characterized by an average particle size of about 10 nm to about 80 nm.

[0109] According to some embodiments, the nanoparticles are characterized by a hydrodynamic diameter of about 80 nm to about 110 nm.

[0110] According to some embodiments, the nanoparticles are characterized by a polydispersity index of about 0.2 to about 0.3.

[0111] According to some embodiments, the above method a) surface oxidizing the above material, b) sonicating the surface-oxidized material of step a) in the presence of an acid, and c) performing bioleaching of the sonicated material of step b) in the presence of cyanide formed from cyanide-producing microorganisms in the presence of glycine to form a leachate containing PGM, The above cyanide-producing microorganisms are characterized by the HCN synthase operon (hcnA, hcnB, and hcnC) in the genome.

[0112] According to some embodiments, the method comprises: a) surface oxidizing the material; b) sonicating the surface-oxidized material of step a) in the presence of an acid; and c) bioleaching the sonicated material of step b) in the presence of cyanide formed from cyanide-producing microorganisms in the presence of glycine to form a leachate containing PGM. The cyanide-producing microorganisms are selected from Chromobacterium violaceum, Pseudomonas fluorescens, Bacillus megaterium, or combinations thereof.

[0113] The present invention also provides a method for pretreating a material containing a platinum group metal (PGM), the method comprising: sonicating the material in the presence of an acid, wherein at least the surface of the material is oxidized.

[0114] According to some embodiments, the oxidized surface of the material contains PtO 2 , PdO, and / or Rh 2 O 3 and includes.

[0115] According to some embodiments, the acid is nitric acid.

[0116] The present invention provides a method for pretreating a material containing a platinum group metal (PGM), the method comprising: reducing the material in the presence of a reducing agent, wherein at least the surface of the material is oxidized.

[0117] According to some embodiments, the reducing agent is an organic acid.

[0118] According to some embodiments, the reducing agent is selected from formic acid and / or ascorbic acid.

[0119] Depending on the embodiment, the above method further includes a step of subjecting the above material to ultrasonic treatment in the presence of an acid before the reduction step.

[0120] The present invention also provides a method for extracting platinum group metals (PGMs) from a material, the method comprising: a) bioleaching the above material in the presence of cyanide formed from cyanide-producing microorganisms in the presence of a cyanide precursor to form a leachate containing PGM.

[0121] Depending on the embodiment, the above method includes: a) bioleaching the above material in the presence of cyanide formed from cyanide-producing microorganisms in the presence of a cyanide precursor, and further in the presence of a ROS scavenger and / or a dispersant, to form a leachate containing PGM.

[0122] Depending on the embodiment, the above method includes: a) bioleaching the above material in the presence of cyanide formed from cyanide-producing microorganisms in the presence of a cyanide precursor, and further in the presence of a ROS scavenger and / or a dispersant, to form a leachate containing PGM, wherein the concentration of the above ROS scavenger is about 0.2 g / L to about 2 g / L, and the concentration of the above dispersant is about 0.2 g / L to about 1 g / L.

[0123] Depending on the embodiment, the above method includes: a) bioleaching the above material in the presence of cyanide formed from cyanide-producing microorganisms in the presence of a cyanide precursor, and further in the presence of a ROS scavenger and / or a dispersant, to form a leachate containing PGM, wherein the concentration of the above ROS scavenger is about 0.2 g / L to about 2 g / L, and the concentration of the above dispersant is about 0.2 g / L to about 1 g / L, and the above ROS scavenger is added after mixing the above material with the above cyanide-producing microorganisms for at least 10 hours.

[0124] The following presents an exemplary description of the functions of the present invention. In the following embodiments, the present invention is described in relation to several conditions for the sake of consistency in introducing the present invention. However, those skilled in the art will understand that the present invention is not limited to such.

Example

[0125] 1. Ultrasonic-assisted nitric acid pretreatment to enhance the bio-recovery of PGM The elemental composition of the metal-supported solid is an issue for sensitive biological processes. SAC contains a mixture of metal oxide additives and other metals excluding PGM. Unfortunately, metals, especially copper, zinc, iron, and titanium, are present at high concentrations and compete to form stable metal-cyanide complexes that reduce the recovery efficiency of PGM. In addition to these metals, other metals present in SAC are toxic and directly affect bacterial growth and activity. Also, the presence of metal oxides forms a passivation layer on the surface, making it difficult for the leaching agent to extract the desired metal. Therefore, it is necessary to remove these interfering metals at the initial stage of the metal recovery process. Since the use of ultrasonic-assisted nitric acid pretreatment and its effect on the bio-extraction of PGM from SAC have not been reported, the inventors examined the effect of ultrasonic-assisted nitric acid pretreatment of SAC to remove the competing metals copper, zinc, iron, and titanium to the maximum extent.

[0126] To avoid the loss of Pd during ultrasonic treatment, the surface oxidation of Pd present in SAC was carried out before ultrasonic treatment. This is because Pd is soluble in nitric acid, while Pt and Rh have the lowest solubility in nitric acid. By removing a large amount of Pd before pretreatment, the amount of Pd that can be recovered during bioleaching is effectively reduced. In the laboratory atmosphere, the surface oxidation of Pd was carried out by heating SAC in a furnace to form a protective oxide layer on the surface and reduce its dissolution in the ultrasonic-assisted nitric acid pretreatment. The oxidation of PGM at a lower temperature is characterized by the formation of a thin cloudy film, visible or invisible, on their surfaces. Generally, PGM shows resistance to oxidation in air. Since Pd is soluble in nitric acid and leaches out during ultrasonic pretreatment with nitric acid, the surface oxidation of Pd was carried out before ultrasonic treatment to minimize its removal during the pretreatment of SAC.

[0127] Multivariate optimization is a rapid, accurate, and time-saving approach that reduces the total number of experiments required to develop a mathematical model and evaluate the statistical significance of the factors being studied. The Response Surface Methodology (RSM) is a powerful statistical tool used to evaluate interaction effects and build a functional relationship between the response of interest and the process variables. RSM identifies the optimal conditions of the variables that result in the minimum or maximum value of the response over the desired range. To investigate the effects of various process variables on the ultrasonic-assisted pretreatment of SAC, the Central Composite Design (CCD) of RSM was applied to construct a statistical model for the optimal removal rates of Cu, Zn, Fe, and Ti. A multi-objective approach was used to optimize various process parameters for the SAC pretreatment. The selected parameters and their levels designed by the CCD of RSM are shown in Table 1.

[0128]

Table 1

[0129] The experimental relationships for the extraction of Cu, Zn, Fe, and Ti with respect to the coded factors are given by the following quadratic polynomials 1, 2, 3, and 4, respectively. Cu extraction rate (%) = 64.32 - 1.47A + 4.59B - 5.64C - 3.32D + 3.52E + 0.28AB - 0.19AC - 0.45AD - 0.42AE - 0.33BC - 0.61BD + 0.01BE - 1.02CD - 1.52CE - 0.95DE - 4.14A 2 - 2.54B 2 - 5.27D 2 - 2.46E 2 (1) Zn extraction rate (%) = + 71.24 - 1.83A + 4.09B - 6.19C - 3.37D + 3.64E + 0.32AB - 0.80AC - 0.59AD - 0.85AE - 0.50BC - 0.78BD + 0.55BE - 0.56CD - 1.47CE - 0.78DE - 4.01A 2 - 3.35B 2 - 5.80D 2 - 2.95E 2 (2) Fe extraction rate (%) = + 47.11 - 1.06A + 3.91B - 4.91C - 1.86D + 4.42E + 0.38AB - 0.69AC - 0.24AD - 0.71AE - 0.09BC - 0.24BD + 0.90BE - 0.93CD - 1.16CE - 0.73DE - 3.31A 2 - 2.02B 2 - 3.85D 2 - 1.91E 2 (3) Ti extraction rate (%) = + 58.42 - 0.91A + 3.96B - 4.86C - 1.37D + 5.01E - 0.41AB - 0.20AC - 0.09AD - 0.12AE - 1.29BC + 0.99BD - 0.31BE - 0.09CD - 0.41CE - 0.66DE - 4.79A 2 - 2.84B 2 - 2.93D 2 - 1.44E 2 (4)

[0130] A, B, C, D, and E represent the duration (minutes) of ultrasonic treatment, ultrasonic output (%), ultrasonic frequency (kHz), nitric acid concentration (M), and temperature (°C), respectively. The coefficients of each of the factors (A, B, C, D, and E) in the experimental models (Equations 1 - 4) directly indicate their effects on the overall process. Examining the linear coefficients of these variables in these equations, which were developed by fitting the experimental results, the following was shown. i. For the copper removal rate (%), the order of parameter significance was in the order of ultrasonic frequency > ultrasonic output > temperature > nitric acid > ultrasonic duration. Among these, ultrasonic frequency, nitric acid, and ultrasonic duration had negative linear effects, while ultrasonic output and temperature had positive linear effects. ii. For the zinc removal rate (%), the order of parameter significance was in the order of ultrasonic frequency > ultrasonic output > temperature > nitric acid > ultrasonic duration. Among these, ultrasonic frequency, nitric acid, and ultrasonic duration had negative linear effects, while ultrasonic output and temperature had positive linear effects. iii. For the iron removal rate (%), the order of parameter significance was in the order of ultrasonic frequency > temperature > ultrasonic output > nitric acid > ultrasonic duration. Among these, ultrasonic frequency, nitric acid, and ultrasonic duration had negative linear effects, while ultrasonic output and temperature had positive linear effects. iv. For the titanium removal rate (%), the order of parameter significance was in the order of temperature > ultrasonic frequency > ultrasonic output > nitric acid > ultrasonic duration. Among these, ultrasonic frequency, nitric acid, and ultrasonic duration had negative linear effects, while ultrasonic output and temperature had positive linear effects.

[0131] Analysis of variance (ANOVA) is required to test the significance and validity of the quadratic model and to examine the effects of process parameters on each response.

[0132] Selective extraction of copper, zinc, iron, and titanium Using the analysis of variance (ANOVA), the effects of process parameters on the extraction of Cu, Zn, Fe, and Ti were examined. Statistical analysis was applied to determine significant terms and process variables, as well as their individual and interaction effects on Cu, Zn, Fe, and Ti removal.

[0133] For the extraction of Cu, Zn, and Fe, the F-values of the model (21.25 for Cu, 26.61 for Zn, and 23.69 for Fe) and the P-values (<0.0001) indicate that the model is highly significant, and the accuracy of the model is demonstrated when the likelihood of error occurrence is less than 0.01%. In these models, parameters A, B, C, D, E, CE, A 2 、B 2 、C 2 、D 2 、and E 2 are significant terms with high F-values and P-values less than 0.05. In other words, the linear effects of variables A, B, C, D, and E, the quadratic terms A 2 、B 2 、C 2 、D 2 、and E 2 、and the interaction between the interaction term CE were significant in the extraction of Cu, Zn, and Fe. The coefficients of variation for Cu, Zn, and Fe were 7.68%, 6.57%, and 8.41%, respectively, suggesting that the model has reproducibility and appropriately explains the response.

[0134] For the extraction of Ti, the F-value of the model (38.15) and the P-value (<0.0001) indicate that the model is highly significant, and the accuracy of the model is demonstrated when the likelihood of error occurrence is less than 0.01%. In this model, parameters A, B, C, D, E, BC, BD, A 2 、B 2 、C 2 、D 2 、and E 2 are significant terms with F-values and P-values less than 0.05. The coefficient of variation for Ti is 5.22%, suggesting that the model has reproducibility and appropriately explains the response. In all models, R 2The values are relatively high (Cu is 0.94, Zn is 0.95, Fe is 0.94, Ti is 0.96).

[0135] Effect of individual process variables on metal extraction The effects of each process variable on the extraction of Cu, Zn, Fe, and Ti are shown in Figures 1, 2, 3, and 4, respectively.

[0136] Ultrasonic duration: The one-factor plots in Figures 1a, 2a, 3a, and 4a show the relationship between the ultrasonic duration and the extraction rates of Cu, Zn, Fe, and Ti, respectively, when other parameters are kept constant at their median values. When the ultrasonic duration was increased up to 70 - 80 minutes, the extraction rates of these metals reached their maximum. By performing ultrasonic treatment, the dissolution of metals increases due to the deaggregation and destruction of SAC particles, the contact between SAC and the liquid medium increases, and the heat generated by the ultrasonic waves increases. However, when exposed to ultrasonic treatment for a long time, the concentration of dissolved metals decreases. This is because the dissolved metals adsorb on the surface of the oxide. This phenomenon has been observed in conventional leaching processes of metals such as nickel, copper, and cobalt.

[0137] Ultrasonic Output (%): The ultrasonic output is the second most important parameter in the extraction of Cu and Zn and the third most important parameter in the extraction of Fe and Ti. The one-factor plots in Figures 1b, 2b, 3b, and 4b show the relationship between the ultrasonic output and the extraction rates of Cu, Zn, Fe, and Ti, respectively, when other parameters are kept constant at their median values (however, the ultrasonic frequency was kept at 37 kHz). Increasing the ultrasonic output up to 80%, the acoustic cavitation increased with the increase in output, resulting in the maximum metal extraction rate. Further increasing the output, the metal extraction rate decreased, probably because the vibration amplitude became larger at a high ultrasonic output, separating the diaphragm from the solution and reducing the energy transfer efficiency between the ultrasonic processor and the liquid medium. As a result, the intensity of acoustic cavitation decreased. Such an effect on metal extraction has been reported for Cu recovery. Another possible reason is that at a high output of ultrasonic treatment, metals precipitate, resulting in a reduction in the metal extraction rate.

[0138] Ultrasonic Frequency: The one-factor plots in Figures 1c, 2c, 3c, and 4c show the relationship between the ultrasonic frequency and the extraction rates of Cu, Zn, Fe, and Ti, respectively, when other parameters are kept constant at their median values. The metal extraction rate was maximized when the ultrasonic frequency was low. The relationship between the ultrasonic intensity (I) and the frequency (f) is shown in Equation 5. I=(1 / 2)ρcf 2 A 2 (5) Here, A is the vibration amplitude, and ρ and c are the density and the speed of sound in the liquid medium, respectively. When the intensity I is constant, a lower ultrasonic frequency results in a larger amplitude, generating large cavitation bubbles, and consequently, strong hydrodynamic shear forces are generated, enhancing the metal extraction rate.

[0139] The models described by Equations 1 - 3 indicate that the ultrasonic frequency has the most significant effect on the extraction of Cu, Zn, and Fe, and the model described by Equation 4 indicates that the ultrasonic frequency is the second most significant parameter in the extraction of Ti.

[0140] Nitric acid concentration: The one-factor plots in Figures 1d, 2d, 3d, and 4d show the relationship between the nitric acid concentration and the extraction rates of Cu, Zn, Fe, and Ti, respectively, when other parameters are kept constant at their median values. The maximum metal extraction rate was obtained when the nitric acid concentration was 8 - 8.5 M. As the nitric acid concentration increases, the metal recovery rate decreases, which is due to the oxidation potential of nitric acid. As a result, the metal is oxidized and a passive layer is formed on the SAC, thereby reducing the metal extraction rate. Similar findings have been reported regarding the role of nitric acid concentration in metal leaching.

[0141] Temperature: The one-factor plots in Figures 1e, 2e, 3e, and 4e show the relationship between the ultrasonic treatment temperature and the extraction rates of Cu, Zn, Fe, and Ti, respectively, when other parameters are kept constant at their median values. As the temperature increases, there is a positive effect on the metal extraction rate, that is, the metal extraction rate increases as the temperature increases, and the maximum metal extraction rate was obtained at a temperature of 70 °C. As the temperature increases, the reaction between the metal and the acid is accelerated, the reaction rate increases, and the metal extraction rate increases.

[0142] Interaction effects of process variables on metal extraction Response surfaces were plotted between two independent process variables while keeping other independent variables constant at their median values. Plots regarding the interaction effects of process variables on the extraction of Cu, Zn, Fe, and Ti are shown in Figures 5, 6, 7, and 8, respectively. These plots are based on the experimental conditions set by CCD for each response. The 3D surface graphs show the interaction effects between two parameters (variables) and each response. In the 3D surface graphs, the two variable parameters are shown on the x-axis, and all other parameters are kept constant at their median values. The interaction plots show the interaction effects between significant interaction terms and each response.

