A composite microbial community and a method for extracting copper by coupling electric field assisted bioleaching

Abstract

This application provides a composite microbial community and a method for copper extraction by bioleaching assisted by a coupled electric field. The composite microbial community includes *Thiobacillus ferrooxidans*, *Aspergillus niger*, and *Gnaphalium affine*. The composite microbial community is obtained by inoculating *Thiobacillus ferrooxidans*, *Aspergillus niger*, and *Gnaphalium affine* in a composite culture medium at an inoculation ratio of 2:(0.5~2):(0.5~2) and co-culturing them. Under the assistance of an electric field, the composite microbial community synergistically promotes the leaching and extraction of copper from copper-containing substances. This method, through the coupling and coordination of biological and electric field effects, improves the copper leaching and recovery efficiency, and has the advantages of mild conditions, environmental friendliness, and high resource utilization efficiency.

Description

Technical Field

[0001] This application relates to the field of bioleaching, specifically to a composite microbial community and its coupled electric field-assisted bioleaching method for copper extraction. Background Technology

[0002] The electroplating industry generates a large amount of electroplating sludge containing heavy metals during surface treatment. Copper, as one of the main valuable metals, has high recycling value. How to achieve efficient and green recovery of copper from electroplating sludge has always been an important research direction in the field of solid waste resource utilization.

[0003] Currently, the main methods for treating copper in electroplating sludge include chemical leaching, pyrometallurgy, and bioleaching. Among these, bioleaching has received widespread attention due to its mild conditions and environmental friendliness. Existing bioleaching technologies often employ single-species bacteria such as *Acidithiobacillus ferrooxidans*, which promote the transfer of metal from the solid to the liquid phase through their oxidative metabolism. However, electroplating sludge has a complex composition, typically containing organic matter, various heavy metals, and inhibitory substances. Single-species systems have limited adaptability to complex matrices, resulting in low metal leaching efficiency and insufficient stability. Furthermore, in existing technologies, the bioleaching process and metal recovery process are usually independent, leading to a long overall process flow and high energy consumption. Simultaneously, the accumulation of metal ions in the system may inhibit microorganisms, further affecting the sustainability and efficiency of the bioleaching process.

[0004] Therefore, how to improve the bioleaching efficiency of copper in complex systems and achieve effective synergy and enhancement of the leaching and recovery processes is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] This application provides a composite microbial community and its coupled electric field-assisted bioleaching copper extraction method, aiming to solve the problems of insufficient adaptability of the microbial system, low leaching efficiency, and the separation of leaching and recovery processes in the prior art.

[0006] In a first aspect, this application provides a complex microbial community, including: Acidithiobacillus ferrooxidans ATCC 23270; Aspergillus niger ATCC 6275; and Meyerozyma guilliermondii ATCC 14242. The composite microbial community was obtained by inoculating the *Acidithiobacillus ferrooxidans*, *Aspergillus niger*, and *Gynostemma pentaphyllum* in a composite culture medium at an inoculation ratio of 2:(0.5~2):(0.5~2) and then co-culturing them. The composite culture medium comprises: 2.5–3.5 g / L (NH₄)₂SO₄, 0.05–0.2 g / L KCl, 0.4–0.6 g / L K₂HPO₄, 0.4–0.6 g / L MgSO₄·7H₂O, 0.005–0.02 g / L Ca(NO₃)₂·4H₂O, 40–50 g / L FeSO₄·7H₂O, and 0.5–2 g / L MgSO₄·7H₂O. 0 The compound culture medium contains 0.5-1.0 g / L glucose and 0.05-0.2 g / L yeast extract; the pH of the compound culture medium is 2.5-2.8.

[0007] This application constructs a complex microbial community system with complementary metabolic functions by combining *Thiobacillus ferrooxidans*, *Aspergillus niger*, and *Gynostemma pentaphyllum*, which is suitable for efficient bioleaching of copper in copper-containing electroplating sludge.

[0008] Specifically, *Acidithiobacillus ferrooxidans* (Af), as an autotrophic acidophilic bacterium, can utilize reducing sulfides in substrates to oxidize and produce acid (sulfuric acid), while simultaneously converting Fe... 2+ Oxidized to Fe 3+ This creates an acidic environment with a high redox potential, promoting the oxidative dissolution of copper-containing substances. However, in complex electroplating sludge systems, *Acidithiobacillus ferrooxidans* has relatively limited adaptability to organic matter and inhibitory components, and its effect on copper in complexed or insoluble forms is limited, potentially affecting overall leaching efficiency. Therefore, *Aspergillus niger* (An) is introduced as an acid-producing fungus. During its growth, it secretes low-molecular-weight organic acids (such as citric acid and oxalic acid), which helps lower the initial pH and, through acid dissolution and organic acid complexation, disrupts the recalcitrant organic-heavy metal complex structure in the sludge, promoting copper release. This, to some extent, compensates for the insufficient effect of *Acidithiobacillus ferrooxidans* on complex substrates. However, in the above system, the interaction between metal components and microorganisms and their metabolites may still be limited due to solid-liquid interface contact and mass transfer processes. Therefore, *Meyerozyma jimoides* is further introduced. Guilliermondii (Mg) can secrete biosurfactants under heavy metal stress. Since electroplating sludge is usually hydrophobic and viscous, conventional bacteria have difficulty fully contacting the target substances. The surfactants secreted by this yeast can reduce the solid-liquid interfacial tension, improve the wettability and dispersibility of sludge particles, and may increase the contact area between ferrooxidizobacillus and Aspergillus niger and copper-containing components, thereby synergistically promoting the transformation, desorption and release of copper components.

