Method for eliminating passivation layer of bioleaching, phage and application thereof
By using bacteriophages to specifically cleave the passivation layer on the ore surface under extreme metallurgical conditions, the problem of passivation layer obstruction during bioleaching was solved, resulting in a significant increase in copper ion leaching rate and an environmentally friendly bioleaching effect.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- CENT SOUTH UNIV
- Filing Date
- 2026-02-11
- Publication Date
- 2026-05-19
AI Technical Summary
In existing bioleaching technologies, the formation of a passivation layer on the ore surface hinders the attachment of microorganisms and the efficiency of oxidation reactions, and there is a lack of effective pollution-free methods to destroy the passivation layer.
By using bacteriophages to specifically lyse aged or over-proliferated bacteria under extreme metallurgical conditions, the passivation membrane structure is destroyed, fresh mineral surfaces are exposed, and bioleaching efficiency is improved.
It significantly improved the leaching rate of copper ions by 63.1%, and maintained high activity even under extreme conditions. It is environmentally friendly and does not change the acid-base balance of the leaching system.
Smart Images
Figure CN121718696B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biohydrometallurgical technology, specifically relating to a method for eliminating the passivation layer in bioleaching, bacteriophages and their applications. Background Technology
[0002] With the ever-increasing demand for mineral resources, traditional ore extraction methods face numerous challenges, including high energy consumption, heavy pollution, and low resource utilization. Although bioleaching, as a green and low-carbon emerging technology, has been widely applied in industry, it is still limited by issues such as slow microbial metabolic rates, limited strain selection, and sensitivity to environmental fluctuations. In particular, in chalcopyrite systems, the formation of passivation films severely hinders microbial adhesion and oxidation reaction efficiency. An effective method to specifically destroy the passivation layer on the mineral surface without introducing chemical pollution is still lacking. Therefore, there is an urgent need to develop new biological regulation methods to solve this industry problem. Summary of the Invention
[0003] The primary objective of this invention is to address the technical problem of low leaching efficiency caused by the formation of passivation layers during existing ore bioleaching processes, by providing a method for eliminating bioleaching passivation layers using bacteriophages. This method can effectively disrupt the passivation film barrier on the mineral surface, thereby significantly improving the bioleaching efficiency of minerals.
[0004] To achieve the above objective, a method for eliminating the passivation layer during bioleaching is proposed, wherein bacteriophages are added during the bioleaching of the ore.
[0005] By utilizing the specific lysis of aged or over-proliferated bacteria adsorbed on the ore surface by bacteriophages, the passivation film structure composed of biofilm and sulfur-containing precipitates is disrupted, exposing the fresh mineral surface, thereby restoring and improving the leaching efficiency of copper ions. Therefore, the bacteriophage is characterized by its ability to lyse the bacteria used in bioleaching.
[0006] Furthermore, the bacteriophage can maintain its lytic activity in environments with pH < 2.0 and in the presence of bioleached metal ions. This avoids the drawback of bacteriophages being easily inactivated or unstable under extreme metallurgical conditions, leading to their failure.
[0007] The method involves adding bacteriophages to the leaching system when passivation precipitation occurs on the mineral surface and / or the copper leaching rate begins to show a downward inflection point.
[0008] When passivation deposits appear on the mineral surface, it is basically the inflection point where the copper leaching rate begins to decline. This is because the appearance of the passivation layer hinders the leaching process, which leads to a decrease in the copper leaching rate. Therefore, it is important to optimize the removal of the passivation layer at the appropriate time.
[0009] In the early stages of leaching, the passivation layer has not yet formed, and the pulsed intervention of bacteriophages lacks a target, so the effect of addition at this stage is limited. In the later stages of leaching, the passivation layer has formed and tends to be dense, and the target of bacteriophage action—the embedded bacteria—is spatially restricted, resulting in a significant reduction in the phage lysis effect.