[0143] The ultrasonic output (B) and the ultrasonic frequency (C) are the most important parameters for the Cu extraction rate, and the coefficient of CE shows a significant interaction effect on the Cu extraction rate (Equation 1). Figures 5(a - e) show the interaction between the most significant parameter, i.e., the ultrasonic frequency, and all other parameters. The Cu extraction rate (%) was maximized at an ultrasonic frequency of 37 kHz and a temperature of 70 °C [Figures 5(a and e)]. Figures 5(b and d) show that the Cu extraction rate (%) was maximized with nitric acid of 8 - 8.5 M and an ultrasonic treatment duration of 80 minutes. When the ultrasonic treatment duration was further increased, dissolved species adsorbed onto the SAC particles, resulting in a decrease in the Cu removal rate, and the loss increased with a higher acid concentration. At a high acid concentration (12 M), the Cu removal rate was maximized with an ultrasonic treatment duration of 75 minutes. The decrease in the metal extraction rate is due to the high oxidation potential of high - concentration nitric acid, as a result of which the metal is oxidized and a passivation layer is formed on the SAC particles. As the ultrasonic treatment duration and the nitric acid concentration increase, the loss of the Cu extraction rate becomes larger, which is due to the coupling effect of the adsorption of dissolved species and the passivation of the SAC particles. It has been reported that the copper extraction rate similarly decreases as the ultrasonic treatment duration increases.

[0144] The Cu extraction rate was maximized at a low ultrasonic frequency, but a higher concentration of nitric acid is required to extract a sufficient amount of Cu at a high ultrasonic frequency. At a high frequency, the Cu removal rate is low due to the adsorption of dissolved species and the small cavitation effect. Therefore, it is concluded that the synergetic effect between SAC passivation, small vibration amplitude, and weak hydrodynamic shear force is the cause of the significant decrease in the Cu extraction rate.

[0145] Figure 5c shows the interaction between the ultrasonic frequency and the ultrasonic output. The extraction rate (%) of Cu reached its maximum when the ultrasonic output was 80%. Similar findings have been reported regarding the effects of ultrasonic frequency and ultrasonic output on the Cu removal rate. The interaction between the ultrasonic output and the ultrasonic duration at various nitric acid concentrations indicates that the removal rate of Cu was maximized at an optimal nitric acid concentration of 8.5M. As the acid concentration increased, the removal rate of Cu decreased, probably due to a significant passivation effect. Overall, several conclusions can be drawn from Figures 5(a - e). (i) The extraction rate (%) of Cu reached its maximum with a 80 - minute ultrasonic treatment duration. (ii) As the acid concentration increased, especially when the ultrasonic treatment was prolonged, the metal extraction rate decreased. (iii) The optimal extraction rate (%) of Cu was obtained at an ultrasonic output of 80%. (iv) As the temperature increased, the Cu extraction rate increased. (v) The optimal Cu removal rate was obtained at a low ultrasonic frequency, but a lower acid concentration was required.

[0146] Regarding the Zn extraction rate, the ultrasonic output (B) and the ultrasonic frequency (C) are the most important parameters, and the coefficient of CE indicates a significant interaction effect on the Zn extraction rate (Equation 2). Figures 6(a - e) show the interaction between the most significant parameter, i.e., the ultrasonic frequency, and all other parameters. Similar trends were observed for the extraction of Cu and Zn. A low acid concentration (8.5M) is favorable for the dissolution and extraction of Zn with an 80 - minute ultrasonic treatment time before it begins to decline due to adsorption and precipitation on the SAC particles. Both Zn and Cu had higher removal rates at low acid concentrations, probably because the passivation effect was low. As the temperature increased, the extraction rates of Zn and Cu increased.

[0147] Figure 6c shows the relationship between ultrasonic output and frequency at a constant ultrasonic treatment duration and a constant nitric acid concentration. When the ultrasonic frequency was high, the Zn removal rate decreased. This decrease was more evident when the ultrasonic output was low and the ultrasonic treatment duration was short. This low Zn removal rate was due to the small vibration amplitude and small cavitation effect at high frequencies, low ultrasonic intensity, and insufficient metal dissolution time. Previous studies have reported that although the dissolution rate of metal decreases as the ultrasonic treatment time increases, it does not affect the dissolution rate of specific metals including zinc. In the findings of the present inventors regarding the effect of ultrasonic treatment duration on the Zn removal rate, the ultrasonic treatment duration as an individual parameter has the least effect on the Zn removal rate, but it has been shown that its effectiveness increases due to interaction with other parameters such as ultrasonic output, especially when the ultrasonic frequency is high. It is concluded that the optimal conditions for Cu extraction are advantageous for the Zn removal rate.

[0148] Regarding the extraction rates of Fe and Ti, the ultrasonic frequency (C) and temperature (E) are the most important parameters. The coefficient of CE shows a significant interaction effect on the extraction rate of Fe, and the coefficients of BC and BD show significant interaction effects on the extraction rate of Ti (Equations 3 and 4). Figures 7(a - d) and 8(a - d) show the interactions between ultrasonic frequency and all other parameters for the extraction of Fe and Ti, respectively. Both Fe and Ti show similar behavior for individual parameters as seen for the extraction of Cu and Zn. Similar to the extraction of Cu and Zn, the coefficient of CE shows the most significant interaction effect on the Fe extraction rate [Equation 3, Figure 7(e)]. However, the coefficients of BC and BD show the most significant interaction effect on the Ti extraction rate [Equation 4, Figures 8(e and f)]. The reaction temperature shows a positive effect on the extraction of Cu, Zn, Fe, and Ti, that is, as the temperature increases, the metal removal rate increases due to the increase in reaction rate and the increase in reaction kinetics, and the optimal metal removal rate was observed at a temperature of 70°C.

[0149] To determine the normality of the dataset, normal probability plots as shown in Figs. 9(a, c, e, and g) were obtained. The presence of points mainly on the straight line indicated a normal distribution and supported the validity of the least-squares fitting of the developed model. Relatively high R 2 values for Cu, Zn, Fe, and Ti indicated that the quadratic model was suitable for representing the experimental data.

[0150] Figs. 9(b, d, f, and h) show the predicted data versus the actual data for the metal extraction rate. A strong correlation between the predicted response and the experimental response indicated good model fitting and high significance for all models. The proximity of most points to the 45° line demonstrated the accuracy of the models for predicting the response.

[0151] Optimization and verification The significant factors and optimal conditions for the extraction of Cu, Zn, Fe, and Ti from SAC in ultrasonic-assisted pretreatment were established from the fitted regression model. Based on numerical optimization, the maximum extraction rates of Cu, Zn, Fe, and Ti (80.74%, 86.68%, 59.14, and 70.44, respectively) were obtained under the optimal conditions (i.e., ultrasonic power 80%, nitric acid concentration 8.5 M, ultrasonic duration 80 min, ultrasonic frequency 37 kHz, and temperature 70 °C). From the verification experiments under these optimal conditions, the extraction rates of Cu (82%), Zn (88%), Fe (60), and Ti (72) were shown, which were consistent with the model. The metal removal rates during the ultrasonic-assisted pretreatment of the optimized SAC are shown in Table 2.

[0152]

Table 2

[0153] As is evident, a substantial amount of most non-PGMs (which, if not removed, could interfere with PGM recovery by forming stable complexes with cyanide) was removed by ultrasonic irradiation. The particle size was reduced by ultrasonic pretreatment. This is because the particles are destroyed by acoustic cavitation. At lower frequencies, this pretreatment increases particle collisions, thereby reducing the particle size, increasing the surface area, and enhancing subsequent metal leaching.

[0154] 2.C. violaceum-mediated improvement of PGM bio-recovery rate from SAC To enhance cyanide production and bioleaching efficiency, C. violaceum, a BSL-2 organism, was used. C. violaceum is a Gram-negative facultative anaerobe that does not form spores. This bacterium can utilize a wide range of energy sources using appropriate oxidase and reductase enzymes and is used for metal bio-dissolution due to its cyanide-related metabolic activity. The HCN synthase operon (hcnA, hcnB, and hcnC) in its genome encodes formate dehydrogenase and two amino acid oxidases, respectively, and is involved in cyanate synthesis. Cyanide synthesis occurs in the presence of low levels of oxygen, where four electrons generated by HCN synthase are transferred to oxygen. Thus, C. violaceum produces HCN under aerobic conditions, and cyanide is produced as a secondary metabolite. Cyanide-producing bacteria inherently have cyanide-degrading ability. C. violaceum and B. megaterium synthesize β-cyanoalanine synthase, an enzyme that converts cyanide to β-cyanoalanine during the late stationary and early death phases. Most transition metals (excluding lanthanoids and actinoids) form stable water-soluble complexes with cyanide, and these complexes exhibit very high chemical stability.

[0155] C. violaceum wild strains have low cyanide production yields and strict regulation under stoichiometric control, so modified strains were constructed to increase cyanide production. Tay et al., 2013 (Department of Biochemistry, Faculty of Medicine, NUS (incorporated herein by reference)) constructed a metabolically modified strain of C. violaceum. The modified strain named C. violaceum pBAD hcnABC has two sets of cyanide-producing operons (whereas the wild strain has one set of cyanide-producing operons) and requires L-(+)-arabinose as an inducer to induce the expression of the double cyanide-producing operons. The metabolically modified strain of C. violaceum was used in bioremediation research. Pretreated SAC was used for two-stage bioremediation.

[0156] Growth (OD 600nm ) and cyanide production of C. violaceum pBAD hcnABC strains with initial pH values of 7.5, 8, 9, 10, and 11 were observed. Optimal bacterial growth was observed at pH 7.5, and long lag phases were observed at pH 10 and 11 due to the alkaline medium. Bacterial growth was high at pH 7.5, but the highest cyanide production rate was achieved at pH 9. The cyanide production rate peaked towards the initial stationary phase. After 30 hours, the cyanide produced by C. violaceum pBAD hcnABC at pH 7.5, 8, 9, 10, and 11 was 30.23 mg / L, 33.87 mg / L, 40.44 mg / L, 21.57 mg / L, and 10.18 mg / L, respectively.

[0157] Dissolved oxygen (DO) is an important parameter in biological processes because it directly affects bacterial growth, cyanide production, and leaching. Oxygen consumption is regarded as an indicator of metabolic activity. This is because DO plays an important role during bacterial growth and leaching, as the DO level decreased significantly within 24 hours due to bacterial respiration. To investigate the role of DO in bacterial growth and cyanide production, the cell count (CFU / mL) and cyanide production rate were measured over 6 days with initial pH values of 7.5, 8, 9, 10, and 11. The pH value was set before adding the inoculum, and glycine was added in the mid-log phase. Hydrogen peroxide (H 2 O 2 , 35% w / w) was used as an additional oxygen source for the bacteria. A predetermined amount of H 2 O 2 (0.04% v / v) was added to the culture 12 hours before sample collection on days 1 to 6. The bacteria grew better and produced more cyanide in the presence of H 2 O 2 . After 6 days, in the absence of H 2 O 2 , the cyanide produced by C. violaceum pBAD hcnABC at pH 7.5, 8, 9, 10, and 11 was 33.29 mg / L, 38.27 mg / L, 48.30 mg / L, 35.26 mg / L, and 20.17 mg / L, respectively. After 6 days, in the presence of H 2 O 2 , the cyanide produced by C. violaceum pBAD hcnABC at pH 7.5, 8, 9, 10, and 11 was 35.33 mg / L, 41.36 mg / L, 52.20 mg / L, 39.30 mg / L, and 24.37 mg / L, respectively.

[0158] Two-stage bioleaching was used to recover PGM from SAC using metabolically engineered C. violaceum pBAD hcnABC. Multivariable optimization and RSM techniques were used to evaluate the individual and interaction effects and to establish the functional relationship between the objective response and the process variables.

[0159] Two - stage bioleaching using central composite design (CCD) Two - stage bioleaching was carried out in 250 mL Erlenmeyer flasks containing 100 mL of culture medium. In the two - stage bioleaching, first, bacteria were grown at pH 7.5 (in the absence of SAC) to achieve optimal bacterial growth, and then the pH was changed (before adding SAC) to the optimal cyanide concentration. Then, a predetermined amount of glycine and pretreated SAC were added to the culture medium at the mid - logarithmic phase of bacterial growth and 24 hours after inoculation (day 0), respectively. Samples were taken daily from day 0 to day 5 to monitor cell density, pH, free cyanide concentration, and PGM recovery rate. Hydrogen peroxide (H 2 O 2 , 35% w / w) was used as an additional oxygen source for the bacteria. A predetermined amount of hydrogen peroxide (H 2 O 2 ) was added to the culture 3 hours before sampling from day 1 to day 5. When the cell density and cyanide yield were optimized, the pretreated SAC was added to the culture medium. The effects of the following five parameters (glycine concentration, pulp density, pH, H 2 O 2 concentration, and temperature) on the bioleaching efficiency with respect to PGM recovery rate were investigated. The central composite design (CCD) of RSM was applied to construct a statistical model for the optimal recovery rates of Pt, Pd, and Rh from SAC. The selected parameters and their levels designed by the CCD of RSM are shown in Table 3.

[0160]

Table 3

[0161] A second - order model for the recovery rates of Pt, Pd, and Rh was developed, and the experimental relationships between the metal recovery rates and the coded factors are shown in quadratic polynomials 6, 7, and 8, respectively. Pt recovery rate (%) = +55.63 + 1.49A - 10.92B + 2.60C + 1.04D + 0.23E + 0.09AB - 0.25AC - 0.25AD + 0.13AE - 0.26BC - 0.18BD - 0.16BE - 0.53CD - 6.55A 2 + 0.73B 2 - 2.59C 2 - 1.29D 2 - 2.74E 2 (6) Pd recovery rate (%) = +62.32 + 1.45A - 9.35B + 2.51C + 1.27D + 0.29E - 0.48AB - 0.25AC - 0.13AD + 0.25AE - 0.03BC - 0.34BD - 0.43BE - 0.49CD - 6.58A 2 + 0.21B 2 - 2.29C 2 - 1.13D 2 - 2.30E 2 (7) Rh recovery rate (%) = +90.42 + 0.76A - 7.56B + 1.95C + 0.74D + 0.64E + 0.04AB - 0.56AC - 0.19AD + 0.62AE + 1.18BC + 0.71BD + 0.16BE - 0.04CD - 4.09A 2 - 0.28B 2 - 3.21C 2 - 0.36D 2 - 2.08E 2 (8)

[0162] A, B, C, D, and E represent glycine concentration (g / L), pulp density (% w / v), pH, H 2 O 2 concentration (% v / v), and temperature (°C), respectively. The coefficients of each factor (A, B, C, D, and E) in the experimental models (Equations 6 - 8) directly indicate their effects on the overall process. When examining the linear coefficients of the variables in the equations developed by fitting the experimental results, the order of significance of the parameters was shown as pulp density > pH > glycine concentration > H 2 O 2 concentration > temperature. Among these, pulp density has a negative linear effect, and pH, glycine concentration, H 2 O2 Concentration and temperature have a positive linear effect. For Pt and Pd, glycine concentration, pulp density, pH, and H 2 O 2 The parameter of concentration showed a significant (p < 0.001) linear effect, while for Rh, all parameters showed a significant (p < 0.001) linear effect.

[0163] Analysis of variance (ANOVA) is required to test the significance and validity of the quadratic model and to examine the effects of process parameters on each response.

[0164] Selective Extraction of Platinum, Palladium, and Rhodium Using the analysis of variance (ANOVA) of the quadratic polynomial, the effects of process parameters on the recovery rates of Pt, Pd, and Rh were examined. Statistical analysis techniques were applied to determine significant terms and process variables, and their individual and interaction effects on the PGM recovery rates.

[0165] The F-values of the models (60.40 for Pt, 76.85 for Pd, and 80.15 for Rh) and the P-values (<0.0001) indicate that the models are highly significant, and the accuracy of the models is demonstrated when the likelihood of error occurrence is less than 0.01%. In the quadratic models of the recovery rates of Pt and Pd, the parameters A, B, C, D, E, A 2 、B 2 、C 2 、D 2 、and E 2 are significant terms with high F-values and P-values less than 0.05. The coefficient of variation (CV) is a measure of the residual variation of the data with respect to the average particle size. Relatively low CV values indicate that the experimental data are more accurate and reliable. The CVs of the Pt model and the Pd model being 4.08% and 2.92% respectively suggest that the models are reproducible and can appropriately explain the responses. In the quadratic model of Rh recovery, the parameters A, B, C, D, E, BC, BD, A 2 、C 2 、and E 2is a significant term with a high F-value and a P-value less than 0.05. The CV of the Rh recovery model being 1.56% suggests that the model has reproducibility and appropriately explains the response. All models have relatively high R 2 values (Pt is 0.99, Pd is 0.98, Rh is 0.99).