[0009] By controlling the inoculum ratio of *Thiobacillus ferrooxidans*, *Aspergillus niger*, and *Gnaphalium affine* to 2:(0.5~2):(0.5~2), this approach is not based on single-species activity optimization, but rather on a limited coexistence ratio window established to meet the requirements for the relatively stable operation of the complex microbial community. *Thiobacillus ferrooxidans*, as the dominant bacterium in the system, serves as the core driving force for maintaining the system's strong oxidizing power and continuous leaching capacity; therefore, its dominant inoculum ratio must be maintained. *Aspergillus niger* and *Gnaphalium affine* are both heterotrophic auxiliary bacteria. This application limits the above-mentioned inoculum ratio range to maintain the heterotrophic bacteria in an auxiliary metabolic state, thereby achieving the desired effect of *Thiobacillus ferrooxidans*. The long-term restricted coexistence of the ternary microbial community under the dominance of bacilli ultimately constructs a complex microbial community system that combines high copper release capacity with long-term operational stability. Through co-cultivation, the environmental adaptation and interaction relationships of the microbial community can be established before formal contact with copper-containing sludge. On the one hand, the low-molecular-weight organic acids secreted by Aspergillus niger can promote the initial acidification of the system, creating suitable precursor conditions for the proliferation of Acidobacterium ferrooxidans. On the other hand, the surfactants secreted by Saccharomyces cerevisiae may coat the surface of the cells, which can enhance the buffering capacity and stress resistance of the complex microbial community when introduced into the electroplating sludge environment with high concentrations of heavy metals, and shorten the adaptation period to the toxic environment.

[0010] Adding 0.5–2 g / L of elemental sulfur to the composite culture medium, which serves as an auxiliary energy substrate for *Thiobacillus ferrooxidans*, allows for the continuous generation of sulfuric acid through oxidation, thus enabling the slow-release regulation of the system's acidity. The resulting acidic and redox environment helps regulate the acidic metabolic response of *Aspergillus niger*, promoting the secretion and release of organic acids and enhancing the complexation and dissolution of copper ions. Simultaneously, this acid production process helps improve the adaptability and synergistic stability of *Aspergillus niger* and *Gnaphalium affine* during co-culture.

[0011] This application addresses the problem of the difficulty in coexistence of microorganisms with different nutritional types by restrictively regulating the culture system. Only low concentrations of glucose and yeast extract are introduced, limiting the organic components to a low concentration range, thus maintaining an oligotrophic environment dominated by inorganic energy. On one hand, the organic nutrient source can meet the basic growth requirements of *Aspergillus niger* and *Gnaphalium affine* in the early stages of cultivation, promoting their environmental adaptation and the formation of the initial microbial community structure. On the other hand, this concentration reduces the impact on the oxidative metabolic activity of the strictly autotrophic bacterium *Acidithiobacillus ferrooxidans*, helping to ensure its normal growth and metabolism. As the cultivation process progresses, the initial organic components are gradually consumed, and the system gradually transforms into a restricted environment dominated by inorganic energy. The overall metabolic activity of heterotrophic bacteria decreases, and they can maintain basic physiological homeostasis by relying on residual trace organic matter, microbial extracellular polymers, and intermediate products of cell lysis, forming a certain degree of synergistic effect with autotrophic bacteria. Simultaneously, the trace organic nutrient source can promote the metabolic activity and secretion of surfactants in yeast without excessively increasing the organic load of the system, contributing to the construction of a multi-microbial complex system.

[0012] In some embodiments, the complex microbial community is prepared by the following method: Single-strain cultures of *Thiobacillus ferrooxidans*, *Aspergillus niger*, and *Gnaphalium affine* were added to a composite culture medium at a volume ratio of 2:(0.5~2):(0.5~2), with an inoculum size of 5%~20% of the medium volume. The medium was incubated with shaking at 25~35℃ and 120~180 rpm until the viable bacterial concentration of the composite culture reached (0.8~1.2) × 10⁻⁶. 8 CFU / mL; The viable cell concentrations of the single-strain seed cultures of *Thiobacillus ferrooxidans*, *Aspergillus niger*, and *Gnaphalium affine* were each independently (0.8~1.2) × 10⁻⁶. 8 CFU / mL.

[0013] In some of the above embodiments, under the above conditions, the single-strain seed liquids of *Thiobacillus ferrooxidans*, *Aspergillus niger*, and *Gnaphalium affine* are inoculated into the composite culture medium in the above proportions. This can promote the synchronous establishment of the metabolic functions of the three strains, inhibit the imbalance of the bacterial community, and improve the stability of the composite culture, thereby providing a stable source of bacteria for the subsequent bioleaching system.

[0014] In some embodiments, the single-strain seed culture of *Thiobacillus ferrooxidans* is obtained by inoculating *Thiobacillus ferrooxidans* in 9K liquid medium and culturing it. The single-strain seed culture of Aspergillus niger was obtained by inoculating Aspergillus niger into PDB liquid medium and culturing it. The single-strain seed culture of *Gynostemma pentaphyllum* was obtained by inoculating *Gynostemma pentaphyllum* into YPD liquid medium and culturing it.

[0015] In some of the above embodiments, the composition of 9K liquid culture medium includes: 2~4 g / L (NH4)2SO4, 0.05~0.2 g / L KCl, 0.4~0.6 g / L K2HPO4, 0.4~0.6 g / L MgSO4·7H2O, 0.005~0.02 g / L Ca(NO3)2·4H2O, and 40~50 g / L FeSO4·7H2O; the composition of PDB liquid culture medium includes: 150~250 g / L potato extract and 15~25 g / L glucose; the composition of YPD liquid culture medium includes: 10~30 g / L peptone, 5~15 g / L yeast extract, and 15~25 g / L glucose.

