Application of vanillin in promoting leaching efficiency of acidophilic bacteria

Abstract

The application of vanillin in promoting the leaching efficiency of acidophilic bacteria is disclosed. Through the experiments of different concentrations of vanillin on the physicochemical parameters of bioleaching, mineral adhesion and EPS production of acidophilic bacteria, it is proved that the addition of 10 mg / L vanillin is beneficial to the leaching of minerals; the addition of 50 mg / L vanillin promotes the mineral leaching and the growth of strains in long-term leaching (>9 days). This strategy of improving the iron and sulfur oxidation ability of acidophilic bacteria through a new type of signal molecule lays a theoretical foundation for improving the leaching efficiency of strains in the bioleaching industry, provides a new idea for solving the environmental pollution problems such as acid mine drainage, and has a broad development prospect.

Description

Technical Field

[0001] This invention relates to the field of bioleaching technology, and more specifically to a method for promoting the leaching efficiency of acidophilic bacteria with vanillin. Background Technology

[0002] *Acidithiobacillus ferrooxdians* (A. ferrooxdians) is an aerobic, acidophilic, chemoautotrophic Gram-negative bacterium. As the type strain in bioleaching, it is widely found in iron-containing or sulfuric acid mine waters. Its growth and reproduction primarily obtain energy through the oxidation of ferrous iron. *A. ferrooxdians* is considered an important member of the bioleaching bacteria used for metal recovery and has been used in various biomining processes such as pond leaching, heap leaching, and tank leaching. Another type is *Acidithiobacillus thioooxidans* (A. thioooxidans), an extremely acidophilic, chemoautotrophic, Gram-negative rod-shaped microorganism. *A. thioooxidans* grows and survives autotrophically, utilizing elemental sulfur and reduced inorganic sulfur compounds as energy sources. It is one of the most critical sulfur-oxidizing microorganisms associated with the bioleaching process.

[0003] Biometallurgy is a new technology that utilizes microorganisms to selectively leach valuable elements from ores, directly producing high-purity metals and their materials. It features a short process, low cost, environmental friendliness, and low pollution, and has become a cutting-edge technology in global mineral processing. However, due to the long growth cycle of microorganisms and the lack of control methods, slow leaching rates and low leaching rates are major challenges restricting the industrial application of biometallurgy. Therefore, enhancing the role of microorganisms, particularly by improving the iron-sulfur oxidation capacity of the strains, is a key factor in improving leaching efficiency.

[0004] Intercellular signaling systems in Gram-negative bacteria rely on diffusible small molecules, such as N-acylhomoserine lactones (AHLs). Vanillin is a promising AHL molecular mimic that inhibits different acyl chains and may have inhibitory effects on mixed biofilms in the environment; it has already been used in activated sludge systems. Utilizing signaling molecule mimics to enhance the iron-sulfur oxidation capacity of bacterial strains holds promise for laying a theoretical foundation for improving the leaching efficiency of strains in the biometallurgical industry. A search revealed no reports on the application of vanillin in promoting the leaching efficiency of acidophilic bacteria. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide the application of vanillin in promoting the leaching efficiency of acidophilic bacteria.

[0006] The vanillin described in this invention has the structural formula 4-(HO)C6H3-3-(OCH3)CHO.

[0007] In this invention, vanillin is added during the acidophilic leaching process.

[0008] The mineral types include at least one of chalcopyrite and pyrite.

[0009] Acidophilic bacteria include at least one of the following: ferrooxdians (a thermophilic bacterium) and thiooxidans (acidic sulfur bacterium).

[0010] The amount of vanillin added should not exceed 100 mg / L.

[0011] Furthermore, the amount of vanillin added is 10-50 mg / L.

[0012] Furthermore, when the leaching time is ≤9 days, the amount of vanillin added is 8-12 mg / L, and when the leaching time is >9 days, the amount of vanillin added is 45-55 mg / L.

[0013] The leaching system temperature was 28-30℃, pH 1.5-2.0, and the inoculum size was 2×10⁻⁶. 7 -2.5×10 7 Cells / ml, pulp concentration: 1.5-2.5%, leaching time: 22-26 days, rotation speed: 150-180 rpm.