[0166] Individual effects of process variables on PGM recovery rate The individual effects of process variables on the recovery rates of Pt, Pd, and Rh are shown in Figures 10, 11, and 12, respectively. These figures represent one-factor plots showing the relationship between the individual process variables and the PGM recovery rate. In the one-factor plots, one variable was varied over the range of experimental conditions while the other parameters were kept constant at their median values.

[0167] Glycine concentration: One-factor plots Figures 10a, 11a, and 12a show the relationship between glycine concentration and the recovery rates of Pt, Pd, and Rh, respectively. As the glycine concentration increased, the recovery rates of Pt, Pd, and Rh increased. The PGM recovery rate reached its maximum at a glycine concentration of 10 g / L. Further increase in glycine concentration led to a decrease in the PGM recovery rate. As mentioned above, glycine is a precursor in cyanide production. High concentrations of glycine are favorable for cyanide production and directly affect the PGM recovery rate, but when the glycine concentration becomes too high, bacterial growth is inhibited, thereby reducing the PGM recovery rate. Previous studies have reported that glycine beyond the optimal concentration inhibits the synthesis of peptidoglycan in the bacterial cell wall.

[0168] Pulp density: Pulp density shows a significant negative linear effect and is the most important parameter for PGM recovery. The one-factor plots in Figures 10b, 11b, and 12b show the relationship between pulp density and the recovery rates of Pt, Pd, and Rh, respectively. The PGM recovery rate reached its maximum at a pulp density of 0.5% w / v, and as the pulp density increased, the PGM recovery rate decreased significantly. The decrease in the PGM recovery rate at high pulp density may be due to several reasons as follows.

[0169] (1) When heavy metals are present at higher concentrations, the toxicity of the metals increases and the number of viable cells decreases. The increased toxicity inhibits bacterial growth and thus cyanide production. Although two-stage bioleaching significantly reduced the negative effect of metal toxicity on bacterial growth, a decrease in the number of viable cells and cyanide production through bioleaching was inevitable. Similar findings regarding the bioleaching of gold from electronic waste, namely that high pulp density has a negative effect on the gold recovery rate, have been reported.

[0170] (2) The presence of higher concentrations of Cu, Zn, Fe, and Ti competes with the cyanide leaching agent, thus reducing the concentration of cyanide available for the formation of PGM-cyanide complexes and resulting in a decrease in the recovery rate. Figures 13(a - c) show the effect of pulp density on the PGM recovery rate. As is evident, the recovery rates of Pt, Pd, and Rh were maximized at a pulp density of 0.5% w / v, and the metal recovery rate decreased as the pulp density increased.

[0171] pH: pH shows a significant positive linear effect and is the second most important parameter for PGM recovery. The one-factor plots in Figures 10c, 11c, and 12c show the relationship between pH and the recovery rates of Pt, Pd, and Rh, respectively. As pH increased from 7 to 9, the PGM recovery rate increased significantly. The PGM recovery rate was maximized at pH 9 - 9.5 and decreased as pH increased (above pH 10). The following several reasons are hypothesized for the effect of pH.

[0172] (1) Optimal bacterial growth was observed at pH 7.5 and 8, while the maximum free cyanide was observed at pH 9 - 9.5. The cyanide leaching agent is undissociated hydrocyanic acid (HCN) and cyanide anion (CN -) is dissolved as free cyanide and generated. At pH 7.5 and 8, the cyanide in the system mainly exists as (volatile) HCN. Therefore, the amount of cyanide available for PGM-cyanide complex formation decreases. Despite the optimal bacterial growth at pH 7.5 and 8, the volatility of cyanide results in a decrease in metal recovery rate. However, at pH 9 - 9.5, the free cyanide mainly exists as cyanide anions that form PGM-cyanide complexes. Therefore, an optimal PGM recovery rate was achieved at pH 9 - 9.5.

[0173] (2) In alkaline media (pH 10 and 11), little bacterial growth with a long lag phase was observed. Alkaline media inhibit bacterial growth, thereby limiting the production of cyanide for PGM-cyanide complex formation.

[0174] Figures 14(a - c) show the effect of pH on the PGM recovery rate. As is evident, the recovery rates of Pt, Pd, and Rh were maximized at pH 9.5.

[0175] H 2 O 2 Concentration: DO decreases significantly within 24 hours due to bacterial respiration, but oxygen is also necessary for PGM-cyanide complex formation. Figures 10d, 11d, and 12d show that DO plays an important role during bioleaching, and adding H 2 O 2 significantly enhances the bioleaching efficiency. The PGM recovery rate was maximized at the optimal H 2 O 2 concentration of 0.08% v / v. Similar findings that the addition of H 2 O 2 increased the gold recovery rate have been reported in previous studies. The effect of H 2 O 2 on the PGM recovery rate is shown in Figures 15(a - c), and as is evident, adding H 2 O 2 significantly increases the PGM recovery rate at the optimal pH 9.5.

[0176] Temperature: From Equations 6, 7, and 8, it is clear that among the five parameters investigated, temperature is the least significant parameter during bacterial growth and bioleaching, and it shows a positive linear effect on bacterial growth and PGM recovery rate. Figures 10e, 11e, and 12e show that within the temperature range investigated (i.e., 22 °C to 38 °C), the PGM recovery rate was maximized at 30 °C, which is also the optimal temperature for the growth of C. violaceum.

[0177] Interaction effects of process variables on PGM recovery rate Response surfaces were plotted between two independent process variables while keeping all other independent variables constant at their median values. Plots regarding the interaction effects of process variables on the recovery of Pt, Pd, and Rh are shown in Figures 16, 17, and 18, respectively. The 3D surface graphs show the interaction effects between two parameters (variables) and their respective responses. In the 3D surface graphs, the two parameters are shown on the x-axis and all other parameters are kept constant at their median values. Interaction plots show the interaction effects between significant interaction terms and their respective responses.

[0178] Figures 16(a - d), 17(a - d), and 18(a - d) show the interactions between the most significant parameter (pulp density) and all other parameters and their related responses for Pt, Pd, and Rh, respectively. As the glycine concentration increased up to 10 g / L, the PGM recovery rate increased, and this effect was more evident at alkaline pH. Further increase in the glycine concentration led to a decrease in the PGM recovery rate, probably due to its negative effect on bacterial growth and cyanide production. Also, H 2 O 2When the concentration of 2 O 2 was low, a significant decrease in PGM recovery was observed, and this effect was more evident at pH 8. Similarly, at an optimal glycine concentration of 10 g / L and a pulp density of 0.5% w / v, the PGM recovery was maximized. As the pulp density increased, the PGM recovery decreased significantly, and this effect was more evident with increasing glycine concentration. This is because high pulp density and glycine concentration have a negative impact on bacterial growth and thus on bioleaching efficiency. The interaction effect between glycine and H 2 O 2 concentration and the interaction effect between glycine and temperature were more evident at low glycine concentrations. When the glycine concentration was low in the presence of a smaller amount of H 2 O 2 , the PGM recovery decreased. Similarly, at lower glycine concentrations and extreme conditions, a significant decrease in PGM recovery was observed. The perturbation plots in Figures 16(e), 17(e), and 18(e) show the sensitivity of the process variables and represent the comparative effects of the factors at a specific point (the central point). A steep gradient or curvature of a factor indicates that the response is sensitive to that factor. A relatively flat line indicates insensitivity to changes in that factor. The perturbation plots and the quadratic polynomials 6, 7, and 8 show that pulp density, pH, and glycine concentration have the most significant effect on PGM recovery. Temperature and H 2 O 2 showed the least effect over the range of values investigated.

[0179] To determine the normality of the dataset, normal probability plots were obtained and are shown in Figures 19(a, c, and e). The presence of points mainly on the line indicates a normal distribution, supporting the validity of the least-squares fitting of the developed model. High R 2The values indicate that the quadratic model is suitable for representing the experimental data. Figures 19(b, d, and f) show the predicted data versus the actual data for the metal extraction rate. Good model fitting and high significance were shown for all the models due to the strong correlation between the predicted responses and the experimental responses. The accuracy of the model for predicting the responses was demonstrated by the fact that most of the points were close to the 45° line.

[0180] Optimization and verification The significant factors and optimal conditions for the recovery of Pt, Pd, and Rh from SAC were established from the fitted regression model. Based on numerical optimization, the maximum extraction rates of Pt, Pd, and Rh (68.47%, 73.10%, and 97.51% respectively) were predicted under the optimal conditions, i.e., glycine concentration 10 g / L, pulp density 0.5% w / v, pH 9.4, H 2 O 2 2 concentration 0.08% v / v, and temperature 30 °C. Verification experiments were conducted in 3 replicates under the optimal conditions and the developed surface response model was verified by comparing the results with those predicted by the model. The verification experiments under these optimal conditions resulted in the extraction of Pt (69%), Pd (74%), and Rh (99%), which were in agreement with the model.

[0181] Spent media leaching at alkaline pH Spent media leaching under alkaline conditions resulted in higher metal recovery rates compared to two-stage bioleaching. This is due to the following reasons.

[0182] (1) In two-stage bioleaching, bacteria consume oxygen for respiration, leaving little oxygen available for metal complex formation. In spent media leaching, by separating bacterial growth from metal complexation, more DO becomes available for metal complexation. This is the result of separating cells from the culture after reaching maximum cell density and cyanide production and using only cell-free metabolites (spent media) for leaching. For example, bacteria were grown at pH 9 until the cyanide concentration reached its maximum, after which the cell-free media (spent media) was recovered (by filtration), the pH was adjusted to pH 10.5, and then the pretreated SAC was added. The absence of cells in the leaching medium ensured a high concentration of DO available for PGM-cyanide complexation.

[0183] (2) Bacteria produce cyanide in the late logarithmic and early stationary phases. In the stationary phase, bacteria produce and consume cyanide simultaneously. In the late stationary phase, bacteria convert cyanide to non-toxic β-cyanoalanine. In cell-free spent media leaching, there is no consumption of cyanide by cells, thus increasing the PGM recovery rate.

[0184] (3) pH affects the dissociation of free cyanide ions (CN - ) and hydrogen cyanide as H + (HCN, pKa: 9.3). A low pH favors the reduction of cyanide by volatilization (as HCN), and a high pH favors the increase in dissociation and free cyanide ion (CN - ) concentration. Leaching of spent media under alkaline conditions minimized the reduction of the cyanide leaching agent and increased the PGM recovery rate.

[0185] Overall, in spent media leaching, the recovery rates of Pt, Pd, and Rh are increased due to the absence of (i) the conversion of cyanide to β-cyanoalanine, (ii) the adsorption of metal ions to biomass, and (iii) the reduction of cyanide by gas evolution. The spent media leaching experiment was carried out with H 2 O 2It was carried out under the optimal conditions of glycine concentration (10 g / L) and temperature (30 °C) in the absence of . The maximum PGM recovery rates [Pt (76%), Pd (81%), and Rh (100%)] were achieved at an alkaline pH with a pulp density of 0.5% w / v.

[0186] In Table 4, the PGM recovery rates in two-stage bioleaching and spent media leaching are compared. The spent media leaching at an alkaline pH results in the highest PGM recovery rate.

[0187]

Table 4

[0188] Scale-up of PGM recovery in a bioreactor The present invention also provides a scale-up of PGM recovery (from a working volume of 100 mL to 1 L) by the overall optimization of significant factors. As shown above, it is not easy to transfer from small-scale experiments to a large-scale setup feasible for industrial applications. The recovery process involves a number of factors that act with and / or against each factor and can thus be antagonistic. Through this study, the inventors have found a range of conditions that can bring about a synergistic (or at least additive) effect. As an example, the scale-up study was carried out in a bioreactor using a modified strain of C. violaceum with a pulp density of 1% w / v under the optimized conditions (glycine concentration 10 g / L, pH 9.4, and temperature 30 °C) in two-stage bioleaching. In the shake flask experiment, H 2 O 2were added at specific intervals. In the scale-up study, dissolved oxygen was supplied during bioreaching and maintained using purified oxygen continuously (DO measured as % saturation). The scale-up study was conducted under batch culture mode and fed-batch culture mode. In the batch culture mode, after inoculating 1 L of the culture, only base and acid (for pH control), oxygen, and antifoaming agent were added to the sterilized culture medium. In the fed-batch culture mode, fresh medium was added during bioreaching (along with the addition of acid, base, DO, and antifoaming agent), with an initial working volume of 0.5 L, and an additional 0.5 L of fresh medium was added 2 days after adding SAC. By using the fed-batch culture mode and providing new nutrients, bacterial growth, cyanide production, and metal-cyanide complexation were enhanced. The scale-up study was conducted with DO saturations of 10%, 20%, 30%, 40%, and 50%.

[0189] Samples were taken daily from day 0 to day 5 to monitor cell density, pH change, free cyanide concentration, and PGM recovery rate. The results of bacterial growth and cyanide concentration in two-stage bioreaching are reported in Figure 20, which also shows the effect of DO on bacterial growth and cyanide production. The optimal cell number and cyanide concentration in two-stage bioreaching were obtained in the presence of DO at 30% saturation. As the DO concentration increases, bacterial growth decreases and thus cyanide production decreases for the following reasons.

[0190] (1) High concentrations of DO were obtained with high air flow rates and stirrer speeds. Shearing force from the impeller generates shear stress, which damages the cell wall and thus impairs cell viability.

[0191] (2) When the DO concentration is high, reactive oxygen species (ROS) such as superoxide (O 2 - ) accumulate as by-products of aerobic metabolism, causing oxidative stress. Since these ROS are more reactive than oxygen molecules, they are toxic to cells.

[0192] Regarding the shaking flask experiment and the scale-up study in a bioreactor, when comparing the PGM recovery rates in two-stage bioleaching, the result showed that the PGM recovery rate of the latter was slightly higher. When using a shaking flask, the maximum PGM recovery rates were obtained for Pt (62%), Pd (69%), and Rh (96%) at a pulp density of 1% w / v. In the scale-up study under the batch culture mode, under the same operating conditions and with a DO saturation of 30%, the maximum PGM recovery rates of Pt (64%), Pd (70%), and Rh (98%) were obtained. Under the fed-batch culture mode, the maximum PGM recovery rates of Pt (65%), Pd (72%), and Rh (100%) were obtained. A slightly higher PGM recovery rate was obtained in the fed-batch culture mode compared to the batch culture mode. By leaching the used medium under the batch culture mode under the optimal conditions, when the pulp density was 1% w / v, the maximum PGM recovery rates of Pt (70%), Pd (75%), and Rh (100%) were obtained. The study of 1 liter in a bioreactor not only demonstrated the results of the shaking flask experiment but also showed that slightly higher PGM recovery rates could be achieved by better controlling DO in the process.

[0193] 3. Pretreatment by Reduction for Rapid and Improved Recovery of PGM As described above, the main drawbacks of bioleaching include the toxicity of the substrate, low leaching rate, and low efficiency when operating at high pulp densities. The low leaching rate and low leaching efficiency were caused by the presence of other competing metals interfering with the leaching process and the presence of a passivating oxide film on the solid. To overcome the former, competing metals such as Cu, Zn, Ti, and Fe were removed by pretreatment with ultrasonic treatment. However, the latter hinders PGM recovery, slows down the process, and makes it more inefficient. When heated up to 350 °C, Pt is converted to PtO by forming a thin solid oxide film that limits the bio-mobilization of Pt. 2 PdO and Rh 2 O 3 formation occurs at 800 - 840 °C and 600 °C respectively. The formation of PdO and Rh 2 O 3All of them form a protective layer on the surface, which hinders biocatalysis. Such temperatures generally occur in catalytic converters, and as a result, a part of the PGM is converted into their oxides. Although these PGM oxides do not exist in significant amounts, they form a protective layer on the PGM, resulting in a low metal recovery rate. To overcome this problem, a SAC reduction pretreatment was carried out before leaching to reduce the PGM oxides into elemental form, thereby increasing the recovery rate and speed. In the reduction pretreatment, three parameters, namely reducing agent concentration, reduction time, and reduction temperature, were investigated. Two reducing agents, namely formic acid and ascorbic acid, were used for the reduction pretreatment. The reduction pretreatment was carried out under experimental conditions of reducing agent concentration (1 vol%, 5 vol%, 10 vol%, 15 vol%), reduction time (30 minutes, 60 minutes, 90 minutes, 120 minutes), and reduction temperature (50 °C, 60 °C, 70 °C, 80 °C, 90 °C). The reduction experiments were carried out in a shaking water bath at a stirring speed of 250 rpm. A schematic diagram of the experimental design used in this study is shown in Figure 21. A series of pretreatment techniques were applied. First, the SAC samples were oxidized thermally (pre-oxidized), and then nitric acid treatment (optimization) assisted by ultrasound was carried out. Then, the SAC samples were reduced using formic acid or ascorbic acid, and thereafter, spent media leaching of the reduced SAC was carried out under optimized conditions (pH 10.5, glycine concentration 10 g / L, and temperature 30 °C).