[0016] By using the above-mentioned culture medium and cultivating the strains to the active growth phase under suitable temperature and pH conditions, it is helpful to maintain the activity and metabolism of the strains after inoculation and during the co-culture stage of the complex microbial community, thereby enabling the subsequent bioleaching reaction to be initiated and carried out more stably.

[0017] Secondly, this application provides a method for copper extraction by bioleaching assisted by a composite microbial community coupled with an electric field, comprising: S1: Provide a dual-chamber bioelectrochemical reactor, with the anode and cathode chambers separated by a cation exchange membrane. Add copper-containing electroplating wet sludge with a water content of 70-85% to the anode chamber and add the composite microbial community obtained from any of the first aspects to form a bioleaching slurry and maintain an aerobic environment in the anode chamber. Inject an electrolyte solution into the cathode chamber. S2: Connect the graphite electrode in the anode chamber and the titanium sheet electrode in the cathode chamber to the positive and negative terminals of a DC regulated power supply, respectively, and apply a DC voltage to carry out the bioleaching reaction; S3: Under the action of a DC electric field, copper ions leached from the composite bacterial community in the anode chamber migrate through the cation exchange membrane to the cathode chamber, where a reduction reaction occurs on the surface of the cathode titanium sheet to deposit metallic copper, and the copper-containing products are collected.

[0018] This application addresses the core contradiction in traditional bioleaching systems: the difficulty in simultaneously achieving high copper release efficiency and long-term stable microbial community operation. It constructs a confined coexistence micro-electric field-coupled dynamic steady-state bioleaching system. In traditional systems, improving copper leaching efficiency typically requires enhancing acidification capacity, mineral disruption capacity, and mass transfer efficiency. However, with the continuous release of copper, the Cu content in the system... 2+ Continuous accumulation, high concentration of Cu 2+ It also inhibits the metabolism of *Thiobacillus ferrooxidans* and is toxic to heterotrophic fungi and yeasts, leading to flora instability and stagnation of late-stage leaching. This application introduces a micro-electric field system, driving Cu through an electric field... 2+ The bacteria migrate across the membrane and deposit at the cathode, continuously reducing the toxicity of copper ions in the anode chamber and constructing a "release-migration-deposition" system, which is conducive to maintaining the ecological stability of the microbial community. Therefore, the composite microbial community enhances the copper release capacity, and the micro-electric field dynamically removes metabolic end products, thus jointly achieving the long-term operation of the high copper release system in the bioleaching process.

[0019] Furthermore, under the action of a continuous electric field, copper ions generated by bioleaching in the anode chamber migrate to the cathode chamber through the cation exchange membrane and undergo a reduction reaction on the surface of the cathode titanium sheet to deposit as metallic copper. This achieves the directional enrichment and direct electrochemical recovery of copper, and the obtained metallic copper has high purity, which is convenient for subsequent resource utilization.

[0020] In some implementations, after the system has been running for 24 hours, the pH of the bioleaching slurry can be decreased and maintained at 1.8 to 2.8 without the need for additional acid addition.

[0021] It is worth noting that the inventors discovered that in the system of this application, under the combined metabolic action of the three bacterial groups and the synergistic effect of the electric field, the pH of the system can spontaneously decrease and be maintained within a suitable acidic range without the need for additional acid regulators. This may be because, under aerobic conditions, *Thiobacillus ferrooxidans* in the combined bacterial group can continuously oxidize ferrous ions and reduced sulfides in the system, generating ferric ions and sulfate ions respectively, accompanied by the production of H+. + This drives a gradual increase in the acidity of the system; simultaneously, *Aspergillus jimoyense* and *Aspergillus niger* can secrete organic acids during metabolism, further replenishing the acid source and forming a multi-pathway synergistic acid production mechanism; at the same time, the electric field can accelerate H+ production. + The migration of metal ions allows the acid to diffuse rapidly and uniformly throughout the system. Based on the synergistic effect of bio-oxidation producing acid and electrochemical acid-promoting effects, the system pH can gradually decrease in the early stages of the reaction, achieving efficient metal leaching without the need for external acid regulators, thus helping to reduce operating costs.

[0022] In some embodiments, the electroplating wet sludge can be pretreated before use, including removing large particulate impurities and homogenizing and stirring, to improve the uniformity of the system.

[0023] In some embodiments, in S1, based on the total volume of the final reaction system in the anode chamber, a composite microbial community is added at a volume ratio of 5% to 15%, and then deionized water is used to make up the system volume so that the slurry solid content is 5% to 15%.

[0024] In some of the above embodiments, controlling the inoculum amount and solid content within the above range helps the microorganisms to adapt quickly, grow well and metabolize after entering the reactor, while ensuring that the slurry has good mass transfer characteristics and electric field response, laying the foundation for subsequent leaching and recovery.

[0025] In some embodiments, the electrolyte solution in S1 is a 0.05~0.2 mol / L Na2SO4 aqueous solution.

[0026] In some of the above embodiments, the Na2SO4 aqueous solution within the specified concentration range can stabilize the conductivity of the system, provide moderate resistance, and simultaneously ensure both electric field efficiency and biocompatibility.

[0027] In some embodiments, in step S1, silver sulfide is also added to the anode chamber at a dosage of 0.01~0.2 g / L, the dosage being based on the total volume of the reaction system in the anode chamber.

