Method for recovering germanium element from fly ash by microorganism

Abstract

This invention discloses a microbial method for the recovery of germanium from fly ash, relating to the field of fly ash biometallurgy. The method includes: pre-treating the fly ash by grinding and drying; culturing *Thiobacillus acidophilus*, *Pseudomonas putida*, and *Saccharomyces cerevisiae*, respectively; subjecting the *Thiobacillus* to osmotic gradient acclimation; mixing the pre-treated fly ash with a basic leaching medium, and then sequentially inoculating the acclimated *Thiobacillus* with sulfur powder, inoculating *Pseudomonas putida* with glucose, and inoculating *Saccharomyces cerevisiae* with sucrose in three stages, maintaining pH values ​​of 2.5–3.5, 4.0–5.0, and 5.5–6.5 respectively in each stage; periodically adding the corresponding bacterial strains and carbon / energy sources at each stage; and centrifuging to collect the germanium-containing leachate and recover the germanium element. This invention achieves high germanium leaching rates through the synergistic effect of the three bacteria, the synergistic effect of spatial stepwise inoculation and temporal gradient replenishment, osmotic gradient acclimation, and staged pH control, while maintaining a mild and environmentally friendly process.

Description

Technical Field

[0001] This invention relates to the field of fly ash biometallurgical technology, specifically to a microbial method for recovering germanium from fly ash. Background Technology

[0002] Fly ash is a major solid waste generated by coal-fired power plants, with a huge global annual output that is increasing year by year. Open-air storage of fly ash not only occupies a large amount of land, but also leaches out heavy metals and other harmful substances, posing a serious threat to ecological environment safety and human health.

[0003] On the other hand, fly ash contains abundant rare metal elements, among which germanium (Ge), as a strategic rare metal, has irreplaceable and important application value in fields such as information and communication, modern aviation, military equipment, new energy, and semiconductors. Recovering germanium from fly ash can not only alleviate the shortage of germanium resources but also achieve high-value utilization of solid waste.

[0004] Currently, the main methods for recovering germanium from fly ash include chemical leaching and microbial leaching. Chemical leaching methods, such as chloride distillation, consume large amounts of chloride, resulting in high energy consumption, demanding equipment requirements, complex processes, and the generation of large amounts of waste liquid and residue, causing secondary pollution. Although wet germanium extraction processes are relatively simple to operate, they require the use of chemical reagents such as ammonium fluoride and sulfuric acid, which still pose environmental pollution problems.

[0005] Microbial leaching has advantages such as low energy consumption and environmental friendliness, and has received widespread attention in recent years. Existing methods for recovering germanium by microorganisms mainly use a single strain for leaching, utilizing organic or inorganic acids produced by microbial metabolism to dissolve germanium. However, existing methods still have the following technical defects: (1) Germanium in fly ash mainly exists in the aluminosilicate glass phase in a isomorphic form, which is wrapped by the Si-O-Al network structure, making it difficult for microorganisms and their metabolites to fully contact germanium; (2) The high salt and high alkalinity environment of fly ash has a significant inhibitory effect on microbial growth, making it difficult for the strain to maintain high activity in this environment for a long time, resulting in unstable leaching efficiency; (3) Existing methods have complex pretreatment of fly ash, usually requiring high temperature and high pressure or chemical activation treatment, which increases process cost and operation difficulty; (4) The leaching rate of germanium is generally low, which is difficult to meet the requirements of industrial production.

[0006] Therefore, developing a microbial method for recovering germanium from fly ash that can overcome the above-mentioned technical defects, has higher germanium recovery efficiency, and is environmentally friendly has important industrial application value and environmental significance. Summary of the Invention