[0194] Effect of process parameters on SAC reduction and PGM recovery Not only were the competing metals removed from the SAC by ultrasonic-assisted nitric acid pretreatment, but also the particle size became smaller, thus increasing its surface area, and as a result, a higher leaching efficiency was obtained. Figures 22(a - c) show the effect of SAC reduction on the leaching efficiency at a pulp density of 0.5% w / v for the recovery of Pt and Pd and 4% w / v for the recovery of Rh. For non-reduced SAC, the recovery rates of Rh at pulp densities of 0.5% w / v, 1% w / v, and 2% w / v were 100%, 98%, and 93% respectively. Therefore, under spent media leaching, the effect of SAC reduction on the recovery rate of Rh using a high pulp density was determined. The spent media leaching of the reduced SAC was carried out at pH 10.5, glycine concentration 10 g / L, and 30 °C.

[0195] Reducing agent concentration: Either formic acid or ascorbic acid can reduce PGM oxides to the metallic form. Since both are biodegradable, these acids were used. Figure 22(a) shows that the optimal concentrations of the reducing agents for formic acid and ascorbic acid are 5% vol and 10% vol, respectively, and the leaching efficiency did not increase at higher concentrations. The leaching efficiencies of Pt, Pd, and Rh increased from (76%, 81%, and 65%, respectively) to 81%, 85%, and 69% for formic acid, and to 84%, 87%, and 69% for ascorbic acid. Formic acid is a stronger reducing agent than ascorbic acid. Therefore, formic acid shows high activity at a lower concentration, and the passive oxide layer of PGM oxides is reduced. On the other hand, ascorbic acid is a mild reducing agent.

[0196] Reduction time: Figure 22(b) shows the effect of reduction time on SAC reduction and leaching efficiency. Ascorbic acid required 60 minutes, probably due to its higher concentration, compared to 90 minutes required for formic acid to achieve optimal leaching. For SAC reduction with formic acid, the leaching efficiencies of Pt, Pd, and Rh increased slightly from 81%, 85%, and 69% to 85%, 88%, and 71%, respectively, as the reduction time increased from 60 minutes to 90 minutes. The leaching efficiency did not increase further even when the reduction duration was extended (formic acid > 90 minutes, ascorbic acid > 60 minutes). This is probably because the reduction was completed within these durations.

[0197] Reduction temperature: Figure 22(c) shows that increasing the reduction temperature from 50 °C to 80 °C for formic acid and from 50 °C to 60 °C for ascorbic acid increased the reduction of PGM oxides and improved subsequent leaching. The leaching efficiencies of Pt, Pd, and Rh increased from 83%, 87%, and 71% to 91%, 95%, and 74% for formic acid, and from 86%, 88%, and 69% to 88%, 90%, and 72% for ascorbic acid, respectively. Further increasing the reduction temperature decreased the PGM recovery rate because the decomposition rate of the reducing agent increased.

[0198] The optimal conditions obtained for SAC reduction were a reducing agent concentration of 5 vol% and 10 vol% for formic acid and ascorbic acid, respectively, a reduction time of 90 minutes and 60 minutes for formic acid and ascorbic acid, respectively, and a reduction temperature of 80 °C and 60 °C for formic acid and ascorbic acid, respectively. Overall, SAC reduction with formic acid increased the PGM recovery rate. Figure 23 compares the PGM recovery rates from SAC with and without reduction in spent media leaching at different pulp densities (0.5% w / v, 1% w / v, 2% w / v, and 4% w / v) under optimized conditions. The reduced SAC showed a higher recovery rate compared to the non-reduced SAC. The maximum PGM recovery rates achieved under spent media leaching (day 2) with reduction by formic acid, i.e., the maximum recovery rates of Pt, Pd, and Rh at a pulp density of 0.5% w / v were 91%, 95%, and 100%, respectively, the maximum recovery rates of Pt, Pd, and Rh at a pulp density of 1% w / v were 87%, 91%, and 100%, respectively, the maximum recovery rates of Pt, Pd, and Rh at a pulp density of 2% w / v were 61%, 68%, and 100%, respectively, and the maximum recovery rates of Pt, Pd, and Rh at a pulp density of 4% w / v were 39%, 45%, and 74%, respectively.

[0199] 4. Control measures for microbial-metal interactions and oxidative stress for improving bioleaching at high pulp densities The main drawbacks of bioleaching are low leaching rates and low efficiency, which are due to the toxicity of the substrate that occurs especially when operating at high pulp densities. To overcome the low leaching efficiency, ultrasonic-assisted nitric acid pretreatment of SAC was applied and bioleaching was enhanced using a modified strain of C. violaceum. SAC reduction was also carried out prior to bioleaching to optimize the process. Interfering metals at concentrations that are toxic to bacteria and have an adverse effect on bioleaching efficiency were removed by ultrasonic pretreatment. The modified strain of C. violaceum produces higher concentrations of cyanide to enhance metal mobilization, and by SAC reduction, the passivation oxide film present on the substrate was removed, increasing the PGM extraction rate. By SAC reduction, the PGM recovery rates at pulp densities of 0.5% w / v, 1% w / v, 2% w / v, and 4% w / v were significantly increased.

[0200] Known strategies used to enhance the leaching efficiency from municipal waste include the use of mixed cultures of cyanogenic bacteria and the application of ultrasound. Mixed cultures of Pseudomonas aeruginosa and C. violaceum have been reported to exhibit higher leaching ability than other combinations of mixed cultures or single cultures examined, probably due to their high resistance to metal toxicity. In other studies on the use of pure and mixed cultures of cyanogenic bacteria, although the mixed culture of C. violaceum and P. aeruginosa showed the highest gold recovery rate per unit cyanide produced, probably due to its high resistance to metal toxicity, it has been reported that a pure culture of C. violaceum produced more cyanide (20 mg / L) than the mixed culture of C. violaceum and P. aeruginosa (15 mg / L). However, no information has been shown on how the mixed culture microbial consortium expressed higher resistance to metal toxicity. Sonication can improve bacterial growth and bioleaching by reducing the resistance at the boundary layer between the bacterial cell wall and nutrients, enhancing the transport of oxygen and nutrients, and increasing the transport of cell excretions from the cells. Here too, no explanation has been shown on how sonication can enhance bioleaching efficiency without exerting harmful toxic effects on the bacteria. This is because local high temperature and pressure occur during sonication, causing stress that harms the growth of the bacteria. Also, in these studies, no examination has been made on how the mixed culture of cyanogenic bacteria and sonication during bioleaching can improve the bioleaching efficiency at high pulp density.

[0201] Although SAC reduction has been shown to be effective in improving and enhancing PGM recovery rates, further research was conducted to develop a more efficient bioleaching technology that can overcome pulp density constraints, reduce microbial-metal interactions, and recover PGM with high efficiency at higher pulp densities. This research aims to investigate microbial-metal interactions and pulp density constraints for the recovery of Pt, Pd, and Rh from SAC at high pulp densities. The specific objectives of this research are to examine the oxidative stress imposed on bacteria at high pulp densities, develop strategies to reduce the cytotoxicity and oxidative stress of metal ions that harm bacterial growth, minimize the adsorption of particles on bacterial cells, and enhance bacterial growth, cyanide production, and PGM recovery rates.

[0202] Oxidative stress and reactive oxygen species (ROS) Oxygen is essential for aerobic bacteria. However, when its concentration drops below a certain level, it causes hypoxia leading to cell death, and when the oxygen concentration exceeds a certain level, it causes oxidative stress. In addition, other factors such as ionizing radiation, UV light, ozone, nitrogen oxides, and metals cause oxidative stress in bacteria. High concentrations of metals lead to oxidative stress, osmotic stress, and metal ion stress in bacteria due to the cytotoxicity of metal ions. Many publications deal with the oxidative stress caused by high levels of oxygen. Microorganisms have been reported to have developed defense reactions to withstand oxygen concentrations in the environment. When the oxygen concentration exceeds the air saturation level, reactive oxygen species (ROS) accumulate as by-products of aerobic metabolism. These ROS, which can be generated by endogenous and exogenous sources, include superoxide anion radicals, hydroxyl radicals, and hydrogen peroxide. These ROS are more reactive than oxygen molecules and are toxic to bacteria.

[0203] H in shaking flask experiments 2 O 2、and when the concentration of oxygen molecules increases during scale-up in a bioreactor, it has a negative effect on the overall bioleaching efficiency. This is caused by the oxidative stress resulting from these molecules being at high concentrations. Previous studies have focused on the reaction mechanism in which these components are generated intracellularly and damage cell components. There have been no reports on the physiological responses of bacteria exposed to high concentrations of metals, metal toxicity, and osmotic stress that cause oxidative stress as a secondary reaction to survive in the presence of high concentrations of heavy metals. High concentrations of metals cause oxidative stress, creating an imbalance between the generation and elimination of ROS (by endogenous and exogenous antioxidants). As a result, the concentration of ROS increases, having a negative effect on bacterial growth, metabolism, enzyme activity, and gene expression, damaging DNA, proteins, and lipids, and ultimately leading to cell damage and cell death.

[0204] Bacteria have enzymatic and non-enzymatic defense reaction mechanisms and repair systems that protect them from damage caused by oxidative stress and control damage by inactivating ROS. The innate antioxidant defense reaction mechanisms are generally insufficient to protect against oxidative damage, and antioxidant additives are generally used. When exposed to high concentrations of ROS, the endogenous antioxidant system is impaired, and exogenous antioxidants are required to compensate for the lack of antioxidants. Although studies on the harmful effects of ROS on bacterial growth and cell function have been reported, its role during bioleaching at high pulp densities seems to have been mostly overlooked. In one such study, copper ions (Cu +2) It has been reported that the accumulation of [[ID=]] can increase intracellular ROS and cause the death of Acidithiobacillus ferrooxidans. As reported, high concentrations of iron lead to oxidative stress, damage biomolecules (carbohydrates, lipids, proteins, and nucleic acids), and result in cell death. SAC contains many heavy metals at high concentrations and has been shown to cause cell death at a pulp density of 4% w / v. However, what remains unclear is the toxic effect of metals on bacterial growth, cyanide production, and bioleaching efficiency at high pulp densities with respect to ROS and oxidative stress. Therefore, it is important to investigate the effects of ROS and oxidative stress on bioleaching efficiency.

[0205] Microbe-metal interactions Microbe-metal interactions also affect metal recovery, especially in high-pulp-density systems. In bioleaching, metal particles not only aggregate by gravity but also undergo specific adsorption of these particles onto cells. Since bioleaching is a diffusion-controlled reaction mechanism, the adsorption of cells onto metal particles and the aggregation of particles limit bioleaching and restrict metal recovery. Extracellular polymeric substances (EPS) embedded with biofilm microorganisms are responsible for the attachment of the biofilm to the particle surface. Although the complete profile of EPS has not been fully established, EPS is thought to consist of polysaccharides, proteins, glycoproteins, and glycolipids. The presence of reactive groups in EPS causes this polymer to be affected by chelating agents, ions, and surface-active compounds. Adsorptive EPS consists of charged groups and binds to various metals, nutrients, ROS, and contaminants. EPS plays an important role in removing heavy metals from the environment by its ability to bind metal ions in solution (thereby preventing metal ions from entering bacterial cells and protecting bacteria from environmental stress), but the adsorption of cells and EPS onto the particle surface effectively reduces bioleaching efficiency. Therefore, it is considered that bioleaching efficiency can be enhanced by minimizing the attachment of cells and EPS to metal particles.

[0206] Therefore, the present inventors adopted a strategy to further enhance the bioleaching efficiency at higher pulp densities by simultaneously controlling the oxidative stress and the negative effects of microorganism-metal interactions. A two-stage bioleaching technique was developed that minimized oxidative stress in the presence of high concentrations of metals by introducing the antioxidant glutathione (GSH) and minimized bacteria-metal interactions by adding the dispersant polyvinylpyrrolidone (PVP). The two-stage bioleaching was carried out in 250 mL Erlenmeyer flasks containing 100 mL of culture medium. The bioleaching experiments were conducted without (control) and with GSH and PVP. PVP was added to the culture when adding SAC, while GSH was added 12 hours after adding SAC. The overproduction of ROS in cells was determined using three different oxidative stress-sensitive probes commonly used to measure intracellular ROS. Three probes, namely dihydrorhodamine 123 (DHR123), 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA), and dihydroethidium (DHE) were used. The effect of PVP on bioleaching was determined by measuring the zeta potential.

[0207] Two-stage bioleaching at pulp densities of 0.5% w / v, 1% w / v, 2% w / v, and 4% w / v To examine the effect of pulp density on bacterial growth, cyanide production, pH change, and bioleaching efficiency at pulp densities of 0.5% w / v, 1% w / v, 2% w / v, and 4% w / v, two-stage bioleaching with C. violaceum was carried out. Figures 24(a-c) show the effect of pulp density on PGM recovery. This figure shows that the recovery rates of Pt, Pd, and Rh are maximized at a pulp density of 0.5% w / v, and the PGM recovery rate decreases as the pulp density increases. At low pulp densities of 0.5% w / v and 1% w / v, a slight increase in the recovery rate was observed from day 1 to day 4, indicating bacterial activity and cyanide production. At pulp densities of 2% w / v and 4% w / v, generally only a very slight increase in the PGM recovery rate was observed after day 1, indicating a decrease in bacterial activity (see Figure 25). The bioleaching efficiencies of Pt, Pd, and Rh at pulp densities of 0.5% w / v, 1% w / v, 2% w / v, and 4% w / v were 69%, 74%, and 99%; 62%, 67%, and 95%; 42%, 48%, and 84%; and 30%, 33%, and 81%, respectively. This is because high concentrations of metals have an adverse effect on viable cells, resulting in low cyanide production and low metal recovery rates.

[0208] The results shown in Fig. 24 are consistent with the data in Fig. 25 showing the free cyanide concentration and cell number over 5 days during two-stage bioleaching. Figs. 25(a - b) show that a gradual decrease in cell number and free cyanide concentration was observed from day 1 to day 4 at pulp densities of 0.5% w / v and 1% w / v, indicating the presence of viable cells, bacterial activity, and cyanide production. At a pulp density of 2% w / v, few viable cells were observed after day 3, while at a pulp density of 4% w / v, no viable cells were observed from day 3. The free cyanide concentrations at pulp densities of 0.5% w / v, 1% w / v, 2% w / v, and 4% w / v were 6.44 mg / L, 4.35 mg / L, 2.14 mg / L, and 0.80 mg / L on day 4. The decrease in viable cells and cyanide production due to an increase in pulp density during bioleaching indicated a decrease in bacterial activity and suppressed bioleaching efficiency.

[0209] The toxicity of the metal was the direct cause of the decrease in bacterial activity and bioleaching efficiency. Unfavorable conditions (such as changes in pH, lack of nutrient supply, and dissolved oxygen) could be another cause for the decrease in bioleaching efficiency. Similar findings were shown in studies in a bioreactor (1 liter) under fed-batch culture mode where pH and high levels of oxygen were maintained throughout bioleaching. Therefore, it was concluded that the toxicity of the metal at high pulp densities limited the bioleaching efficiency. To determine the effect of metal toxicity on bioleaching, especially at high pulp densities, all subsequent two-stage bioleaching experiments were conducted at pulp densities of 4% w / v, 8% w / v, and 12% w / v. Notably, SAC was acidic, and when added to the culture, the pH decreased, especially at high pulp densities. The pH of the culture after adding SAC at pulp densities of 8% w / v and 12% w / v was between pH 3 and pH 4. After adding SAC to the culture, the pH of the culture during two-stage bioleaching was maintained at pH 9.4 (i.e., the optimal pH for maximum cyanide production and PGM recovery rate).

[0210] Intracellular ROS Generation during Bioleaching The SAC has a very complex composition and contains various metals (as shown in Table 2). At high concentrations, these metals are toxic to bacteria, leading to a decrease in bacterial growth and cyanide production, and thus a decrease in bioleaching activity. The toxicity imposed by the presence of multiple metals causes metal stress in bacteria, which leads to oxidative stress, induces excessive intracellular ROS, and results in cell death. ROS are generated as by-products of aerobic metabolism and exposure to various natural and synthetic toxins. It has been reported that oxidative stress occurs when microorganisms are exposed to clinical antibiotics, solvents, metals, and heat. It is hypothesized that stress that physically damages redox enzymes can result in oxidative stress. High levels of ROS can damage proteins, lipids, and nucleic acids. At low pulp densities, bacterial growth and cyanide production are high, and the adverse effects of metal toxicity, metals, and oxidative stress are low. However, at high pulp densities, the synergistic effects of metal toxicity, metals, and oxidative stress have an adverse effect on bioleaching efficiency.