[0010] The ore in this invention system includes metal sulfide ores; the bacteria used in bioleaching include: *Thiobacillus acidophilus* (…). Acidithiobacillus thiooxidans ), Thiobacillus ferrooxidans ( Acidithiobacillus ferrooxidans ) and iron-loving spirochetes ( Leptospirillum ferriphilum At least one of the following.
[0011] The preferred phage preservation number used in this invention is CCTCC NO:M 20252758, and the ore mentioned includes chalcopyrite.
[0012] The method of this invention can be used not only for the bioleaching of chalcopyrite, but also for other ores that undergo passivation during the bioleaching process.
[0013] Furthermore, the phage dosage was 1×10⁻⁶. 8 ~10 9 The dosage is pfu / mL, and the dosage is 0.1%-5% of the leaching system volume.
[0014] Preferably, the supernatant after complete lysis of the host bacteria is concentrated to 1 / 100 of its original volume (phage concentration 10) using PEG precipitation. 8 The concentration of pfu (approximately 1 pfu / mL) was obtained after adding 1% of the leaching system volume.
[0015] Furthermore, when bacteriophages are added as a formulation to the leaching system, the formulation preparation process is as follows:
[0016] During the culture of mineralizing bacteria, mitomycin C, an inducer, was added. After successful induction, the induced culture was centrifuged at 9000 rpm and 4°C for 30 min to remove host cells. The phage lysis buffer was mixed with 10% (w / v) PEG8000 and 1M NaCl (both final concentrations after mixing) and incubated overnight at 4°C. The next day, the precipitate was collected by centrifugation at 9000 rpm and 4°C for 30 min. The precipitate was resuspended in 1-2 mL of SM buffer (50 mM Tris-HCl, 100 mM NaCl, 10 mM MgSO4·7H2O, 0.01% gelatin).
[0017] The specific steps for phage purification are as follows:
[0018] Add 2 mL of chloroform to the above resuspension, shake vigorously, centrifuge at 3000 rpm for 15 min, and collect the supernatant, which contains phage particles.
[0019] The supernatant was further purified by CsCl density gradient ultracentrifugation (1.35–1.70 g / mL, 180,000 × g, 1 h, 4 °C). The milky white phage bands were collected, extracted with a syringe, and dialyzed into 500 mL of SM buffer (4 °C, 5 h). The final concentration was 10... 8 Approximately pfu / mL.
[0020] The second objective of this invention is to provide a bacteriophage, accession number: CCTCC NO: M 20252758. vConTACT2 analysis shows that it belongs to... Caudovirales Head Myoviridae The family has a typical icosahedral head (head diameter ~70 nm) and a contractile tail sheath.
[0021] The aforementioned bacteriophages can lyse the commonly used bacteria in bioleaching and are also adapted to the high acidity and high metal ion concentration environment of bioleaching.
[0022] A third objective of this invention is to provide the application of the aforementioned bacteriophage for eliminating the passivation layer during bioleaching of ores.
[0023] The ore includes metal sulfide ores, and further includes chalcopyrite.
[0024] Compared with the prior art, the present invention has the following beneficial effects:
[0025] (1) Significant breakthrough in chalcopyrite leaching efficiency bottleneck: This invention utilizes bacteriophage to precisely break the passivation layer. Under the same experimental conditions, the copper leaching rate is increased by 63.1% compared to the control group without bacteriophage.
[0026] (2) Specific removal of passivation layer with a clear mechanism: This invention specifically solves the industry problem of "passivation" of chalcopyrite. By using bacteriophage to specifically lyse the aged or over-proliferated bacteria on the mineral surface, the passivation film structure formed by the interweaving of biofilm and sulfur-containing precipitates is effectively broken down, exposing the fresh mineral surface and continuously providing active sites for oxidation reactions.