[0028] In some of the above embodiments, during the bioleaching process, as the complex microbial community continuously produces acid and the oxidizing environment gradually intensifies, Ag is gradually released from silver sulfide under acidic, electric field, and oxidizing conditions. +It can further undergo interfacial displacement reactions with refractory minerals such as sulfides in sludge, which helps to break down the mineral passivation layer and enhance continuous leaching capacity, maintain the catalytic activity of the anolyte interface, and together with the composite microbial community and micro-electric field, form a dynamic regulation system suitable for continuous bioleaching. At the same time, the low solubility of silver sulfide helps to inhibit Ag in the system. + Excessively high instantaneous concentrations can cause toxic effects on heterotrophic bacteria, while simultaneously slowing down the early migration and loss of silver ions under the influence of an electric field, thus improving the utilization rate of silver components in the anodic bioleaching system.

[0029] In some embodiments, in step S2, a DC voltage of 0.4 to 0.8 V is applied, and the temperature for the bioleaching reaction is 25 to 35°C.

[0030] In some of the above embodiments, the conditions described help maintain the appropriate metabolic activity of the three bacteria, *Thiobacillus ferrooxidans*, *Aspergillus niger*, and *Gnaphalium affine*, thus ensuring leaching efficiency.

[0031] Compared with the prior art, the beneficial effects of this application are at least as follows: This application constructs a dynamic system suitable for continuous bioleaching through the synergistic effect of a composite microbial community, a restricted composite culture medium, a micro-electric field, and silver sulfide. Specifically, the composite microbial community enhances the release of copper from sludge; the composite culture medium maintains a restricted coexistence state dominated by autotrophic bacteria and assisted by heterotrophic bacteria, inhibiting excessive heterotrophic bacteria proliferation that could lead to system instability; and the micro-electric field continuously drives the release of copper from the sludge. 2+ The bacteria migrate and deposit at the cathode, reducing the toxicity of accumulated copper ions in the anode chamber, thereby maintaining the long-term stable operation of the bacterial community; silver sulfide, on the other hand, gradually releases Ag. + It activates the interface of refractory minerals and inhibits silver salt loss and microbial stress, which helps to balance the stable operation of microbial communities, high copper release and migration recovery, and is conducive to improving the efficiency of continuous bioleaching. Detailed Implementation

[0032] The various embodiments or implementation schemes in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments.

[0033] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with an embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0034] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0035] In this specification, unless otherwise specified, "parts" refers to "parts by weight".

[0036] The following describes embodiments of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0037] Yeast extract: Total nitrogen content ≥9%; Penicillium chrysogenum: ATCC 10106; Brewing yeast: CGMCC 2.119; Copper-containing electroplating wet sludge: with a moisture content of 80%, after removal of large particulate impurities and homogenization pretreatment, the initial copper content is 2.1%, nickel content is 0.9%, and chromium content is 1.5% on a dry basis.

[0038] 9K liquid culture medium: Each liter of medium contains 3 g (NH4)2SO4, 0.1 g KCl, 0.5 g K2HPO4, 0.5 g MgSO4·7H2O, 0.01 g Ca(NO3)2·4H2O, and 44.2 g FeSO4·7H2O. The pH is adjusted to 2-3 with 1M sulfuric acid aqueous solution and then sterilized before use.

[0039] Preparation of single-strain seed culture of *Thiobacillus ferrooxidans*: On a sterile operating table, using a sterile inoculation needle, pick an appropriate amount of single colonies of *Thiobacillus ferrooxidans* from the slant culture and inoculate them into a sterile Erlenmeyer flask containing sterile 9K liquid medium. After inoculation, place the Erlenmeyer flask in a constant temperature shaker at 30°C and 150 rpm for shaking culture; until the viable bacterial concentration reaches 1 × 10⁻⁶. 8 CFU / mL, store at 4℃ for short-term use.

[0040] PDB liquid culture medium: Each liter of medium contains 200 g potato extract and 20 g glucose. The pH is adjusted to 3.8-4.2 with 1M sulfuric acid aqueous solution and then sterilized before use.

[0041] Preparation of Aspergillus niger seed culture: On a sterile operating table, use a sterile inoculation needle to pick up an appropriate amount of Aspergillus niger spores from the slant culture and inoculate them into a sterile Erlenmeyer flask containing sterile PDB liquid culture medium. After inoculation, place the Erlenmeyer flask in a constant temperature shaker at 30°C and 150 rpm for shaking culture; until the viable bacterial concentration reaches 1×10⁻⁶. 8 CFU / mL, store at 4℃ for short-term use.

[0042] Preparation of seed culture of Penicillium chrysogenum: On a sterile operating table, using a sterile inoculation needle, pick an appropriate amount of *Penicillium chrysogenum* from the slant culture and inoculate it into a sterile Erlenmeyer flask containing sterile PDB liquid medium. After inoculation, place the Erlenmeyer flask in a constant temperature shaker at 30°C and 150 rpm for shaking culture; until the viable bacterial concentration reaches 1 × 10⁻⁶. 8 CFU / mL, store at 4℃ for short-term use.

[0043] YPD liquid culture medium: Each liter of medium contains 20 g peptone, 10 g yeast extract, and 20 g glucose. The pH is adjusted to 3.8-4.2 with 1M sulfuric acid aqueous solution and then sterilized before use.