[0014] The application described in this invention specifically includes the following steps:

[0015] (1) The mineral sample was crushed to a particle size of 1 mm by hammering, then ground by a pulverizer and dry sieved to obtain a mineral sample with a particle size of less than 74 μm;

[0016] (2) Selection and cultivation of acidophilic bacteria: At least one of the following aurophilic bacteria, *Acidithiobacillus ferrooxdians* and *Acidithiobacillus thiooxidans*, was selected. The culture medium was 9K medium containing ferrous iron, with the initial pH adjusted to 2.0 by 10% H2SO4. The substrate amounts were 44.7 g / L FeSO4·7H2O, 10 g / L sulfur powder, and 2% chalcopyrite. The temperature was set at 30℃ and the rotation speed was set at 180 r / min. The bacterial culture samples were collected at mid-logarithmic time for later use. The substrate used for *Af* bacteria was FeSO4·7H2O, and the energy substrate used for *At* bacteria was sulfur powder.

[0017] (3) Add vanillin for bioleaching: Weigh 2g of chalcopyrite that has passed through a 200-mesh sieve into a 250ml conical flask, add 100ml of 9K culture medium to form a leaching system with a slurry concentration of 2%, adjust the pH with 10% sulfuric acid, and after 72h the pH stabilizes to 2.0, inoculate the strains Acidithiobacillus ferrooxdians and Acidithiobacillus thioooxidans cultured in step (2) in equal proportions, with a community inoculation amount of 2×10 7 Cells / ml were collected, and then 50 mg / L vanillin was added. The mixture was placed in a shaker at 30°C and cultured at 180 rpm for 24 days.

[0018] When applying this invention, the supernatant is taken for testing every 3 days; after 24 days of culture, the bacterial cells and slag are taken for testing.

[0019] Specifically, this involves the determination of leaching parameters, including the concentrations of total copper and total iron in the leaching solution, the concentration of attached bacteria, the extraction and determination of extracellular polymeric substances (EPS), and the characterization of minerals.

[0020] The results showed that throughout the leaching process, the total iron and copper ion concentrations in the leachate from the 10 mg / L vanillin experimental group were consistently higher than those from the 0 mg / L experimental group. In the 50 mg / L experimental group, the total copper ion concentration exceeded that of the 0 mg / L group on day 6, and the total iron ion concentration exceeded that of the 0 mg / L group on day 9. In the 100 mg / L experimental group, the total copper ion concentration exceeded that of the 0 mg / L group on day 12. However, the addition of 10 mg / L vanillin was beneficial to mineral leaching. During long-term leaching (>9 days), the addition of 50 mg / L vanillin promoted mineral leaching.

[0021] In the initial stage of leaching, the concentration of attached bacteria decreased with increasing vanillin concentration; in the later stage, the concentration of attached bacteria in the 50 mg / L experimental group continued to increase to its highest level. The concentrations of humic acid compounds and polysaccharides in EPS of the 50 mg / L treatment group were higher than those of other experimental groups. The addition of 50 mg / L vanillin weakened the diffraction peaks of chalcopyrite, with more obvious adsorption peaks of functional groups such as -OH and amide groups, a distinct potassium vanadium iron oxide structure, and more severe corrosion of the mineral surface. In contrast, the 100 mg / L experimental group showed stronger chalcopyrite diffraction peaks and a distinct potassium vanadium iron oxide structure compared to the bacterial control group.

[0022] In the above applications, the vanillin concentration is preferably 10 mg / L, which is beneficial for mineral leaching. In long-term leaching (>9 days), the preferred final concentration of vanillin is 50 mg / L, which promotes both mineral leaching and bacterial growth.