[0007] To address the aforementioned problems, this invention provides a microbial method for the recovery of germanium from fly ash, which solves the problems of low germanium leaching rate, insufficient microbial activity in the fly ash environment, and complex processes in existing microbial methods for recovering germanium from fly ash.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides a method for the microbial recovery of germanium from fly ash, specifically comprising the following steps: S1: Fly ash pretreatment: Grind the fly ash, sieve it, and dry it to constant weight; S2: Selective culture of functional strains: culture of *Thiobacillus acidophilus* (… Acidithiobacillus thiooxidans ), Pseudomonas putida ( Pseudomonas putida ) and brewer's yeast ( Saccharomyces cerevisiae Selective culture was performed to obtain Thiobacillus culture medium, Pseudomonas suspension and Saccharomyces cerevisiae suspension, respectively; S3: Osmotic gradient acclimatization: The Thiobacillus culture medium obtained in step S2 is subjected to osmotic gradient acclimatization in a culture medium containing a sodium chloride gradient to obtain an osmotically acclimatized Thiobacillus culture medium. S4: Spatial stepwise inoculation and leaching: The pretreated fly ash obtained in step S1 is mixed with the basic leaching medium to obtain a mixture, and then three stages of spatial stepwise inoculation and leaching are performed sequentially: First stage: Inoculate the mixture with the osmotically acclimatized Thiobacillus culture obtained in step S3, add sulfur powder, and carry out the leaching reaction under pH 2.5~3.5 conditions; Second stage: Inoculate the Pseudomonas suspension obtained in step S2 into the reaction system after leaching in the first stage, add glucose, and carry out the leaching reaction under pH 4.0~5.0 conditions; Third stage: Inoculate the Saccharomyces cerevisiae suspension obtained in step S2 into the reaction system after leaching in the second stage, add sucrose, and carry out the leaching reaction under pH 5.5~6.5 conditions; S5: Time-gradient replenishment: In the above three stages, after the start of each stage, the corresponding functional strain of bacterial solution is replenished at regular intervals, and half of the corresponding carbon source or energy substance is replenished before the end of each stage. S6: Separation and Recovery: After the leaching reaction is completed, the reaction mixture is centrifuged and the supernatant is collected as germanium-containing leachate. Germanium element is separated and recovered from germanium-containing leachate.

[0009] Furthermore, in step S1, the sieve size is 200-400 mesh, and the drying temperature is 60-80℃.

[0010] Further, in step S2, the selective culture specifically includes: (a) Inoculate Thiobacillus acidophilus into 9K liquid medium and culture for 48-72 h at pH 2.0-3.0, temperature 28-32℃ and shaking speed 150-200 rpm to obtain Thiobacillus culture medium; (b) Inoculate *Pseudomonas putida* into LB liquid medium and culture for 36-48 h at pH 6.5-7.2, temperature 30-35 °C and shaking speed 150-200 rpm. Collect the bacterial cells by centrifugation and resuspend them in sterile deionized water to obtain a *Pseudomonas putida* bacterial suspension. (c) Inoculate the Saccharomyces cerevisiae into YPD liquid medium and culture it for 24-36 h at pH 5.0-6.0, temperature 28-30℃ and shaking speed 120-180 rpm. Collect the cells by centrifugation and resuspend them in sterile deionized water to obtain a Saccharomyces cerevisiae suspension.

[0011] Furthermore, the 9K liquid culture medium consists of: (NH4)2SO4 3.0 g / L, KCl 0.1 g / L, K2HPO4 0.5 g / L, MgSO4·7H2O 0.5 g / L, Ca(NO3)2 0.01 g / L, FeSO4·7H2O 44.2 g / L, with the pH adjusted to 2.0~3.0 using dilute sulfuric acid; the LB liquid culture medium consists of: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7.0~7.2; and the YPD liquid culture medium consists of: yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L, pH 5.0~6.0.

[0012] Further, in step S3, the osmotic gradient acclimatization specifically involves: transferring the Thiobacillus culture medium sequentially to 9K medium with sodium chloride concentrations of 5 g / L, 10 g / L, 15 g / L, and 20 g / L, respectively, inoculating at a 10% inoculum at each concentration, and subculturing 2-3 times consecutively, with each culture lasting 48-72 hours.

[0013] Further, in step S4, the basic leaching medium is sterilized deionized water, and the solid-liquid ratio of the pretreated fly ash to the basic leaching medium is 1:10~20 (g / mL); the leaching times for the first, second, and third stages are 3~5 days, 4~6 days, and 3~5 days, respectively; in the first stage, the inoculation amount of Thiobacillus culture medium is 5%~15% of the total volume of the mixture, and the amount of sulfur powder added is 0.5%~1.5% of the fly ash mass; in the second ... third stage, the inoculation amount of Thiobacillus culture medium is 5%~15% of the total volume of the mixture, and the amount of sulfur powder added is 0.5%~1.5% of the fly ash mass; in the third stage, the inoculation amount of Thiobacillus culture medium is 5%~15% of the total volume of the mixture, and the amount of sulfur powder added is 0.5%~1.5% of the fly ash mass; in the fourth stage, The inoculation amount of the *Saccharomyces cerevisiae* suspension is 5% to 10% of the total volume of the reaction system after the first stage leaching, and the amount of glucose added is 0.5% to 2.0% of the fly ash mass; in the third stage, the inoculation amount of the *Saccharomyces cerevisiae* suspension is 5% to 10% of the total volume of the reaction system after the second stage leaching, and the amount of sucrose added is 0.3% to 1.0% of the fly ash mass; the reaction temperatures of the first, second, and third stages are 30 to 35°C, 30 to 35°C, and 28 to 32°C, respectively.