[0044] Preparation of single-strain seed culture of *Saccharomyces cerevisiae*: On a sterile operating table, using a sterile inoculation needle, pick an appropriate amount of single colony of *Saccharomyces cerevisiae* from the slant culture and inoculate it into sterile YPD liquid medium. Incubate at 30°C and 150 rpm in a constant-temperature shaker until the viable cell concentration reaches 1 × 10⁻⁶. 8 CFU / mL, store at 4℃ for short-term use.

[0045] Preparation of single-strain seed culture of Saccharomyces cerevisiae: On a sterile operating table, use a sterile inoculation needle to pick an appropriate amount of single colony of *Saccharomyces cerevisiae* from the slant culture and inoculate it into sterile YPD liquid medium. Incubate at 30°C and 150 rpm in a constant-temperature shaker until the viable cell concentration reaches 1 × 10⁻⁶. 8 CFU / mL, store at 4℃ for short-term use.

[0046] Compound culture medium A: Each liter of medium contains 3 g (NH4)2SO4, 0.1 g KCl, 0.5 g K2HPO4, 0.5 g MgSO4·7H2O, 0.01 g Ca(NO3)2·4H2O, and 1 g S. 0 44.2 g FeSO4·7H2O, 0.8 g glucose, and 0.1 g yeast extract were prepared by adjusting the pH to 2.5-2.8 with 1M sulfuric acid aqueous solution and sterilizing before use.

[0047] Compound culture medium B: Each liter of medium contains 3 g (NH4)2SO4, 0.1 g KCl, 0.5 g K2HPO4, 0.5 g MgSO4·7H2O, 0.01 g Ca(NO3)2·4H2O, and 1 g S. 0 44.2 g FeSO4·7H2O and 0.1 g yeast extract were prepared by adjusting the pH to 2.5-2.8 with 1M sulfuric acid aqueous solution and sterilizing before use.

[0048] Compound culture medium C: Each liter of medium contains 3 g (NH4)2SO4, 0.1 g KCl, 0.5 g K2HPO4, 0.5 g MgSO4·7H2O, 0.01 g Ca(NO3)2·4H2O, and 1 g S. 0 44.2 g FeSO4·7H2O and 0.8 g glucose were prepared by adjusting the pH to 2.5-2.8 with 1M sulfuric acid aqueous solution and sterilizing before use.

[0049] Compound culture medium E: Each liter of medium contains 3 g (NH4)2SO4, 0.1 g KCl, 0.5 g K2HPO4, 0.5 g MgSO4·7H2O, 0.01 g Ca(NO3)2·4H2O, and 1 g S. 0 44.2 g FeSO4·7H2O, 1.5 g glucose, and 0.5 g yeast extract were prepared by adjusting the pH to 2.5-2.8 with 1M sulfuric acid aqueous solution and sterilizing before use.

[0050] Preparation Example 1 Preparation of complex microbial communities: The above-prepared seed cultures of *Acidithiobacillus ferrooxidans*, *Aspergillus niger*, and *Saccharomyces cerevisiae* were mixed at a volume ratio of 2:1:1 and inoculated into sterile composite culture medium A at an inoculation volume of 10% (v / v) of the total culture medium volume. The mixture was then placed in a constant-temperature shaker at 30℃ and 150 rpm for 24–72 h to allow the viable bacterial concentration to reach 1 × 10⁻⁶. 8 CFU / mL was used to obtain complex bacterial group A, which was then stored at 4℃ for a short period of time.

[0051] Preparation Example 2 Preparation of complex microbial communities: The above-prepared seed cultures of *Thiobacillus ferrooxidans*, *Aspergillus niger*, and *Saccharomyces cerevisiae* were mixed at a volume ratio of 2:1:1 and inoculated into sterile composite culture medium B at an inoculation volume of 10% (v / v) of the total culture medium volume. The mixture was then placed in a constant-temperature shaker at 30℃ and 150 rpm for 24–72 h to achieve a viable bacterial concentration of 1 × 10⁻⁶. 8CFU / mL was used to obtain complex bacterial group F, which was then stored at 4℃ for short-term use.

[0052] Preparation Example 3 Preparation of complex microbial communities: The above-prepared seed cultures of *Acidithiobacillus ferrooxidans*, *Aspergillus niger*, and *Gnaphalium affine* were mixed at a volume ratio of 2:1:1 and inoculated into sterile composite culture medium C at an inoculation volume of 10% (v / v) of the total culture medium volume. The mixture was then placed in a constant-temperature shaker at 30℃ and 150 rpm for 24–72 h to allow the viable bacterial concentration to reach 1 × 10⁻⁶. 8 CFU / mL was used to obtain complex bacterial group G, which was then stored at 4℃ for short-term use.

[0053] Preparation Example 4 Preparation of complex microbial communities: The above-prepared seed cultures of *Thiobacillus ferrooxidans*, *Aspergillus niger*, and *Saccharomyces cerevisiae* were mixed at a volume ratio of 2:1:1 and inoculated into sterile composite culture medium E at an inoculation volume of 10% (v / v) of the total culture medium volume. The mixture was then placed in a constant-temperature shaker at 30℃ and 150 rpm for 24–72 h to allow the viable bacterial concentration to reach 1 × 10⁻⁶. 8 CFU / mL was used to obtain complex bacterial group I, which was then stored at 4℃ for short-term use.

[0054] Comparative Preparation Example 1 Preparation of complex microbial communities: The seed cultures of *Thiobacillus ferrooxidans* and *Aspergillus niger* prepared above were mixed at a volume ratio of 2:1 and inoculated into sterile composite culture medium A at an inoculation volume of 10% (v / v) of the total culture medium volume. The mixture was then placed in a constant temperature shaker at 30℃ and 150 rpm for 24–72 h to allow the viable bacterial concentration to reach 1 × 10⁻⁶. 8 CFU / mL was used to obtain complex bacterial group B, which was then stored at 4℃ for short-term use.