[0023] This invention addresses the problem of low bioleaching efficiency by providing a method to enhance the leaching efficiency of acidophilic bacteria using a quorum sensing signaling molecule mimic. Experiments were conducted to investigate the effects of adding different concentrations of vanillin on the physicochemical parameters of acidophilic bacterial bioleaching, mineral adhesion, and EPS formation. The addition of 10 mg / L vanillin was found to be beneficial for mineral leaching. In long-term leaching (>9 days), the addition of 50 mg / L vanillin promoted both mineral leaching and bacterial growth. The concentrations of humic acid-like compounds and polysaccharides in EPS were higher than in other experimental groups, thereby improving the iron-sulfur oxidation capacity of the bacteria. This strategy of enhancing the iron-sulfur oxidation capacity of acidophilic bacteria through a novel signaling molecule mimic provides a theoretical foundation for improving the leaching efficiency of bacteria in the biometallurgical industry and offers a new approach to solving environmental pollution problems such as acidic mine water discharge, demonstrating broad development prospects. Attached Figure Description

[0024] Figure 1 XRD diffraction pattern of chalcopyrite;

[0025] Figure 2 The changes in physicochemical parameters of chalcopyrite during leaching with vanillin, a signaling molecule analog, in the co-culture system of Af and At: (a) total copper; (b) total iron;

[0026] Figure 3 Changes in cell density during leaching of chalcopyrite with the addition of vanillin, a signaling molecule mimic, to an Af and At co-culture system;

[0027] Figure 4 Three-dimensional fluorescence spectra of extracellular polymeric substances and concentrations of vanillin, a signaling molecule mimic, added to the co-culture system of As and At, during leaching of chalcopyrite: (a) 0 mg / L vanillin group; (b) 10 mg / L vanillin group; (c) 50 mg / L vanillin group; (d) 100 mg / L vanillin group;

[0028] Figure 5 The concentration of extracellular polymeric polysaccharide during the leaching of chalcopyrite with the signaling molecule mimic vanillin added to the co-culture system of Af and At.

[0029] Figure 6 XRD patterns of leaching residue under the influence of different concentrations of vanillin (c-chalcopyrite, p-pyrite, q-quartz XRD patterns);

[0030] Figure 7 FTIR spectra of the leaching residue surface under the influence of different concentrations (mg / L) of vanillin;

[0031] Figure 8The images are SEM images, in which (a) untreated chalcopyrite, (b) non-biochemical acid leaching residue, (c) 0 mg / L vanillin bioleaching residue, (d) 10 mg / L vanillin bioleaching residue, (e) 50 mg / L vanillin bioleaching residue, and (f) 100 mg / L vanillin bioleaching residue. Detailed Implementation

[0032] The present invention will be described in detail below through specific implementation examples. These specific implementation examples are only illustrative and are not intended to limit the scope of the present invention.

[0033] To address the problems existing in the prior art, the present invention provides a method for improving the leaching efficiency of acidophilic bacteria based on quorum sensing signal molecular mimicry. The content of the present invention will be described in detail below with reference to specific drawings and embodiments.

[0034] Example 1: Selection, processing and composition analysis of mineral raw materials

[0035] (1) Selection of mineral raw materials: The pure chalcopyrite used came from Dongchuan, Yunnan, and was purchased from Mingfa Mineral Trading Company.

[0036] (2) Mineral raw material processing: The mineral sample is crushed to a particle size of about 1 mm by hammer, then ground by a pulverizer and dry sieved to obtain a mineral sample with a particle size of less than 74 μm, which is used for leaching test and analysis.

[0037] (3) Mineral raw material composition analysis: The main components of the mineral sample were analyzed. The XRD (X-ray diffraction) patterns of the mineral are shown below. Figure 1 As shown, the results indicate that the main components of the chalcopyrite sample are chalcopyrite (84.46%), pyrite (8.32%), quartz (2.27%), and dolomite (4.95%).

[0038] Example 2: Selection and Cultivation of Microbial Strains

[0039] (1) Strain selection: One strain is the amphiphilic *Acidithiobacillus ferrooxdians* (hereinafter referred to as *A. ferrooxdian*), an aerobic, acidophilic, chemoautotrophic Gram-negative bacterium commonly found in iron- or sulfuric acid-containing mineral waters. Its growth and reproduction primarily rely on the oxidation of ferrous iron. Another strain is the acidic sulfur-oxidizing *A. thioooxidans*, an extremely acidophilic, chemoautotrophic, Gram-negative rod-shaped microorganism. *A. thioooxidans* utilizes elemental sulfur and reduced inorganic sulfur compounds as energy sources for growth and survival through autotrophy.