[0014] Further, in step S5, the specific operation of the timed replenishment is as follows: 24 hours after the start of each stage, the corresponding functional strain bacterial solution is replenished, and the replenishment amount is 50% of the initial inoculation amount for that stage; for stages with a leaching time of 4 days or more, it is replenished again at 48 hours, and the replenishment amount is 30% of the initial inoculation amount for that stage; the replenishment of half the amount of carbon source or energy substance corresponding to that stage before the end of each stage refers to: half the amount of sulfur powder added before the end of the first stage, half the amount of glucose added before the end of the second stage, and half the amount of sucrose added before the end of the third stage.

[0015] Further, in step S5, the centrifugation speed is 4000~8000 rpm and the centrifugation time is 10~20 min; the method for separating and recovering germanium from germanium-containing leachate is solvent extraction or ion exchange.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The present invention uses a combination of three strains: acidophilic thiobacillus, putrid pseudomonas and brewer's yeast. The three strains respectively produce sulfuric acid to destroy the structure of fly ash, produce organic acid to complex and dissolve germanium ions, and adsorb and desorb germanium ions. Their functions complement each other and produce a significant synergistic effect, which significantly improves the germanium leaching rate.

[0017] (2) This invention employs a spatial stepwise inoculation strategy. Based on the functional requirements of microorganisms at different stages of the leaching process, three strains are sequentially inoculated under optimized pH conditions, allowing each strain to exert its maximum effect under its optimal conditions. Simultaneously, a time-gradient replenishment strategy is adopted, with strains and carbon / energy sources replenished at regular intervals at each stage to maintain the continuous high activity of microorganisms. The combined use of these two strategies produces a synergistic effect, further improving the germanium leaching rate.

[0018] (3) By acclimating Thiobacillus to osmotic pressure gradient, this invention significantly improves Thiobacillus’ adaptability and metabolic activity in the high-salt environment of fly ash, effectively solving the problem of insufficient activity of microorganisms in fly ash environment.

[0019] (4) The present invention adopts phased pH control so that the pH of each phase matches the optimal metabolic conditions of the corresponding strain, thereby further improving the leaching efficiency of each phase.

[0020] (5) This invention requires only simple grinding and drying pretreatment, without the need for high temperature and high pressure or chemical activation treatment. The process conditions are mild, the operation is simple, the energy consumption is low, and the environment is friendly. The germanium leaching rate can reach 92.1%~96.5%, providing a brand-new technical approach for the efficient and green recovery of germanium resources in fly ash. Detailed Implementation

[0021] To make the objectives and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0022] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0023] The fly ash used in the following examples contains 0.32% germanium by mass, and its main chemical composition includes: 52.6% SiO2, 28.3% Al2O3, 7.8% Fe2O3, 3.5% CaO, 1.2% MgO, and 6.6% other components (including Ge).

[0024] 9K liquid culture medium: (NH4)2SO4 3.0g / L, KCl 0.1g / L, K2HPO4 0.5g / L, MgSO4·7H2O 0.5g / L, Ca(NO3)2 0.01g / L, FeSO4·7H2O 44.2g / L, pH adjusted to 2.0~3.0 with dilute sulfuric acid.

[0025] LB liquid medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7.0~7.2.

[0026] YPD liquid culture medium: yeast extract 10g / L, peptone 20g / L, glucose 20g / L, pH 5.0~6.0.

[0027] Germanium content was determined using inductively coupled plasma optical emission spectrometry (ICP-OES), and the germanium leaching rate was calculated using the following formula: Germanium leaching rate (%) = (mass of germanium in the leachate / total mass of germanium in the raw fly ash) × 100%

[0028] Example 1

[0029] This embodiment provides a method for the microbial recovery of germanium from fly ash, specifically including the following steps: S1: Fly ash pretreatment: Grind the fly ash through a 300-mesh sieve and dry it at 70℃ to constant weight; S2: Selective culture of functional strains: culture of *Thiobacillus acidophilus* (… Acidithiobacillus thiooxidans ), Pseudomonas putida ( Pseudomonas putida ) and brewer's yeast ( Saccharomyces cerevisiae Selective cultivation is carried out, specifically as follows: (a) Thiobacillus acidophilus was inoculated into 9K liquid medium and cultured for 60 h at pH 2.5, temperature 30 °C, and shaking speed 180 rpm to obtain a Thiobacillus culture broth (cell concentration approximately 10). 8 (CFU / mL) (b) *Pseudomonas putida* was inoculated into LB liquid medium and cultured for 40 h at pH 7.0, 32 °C, and 180 rpm. The bacterial cells were collected by centrifugation and resuspended in sterile deionized water to obtain a *Pseudomonas* bacterial suspension (bacterial concentration approximately 10⁻⁶). 8 (CFU / mL) (c) Inoculate *Saccharomyces cerevisiae* into YPD liquid medium and culture for 30 h at pH 5.5, temperature 29℃, and shaking speed 150 rpm. Collect the cells by centrifugation and resuspend them in sterile deionized water to obtain a *Saccharomyces cerevisiae* suspension (cell concentration approximately 10). 8 (CFU / mL).