[0211] The production of intracellular ROS during bioleaching was monitored using the oxidative stress-sensitive probes dihydrorhodamine-123 (DHR-123), 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA), and dihydroethidium (DHE). DHR-123 is a specific ROS mitochondrial dye, and DCFH-DA is a cytoplasmic dye. Fluorescent rhodamine 123 is the oxidized product of non-fluorescent DHR-123, is positively charged, and selectively binds to the inner mitochondrial membrane of living cells. The oxidation of DHR-123 is catalyzed by the enzyme peroxidase and measured using an excitation wavelength of 505 nm and an emission wavelength of 535 nm. When taken up by living cells, the acetyl group of DCFH-DA is removed by membrane esterase to form 2’,7’-dichlorodihydrofluorescein (DCFH). Non-fluorescent DCFH is ROS (RO, RO 2 , HOCl, OH, and ONOO -etc.) is very sensitive, oxidized to highly fluorescent 2’,7’-dichlorofluorescein, and measured using an excitation wavelength of 504 nm and an emission wavelength of 524 nm. DHE is specific for O 2 - (the one-electron reduction product of O 2 ), and is minimally oxidized by HOCl, ONOO - , and H 2 O 2 . However, O 2 - can lead to the formation of H 2 O 2 , · OH, and reactive nitrogen species (RNS). Oxidation of DHE by O 2 - generates 2-hydroxyethidium (EOH) and intermediate products. The possibility of oxidation of intermediate products to fluorescent ethidium (E + ) by H 2 O 2 and · OH is low. E + fluorescence is measured with excitation at 500 - 530 nm and emission at 590 - 620 nm, respectively. EOH fluorescence is measured at an excitation wavelength of 480 nm and an emission wavelength of 567 nm. The fluorescence value has a positive correlation with the ROS content.

[0212] The time-course changes in intracellular ROS content during two-stage bioleaching at pulp densities of 4% w / v, 8% w / v, and 12% w / v are shown in Figs. 26(a - c). These figures show that the intracellular ROS content increased over time with the increase in metal concentration. At high pulp densities, the accumulation of intracellular ROS content was more than that at low pulp densities. The increase in intracellular ROS content has an adverse effect on bacterial growth, cyanide production, and bioleaching efficiency. The corresponding cell numbers and free cyanide concentration values during two-stage bioleaching at different pulp densities are shown in Fig. 27(a). The free cyanide concentration was 52.04 mg / L on day 0. In the control experiment, the free cyanide concentration was 56.14 mg / L on day 2, while the free cyanide concentrations at pulp densities of 4% w / v, 8% w / v, and 12% w / v were 4.45 mg / L, 2.31 mg / L, and 1.13 mg / L on day 2, respectively. In the control experiment, a gradual decrease in cell number and a slight increase in cyanide concentration were observed, which indicates bacterial activity. In contrast, in the presence of SAC, a decrease in cell number and cyanide concentration was observed, which indicates a decrease in bacterial activity and cyanide production, as well as the consumption of cyanide within 48 hours of the bioleaching process. Therefore, from Figs. 26(a - c) and Fig. 27(a), it can be concluded that the toxicity of the metal causes oxidative stress in the cells, resulting in the accumulation of intracellular ROS and leading to cell death. The obtained PGM recovery rates are shown in Fig. 27(b). The maximum recovery rates of PGM achieved on day 2, i.e., the maximum recovery rates of Pt, Pd, and Rh at a pulp density of 4% w / v were 29%, 32%, and 61%, respectively; the maximum recovery rates of Pt, Pd, and Rh at a pulp density of 8% w / v were 24%, 26%, and 39%, respectively; and the maximum recovery rates of Pt, Pd, and Rh at a pulp density of 12% w / v were 18%, 23%, and 36%, respectively.

[0213] Effect of exogenous antioxidants on intracellular ROS, bacterial activity, and metal recovery rate The dramatic increase in endogenous ROS due to oxidative stress is a threat to cell viability. However, microorganisms have reaction mechanisms to remove intracellular ROS and maintain the balance between the production and elimination of intracellular ROS. There are non-enzymatic antioxidants and intracellular enzymatic antioxidant systems in microorganisms that remove excessive ROS and minimize intracellular oxidative stress. However, the endogenous ROS removal system may not be sufficient to regulate intracellular ROS and prevent oxidative stress. This is because the endogenous antioxidant system is impaired by high concentrations of ROS. It is important to carefully evaluate the activity and capacity of exogenous antioxidants. The time and antioxidant concentration required to inhibit a defined concentration of ROS are important factors to consider.

[0214] Glutathione (GSH) consists of three amino acids, namely glutamate, cysteine, and glycine, and has antioxidant, immunostimulatory, and cell detoxification properties. It has been used to eliminate intracellular ROS, protect cells from ionic damage, osmotic damage, and oxidative damage, and improve bacterial growth activity. The use of GSH as a ROS scavenger to eliminate intracellular ROS during bioleaching has not been reported.

[0215] After adding SAC (i.e., starting bioremediation), exogenous GSH was added at concentrations of 0, 0.2, 0.4, 0.6, 0.8, 1, 1.5, and 2 (g / L) 12 hours later. A bioremediation experiment without GSH (control) was conducted to identify the effect of GSH on intracellular ROS, bacterial growth, cyanide production, and bioremediation activity. The initial bioremediation experiment was carried out at a pulp density of 4% w / v. Figures 28(a - c) show the intracellular ROS content at a pulp density of 4% w / v and various GSH concentrations during two - stage bioremediation. The intracellular ROS content in the group without GSH continuously increased (from 12 hours to 96 hours) due to the induction of oxidative stress in bacteria by the presence of metals. When the groups containing GSH were examined in more detail, it was found that ROS was most inhibited by adding 0.6 g / L of GSH, and the inhibition of ROS decreased when the GSH concentration was increased or decreased. Although the degree of increase was small compared to the group without GSH, a continuous increase in the ROS content was observed after adding low - concentration (0.2 g / L) and high - concentration (1.5 g / L and 2 g / L) GSH (similar to the GSH - free control). This is probably due to the low activity of GSH at low concentrations and the negative effects on cells at high concentrations. LiCoO 2 In recent studies on the bioremediation of Li and Co from LiCoO 2 it was reported that when 0.3 g / L of GSH was added, the bioremediation efficiency of Li increased from 87.6% to 98.1% and the bioremediation efficiency of Co increased from 87.0% to 96.3% at a pulp density of 5.0% of LiCoO

[0216] The corresponding cell numbers at various GSH concentrations in two-stage bioleaching at a pulp density of 4% w / v are shown in Figure 29. In the group without GSH, a decrease in the number of viable cells was observed, and no viable cells were observed after the third day, while in the group with GSH, cell viability (and thus activity) was observed over four days. The optimal number of viable cells was found in the culture in the presence of 0.6 g / L of GSH, which supported the results shown in Figure 28. That is, the addition of GSH (optimal value of GSH: 0.6 g / L) inhibited the accumulation of ROS, eliminated oxidative stress, and increased bacterial activity (growth and cyanide production). Since bacteria have enzymatic and non-enzymatic defense response mechanisms against oxidative stress, the possible reason for ROS inhibition is presumably that the addition of exogenous GSH enhanced intracellular ROS removal and antioxidant activity of bacteria, and as a result, bacterial activity was enhanced. Another possible reason is that the addition of exogenous GSH may stimulate the production of non-enzymatic small molecule antioxidants.

[0217] Figure 30 shows the PGM recovery rates during two-stage bioleaching at a pulp density of 4% w / v and various GSH concentrations. At high pulp density, metal toxicity induced oxidative stress in bacteria and killed all viable cells within two days of the addition of SAC. The decrease in bacterial activity led to a decrease in cyanide production and accordingly a decrease in bioleaching efficiency. The addition of exogenous GSH significantly enhanced intracellular ROS removal activity, decreased intracellular ROS content, and greatly increased bacterial activity, cyanide production, and thus bioleaching efficiency. As shown in Figure 30, significantly higher bioleaching efficiency was obtained after the addition of GSH. The maximum recovery rates of Pt, Pd, and Rh obtained were 30%, 33%, and 62% respectively in the absence of GSH (control), and 56%, 64%, and 80% respectively in the presence of GSH, showing increases of 87%, 94%, and 29% respectively.

[0218] Effect of Exogenous Dispersants on Microbe-Metal Interactions and Metal Recovery The toxic effects of heavy metals on soil microorganisms have been widely studied. For example, it has been reported that heavy metal pollution has an adverse effect on bacterial respiration and that exposure to metal toxicity leads to a decrease in viable cells. However, the negative impact of metals on bacterial activity during bioleaching at high pulp densities has not been extensively addressed. Complete growth inhibition at high metal concentrations has been shown previously (Figure 25). GSH removes intracellular ROS and enhances bacterial activity, but bioleaching can be inhibited by the formation of biofilms and the adsorption of bacteria onto metal particles. It has been reported that bioleaching decreased when cells adsorbed onto silver particles. The functional groups on the cell wall are responsible for the adsorption of substrates onto the cell surface and inhibit metal-cyanide complexation. Metals can also adhere to the cell surface, causing ion imbalance and altering enzyme function. Bacteria that develop a metal tolerance response mechanism by producing extracellular polymeric substances (EPS) and forming cell surface complexes have low bacterial growth in the presence of metals. This growth inhibition resulting from EPS can occur by reducing the removal of metabolites from bacterial cells or by reducing oxygen transfer. Therefore, EPS binding and the attachment of bacteria to metal particles may suppress the diffusion of cyanide and oxygen in diffusion-controlled bioleaching.

[0219] Many chelating agents are known to be able to reduce metal toxicity. Polyvinylpyrrolidone (PVP) is a chemically inert, biodegradable, biocompatible polymer that is widely used in green chemistry applications. It has a stable pH, temperature resistance, and is used as a capping agent in the synthesis of nanoparticles due to its non-ionic dispersant properties. PVP is a water-soluble, non-toxic, amorphous polymer with film-forming ability, good binding properties, and adhesiveness, and acts as a surface stabilizer and growth regulator, preventing particle aggregation. Therefore, it stands to reason that bioleaching efficiency can be enhanced by suppressing non-specific binding and the adsorption of cells onto particles. PVP may be used to disperse bacteria and SAC and increase the contact between metal particles and cyanide leaching agents.

[0220] Exogenous PVP was introduced at concentrations of 0, 0.2, 0.4, 0.6, 0.8, 1 (g / L) at the time of SAC addition (start of bioleaching). Also, a bioleaching experiment without PVP (control) was conducted to identify the effect of PVP on bacterial growth, cyanide production, and bioleaching activity. The initial bioleaching experiment was carried out at a pulp density of 4% w / v. The effect of PVP on bacterial activity was investigated by measuring the zeta potential of the culture. In the absence of PVP, the negatively charged bacterial surface and the EPS in which biofilm microorganisms were embedded tended to adsorb particles to the biofilm, thus enhancing particle aggregation and reducing the active surface of the particles.

[0221] Table 5 shows the effect of PVP on the zeta potential of bacterial cultures at various periods in two-stage bioleaching at a pulp density of 4% w / v. For the measurement of the zeta potential, each experiment was conducted in triplicate and the average data are shown. Since the bacterial surface is negatively charged, it interacts with PVP in the aqueous solution, and a large decrease in the zeta potential was observed. EPS containing both hydrophilic and hydrophobic sites interacts with particles due to its binding properties, but the addition of PVP suppresses the non-specific binding between particles and EPS, and also suppresses the undesirable adsorption of cells on the particles, resulting in the dispersion of the biofilm. PVP does not have a negative impact on bacterial growth and cyanide production. The decrease in the zeta potential increased with increasing PVP concentration, i.e., at 0.4 g / L, 0.6 g / L, 0.8 g / L, and 1 g / L. Thiocyanate (SCN -) The binding constant of the anion is 5.3, which indicates that PVP has a strong binding affinity for the anion. When SAC is added to the bacterial culture, PGM forms a complex with cyanide in the presence of oxygen. The PGM-cyanide complex is formed during bioleaching, and an increase in zeta potential is observed, which may be due to the greater affinity of PVP for the PGM-cyanide complex (Table 5). The increase in zeta potential was directly related to the release of the negative sites on the bacterial surface that had been occupied by PVP in the aqueous solution until then. PVP not only inhibited the adsorption of cells on SAC particles and the binding of EPS to SAC, but also interfered with the direct contact between SAC and cells, resulting in a reduction in metal toxicity to cells. Therefore, in the presence of PVP, SAC is less likely to adhere to the biofilm, thus enabling PGM-cyanide complexation.

[0222] As reported, PVP interferes with particle aggregation. The dispersed particles enhanced the reaction between the metal and cyanide, resulting in an increased reaction rate. Also, from the reduction in the toxicity of the metal to the cells, cell growth and cyanide production increased. Bioleaching is a diffusion-controlled reaction mechanism, and metal-cyanide complexation occurs at the solid-liquid interface. Therefore, it can be concluded that the addition of PVP improves metal-cyanide complexation, reduces the reaction time, and enhances the bioleaching efficiency. When SAC was added, the increase in the zeta potential of the bacterial culture was maximized at a PVP concentration of 0.4 g / L. In a study on the bioleaching of Ag from printed circuit board waste, it was reported that the recovery rate of Ag increased up to 1.8 times when 0.24 g / L of PVP was added.

[0223] The distribution of SAC particles in the bacterial solution was observed by TEM and shown in Figs. 31(a - f). Figs. 31(a - c) show the adsorption of SAC particles onto bacteria in the absence of PVP. Fig. 31c shows that SAC particles are adsorbed onto bacteria and encapsulated in the EPS matrix. Fig. 31d shows that SAC particles aggregate due to high surface energy, interfacial energy, and gravitational force in the absence of PVP. Figs. 31(e - f) show the distribution of SAC particles in the bacterial solution after adding PVP (0.4 g / L). The addition of PVP inhibited the aggregation of SAC particles and the adsorption of SAC particles onto bacteria. Fig. 32 shows the cell number and free cyanide concentration during two - stage bioleaching at a pulp density of 4% w / v, a GSH concentration of 0.6 g / L, and a PVP concentration of 0.4 g / L. The addition of PVP had a positive effect on bacterial growth and cyanide production during two - stage bioleaching by minimizing the negative effects of metal toxicity.

[0224] Fig. 33 shows the effects of GSH and PVP on the PGM recovery rate during two - stage bioleaching at a pulp density of 4% w / v. GSH and PVP were added at optimal concentrations (0.6 g / L and 0.4 g / L, respectively) at a given time. Apparently, the addition of GSH and PVP increased the PGM recovery rate. The maximum recovery rates of Pt, Pd, and Rh obtained were 30%, 33%, and 62% respectively in the control experiment, 56%, 64%, and 80% respectively in the presence of GSH, and 68%, 74%, and 86% respectively in the presence of GSH and PVP. The overall effects of GSH and PVP increased the duration for bacterial growth and cyanide production, thus bioleaching activity. The removal of intracellular ROS, reduction of oxidative stress, decrease in cell adsorption to particles and EPS binding, and increase in the contact between cyanide leaching agent and metal were proven to be useful strategies for enhancing bioleaching efficiency.

[0225]

Table 5

[0226] Two-stage bioleaching at high pulp density Two-stage bioleaching was carried out at pulp densities of 4% w / v, 8% w / v, and 12% w / v in the absence and presence of GSH and PVP. Figure 34 shows the viable cell counts and free cyanide concentrations at pulp densities of 4% w / v, 8% w / v, and 12% w / v. The results were consistent at all pulp densities, indicating the effectiveness of the strategy. At all pulp densities, the addition of antioxidants and dispersants increased growth and cyanide production. Corresponding data on the PGM recovery rates at pulp densities of 4% w / v, 8% w / v, and 12% w / v are shown in Figure 35. The addition of GSH and PVP increased the PGM recovery rates at all pulp densities. In the control experiment (when GSH and PVP were not added), the bioleaching efficiencies of Pt, Pd, and Rh at pulp densities of 4% w / v, 8% w / v, and 12% w / v were 30%, 33%, and 62%; 25%, 27%, and 40%; and 18%, 23%, and 36%, respectively. However, when GSH and PVP were added, the bioleaching efficiencies of Pt, Pd, and Rh at pulp densities of 4% w / v, 8% w / v, and 12% w / v increased to 68%, 74%, and 86%; 53%, 57%, and 73%; and 37%, 45%, and 61%, respectively. It was shown that the PGM recovery rate was higher with reduced SAC than with non-reduced SAC. This is because the thin oxide layer on the particle surface hinders the mobilization of the metal and thus limits bioleaching. Figure 36 compares the PGM recovery rates in two-stage bioleaching using non-reduced SAC and reduced SAC at a pulp density of 4% w / v in the absence and presence of GSH (0.6 g / L) and PVP (0.4 g / L). The PGM recovery rate was higher overall with reduced SAC than with non-reduced SAC. That is, it was 73% for Pt, 82% for Pd, and 90% for Rh at a pulp density of 4% w / v.