[0055] Comparative Preparation Example 2 Preparation of complex microbial communities: The above-prepared *Thiobacillus ferrooxidans* and *Saccharomyces cerevisiae* single-strain seed cultures were mixed at a volume ratio of 2:1 and inoculated into sterile composite culture medium A at an inoculation volume of 10% (v / v) of the total culture medium volume. The mixture was then placed in a constant-temperature shaker at 30℃ and 150 rpm for 24–72 h to allow the viable bacterial concentration to reach 1 × 10⁻⁶. 8 CFU / mL was used to obtain complex bacterial group C, which was then stored at 4℃ for short-term use.

[0056] Comparative preparation example 3 Preparation of complex microbial communities: The prepared *Thiobacillus ferrooxidans* single-strain seed culture was inoculated into sterile composite culture medium A at an inoculation volume of 10% (v / v) of the total culture medium volume. The medium was then incubated in a constant-temperature shaker at 30°C and 150 rpm for 24–72 h to achieve a viable bacterial concentration of 1 × 10⁻⁶. 8 CFU / mL was used to obtain single bacteria D, which were then stored at 4°C for short-term use.

[0057] Comparative preparation example 4 Preparation of complex microbial communities: The above-prepared seed cultures of *Acidithiobacillus ferrooxidans*, *Penicillium chrysogenum*, and *Saccharomyces cerevisiae* were mixed at a volume ratio of 2:1:1 and inoculated into sterile composite culture medium A at an inoculation volume of 10% (v / v). The mixture was then placed in a constant-temperature shaker at 30℃ and 150 rpm for 24–72 h to achieve a viable bacterial concentration of 1 × 10⁻⁶. 8 CFU / mL was used to obtain complex bacterial group E, which was then stored at 4℃ for short-term use.

[0058] Example 1 S1: Provide a dual-chamber bioelectrochemical reactor, with the anode chamber and cathode chamber separated by a cation exchange membrane. Before use, clean and sterilize all components of the reactor. Add 100ml of copper electroplating wet sludge with a water content of 80%, 20ml of compound bacterial group A, 0.02g of silver sulfide, and 80ml of deionized water to the anode chamber. Stir at 100rpm to maintain uniform suspension of the slurry. At the same time, continuously introduce air into the anode chamber at a rate of 0.5vvm. Add 200ml of 0.1 mol / L Na2SO4 aqueous solution to the cathode chamber. S2: Connect the graphite electrode in the anode chamber and the titanium sheet electrode in the cathode chamber to the positive and negative terminals of a DC regulated power supply, respectively, adjust the voltage to 0.5V, and carry out the bioleaching reaction at 30℃. Maintain stirring and aeration during the reaction. S3: After the reaction is complete, stop the power supply, remove the cathode titanium sheet, collect the deposited product on the cathode surface, and at the same time perform solid-liquid separation of the cathode chamber solution to collect the precipitate; combine the obtained solids, wash with deionized water and dry to obtain the copper-containing product.

[0059] Example 2 S1: Provide a dual-chamber bioelectrochemical reactor, with the anode chamber and cathode chamber separated by a cation exchange membrane. Before use, clean and sterilize all components of the reactor. Add 100ml of copper electroplating wet sludge with a water content of 80%, 20ml of compound bacterial group A, 0.02g of silver sulfide, and 80ml of deionized water to the anode chamber. Stir at 100rpm to maintain uniform suspension of the slurry. At the same time, continuously introduce air into the anode chamber at a rate of 0.5vvm. Add 200ml of 0.1 mol / L Na2SO4 aqueous solution to the cathode chamber. S2: Without applying an electric field, the bioleaching reaction is carried out at 30°C, and stirring and aeration are maintained during the reaction. S3: After the reaction is completed, the system is subjected to solid-liquid separation, and the copper-containing leachate is collected. Iron powder or iron filings are added to the leachate, and a displacement reaction is carried out under stirring conditions to reduce the copper ions in the solution to metallic copper and precipitate out. After the reaction is completed, solid-liquid separation is performed, and the product containing copper is obtained after washing with deionized water and drying.

[0060] Example 3 It is largely the same as Example 1, except that silver sulfide was not added in step S1.

[0061] Example 4 Similar to Example 1, except that complex microbial community A is replaced with complex microbial community F.

[0062] Example 5 Similar to Example 1, except that complex microbial community A is replaced with complex microbial community G.

[0063] Example 6 Similar to Example 1, except that complex microbial community A is replaced with complex microbial community I.

[0064] Comparative Example 1 S1: Provide a dual-chamber bioelectrochemical reactor, with the anode chamber and cathode chamber separated by a cation exchange membrane. Before use, clean and sterilize all components of the reactor. Add 100ml of copper electroplating wet sludge with a water content of 80%, 20ml of compound bacterial group B, 0.02g of silver sulfide, and 80ml of deionized water to the anode chamber. Stir at 100rpm to maintain uniform suspension of the slurry. At the same time, continuously introduce air into the anode chamber at a rate of 0.5vvm. Add 200ml of 0.1 mol / L Na2SO4 aqueous solution to the cathode chamber. S2: Connect the graphite electrode in the anode chamber and the titanium sheet electrode in the cathode chamber to the positive and negative terminals of a DC regulated power supply, respectively, adjust the voltage to 0.5V, and carry out the bioleaching reaction at 30℃. Maintain stirring and aeration during the reaction. Step S3 is the same as step S3 in Example 1.