[0040] (2) Bacterial culture: 9K medium containing ferrous iron was used, with the following components: 3 g / L (NH4)2SO4, 0.5 g / L MgSO4·7H2O, 0.5 g / L K2HPO4, 0.1 g / L KCl, and 0.01 g / L Ca(NO3)2. The initial pH of the medium was adjusted to 2.0 with 10% H2SO4. The substrate amounts added were FeSO4·7H2O (44.7 g / L), sulfur powder (10 g / L), and chalcopyrite (2%). FeSO4·7H2O was used as the substrate for Af bacteria, while sulfur powder was used as the energy substrate for At bacteria. The temperature was set to 30℃, and the rotation speed was set to 180 r / min. Bacterial samples were collected at mid-logarithmic growth for later use.

[0041] Example 3: Application of adding different concentrations of vanillin in promoting the leaching efficiency of acidophilic bacteria

[0042] 1. Experiments on the bioleaching of chalcopyrite with different concentrations of vanillin.

[0043] Weigh 2g of chalcopyrite that has passed through a 200-mesh sieve into a 250ml Erlenmeyer flask, add 100ml of 9K culture medium to form a leaching system with a pulp concentration of 2%. Adjust the pH with 10% sulfuric acid. After the pH stabilizes at 2.0 for 72 hours, inoculate with strains A. ferrooxidans and A. thioooxidans in equal proportions, with an inoculum size of 2×10⁻⁶. 7 cell / ml, with 3 replicates for each experiment.

[0044] Before inoculation, the above-mentioned strains were treated with vanillin standards at concentrations of 0 mg / L, 10 mg / L, 50 mg / L, and 100 mg / L, respectively, while the sterile control group remained unchanged. The strains were incubated in a shaker at 30°C and 180 rpm. The supernatant was collected every 3 days for analysis. After 24 days of incubation, the bacterial cells and slag were collected for determination of physicochemical parameters related to leaching.

[0045] 2. Determination of leaching parameters

[0046] The concentrations of total copper and total iron in the leaching solution were periodically measured. The concentration of copper ions in the solution was determined using the 2,9-dimethyl-1,10-phenanthroline spectrophotometric method. The concentration of total iron in the leaching solution was determined using the o-phenanthroline spectrophotometric method. Evaporated water from the leaching solution was periodically replenished with an equal volume of sterile distilled water, and the consumed solution was compensated with sterile 9K culture medium. Results are as follows: Figure 2As shown, throughout the leaching process, the total iron and copper ion concentrations in the leachate of the 10 mg / L vanillin experimental group were consistently higher than those of the 0 mg / L experimental group. In the 50 mg / L vanillin experimental group, the total copper ion concentration exceeded that of the 0 mg / L experimental group on day 6, and the total iron ion concentration exceeded that of the 0 mg / L experimental group on day 9. In the 100 mg / L vanillin experimental group, the total copper ion concentration exceeded that of the 0 mg / L experimental group on day 12.

[0047] Experimental results showed that adding 10 mg / L vanillin was beneficial to mineral leaching within the range of 0-100 mg / L. During long-term leaching (>9 days), adding 50 mg / L vanillin promoted mineral leaching.

[0048] 3. Changes in mineral attachment and extracellular polymers under the influence of vanillin

[0049] (1) Determination of attached microbial concentration: Centrifuge 2 ml of sample at 3000 r / min for 6 min to separate the bacterial solution from the minerals. Remove the upper free bacterial solution, add sterile 9k solution to the lower mineral layer to 2 mL, vortex for 20 min, and then centrifuge at 3000 r / min for 6 min to separate the adsorbed microorganisms from the minerals. Count the adsorbed microorganisms using a hemocytometer. Figure 3 As shown, during the leaching period from day 0 to day 12, the concentration of attached bacteria gradually decreased with increasing vanillin concentration. However, during the period from day 15 to day 24, the attached bacteria concentration in the 50 mg / L experimental group continued to increase, exceeding that of the control group (i.e., the 0 mg / L group) on day 21. The attached bacteria concentration in the 10 mg / L experimental group gradually decreased, converging with that of the 100 mg / L experimental group.

[0050] (2) EPS extraction: After culturing for 24 days, the culture medium was centrifuged at 10,000 rpm and 4°C for 10 min. The supernatant was collected and filtered under aseptic conditions through a 0.22 μm pore size filter to remove residual bacteria. The obtained supernatant contained EPS.