[0227] 5.C. Bio-reduction of SAC leachate by Metallidurance SAC leachate was used in the green synthesis of Pt and Pd nanoparticles. The initial objective of the reduction study was to identify the optimal conditions for the growth of Cupriavidus metallidurans. For this purpose, the initial pH was set at 4, 6, 7, 8, and 10, and a comprehensive study on bacterial growth was conducted at 30 °C and 150 rpm. Higher bacterial growth was observed at initial pH 6, 7, and 8, and optimal bacterial growth was observed at pH 6. The pH of the bacterial culture increased and tended to stabilize around pH 7.5. The inhibition of growth at pH 10 may be due to cell lysis or denaturation of cellular macromolecules.

[0228] The bio-reduction of Pt(II) ions and Pd(II) ions, as well as the biosynthesis of Pt and Pd nanoparticles were investigated. The spent media leachate was used for the bio-reduction of Pt(II) ions and Pd(II) ions. Two-step bio-reduction was carried out by growing bacteria separately in peptone meat extract (PME) medium at pH 6. Before the bio-reduction experiment, the cultures were transferred to fresh medium (10% vol. / vol. inoculum at the initial stationary phase) and grown to the initial stationary growth phase (in the absence of metal ions) to maximize the amount of metabolically active biomass. Once the initial stationary phase was reached, the cells were harvested by centrifugation at 5000 rpm for 15 minutes, washed three times with a sterile filtered 0.9 wt.% aqueous saline solution. The cell pellet was suspended in 9 g / L aqueous saline solution and used as the inoculation stock. When the cell cultures had grown to the stationary phase, the starting bacterial biomass for bio-reduction was collected. The pH was adjusted to a predetermined value, then a predetermined amount of spent media leachate of SAC was added and incubated for 48 hours. Samples were taken at predetermined sampling intervals, centrifuged at 10,000 rpm for 10 minutes, and the supernatant was analyzed for Pt and Pd uptake. C. The effects of different solution pH and metal ion concentrations on the bio-reduction efficiency of viable cells of C. metallidurans were investigated. The bio-reduction efficiency was determined at solution pH 4, 5, 6, 7, and 8, initial metal ion concentrations (ppm) of Pt(II) 425 ppm, 275 ppm, 200 ppm, 175 ppm, and 150 ppm, and initial metal ion concentrations (ppm) of Pd(II) 300 ppm, 200 ppm, 150 ppm, 125 ppm, and 100 ppm.

[0229] Effect of solution pH Figure 37 shows the effect of solution pH, i.e., 4, 5, 6, 7, and 8, on the bioreduction efficiency in the presence of 150 ppm of Pt(II) and 100 ppm of Pd(II) over 48 hours. The bioreduction of both Pt and Pd was maximized at solution pH 6, followed by pH 7 and 8. The optimal pH value for maximum bacterial growth and bioreduction efficiency was pH 6. The maximum bioreduction efficiencies of Pt(II) and Pd(II) obtained at solution pH 6 were 65% and 52%, respectively. The decrease in the concentrations of Pt(II) and Pd(II) during the bioreduction experiment was associated with the bioprecipitation and formation of Pt and Pd nanoparticles. The uptake of Pt and Pd by viable cells of C. metallidurans was high within 24 hours with the addition of spent SAC medium leachate. When the initial pH was 6, the pH of the solution after 24 hours of bioreduction for both Pt(II) and Pd(II) was approximately pH 7, indicating that the pH increased slightly during the experiment. The lowest bioreduction efficiency was observed when the initial pH was 4, probably due to low bacterial activity.

[0230] Effect of initial metal ion concentration Figure 38 shows the effect of the initial concentrations of Pt(II) and Pd(II) on the bio-reduction efficiency at the optimal pH, i.e., pH 6. Two-stage bio-reduction was carried out to minimize the toxic effects of soluble metal ions (metal ion stress and oxidative stress) on bacteria. The highest bio-reduction efficiency was obtained at the lowest concentration of metal ions, i.e., 150 ppm for Pt(II) and 100 ppm for Pd(II). This was probably due to the fact that metal ion complexes (including PGM-cyanide complexes and other metal-cyanide complexes) interfere with bacterial activity by causing metal ion stress and oxidative stress that kill bacterial functions in cells. Reduction started immediately upon exposure to Pt(II) ions and Pd(II) ions, and the immobilized concentrations of Pt and Pd increased with increasing exposure time. However, the reduction rate after 24 hours of exposure to the SAC leachate was lower than that within the first 24 hours. The plate count method was used to determine the effects of Pt(II)-cyanide and Pd(II)-cyanide on bacterial viability. Reacted cells at a given sampling interval were washed and resuspended in filter-sterilized DDI water. Further, the suspension was serially diluted with filter-sterilized DDI water, spread on PME agar plates, and incubated at 30 °C for 2 days. CFUs were counted to determine the dose-response and toxicity of the metal to cell viability. Figure 39 shows the effect of the initial concentrations of Pt(II) ions and Pd(II) ions on cell viability at pH 6 in two-stage bio-reduction. Before adding the SAC leachate, the number of viable cells in the system was 2.8×10 9 CFU / mL. The number of viable cells decreased significantly after 48 hours of bio-reduction, indicating osmotic pressure, toxic effects of metal complexes, and related oxidative stress on viable cells. On the other hand, the cell viability of the culture without Pt(II) ions and Pd(II) ions decreased slightly and remained almost constant.

[0231] Effect of cell metabolic state Using viable and non-viable cells, the active and passive uptake of Pt and Pd was determined. The pH of the growth medium and the spent SAC medium leachate was adjusted to pH 6. The pH-adjusted spent SAC medium leachate supplemented in the growth medium was added to viable and non-viable cells under predetermined conditions, namely 30 °C and 150 rpm, and incubated for 48 hours. Figure 40 shows the effect of viable cells vs non-viable cells on the bioreduction efficiency of Pt(II) and Pd(II) at pH 6. In viable cells, Pt and Pd were bioprecipitated and nanoparticles of Pt and Pd were formed. Also, in non-viable (dead) cells, passive adsorption of Pt(II)-cyanide complex and Pd(II)-cyanide complex occurred, but no metallic Pt and Pd nanoparticles were seen. In the case of viable cells, immobilization of Pt and Pd and formation of nanoparticles occurred intracellularly, extracellularly, and on the cell surface. Dead cells could not actively bind to Pt and Pd, and passive adsorption occurred on the cell surface. The denatured cell membrane and lysed cytoplasmic contents in dead cells provide active sites for the immobilization of Pt and Pd. Functional groups of the cell envelope, such as carboxyl (R-COOH), amine (R-NH 3 + ), phosphoryl (R-OPO 3 H 2 ), and (RO) 2 -P(OH) 2 ), and hydroxyl (R-OH), control cell surface activity. It has been reported that immobilization of Pt on the cell surface occurred as a Pt-organic complex, which implies that the Pt-organic complex may be preferred over the inorganic complex.

[0232] Antibacterial activity of biosynthesized nanoparticles Using the minimum inhibitory concentration (MIC) technique, the antibacterial activity of biosynthesized Pt nanoparticles and Pd nanoparticles against the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa was evaluated. The MIC concentrations of biosynthesized Pt nanoparticles and Pd nanoparticles for bacterial cells were evaluated using both optical density and CFU techniques. The recommended final inoculum size 5×105 It was inoculated into LB growth medium up to CFU / mL. An aliquot of the bacterial culture was exposed to equimolar concentrations of Pt nanoparticles and Pd nanoparticles. The maximum and minimum concentrations of the Pt nanoparticles and Pd nanoparticles used for the MIC were 30 ppm and 0.5 ppm, respectively. The minimum concentration of the nanoparticles was obtained by serial dilution of the colloid. Aliquots of the bacterial cultures supplemented with nanoparticles were incubated at 37 °C for Escherichia coli and 30 °C for Pseudomonas aeruginosa for 24 hours, and the absorbance was measured at 600 nm. To visualize the effect of the nanoparticles on bacterial growth, the cells were spread on LB agar plates and exposed to serially diluted nanoparticle dispersions. The agar plates were incubated at 37 °C for Escherichia coli and 30 °C for Pseudomonas aeruginosa for 24 hours. Figure 41 shows the viability of bacterial cells after incubation for 24 hours in the presence of various concentrations of nanoparticles. The biosynthesized Pt nanoparticles and Pd nanoparticles showed antibacterial activity against the tested bacterial strains. The antibacterial activity against Pseudomonas aeruginosa was higher than that against Escherichia coli, and the MICs of the nanoparticles (inhibiting the visible growth of bacteria) for Pseudomonas aeruginosa and Escherichia coli were found to be 8 ppm and 12 ppm, respectively. The high antibacterial activity of the biosynthesized nanoparticles was presumably due to their small particle size and spherical morphology. The antibacterial activity of nanoparticles depends on many factors such as particle size, shape, surface area / volume ratio, composition, catalytic activity, and concentration. It has been reported that smaller nanoparticles have a larger surface area, such nanoparticles show higher antibacterial activity at lower concentrations, while larger nanoparticles show antibacterial activity only at higher concentrations. The in vivo and in vitro cytotoxicity of Pt nanoparticles and Pd nanoparticles has not been investigated in detail. Only a limited number of studies have examined the cytotoxicity of Pt nanoparticles, and ROS-induced cytotoxicity has not been shown.

[0233] Characterization of Biosynthesized Pt Nanoparticles and Pd Nanoparticles The electronic, optical, physical, and chemical properties of nanoparticles play an important role in determining the behavior of materials and their use in various applications. Analytical techniques such as field emission transmission electron microscopy (FETEM), energy-dispersive X-ray spectroscopy (EDS), selected area electron diffraction (SAED), dynamic light scattering (DLS), and electrophoretic light scattering (ELS) were used to characterize the biosynthesized nanoparticles in terms of composition, morphology, particle size, hydrodynamic diameter, dispersity index, and zeta potential. Using C. metallidurans, the biosynthesis of Pt nanoparticles and Pd nanoparticles from a multi-metal ion leachate was carried out by bio-reduction and bio-accumulation reaction mechanisms. Characterization by FETEM was performed to determine the morphology and particle size of the synthesized nanoparticles. Since Pt nanoparticles and Pd nanoparticles were produced from a multi-metal ion leachate and the resulting solution contained a mixture of Pt nanoparticles and Pd nanoparticles, characterization by EDS, HRTEM, and SAED was carried out to identify the Pt nanoparticles and Pd nanoparticles. HRTEM and SAED are powerful techniques that can determine the nanostructure interface structure, morphology, crystallography, and chemical composition of nanoparticles.

[0234] Morphology and particle size of nanoparticles TEM analysis was performed to determine the particle size, morphology, and crystal structure of the nanoparticles. For unexposed C. metallidurans cells (control), TEM micrographs of whole mounts of both fixed and stained cells and unstained cells were obtained, and the results are shown in Figure 42. In unstained cells, details inside the cell are lacking, but in stained cells, the cell envelope and cytoplasmic contents are visible. Figure 42(b) also shows that the bacteria are encapsulated in an EPS matrix.

[0235] The TEM micrographs of cells exposed to the SAC leachate are shown in Fig. 43. Figs. 43(a - d) show that biogenic nanoparticles were synthesized inside and on the cell surface. The average particle sizes of the nanoparticles shown in Figs. 43a, b, c, and d are 38 nm, 26 nm, 27 nm, and 37 nm, respectively. The average particle size of all the nanoparticles shown in Figs. 43(a - d) is 31.5 ± 0.32 nm. 10% of the nanoparticles show a diameter of less than 20 nm, 72% of the nanoparticles show a diameter of 20 - 40 nm, and 18% of the nanoparticles show a diameter of more than 40 nm. The frequency distribution (%) of the nanoparticles is shown in Fig. 44. Compared with the nanoparticles synthesized on the surface or extracellularly, the nanoparticles synthesized intracellularly generally have a smaller particle size. The morphology of the biosynthesized nanoparticles was mostly irregular shapes, spherical, and cubic. From the EDX spectra in Fig. 45, the synthesis of Pt nanoparticles and Pd nanoparticles was confirmed. Fig. 46 shows that biogenic nanoparticles with different particle sizes and shapes were synthesized extracellularly. The average particle size of the hexagonal nanoparticles is 45 nm, and the average particle size of the spherical nanoparticles is 51 nm. It should be noted that the initial concentrations of Pt(II) and Pd(II) also play a role in the controlled formation of the particle size of the nanoparticles. When the initial concentrations of the Pt - cyanide complex and the Pd - cyanide complex are high, nanoparticles with a large particle size are observed (Fig. 47). Figs. 47a and b show the biogenic nanoparticles synthesized intracellularly and extracellularly, respectively, when the initial concentrations of the Pt - cyanide complex and the Pd - cyanide complex are high. The average particle sizes of the nanoparticles shown in Figs. 47a and b are 45 nm and 75 nm, respectively.

[0236] Bioreduction of Pt(II) and Pd(II) ions resulted in the biosynthesis of extracellular Pt and Pd nanoparticles, which implies that biomolecules produced by C. metallidurans were oxidized during bioreduction. In studies on the biosynthesis of gold nanoparticles, it has been reported that molecules located in the periplasm or cell membrane of C. metallidurans CH34 cells are oxidized during bioreduction. These findings suggested that the cop gene cluster (methionine residues of CopA and CopB) located within the megaplasmid pMOL30 was involved in the bioreduction of extracellular Au nanoparticles from Au(III). In other studies on Pt immobilization, it has been reported that Pt nanoparticles were formed on the cell membrane and within the cytoplasm. In addition, Pt nanoparticles bound in the cytoplasm caused metal toxicity and lysed the cells. Internalized metals inhibit enzymatic function and the bacterial detoxification response, i.e., complexation or reduction of the metal to a less toxic state, or efflux out of the cell, causing oxidative stress and leading to cell death.

[0237] The nanoparticles were identified using high-resolution transmission electron microscopy (HRTEM) images along with corresponding Fourier transform (FFT) and inverse Fourier transform (IFFT) patterns and selected area electron diffraction (SAED) patterns. The HRTEM images of Pd nanoparticles along with the corresponding FFT and IFFT patterns are shown in Fig. 48. The HRTM image of Pd nanoparticles showed that the fringe spacings of Pd nanoparticles were 2.24 Å and 1.94 Å, which were in good agreement with the spacings between the (111) and (200) planes of rhombic dodecahedral Pd. Also, the presence of the PdO phase was observed along with Pd nanoparticles. The SAED pattern of PdO is shown in Fig. 49. The SAED pattern was indexed according to the (111), (200), (220), (222), (400), (420), and (422) reflections of PdO based on d spacings of 3.24 Å, 2.83 Å, 1.99 Å, 1.62 Å, 1.41 Å, 1.25 Å, and 1.14 Å. The SAED pattern of Pt nanoparticles is shown in Fig. 50. The SAED pattern was indexed according to the (200), (220), (400), (331), (420), and (422) reflections of cubic Pt based on d spacings of 1.95 Å, 1.38 Å, 0.97 Å, 0.91 Å, 0.87 Å, and 0.78 Å.

[0238] The aim of this study was to recover Pt and Pd in metallic form from leachates using bioreduction and bioaccumulation reaction mechanisms. This study demonstrated the possibility of biosynthesis of Pt and Pd nanoparticles with different shapes and particle sizes from multi-metal ion leachates. Therefore, it is speculated that this technique can be used to selectively recover Pt and Pd in the form of Pt and Pd nanoparticles from aqueous metal ions. Since Pt and Pd nanoparticles were simultaneously synthesized from multi-metal ion leachates by bioreduction and bioaccumulation, the possibility of controlled synthesis of bimetallic (core-shell) nanoparticles can also be exploited. Compared to monometallic nanoparticles, bimetallic core-shell nanoparticles exhibit improved stability, activity, and selectivity due to lattice strain and the unique core-shell interface.