[0065] Comparative Example 2 It is largely the same as Comparative Example 1, except that complex microbial community B in S1 is replaced with complex microbial community C.

[0066] Comparative Example 3 It is largely the same as Comparative Example 1, except that the complex bacterial group B in S1 is replaced with a single bacterial group D.

[0067] Comparative Example 4 It is largely the same as Comparative Example 1, except that complex microbial community B in S1 is replaced with complex microbial community E.

[0068] Test section After the reaction, solid-liquid separation was performed on the systems of the examples and comparative examples. The leaching residue was collected, washed with deionized water, stirred at 300 r / min for 1 h, filtered, washed again, and dried at 105 °C to constant weight. A certain amount of sample was acid-dissolved, and the copper concentration (mg / L) was determined using inductively coupled plasma optical emission spectrometry (ICP-OES). The mass m of copper in the leached residue could be calculated by combining the constant volume. r .

[0069] Simultaneously, copper-containing products from the examples and comparative examples were collected, and after acid dissolution, the copper concentration (mg / L) was determined using ICP-OES. The mass m of the cathode-deposited copper could be calculated based on the constant volume. The initial sample copper mass m0 and the residual copper mass m0 were then used as the basis for the calculation. r The copper leaching rate and copper extraction rate are calculated based on the mass of deposited copper, m. The copper leaching rate (%) is calculated as follows: (m0 - m...) r Copper extraction rate (%) = m / m0 × 100%.

[0070] The test results are shown in Table 1.

[0071] Table 1

[0072] As shown in Table 1, the copper leaching rate and copper extraction rate of Example 1 of this application are higher than those of the comparative example, indicating that the copper extraction efficiency of the composite microbial bioleaching method of this application is higher. Specifically, Comparative Example 1 lacks *Aspergillus oryzae*, thus losing its biosurfactant effect on wetting and dispersing sludge particles and enhancing mass transfer, leading to sludge particle aggregation and potentially insufficient contact between some copper sites and the leaching system, resulting in a decrease in the copper leaching rate. Comparative Example 2 lacks *Aspergillus niger*, lacking the organic acid complexation and dissolution of copper and the sludge disintegration effect, which is detrimental to the continuous release of copper components. Furthermore, *Aspergillus oryzae* cells may have a certain adsorption effect on some copper ions; without the synergistic release-promoting effect of *Aspergillus niger*, the overall copper leaching efficiency of the system decreases. Comparative Example 3... The strain is a single bacterium of *Thiobacillus ferrooxidans*, which lacks the synergistic effects of auxiliary bacteria in acid production, copper dissolution, and mass transfer. Its acid production capacity is weak, and the copper sites in the sludge are difficult to break, resulting in a low leaching rate, fewer recoverable copper ions, and a decreased extraction rate. In Comparative Example 4, *Penicillium chrysogenum* and *Saccharomyces cerevisiae* were used to replace *Aspergillus niger* and *Gnaphalium affine*. Although a certain degree of synergistic effect of the complex microbial community could still be formed, *Penicillium chrysogenum* may have a weaker effect on the destruction of sludge structure and the promotion of acidification in the system of this application. At the same time, *Saccharomyces cerevisiae* has low tolerance to heavy metals and low complexation and migration ability, making it difficult to effectively promote the dissolution of metal ions.

[0073] As can be seen from Examples 1 and 2, the effect of the electric field has a certain impact on the efficiency of copper extraction by the composite microbial community. Coupled with the electric field, the efficiency of copper extraction by bioleaching is higher.

[0074] As shown in Examples 1 and 3, the addition of silver sulfide has a certain impact on the efficiency of copper extraction by the composite microbial community. As bioleaching proceeds, silver sulfide may slowly release Ag. + It participates in the oxidation and activation process of mineral interfaces, which helps maintain the long-term stable synergy and continuous leaching capacity of the complex microbial community.

[0075] As shown in Examples 1, 4-6, under the same conditions of complex microbial community composition, the type and concentration of organic nutrient sources in the culture medium have a certain impact on the bioleaching effect. Appropriate amounts of glucose and yeast extract can effectively improve the leaching and extraction rates of copper, indicating that organic carbon and organic nitrogen sources can synergistically promote the metabolic activity and organic acid secretion of the complex microbial community, thereby enhancing the metal leaching process. When any component is lacking, the system performance decreases. When the concentration of organic nutrient sources is too high, it leads to a decrease in leaching and extraction rates, possibly because excessive organic matter inhibits the oxidative activity of *Thiobacillus ferrooxidans* and disrupts the synergistic relationship of the microbial community, thus hindering metal leaching.

[0076] The experimental systems of Example 1 and Comparative Examples 1-4 were selected and operated under the same temperature, aeration and stirring conditions. The pH value of the anode chamber system was measured at 24h, 48h, 96h and 168h to analyze the effect of different bacterial communities on the spontaneous pH decrease behavior.

[0077] The test results are shown in Table 2.