[0027] (3) Excellent industrial environmental adaptability: The bacteriophage preparation constructed in this invention has been screened and can maintain high lysis activity in strong acid (pH <2.0) and high concentration of metal ions, overcoming the defects of conventional biological agents that are easy to inactivate and unstable in extreme metallurgical environments, and has good industrial application prospects.
[0028] (4) Green regulation and no secondary pollution: Compared with chemical leaching aids or physical ultrafine grinding, the present invention uses natural biological agents for regulation, without introducing toxic and harmful chemical reagents, and without changing the acid-base balance of the original leaching system, which is in line with the development direction of green bio-metallurgy.
[0029] (5) Optimize the microbial community structure and maintain reaction activity: use bacteriophages to remove aging and inefficient bacteria in the system, promote the functional metabolism and structural optimization of the microbial community, and keep the mineral leaching microbial community at a high level of oxidation activity.
[0030] Preservation information of the bacteriophage of this invention: Preservation number: CCTCC NO: M 20252758, Classification and nomenclature: Acidithobacillus thiooxidans Phage YH01, deposited on December 1, 2025; depositary institution: China Center for Type Culture Collection; location of depositary institution: Wuhan University, Wuhan, China. Attached Figure Description
[0031] Figure 1 This is a flowchart of the method of the present invention.
[0032] Figure 2 This is a transmission electron microscope image of the bacteriophage of the present invention.
[0033] Figure 3 This is a phage function annotation for the present invention.
[0034] Figure 4 The pH change curve of the bioleaching system in Example 2 is shown.
[0035] Figure 5 The copper ion leaching rate curve is continuously monitored during the operation of the bioleaching system in the control group of Example 2.
[0036] Figure 6 This is a SEM image of the passivated material during the operation of the bioleaching system in the control group of Example 2;
[0037] in: Figure 6 Image a shows the SEM image of the initial accumulation of passivating substances during the early stages of bioleaching. Figure 6 b is an SEM image showing the mineral surface being encased in a dense passivation layer during the later stages of bioleaching.
[0038] Figure 7 This is a SEM comparison image of the chalcopyrite surface morphology of the control group and the phage group on day 27 of bioleaching in Example 2.
[0039] in: Figure 7 a is the SEM image of the control group; Figure 7 b is the SEM image of the phage genome.
[0040] Figure 8This is a comparison diagram of the abundance of sulfur oxidation functional genes in the bioleaching microbial community in Example 2.
[0041] Figure 9 This is a comparison diagram of the abundance of iron oxidation functional genes in the bioleaching microbial community in Example 2.
[0042] Figure 10 The bioleaching rate is the bioleaching rate of the bioleaching system in Example 2. Detailed Implementation
[0043] The following examples are intended to further illustrate the present invention, but not to limit it.
[0044] The acidophilic thiobacillus used in the embodiments of the present invention ( Acidithiobacillus thiooxidans (Preservation number: CBCBSUCSU208051, strain number: JYC-17), thiobacillus ferricyanide ( Acidithiobacillus ferrooxidans (Accession number: CBCBSUCSU208067, strain number: YTW) and iron-loving spirochetes ( Leptospirillum ferriphilum (Accession number: CBCBSUCSU208015, strain number: YSK), all purchased from the China Center for Type Culture Collection.
[0045] Example 1: Induction, isolation and identification of bacteriophages
[0046] Thiobacillus acidophilus ( Acidithiobacillus thiooxidans (Accession number: CBCBSUCSU208051, strain number: JYC-17) was inoculated at a ratio of 1:10 (v / v) into BSM medium (100 mL) and cultured at 30°C with shaking at 180 rpm until the cells entered the exponential growth phase (approximately 10... 8 After the cell density was measured to be (cells / mL), mitomycin C (MMC; final concentration 1.0 µg / mL) was added to induce the phage to enter the lysis cycle. A sharp decrease in cell density was observed approximately 8 hours later, indicating successful induction.