[0239] Dispersion index and zeta potential of nanoparticles The zeta potential, hydrodynamic diameter (Z-Ave), and polydispersity index (PDI) of the colloidal dispersion of the biosynthesized nanoparticles were also determined. The physicochemical properties of the nanoparticles play an important role when using these nanoparticles as safe, efficient, and stable nanocarriers in drug delivery. Uniform (monodisperse) nanoparticles are preferred for in vitro and in vivo applications. To obtain the particle size distribution, the hydrodynamic diameter and polydispersity index of the nanoparticles were measured. Dynamic light scattering (DLS) technology was used for in situ measurement of the particle size and PDI of the nanoparticles, and electrophoretic light scattering (ELS) technology was used for measurement of the zeta potential of the nanoparticles.

[0240] When the nanoparticles were produced by viable cells and dead cells, the hydrodynamic diameters of the nanoparticles measured by DLS were 96 nm and 108 nm, respectively. The particle size distribution of the nanoparticles measured by DLS was larger than the particle size measurement values obtained by TEM. The difference in the particle size measurement values obtained by TEM and DLS can be explained by the characteristics of these techniques. The particle diameter measured by TEM is proportional to the length of the particles in the solid stationary state. The particle size distribution measured by TEM images does not include the width of the capping ligand adsorbed on the surface of the particles. In contrast, the hydrodynamic diameter obtained by DLS is proportional to the volume of the nanoparticles and represents the accumulation of multiple measurement values obtained from the dynamic nanoparticle dispersion. Also, the hydrodynamic diameter measured by DLS is related to the movement of the particles in the suspension and reflects the transport characteristics of the particles, taking into account the hydration sphere and the protective or stabilizing layer that can surround the nanoparticles. The measurement of the particle size distribution of nanoparticles with heterogeneous particle size distributions by DLS has certain limitations such as aggregation. In the case of a polydisperse sample, the particle size distribution by TEM is always smaller than the intensity distribution by DLS. Therefore, it can be concluded that aggregation of the nanoparticles occurs in the aqueous suspension. The PDI of the nanoparticles produced by live cells was 0.21, while the PDI of the nanoparticles produced by dead cells was 0.26. This difference in PDI may be due to the difference in particle size. This is because smaller particles are better dispersed and larger particles aggregate.

[0241] The measurement of the zeta potential of nanoparticles by ELS is related to the electrophoretic mobility of the particles in the dispersion. The magnitude of the zeta potential is directly related to particle stability, and the larger it is, the greater the particle stability and the smaller the particles formed. The zeta potential was measured to determine the stability of the nanoparticle suspension. Before measuring the zeta potential, the sample was centrifuged and filtered using a 0.22 micron filter to remove bacterial cells. The measured zeta potential values of the nanoparticle suspensions formed by live and dead cells were -25.92 and -21.03, respectively. Some biomolecules may cap the nanoparticles, resulting in a net negative charge overall and potentially leading to the stabilization of the nanoparticles. It should be noted that the zeta potential measurement values described here were obtained when bacterial cells were grown and two-step bioreduction was carried out at pH 6 and 30 °C. When two-step bioreduction was carried out at pH 8, a higher zeta potential was observed. Also, when the nanoparticles were produced in the presence of the stabilizer PVP, a lower PDI was observed.

[0242] 6. Conclusions Regarding the cyanide in metal-cyanide complexation, the ultrasonic-assisted nitric acid pretreatment of SAC can effectively remove non-target metals that can compete with PGM if not removed. In the multivariate optimization approach based on the response surface used to determine the optimal conditions for the removal of these interfering metals, it was found that the ultrasonic frequency, ultrasonic power, and temperature are the most important parameters for removing copper, zinc, iron, and titanium in the pretreatment of SAC. When the ultrasonic-assisted pretreatment of SAC was optimized, copper (82%), zinc (88%), iron (60%), and titanium (72%) were removed from SAC. The ultrasonic-assisted pretreatment increased the surface area of the SAC particles and decreased the particle size.

[0243] The central composite design and response surface methodology were used to optimize the bioleaching conditions for PGM recovery. A response surface experimental model was developed to determine the individual and interaction effects of process variables on the PGM recovery rate and to optimize the process. Using the central composite design and two-stage bioleaching, the optimal conditions for maximum PGM recovery rates, namely Pt (69%), Pd (74%), and Rh (99%), were determined to be a glycine concentration of 10 g / L, a pulp density of 0.5% w / v, a pH of 9.4, an H 2 O 2 concentration of 0.08% v / v, and a temperature of 30 °C. SAC reduction was found to increase the rate of the leaching process and the PGM recovery rate. Reducing SAC with formic acid prior to bioleaching resulted in higher and faster PGM recovery, and the maximum PGM recovery rates at a pulp density of 0.5% w / v (the maximum recovery rates of Pt, Pd, and Rh were 91%, 95%, and 100%, respectively) were obtained in spent media leaching under the optimized reduction conditions of a reducing agent concentration of 5 vol%, a reduction time of 90 minutes, and a reduction temperature of 80 °C.

[0244] By controlling the microorganism-metal interaction between cells and SAC particles and eliminating oxidative stress within bacterial cells, the bioleaching efficiency was significantly improved at high pulp density. By adopting the strategy of controlling the microorganism-metal interaction using the non-ionic dispersant PVP and removing intracellular ROS using the antioxidant GSH, bacterial growth was enhanced, the net availability of cyanide ions for metal mobilization was increased, and as a result, the PGM recovery rate was increased. When PVP and GSH were added, the adsorption of metals onto the bacterial cell surface decreased, the binding of EPS to SAC particles decreased, and the metal toxicity and oxidative stress on bacteria that could damage cell function were reduced. The dispersing ability of PVP improved the contact between the metal and the cyanide leaching agent, reduced non-specific binding between particles, increased the conversion rate, ensured efficient mixing of the reaction mixture, and created favorable conditions for bioleaching. Overall, this novel strategy enabled the use of a higher pulp density during bioleaching and accordingly obtained a higher PGM recovery rate. The maximum recovery rates of the obtained Pt, Pd, and Rh were 30%, 33%, and 62% respectively at 4% w / v. The corresponding values of Pt, Pd, and Rh in the presence of GSH and PVP were 68%, 74%, and 86% respectively at 4% w / v. Furthermore, using reduced SAC, in the presence of GSH and PVP, the maximum PGM recovery rates were obtained at a pulp density of 4% w / v, namely 73% for Pt, 82% for Pd, and 90% for Rh.

[0245] In situ synthesis of Pt nanoparticles (NPs) and Pd nanoparticles (NPs) using C. metallidurans was performed with an SAC leachate containing soluble Pt-cyanide and Pd-cyanide complexes. The effects of various process parameters (including solution pH, reaction time, initial metal ion concentration, and cell metabolic state) on the bio-reduction efficiency were investigated. It was found that the optimal pH for the growth and bio-reduction of C. metallidurans was pH 6. Viable cells showed more uptake of Pt and Pd within 24 hours from the start of bio-reduction compared to dead cells. High initial concentrations of Pt(II) and Pd(II) led to less uptake of metals due to the toxic effects of metal complexes and reduced associated oxidative stress on viable cells. The maximum metal uptake of Pt(II) and Pd(II), namely 65% and 52% respectively, was obtained after 48 hours of bio-reduction. Nanoparticles with different particle sizes (average 31.5 ± 0.32 nm) and different shapes (cubes, rhombic dodecahedra, hexagons, spheres, and rod shapes) synthesized by viable cells of C. metallidurans were observed intracellularly, on the cell surface, and extracellularly. The biosynthesized nanoparticles showed excellent antibacterial activity, low polydispersity index, and high zeta potential.

[0246]

Table 6

[0247] Method Used automotive catalyst (SAC) The SAC was obtained from Environmental Solutions (Asia) Pte Ltd. The catalytic converter was ground into a fine gray powder and further mechanically sieved using a standard ASTM sieve to a particle size of less than 45 μm. The samples were placed in plastic containers, sealed with parafilm, and stored in a drying cabinet at a temperature of 20 - 22 °C.

[0248] Quantification of the metal content of SAC The sample (0.1 g) was digested in a Tita MPS microwave (PerkinElmer Inc.) equipped with a standard 75 mL container (maximum temperature: 230 °C, maximum pressure: 40 bar). Aqua regia (2.5 mL of 69% HNO 3 + 10 mL of 37% HCl) was added, and then the temperature was ramped up to 210 °C over 15 minutes and held for 30 minutes (at 210 °C). After the container was cooled to room temperature, 2 mL of H 2 O 2 (35% w / w) was added, the temperature was ramped up to 210 °C over 10 minutes and held for 20 minutes. The digest was cooled to room temperature, diluted with deionized water, and centrifuged at 6000 rpm for 15 minutes (KUBOTA 5100 centrifuge). The supernatant was filtered using a 0.45 μm syringe filter to remove suspended particles and then stored at 4 °C until analysis. The SAC residue was dried at 60 °C, weighed, and the amount of solubilized metal was measured. The metal concentration was measured using an inductively coupled plasma optical emission spectrometer (ICP - AES, Thermo Scientific iCAP6200).

[0249] Pretreatment of SAC in an ultrasonic bath To remove non - PGM, pretreatment was carried out using an ultrasonic processor. The ultrasonic bath (Elmasonic P30H) used in this study had the following specifications: (1) two frequency settings: 37 kHz and 80 kHz, (2) maximum ultrasonic intensity: 2.478 W / cm 2 , (3) temperature varying from 30 °C to 80 °C, and (4) ultrasonic treatment output varying from 30% to 100% (where 100% corresponds to 120 W at 37 kHz and 100 W at 80 kHz). To remove non - PGM, ultrasonic - assisted nitric acid pretreatment of SAC was carried out in the ultrasonic bath in the "Sweep" mode where the ultrasonic field duration in the water bath could be made equal. The pretreatment was carried out in a 250 mL Erlenmeyer flask, where SAC was mixed with nitric acid at a ratio of 1:10 (10 mL of HNO 3To this, 1 g of SAC was added. The flask containing SAC and nitric acid was sonicated under predetermined conditions, and then the mixture was transferred to a 50 mL centrifuge tube and centrifuged at 6000 rpm for 15 minutes. 5 mL of the supernatant was filtered using a 0.45 μm syringe filter to remove suspended particles, and then stored at 4 °C until metal analysis. The residue was washed with deionized water and centrifuged again. This process was repeated until the supernatant became clear. The remaining SAC was dried in an oven at 60 °C overnight, then weighed to determine the proportion of digested SAC. And the dried SAC was used in the following leaching experiment.

[0250] Reduction of pretreated SAC The reduction of SAC using formic acid (HCOOH, 98%) and ascorbic acid was carried out in a shaking water bath at a stirring speed of 250 rpm for a predetermined time. After mixing, the mixture was centrifuged at 6000 rpm for 15 minutes. The residue was washed with deionized water and centrifuged again. The remaining SAC was dried in an oven at 60 °C overnight. And the dried SAC was used in the following leaching experiment of reduced SAC.

[0251] Growth of bacterial cultures for bioleaching Metabolic engineering strains of C. violaceum were obtained from the Department of Biochemistry, Faculty of Medicine, National University of Singapore. C. violaceum pBAD hcnABC has an additional copy of the cyanide hcnABC operon with the pBAD promoter and requires the L(+)-arabinose inducer to induce the expression of the additional gene. Bacterial precultures were prepared in 250 mL Erlenmeyer flasks by transferring 1 mL of frozen bacterial stock into 100 mL of autoclaved culture medium. The flasks were kept in an incubator at 30 °C and 150 rpm for 24 h. To prepare a bacterial subculture as the inoculum, 1 mL of the preculture was inoculated into 100 mL of autoclaved culture medium in a 250 mL Erlenmeyer flask. Gentamicin sulfate antibiotic was added to prevent contamination from wild strains. Gentamicin sulfate was added to the culture to obtain a final working concentration of 15 μg / mL. For this purpose, 150 μL of gentamicin sulfate was added from a stock solution with a concentration of 10 mg / mL. The bacterial cultures were grown in an incubator at 30 °C and 150 rpm until reaching the mid-log phase. The L(+)-arabinose inducer was added to obtain a final working concentration of 0.002% w / v at the mid-log phase to induce the expression of the additional hcnABC operon. For this purpose, 1 mL of 0.2% w / v L(+)-arabinose was added to the bacterial culture. Molecular biology grade glycine (purity 99.6%) was added to all bacterial cultures as a precursor for cyanide production. 5% w / v D(+)-glucose anhydrous (purity 99%) (purchased from Alfa Aesar) was added to LB-Miller broth for all bacterial cultures as an additional energy source for the bacteria.

[0252] Growth of Bacterial Cultures for Bioreduction The C. metallidurans CH34 strain purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) was used for the bio-reduction experiment. C. metallidurans was activated in peptone meat extract medium (PME: 8 g / L) at pH 7. To measure the antibacterial activity of the nanoparticles, freeze-dried cells of Escherichia coli (ATCC 53868) and Pseudomonas aeruginosa were purchased from the American Type Culture Collection (ATCC) and activated in LB-Miller broth at pH 7. Bacterial pre-cultures were prepared in 250 mL Erlenmeyer flasks by transferring 1 mL of the frozen bacterial stock into 100 mL of autoclaved culture medium (pH 7). The flasks were kept in an incubator at 150 rpm at 30 °C for C. violaceum, C. metallidurans, and Pseudomonas aeruginosa, and at 37 °C for Escherichia coli for 24 hours. To prepare the bacterial sub-cultures as inoculum materials, 1 mL of the pre-culture was inoculated into 100 mL of autoclaved culture medium in a 250 mL Erlenmeyer flask.

[0253] Leaching study Leaching experiments were conducted in 250 mL shaking Erlenmeyer flasks placed in a shaking incubator under different operating conditions. An inoculum amount of 5% was used in all leaching experiments. Two leaching techniques were used, namely two-stage bioleaching and spent media leaching.

[0254] In two-stage bioleaching, single cultures of bacteria were grown separately in LB medium at the optimal pH (7.00 - 7.25) in the absence of SAC to reduce the toxic effect of SAC on the bacteria. When the mid-logarithmic phase was reached in the batch culture, glycine was added at a predetermined concentration. When the maximum cyanide production was reached, the pH was adjusted to a predetermined value, and then a predetermined amount of untreated or pretreated SAC (particle size ≤ 45 μm, autoclaved) was added. After the addition of SAC, the two-stage bioleaching experiment was carried out over 6 days. Also, a control experiment (i.e., bioleaching medium and SAC) was carried out under the same conditions in the absence of bacteria.

[0255] In spent media leaching, bacteria were grown under the same conditions as in two-stage bioleaching until maximum cell density and cyanide yield were reached. The culture was then centrifuged at 10,000 rpm for 15 minutes and filtered through a 0.22 μm filter to remove cells, obtaining spent media containing cell-free cyanide. The spent media was then used in the leaching experiment. In the spent media leaching experiment, the experiment was carried out over 3 days after adding SAC. A control experiment was also carried out using the uninoculated medium.

[0256] All leaching experiments were performed in triplicate. Samples were taken daily, centrifuged (10,000 rpm, 15 minutes), filtered (0.45 μm), and then analyzed for cyanide and metals. All samples were kept at 4 °C until analysis.

[0257] Scale-up studies were conducted in a bioreactor using a modified strain of C. violaceum under conditions optimized for two-stage bioleaching. As the bioreactor, a 2-liter glass (single-well glass vessel) autoclavable bioreactor manufactured by Sartorius Stedim Biotech (model: BIOSTAT® Aplus) was used. All bioreactor experiments were carried out with a total volume of 1 liter. Before each experiment, the reactor containing the culture medium was autoclaved at 121 °C for 40 minutes together with reagents, filters, and silicon tubes. The pH electrode and peristaltic pump were calibrated before autoclaving, and the pO 2 electrode was polarized and calibrated after autoclaving. After autoclaving, the vessel was cooled to room temperature and then the process was started. Samples were taken daily, centrifuged (10,000 rpm, 15 minutes), filtered (0.22 μm), and analyzed for cyanide and metals. Samples were kept at 4 °C until analysis.