[0078] Table 2

[0079] As shown in Table 2, in Example 1 of this application, the composite microbial community can jointly achieve a rapid decrease and stable maintenance of pH. In Comparative Example 1, which lacks Aspergillus mutandis, a certain degree of acidification can be achieved in the early stage by relying on Aspergillus niger. However, due to the lack of biosurfactants secreted by yeast, sludge particles are prone to agglomeration, and a large amount of alkaline substances are encapsulated in the early stage. The slow release in the later stage of the reaction causes the pH decrease to stagnate. In Comparative Example 2, which lacks Aspergillus niger, the high concentration of free heavy metals in the early stage of the reaction may inhibit the metabolism of Acidobacterium ferrooxidans, causing a temporary stagnation of biological acid production and a slow decrease in pH. In the later stage, the microbial community gradually adapts to the stress environment and begins to slowly acidify, but the acidification start-up is relatively lagging. In Comparative Example 3, Acidobacterium ferrooxidans alone has weak stress resistance and may be subject to the dual neutralization effect of heavy metal inhibition and alkaline substances in the sludge, resulting in low sulfur oxidation acid production efficiency. In Comparative Example 4, Penicillium chrysogenum has insufficient rapid acid reduction effect in the early stage, and the role of Saccharomyces cerevisiae in improving sludge dispersion and enhancing mass transfer is limited. Therefore, the overall acidification start-up is slow and the acid reduction efficiency is insufficient.

[0080] In summary, the ternary synergistic complex microbial community of this application has a good advantage in acidification regulation. Relying on the metabolic complementarity of functional microbial species, it can quickly start acid reduction in the early stage of the reaction, shorten the acidification adaptation period, effectively seize the optimal kinetic window for copper leaching, and at the same time maintain a stable strongly acidic environment for a long time, enhance the mineral lattice destruction and heavy metal complexation dissociation effect, and optimize solid-liquid mass transfer, thereby improving the copper leaching rate and ensuring the efficient operation of the electric field coupled bioleaching system.

[0081] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A complex microbial community, characterized in that, include: Acidithiobacillus ferrooxidans ATCC 23270; Aspergillus niger ATCC 6275; Meyerozyma guilliermondii ATCC 14242; The composite microbial community was obtained by inoculating the *Acidithiobacillus ferrooxidans*, *Aspergillus niger*, and *Gynostemma pentaphyllum* in a composite culture medium at an inoculation ratio of 2:(0.5~2):(0.5~2) and then co-culturing them. The composite culture medium comprises: 2.5–3.5 g / L (NH₄)₂SO₄, 0.05–0.2 g / L KCl, 0.4–0.6 g / L K₂HPO₄, 0.4–0.6 g / L MgSO₄·7H₂O, 0.005–0.02 g / L Ca(NO₃)₂·4H₂O, 40–50 g / L FeSO₄·7H₂O, and 0.5–2 g / L MgSO₄·7H₂O. 0 The compound culture medium contains 0.5-1.0 g / L glucose and 0.05-0.2 g / L yeast extract; the pH of the compound culture medium is 2.5-2.

8.

2. The complex microbial community according to claim 1, characterized in that, The complex microbial community was prepared by the following method: Single-strain cultures of *Thiobacillus ferrooxidans*, *Aspergillus niger*, and *Gnaphalium affine* were added to a composite culture medium at a volume ratio of 2:(0.5~2):(0.5~2), with an inoculum size of 5%~20% of the medium volume. The medium was incubated with shaking at 25~35℃ and 120~180 rpm until the viable bacterial concentration of the composite culture reached (0.8~1.2) × 10⁻⁶. 8 CFU / mL; The viable cell concentrations of the single-strain seed cultures of *Thiobacillus ferrooxidans*, *Aspergillus niger*, and *Gnaphalium affine* were each independently (0.8~1.2) × 10⁻⁶. 8 CFU / mL.

3. The complex microbial community according to claim 2, characterized in that, The single-strain seed culture of *Acidithiobacillus ferrooxidans* was obtained by inoculating *Acidithiobacillus ferrooxidans* into 9K liquid medium and culturing it. The single-strain seed culture of Aspergillus niger was obtained by inoculating Aspergillus niger into PDB liquid medium and culturing it. The single-strain seed culture of *Gynostemma pentaphyllum* was obtained by inoculating *Gynostemma pentaphyllum* into YPD liquid medium and culturing it.

4. A method for copper extraction by bioleaching assisted by a composite microbial community coupled with an electric field, characterized in that, include: S1: Provide a dual-chamber bioelectrochemical reactor, wherein the anode and cathode chambers are separated by a cation exchange membrane, and copper electroplating wet sludge with a water content of 70-85% is added to the anode chamber along with the composite microbial community obtained according to any one of claims 1-3 to form a bioleaching slurry, and the aerobic environment of the anode chamber is maintained, while an electrolyte solution is injected into the cathode chamber. S2: Connect the graphite electrode in the anode chamber and the titanium sheet electrode in the cathode chamber to the positive and negative terminals of a DC regulated power supply, respectively, and apply a DC voltage to carry out the bioleaching reaction; S3: Under the action of a DC electric field, copper ions leached from the composite bacterial community in the anode chamber migrate through the cation exchange membrane to the cathode chamber, where a reduction reaction occurs on the surface of the cathode titanium sheet to deposit metallic copper, and the copper-containing products are collected.

5. The method according to claim 4, characterized in that, In step S1, based on the total volume of the final reaction system in the anode chamber, a composite microbial community is added at a volume ratio of 5% to 15%, and then deionized water is used to replenish the system volume so that the slurry solid content is 5% to 15%.

6. The method according to claim 4, characterized in that, The electrolyte solution mentioned in S1 is a 0.05~0.2 mol / L Na2SO4 aqueous solution.

7. The method according to claim 4, characterized in that, In step S1, silver sulfide is also added to the anode chamber at a dosage of 0.01~0.2 g / L, based on the total volume of the reaction system in the anode chamber.

8. The method according to claim 4, characterized in that, In step S2, a DC voltage of 0.4~0.8V is applied, and the temperature for the bioleaching reaction is 25~35℃.

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