[0047] BSM culture medium components: 3.0 g / L (NH4)2SO4, 0.5 g / L MgSO4·7H2O, 0.15 g / L Na2SO4·10H2O, 0.05 g / L KH2PO4, 0.1 g / L KCl and 0.014 g / L Ca(NO3)2·4H2O, pH adjusted to 2.0 with 3 mol / L sulfuric acid, sterilized at 121℃ for 20 min, and 10.0 g / L UV-sterilized sublimed sulfur added.
[0048] The induced culture was centrifuged at 9000 rpm and 4°C for 30 min to remove host cells. The phage lysis buffer was mixed with 10% (w / v) PEG8000 and 1 M NaCl (both final concentrations after mixing) and incubated overnight at 4°C. The next day, the precipitate was collected by centrifugation at 9000 rpm and 4°C for 30 min. The precipitate was resuspended in 1–2 mL of SM buffer (50 mM Tris-HCl, 100 mM NaCl, 10 mM MgSO4·7H2O, 0.01% gelatin).
[0049] The specific steps for phage purification and molecular biological identification are as follows:
[0050] Add 2 mL of chloroform to the resuspension, shake vigorously, centrifuge at 3000 rpm for 15 min, and collect the supernatant, which contains phage particles.
[0051] The supernatant was further purified by CsCl density gradient ultracentrifugation (1.35–1.70 g / mL, 180,000 × g, 1 h, 4 °C). The milky white phage bands were collected, extracted with a syringe, and dialyzed into 500 mL of SM buffer (4 °C, 5 h) to remove CsCl. The final concentration was 10... 8 Approximately pfu / mL.
[0052] The purified bacteriophages were adsorbed onto a carbon film copper grid, negatively stained with 2% uranium acetate, and their morphology was observed using a transmission electron microscope (TEM, Thermo Fisher Talos L120C) at 80 kV. Results are shown below. Figure 2 .
[0053] Results analysis and discussion:
[0054] The above method can successfully obtain infection. Acidithiobacillus thiooxidans bacteriophages. vConTACT2 analysis showed that it belonged to... Caudovirales Head Myoviridae The bacteriophages possess a typical icosahedral head (~70 nm) and a contractile tail sheath. The bacteriophage bands obtained by CsCl density gradient centrifugation are milky white and of high purity.
[0055] For functional annotations of the bacteriophages of this invention, see [link to relevant documentation]. Figure 3 This phage lysis module is highly enriched, containing a variety of key enzymes that degrade the host cell wall, including glycosyl hydrolases (such as the glycosyl hydrolase 108 family) and Gene 25-like lysozymes. In addition, the genome also encodes a sophisticated DNA packaging, structural protein assembly, and integration transfer system. Through the synergistic effect of these multifunctional modules, the phage effectively infects and regulates host bacteria under extremely acidic conditions.
[0056] The bacteriophage of this invention can maintain lytic activity in an environment with pH < 2.0, as shown in the following results. Figure 4 .
[0057] Example 2: Validation of phage-enhanced chalcopyrite bioleaching process
[0058] Constructing a chalcopyrite leaching system
[0059] Weigh a chalcopyrite sample (iron, sulfur, and copper content 30.7%, 34.3%, and 32.7%, respectively) through a 200-mesh sieve, adjust the slurry concentration to 1% (w / v), and load it into a 1 L bioleaching reactor. Inoculate with an iron oxide / sulfur bacteria complex at an initial concentration of approximately 8 × 10⁻⁶. 8 cells / mL. This bacterial community consists of the following three strains in a 1:1:1 ratio: *Thiobacillus acidophilus* (…). Acidithiobacillus thiooxidans (Preservation number: CBCBSUCSU208051, strain number: JYC-17), thiobacillus ferricyanide ( Acidithiobacillus ferrooxidans (Accession number: CBCBSUCSU208067, strain number: YTW) and iron-loving spirochetes ( Leptospirillum ferriphilum (Accession number: CBCBSUCSU208015, strain number: YSK). This complex microbial community exhibits good oxidation performance and environmental adaptability. The reaction temperature was 30℃, and the stirring speed was 180 rpm.