[0258] Bioreduction studies Bio-reduction experiments were conducted using C. metallidurans in 250 mL shaking Erlenmeyer flasks placed in a shaking incubator with spent SAC medium leachate. An inoculum size of 5% was used in all bio-reduction experiments. C. metallidurans was grown to the mid-logarithmic growth phase at an optimal pH (pH 6), 30 °C, and 150 rpm (in the absence of SAC leachate to reduce the toxic effect on bacteria) to maximize the amount of metabolically active biomass. An aliquot of the bacterial suspension was collected by centrifugation at 12,000×g for 5 minutes. After centrifugation, the cells were washed with filter-sterilized deionized water to remove the remaining culture medium and stored in the relevant growth medium as a stock of inoculum material. Three different experimental approaches were used for the bio-reduction experiments. (1) The bacterial suspension was centrifuged again under the same centrifugation conditions, and the bacterial pellet was resuspended in spent SAC medium leachate (i.e., a metal ion solution in which PGM is in the form of the PGM-CN - complex) under various conditions to investigate the role of inactivated bacteria in the bio-reduction of Pt and Pd. (2) Bacteria were grown to the early stationary growth phase at an optimal pH, the pH was adjusted to various values, and the pH-adjusted spent SAC medium leachate was added to the bacterial culture to examine the effects of the initial pH and initial metal ion concentration on the bio-reduction efficiency. (3) After bacteria were grown to the early stationary growth phase at an optimal pH, the cells were heat-sterilized to obtain non-viable cells. The pH was adjusted to the optimal value, and the pH-adjusted spent SAC medium leachate was added to the non-viable cells to examine the effect of the metabolic state of the cells on the bio-reduction efficiency. After the addition of the SAC leachate, the bacterial culture was held for 2 days. Also, control experiments were conducted using uninoculated (abiotic) medium. All bio-reduction experiments were performed in triplicate. Samples were collected at predetermined sampling intervals, centrifuged (10,000 rpm, 15 minutes), filtered (0.22 μm), and subjected to metal analysis.

[0259] Free cyanide analysis A Thermo Scientific Orion cyanide electrode (model 9606BNWP) was connected to an ion-selective electrode (ISE) meter.

[0260] Metal analysis Metal analysis was performed using an inductively coupled plasma optical emission spectrometer (ICP-AES, Thermo Scientific iCAP6200).

[0261] UV-Vis spectroscopy To measure OD and cyanide concentration, a Shimadzu Biospec Mini UV-VIS spectrometer was used. To measure cyanide concentration, first, the sample was centrifuged at 6000 rpm for 15 minutes to remove bacterial cells.

[0262] Measurement of reactive oxygen species (ROS) Intracellular reactive oxygen species (ROS) were measured by the reported method. The method was modified according to the experimental conditions. Three different oxidation stress-sensitive probes, namely, dihydrorhodamine 123 (DHR123), 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), and dihydroethidium (DHE) were purchased from Sigma Aldrich.

[0263] After the bioremediation experiment at high pulp density, 5 μg / mL of DHR123 was added to 500 μl aliquots of the sample, and then incubated at 30 °C for 2 hours in the dark. The oxidation of non-fluorescent DHR123 was catalyzed by enzymatic peroxidase to fluorescent rhodamine 123. The mixture was transferred to a clear 96-well plate, and the oxidation of DHR123 at the excitation wavelength and emission wavelength (505 nm and 535 nm, respectively) was measured using a microplate reader.

[0264] To measure the oxidation of DCFH-DA, 5 mL of pulp was collected and bacteria were removed using 0.1% isomer Tween-20 solution (purchased from Sigma Aldrich). The bacterial suspension was centrifuged at 10,000 rpm for 10 minutes, and the cell pellet was resuspended in 2 mL of deionized water. DCFH-DA was added from a 1 mM ethanol stock solution to a final concentration of 10 μM and then incubated at 30 °C for 1 hour in the dark. When taken up by living cells, the acetyl group of DCFH-DA is removed by membrane esterase to form 2’,7’-dichlorodihydrofluorescein (DCFH). When taken up by viable cells, DCFH-DA forms non-fluorescent 2,7-dichlorodihydrofluorescein (DCFH). Non-fluorescent DCFH is very sensitive to OH, HOCl, RO, RO 2 , and ONOO - and is oxidized to the highly fluorescent compound 2,7-dichlorofluorescein. The mixture was transferred to a clear 96-well plate, and the oxidation of DCFH-DA was measured at the excitation wavelength and emission wavelength (504 nm and 524 nm, respectively) using a microplate reader.

[0265] DHE is specific for O 2 - (the one-electron reduction product of O 2 ) and is induced to undergo only minimal oxidation by HOCl, ONOO - , and H 2 O 2 . However, O 2 - can lead to the formation of H 2 O 2 , · OH, and reactive nitrogen species (RNS). Oxidation of DHE by O 2 - produces 2-hydroxyethidium (EOH) and intermediate products. The possibility of oxidation of intermediate products to fluorescent ethidium (E 2 O 2 and · OH is low. E + ) +Fluorescence was measured with excitation at 500 - 530 nm and emission at 590 - 620 nm, respectively. EOH fluorescence was measured at an excitation wavelength of 480 nm and an emission wavelength of 567 nm.

[0266] The fluorescence values (relative fluorescence intensity) measured by a microplate reader were positively correlated with ROS. The intracellular ROS content was measured in relative fluorescence units (RFU). The RFU of the solution was measured in the presence and absence of SAC (control). Bacterial growth was measured using the optical density at 600 nm (OD 600 ) and the number of cells.

[0267] Distribution of SAC particles during bioremediation The distribution of SAC particles in the bacterial solution was examined by field emission transmission electron microscopy (FETEM; JOEL JEM - 2100F).

[0268] Characterization of nanoparticles For the bioreduction experiment, bacterial cells and the obtained intracellular and extracellular nanoparticles were examined by field emission transmission electron microscopy (FETEM; JOEL JEM - 2100F). The surface elemental composition of the bioreduced samples was examined using energy - dispersive X - ray spectroscopy (EDX; OXFORD Instruments 6647). EDX analysis was performed using INCA software. Selected area electron diffraction (SAED), a crystallographic technique used to identify crystal structures and analyze lattice matching, was used to examine the formation of nanoparticles. For SAED analysis, ImageJ and Gatan Microscopy Suite (registered trademark) (GMS) software were used. For intracellular nanoparticle formation, Pt nanoparticles and Pd nanoparticles were extracted using the B - PER Bacterial Extraction Reagent kit (Thermo Fisher Scientific).

[0269] Measurement of zeta potential and hydrodynamic diameter For the hydrodynamic diameter and zeta potential, the dynamic light scattering method (DLS) and the electrophoretic light scattering method (ELS) were used respectively, and the zeta potential of the bacterial culture during bioremediation and the nanoparticles during bio-reduction, as well as the hydrodynamic diameter (Z-Ave) of the colloidal dispersion of the biosynthesized nanoparticles were measured by Zetasizer Pro (Malvern panalytical) equipped with the "ZS Xplorer software suite".

[0270] Statistical analysis and optimization Statistical analysis of the data was performed by the analysis of variance method (ANOVA). A 5% significance level was used for all statistical analyses in this study. Using Design Expert (version 11.0.3), Minitab (version 18.1), Origin (version 2019b), and JMP (version 14.0.0), the central composite design of the response surface method was used to perform statistical optimization of the SAC pretreatment and leaching experiments. Statistical optimization was carried out to identify the significance of individual factors and the interactions between various factors. Each run in the design matrix was performed 3 times.

[0271] Experimental error To ensure the accuracy of the results, all experiments were performed in triplicate, and the experimental error was quantified using the standard deviation of the mean. The error bars in the graphs represent the standard deviation.

[0272] It will be understood that many further modifications and variations are possible to the various aspects of the described embodiments. Accordingly, the described aspects are intended to embrace all such changes, modifications, and variations that fall within the spirit and scope of the appended claims.

[0273] Throughout this specification and the following claims, unless the context otherwise requires, the term "comprising" and its variations such as "comprises" and "comprising" are to be interpreted as implying the inclusion of the stated integer or step, or group of integers or steps, but not the exclusion of any other integer or step, or group of integers or steps.

[0274] Throughout this specification and the following claims, unless the context requires otherwise, the phrase "consisting essentially of" and its variations such as "consists essentially of" are to be understood as indicating that the recited elements are essential, i.e., are elements required for the present invention. This phrase admits the presence of other unrecited elements that do not substantially affect the characteristics of the present invention, but excludes additional unrecited elements that may affect the basic and novel characteristics of the defined method.

[0275] References in this specification to prior publications (or information derived therefrom) or to known matters shall not be construed as an admission, approval, or any kind of suggestion that such prior publications (or information derived therefrom) or known matters form part of the common general knowledge in the field of endeavor to which this specification pertains, and shall not be so construed.

Claims

1. A method for extracting platinum group metals (PGM) from a material, comprising: a) subjecting the material to surface oxidation; b) sonicating the surface-oxidized material of step a) in the presence of an acid; and c) performing bioleaching of the sonicated material of step b) in the presence of cyanide formed from cyanide-producing microorganisms in the presence of a cyanide precursor to form a leachate containing PGM.

2. The method according to claim 1, wherein the surface oxidation is carried out at about 700 °C to about 900 °C, preferably about 850 °C.

3. The method according to claim 1 or 2, wherein the surface-oxidized material of step a) is sonicated at a frequency of about 30 kHz to about 80 kHz, preferably about 37 kHz to about 80 kHz.

4. The method according to any one of claims 1 to 3, wherein the surface-oxidized material of step a) is sonicated at an output of about 30 W to about 120 W.

5. The method according to any one of claims 1 to 4, wherein the surface-oxidized material of step a) is sonicated for a duration of about 10 minutes to about 150 minutes, preferably about 70 minutes to about 80 minutes.

6. The method according to any one of claims 1 to 5, wherein the surface-oxidized material of step a) is sonicated at a temperature of about 30 °C to about 80 °C, preferably about 70 °C.

7. The method according to any one of claims 1 to 6, wherein the acid is selected from nitric acid, hydrochloric acid, sulfuric acid, or a combination thereof.

8. The method according to any one of claims 1 to 7, wherein the concentration of the acid is about 2 M to about 16 M, preferably about 8 M to about 9 M.

9. The method according to any one of claims 1 to 8, wherein the surface-oxidized material of step a) is sonicated at a frequency of about 37 kHz, a temperature of about 70 °C, an output of about 100 W, and a duration of about 80 minutes, and the acid is about 8 M to about 8.5 M nitric acid.

10. The method according to any one of claims 1 to 9, further comprising, after step b), reducing the sonicated material of step b) in the presence of a reducing agent.

11. The method according to claim 10, wherein the reducing agent is selected from formic acid, ascorbic acid, glycolic acid, malonic acid, or a combination thereof.

12. The method according to claim 10 or 11, wherein the concentration of the reducing agent is about 1% v / v to about 15% v / v, preferably about 5% v / v to about 10% v / v.

13. The method according to any one of claims 10 to 12, wherein the ultrasonic-treated material in step b) is reduced for a duration of about 30 minutes to about 120 minutes, preferably about 60 minutes to about 90 minutes.

14. The method according to any one of claims 10 to 13, wherein the ultrasonic-treated material in step b) is reduced at a temperature of about 50 °C to about 90 °C, preferably about 50 °C to about 80 °C.

15. The method according to any one of claims 10 to 14, wherein the ultrasonic-treated material in step b) is reduced at a concentration of the reducing agent of about 5 vol% for a duration of about 90 minutes and at a temperature of about 80 °C.

16. The method according to any one of claims 1 to 15, wherein the concentration of the cyanide precursor is about 0.5 g / L to about 20 g / L, preferably about 10 g / L.

17. The method according to any one of claims 1 to 16, wherein bioleaching of the ultrasonic-treated material in step b) is carried out at a pulp density of about 0.1% w / v to about 12% w / v, or about 0.5% w / v.

18. The method according to any one of claims 1 to 17, wherein bioleaching of the ultrasonic-treated material in step b) is carried out at a pH of about 7 to about 11, preferably at a pH of about 9 to about 10.

5.

19. The method according to any one of claims 1 to 18, wherein bioleaching of the ultrasonic-treated material in step b) is carried out at a temperature of about 22 °C to 38 °C, preferably about 30 °C.

20. The method according to any one of claims 1 to 19, wherein bioleaching of the ultrasonic-treated material in step b) is carried out at a concentration of the cyanide precursor of about 10 g / L, a pulp density of about 0.5% w / v, a pH of about 9 to about 10.5, and a temperature of about 30 °C.

21. The method according to any one of claims 1 to 20, wherein bioleaching of the ultrasonic-treated material in step b) is carried out under aerobic conditions in the presence of the cyanide-producing microorganism characterized by the HCN synthase operon (hcnA, hcnB, and hcnC) in the genome.

22. The bioleaching step comprises i) pre-incubating the cyanide-producing microorganism and the cyanide precursor under aerobic conditions to produce the cyanide, and ii) mixing the cyanide-producing microorganism and the cyanide precursor of step i) with the ultrasonic-treated material of step b). The cyanide-producing microorganism is the method according to any one of claims 1 to 21, characterized by an HCN synthase operon (hcnA, hcnB, and hcnC) in the genome.

23. The bioleaching step is i) pre-incubating the cyanide-producing microorganism and the cyanide precursor under aerobic conditions to produce the cyanide, and ii) isolating the cyanide from the cyanide-producing microorganism to form a cell-free medium, and mixing the cell-free medium with the ultrasonicated material of step b), The cyanide-producing microorganism is the method according to any one of claims 1 to 20, characterized by an HCN synthase operon (hcnA, hcnB, and hcnC) in the genome.

24. The cyanide-producing microorganism is selected from C. violaceum, P. fluorescens, B. megaterium, or a combination thereof, and is the method according to any one of claims 1 to 23.

25. The step of pre-incubating the cyanide-producing microorganism includes incubating the cyanide-producing microorganism at a pH of about 7.5 and then incubating the cyanide-producing microorganism at a pH of about 9, and is the method according to any one of claims 22 to 24.

26. The aerobic condition is O ２ The method according to claim 22 or 23, wherein the O saturation % is about 30%.

27. The ultrasonic-treated material of step b) is subjected to bioremediation in the presence of the above microorganisms and H ２ O ２ , and the concentration of H ２ O ２ is from about 0.02% v / v to about 0.16% v / v, preferably about 0.08% v / v, according to the method of claim 21 or 22.

28. The ultrasonicated material in step b) is bioleached in the presence of a ROS scavenger and / or a dispersant, and is the method according to any one of claims 21 to 27.

29. The ROS scavenger is added at least 10 hours after mixing the reduced material with the microorganism, and is the method according to claim 28.

30. The concentration of the ROS scavenger is about 0.2 g / L to about 2 g / L, preferably about 0.6 g / L, and is the method according to claim 28 or 29.

31. The concentration of the dispersant is about 0.2 g / L to about 1 g / L, preferably about 0.4 g / L, and is the method according to any one of claims 28 to 30.

32. The pH of the cell-free medium is about 10.5, and is the method according to claim 23.

33. The material is a used catalyst converter or a used automobile catalyst, and is the method according to any one of claims 1 to 32.

34. The material further contains Cu, Zn, Fe, Ti, or a combination thereof, and is the method according to any one of claims 1 to 33.

35. The method according to any one of claims 1 to 34, characterized by a volume of at least 1 L.

36. The method according to any one of claims 1 to 35, further comprising a step of bioreducing the leachate of step c) to form nanoparticles.

37. The method according to claim 36, wherein the leachate of step c) is bioreduced using C. metallidurans.

38. The method according to claim 37, wherein C. metallidurans is pre-incubated.

39. The method according to any one of claims 36 to 38, wherein the leachate of step c) is bioreduced at a pH of about 4 to about 8, preferably about 6.

40. The method according to any one of claims 36 to 39, wherein the nanoparticles are characterized by an average particle size of about 10 nm to about 80 nm.

41. The method according to any one of claims 36 to 40, wherein the nanoparticles are characterized by a hydrodynamic diameter of about 80 nm to about 110 nm.

42. The method according to any one of claims 36 to 41, wherein the nanoparticles are characterized by a polydispersity index of about 0.2 to about 0.

3.

43. A method for extracting platinum group metals (PGMs) from a material, comprising bioleaching the material in the presence of cyanide produced from cyanide-producing microorganisms in the presence of a cyanide precursor, and further in the presence of a ROS scavenger and / or a dispersant to form a leachate containing PGMs.

44. A method for pretreating a material containing platinum group metals (PGMs), comprising subjecting the material to ultrasonic treatment in the presence of an acid, wherein at least the surface of the material is oxidized.

45. A method for pretreating a material containing platinum group metals (PGMs), comprising reducing the material in the presence of a reducing agent, wherein at least the surface of the material is oxidized.

46. The method according to claim 45, further comprising a step of subjecting the material to ultrasonic treatment in the presence of an acid before the reduction step.

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