[0051] Three-dimensional fluorescence characterization: A Hitachi F-7000 fluorescence spectrophotometer (Hitachi Scientific Instruments Beijing Co., Ltd., Japan) was used to determine the composition of extracellular polymers. Sampling intervals were 5 nm, with an excitation wavelength range of 200-600 nm; the emission spectrum from 200-600 nm was scanned in 5 nm increments at a scan rate of 3000 nm / min. Figure 4 As shown, the mixed-culture EPS from different vanillin treatment groups all contained humic acid-like compounds. Based on fluorescence intensity, it can be seen that the content of humic acid-like compounds in the EPS from the 50 mg / L treatment group was significantly increased compared to other concentrations.

[0052] (3) Polysaccharide determination: Using glucose as a standard, 300 μL of sample was mixed with 900 μL of anthrone sulfate in an EP tube, placed in a constant temperature water bath, and reacted at 98℃ for 20 min. The OD at 625 nm was then measured. Results are as follows: Figure 5 As shown, increased polysaccharide content helps cells adhere to the mineral surface, forming a protective layer. This protective layer reduces the entry of oxygen and other harmful substances, providing a favorable environment for bioleaching.

[0053] 4. Changes in mineral characteristics during bioleaching under the influence of vanillin

[0054] After the leaching experiment, all solutions were filtered through neutral filter paper, and the slag was washed three times with acidified water (pH=2.0), dried in an oven at 40℃, and then subjected to subsequent testing. Figure 6-8 As shown, X-ray diffraction, Fourier transform infrared spectroscopy, and field emission scanning electron microscopy were used to analyze the changes in mineral phases, surface functional groups, and morphological characteristics after leaching.

[0055] (1) XRD results showed that the main components of the mineral leaching and dissolution residue were quartz, pyrite, and chalcopyrite. Compared with the control group with bacteria, the characteristic peak of chalcopyrite in the 50 mg / L experimental group was significantly weakened, while the 100 mg / L experimental group showed a stronger chalcopyrite diffraction peak compared with the control group with bacteria. XRD results showed that the main components of the mineral leaching and dissolution residue were quartz, pyrite, and chalcopyrite. Compared with the original ore, the diffraction peak of chalcopyrite in the non-bioleaching group was not significantly weakened, indicating that the effect of acid leaching on mineral dissolution was limited. Conversely, compared with the non-bioleaching group, the diffraction peak of chalcopyrite in the bioleaching group was significantly weakened, indicating that during the bioleaching process, the microbial community oxidized and destroyed the chalcopyrite, resulting in the release of a large number of copper ions. Compared with the control group with bacteria, the characteristic peak of chalcopyrite in the 50 mg / L experimental group was significantly weakened, while the 100 mg / L experimental group showed a stronger chalcopyrite diffraction peak compared with the control group with bacteria.

[0056] (2) Further FTIR spectral analysis showed that all experimental groups with different vanillin concentrations contained functional groups such as -OH and amide groups. The hydroxyl and amino groups were highly copolymerized, which affected the surface properties of the microorganisms, including surface charge, adsorption properties, and hydrophobicity, thus providing more binding sites. Further FTIR spectral analysis showed that all experimental groups with different vanillin concentrations contained functional groups such as -OH and amide groups. The hydroxyl and amino groups were highly copolymerized, which affected the surface properties of the microorganisms, including surface charge, adsorption properties, and hydrophobicity, thus providing more binding sites. Some absorption peaks in the spectra of different experimental groups showed significant differences, indicating that the concentration of vanillin affected the interaction between the strain and the minerals during the leaching process. A particularly significant difference was observed at 3334.53 cm⁻¹. -1The peak areas of the absorption peaks differed, with the 50 mg / L group showing the largest peak area, followed by the 100 mg / L and 10 mg / L experimental groups, indicating differences in the content of phenols, alcohol hydroxyl groups, and polysaccharides; 1620 cm⁻¹ -1 The absorption peaks also showed similar differences, indicating that the amide groups of the proteins differed. At 1084 cm⁻¹ -1 The adsorption peak at that location can be attributed to SO4. 2- γ3 bending vibration; 1028 cm -1 The adsorption peak at that location belongs to SO4. 2- γ1 bending vibration. 528 and 478 cm -1 This represents the stretching vibration of the FeO6 octahedron. The above band represents the typical adsorption peaks of potassium vanadium iron oxide (PVB). As shown in the figure, the leaching residues from both the 50 mg / L and 100 mg / L experimental groups exhibit a distinct KPB iron oxide structure. At 425 cm⁻¹... -1 The stretching at this point is due to the presence of polysulfides and disulfides in chalcopyrite. The increased stretching in the leaching residue from the 100 mg / L experimental group indicates that under the influence of high concentrations of vanillin, the chalcopyrite underwent oxidation, producing products such as copper sulfide.