[0060] Phage enhancement operation
[0061] The copper ion leaching rate and mineral surface condition were continuously monitored during the reaction system operation. Around day 18-24, when a decreasing inflection point was observed in the copper ion leaching rate and significant precipitate formation on the mineral surface, 5% of the purified and concentrated phage preparation from Example 1 was added to the system, and the reaction continued for 60 days. The control group did not receive any phage.
[0062] Depend on Figure 5 It can be seen that, in the control group, the copper ion leaching rate began to decrease after 18-24 days due to the accumulation of passivating substances; and after 36-39 days, the leaching rate tended to stagnate due to severe passivation reaction. Figure 6 It is evident that the passivating material has just begun to accumulate. Figure 6 b represents the mineral surface being encased in a dense passivation layer during the later stages of the reaction.
[0063] Comparison of leaching effects
[0064] The phage group of this invention was compared with the control group:
[0065] The passivation layer on the surface of the phage group minerals was clearly peeled off (see...) Figure 7b), which exposes the ore matrix;
[0066] The abundance of iron-sulfur oxidation-related genes (RPKM) in the microbial community was significantly enriched on day 33 (P < 0.05). Figure 8-9 This indicates that phage intervention enhanced the potential of microbial iron-sulfur oxidation. The copper ion release concentration was significantly increased, with the final copper leaching rate increasing by 63.1% on day 60. Figure 10 ).
[0067] The results showed that bacteriophages could effectively disrupt the passivation barrier, regulate the microbial community structure, and enhance microbial oxidation activity, thereby significantly improving the bioleaching efficiency of chalcopyrite.
[0068] Example 3
[0069] This embodiment examines the effect of phage addition timing on chalcopy leaching efficiency in order to optimize the addition timing.
[0070] Experimental setup:
[0071] Under the same conditions as in Example 2, the following three experimental groups were set up respectively:
[0072]
[0073] Experimental results
[0074]
[0075] Example 4: Optimization of phage dosage ratio
[0076] This embodiment examines the effect of different volume ratios of bacteriophages on the leaching efficiency of chalcopyrite in order to optimize the dosage ratio.
[0077] Experimental setup:
[0078] Under the same conditions as in Example 2, the following three experimental groups were set up respectively:
[0079]
[0080] Experimental results:
[0081]
[0082] in conclusion:
[0083] Good leaching effect was observed in the range of 0.1%-5% addition ratio, with the optimal ratio being 1%.
[0084] Example 5: Investigation of Slurry Concentration Adaptability
[0085] This embodiment investigates the stability and efficiency changes of a phage-enhanced bioleaching system under different slurry concentrations in order to determine the appropriate mineral loading range.
[0086] Experimental setup:
[0087] Based on Example 2, the pulp concentration is set at the following three levels:
[0088]
[0089] Experimental results:
[0090]
[0091] in conclusion:
[0092] A pulp concentration of 1-10% is more suitable for the technical solution of this invention. At low and medium concentrations, phage regulation can maintain high leaching efficiency. When the pulp concentration is increased to 20%, the leaching rate decreases (20.5%), mainly due to the increased gas-liquid mass transfer resistance caused by the high-concentration pulp and the inhibitory effect of shear force on microbial activity, rather than phage failure. Therefore, this method works best in a pulp concentration range of 1%-15%, and it is recommended to control the pulp concentration within this range in industrial applications.
Claims
1. A bacteriophage ( Acidithobacillus thiooxidans phage YH01, characterized in that, Accession number: CCTCC NO:M 20252758.
2. The application of the bacteriophage according to claim 1, characterized in that, Used to remove the passivation layer during bioleaching of ores.
3. The application according to claim 2, characterized in that, The ore mentioned includes metal sulfide ores.