[0057] (3) By observing the surface morphology of the original minerals, biodissolution residues, and acid leaching residues using SEM, it can be inferred that the EPS concentration is positively correlated with the degree of mineral corrosion. (The surface morphology of the original minerals, biodissolution residues, and acid leaching residues was observed using SEM.) Figure 8 It can be seen that the surface of the original mineral is clean and smooth. Figure 8 a). In the abiotic group, the mineral surface is almost smooth and dense, while some debris is present on the surface ( Figure 8 (b) This indicates that acid leaching has a relatively weak destructive effect on minerals. In the 0 mg / L group, the corrosion pits were small and few, indicating mild corrosion; however, after the addition of vanillin, the corrosion pits became larger and more numerous, and the corrosion became more severe. Compared to other concentrations, at 50 mg / L vanillin, obvious corrosion pits were observed, and the chalcopyrite surface was rougher at this concentration. Therefore, it is evident that 50 mg / L vanillin caused more severe corrosion of the mineral surface, possibly due to a higher content of EPS (extracellular polymeric substances) at this concentration. Thus, it can be inferred that there is a positive correlation between the EPS concentration and the degree of mineral corrosion. Figure 8 ce).

[0058] In summary, the effects of different concentrations of vanillin on the leaching of minerals by acidophilic bacteria were investigated. Within the range of 0-100 mg / L, the addition of 10 mg / L vanillin was beneficial for mineral leaching. For long-term leaching (>9 days), the addition of 50 mg / L vanillin promoted both mineral leaching and bacterial growth.

Claims

1. Application of vanillin in promoting the leaching efficiency of acidophilic bacteria. The molecular formula of vanillin is: 4-(HO)C6H3-3-(OCH3)CHO; mineral types include: At least one of chalcopyrite and pyrite; characterized in that: Specifically, the following steps are included: (1) The mineral sample was crushed to a particle size of 1 mm by hammer, then ground by a pulverizer and dry sieved to obtain a mineral sample with a particle size of less than 74 μm; (2) Selection and culture of acidophilic bacteria: *Thiobacillus ferrooxidans* (a thermophilic bacterium) was selected. Acidithiobacillus ferrooxdians ), acidic sulfur-oxidizing thiobacillus ( Acidithiobacillus thioooxidans At least one of the following was used: the culture medium was 9K medium containing ferrous iron, wherein the initial pH of the culture medium was adjusted to 2.0 with 10% H2SO4, and the substrate added was 44.7 g / L FeSO4·7H2O, 10 g / L sulfur powder, and 2% chalcopyrite; the temperature was set to 30℃, the rotation speed was set to 180 r / min, and the bacterial culture sample was taken for later use at the mid-logarithmic stage. Af The substrate used by the bacteria is FeSO4·7H2O. At The energy substrate used by the bacteria is sulfur powder; (3) Add vanillin for bioleaching: Weigh 2 g of chalcopyrite that has passed through a 200-mesh sieve into a 250 mL conical flask, add 100 mL of 9K culture medium to form a leaching system with a slurry concentration of 2%, adjust the pH with 10% sulfuric acid, and after 72 h the pH stabilizes to 2.0, inoculate the strain of *Thiobacillus ferrooxidans* (Ceratophyllum demersum) cultured in step (2) in equal proportion. Acidithiobacillus ferrooxdians ) and acidic sulfur-oxidizing thiobacillus ( Acidithiobacillus thioooxidans The community inoculation amount was 2×10 7 Cells / mL were collected, then 50 mg / L vanillin was added, and the mixture was placed in a shaker at 30°C and cultured at 180 rpm for 24 days.

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