[0030] S3: Osmotic gradient acclimatization: The Thiobacillus culture obtained in step S2 was sequentially transferred to 9K medium with sodium chloride concentrations of 5 g / L, 10 g / L, 15 g / L and 20 g / L for osmotic gradient acclimatization. At each concentration, 10% inoculum was used, and the culture was passaged twice for 60 h each time to obtain osmotically acclimatized Thiobacillus culture. S4: Spatial stepwise inoculation and leaching: 10g of pretreated fly ash obtained in step S1 was mixed with the basic leaching medium (sterile deionized water) at a ratio of 1:15 (g / mL) to obtain a mixture, and then three stages of spatial stepwise inoculation and leaching were performed sequentially: The first stage (structural destruction stage): The osmotically acclimated Thiobacillus culture obtained in step S3 is inoculated into the mixture at a volume of 10% of the total volume of the mixture, and sulfur powder is added at a volume of 1.0% of the fly ash mass. The leaching reaction is carried out under the conditions of pH 3.0 (maintained by dilute sulfuric acid), temperature of 32℃, and shaking speed of 150 rpm for 4 days. This stage mainly utilizes the sulfuric acid produced by the metabolism of Thiobacillus acidophilus to destroy the Si-O-Al network structure of the fly ash, thereby exposing the encapsulated germanium element.

[0031] The second stage (complexation leaching stage): The Pseudomonas suspension obtained in step S2 is inoculated into the reaction system after the leaching in the first stage. The inoculation amount is 8% of the total volume of the reaction system after the leaching in the first stage. Glucose is added at a rate of 1.0% of the mass of fly ash. The leaching reaction is carried out under the conditions of pH 4.5 (maintained by dilute sodium hydroxide solution), temperature of 32℃, and shaking speed of 150 rpm for 6 days. This stage mainly utilizes the various organic acids (such as citric acid, oxalic acid, gluconic acid, etc.) produced by Pseudomonas metabolism to form soluble complexes with the exposed germanium ions, preventing the germanium ions from redepositing or being adsorbed again, and promoting the dissolution and stabilization of germanium.

[0032] The third stage (adsorption-desorption stage): The *Saccharomyces cerevisiae* suspension obtained in step S2 was inoculated into the reaction system after the second stage leaching. The inoculation amount was 8% of the total volume of the reaction system after the second stage leaching. Sucrose was added at a rate of 0.5% of the fly ash mass. The leaching reaction was carried out under the conditions of pH 6.0 (maintained by dilute sodium hydroxide solution), temperature of 30℃, and shaking speed of 150 rpm for 3 days. In this stage, the residual germanium ions were mainly adsorbed by the functional groups such as carboxyl and hydroxyl groups on the cell wall of *Saccharomyces cerevisiae*, and their complete desorption from the solid surface was promoted through competitive adsorption.

[0033] S5: Time-gradient replenishment: In the above three stages, the corresponding functional strain bacterial solution is replenished 24 hours after the start of each stage, and the replenishment amount is 50% of the initial inoculum amount for that stage; for stages with a leaching time of 4 days or more, a second replenishment is made at 48 hours, and the replenishment amount is 30% of the initial inoculum amount for that stage; in addition, half the amount of sulfur powder is added before the end of the first stage, half the amount of glucose is added before the end of the second stage, and half the amount of sucrose is added before the end of the third stage. S6: Separation and Recovery: After the leaching reaction, the reaction mixture was centrifuged at 6000 rpm for 15 min. The supernatant was collected as the germanium-containing leachate. Germanium was separated and recovered from the germanium-containing leachate using solvent extraction. Specifically, the pH of the germanium-containing leachate was adjusted to 2.0, and an extractant (10% v / v sulfonated kerosene + 5% v / v isooctanol + 0.5 mol / L citric acid) was added. The mixture was shaken for 20 min at a volume ratio of 1:2 for the organic phase to the aqueous phase. After standing and separating the layers, the organic phase was separated. Then, a 1.0 mol / L sodium hydroxide solution was mixed with the organic phase at a volume ratio of 1:1 and shaken for 15 min. After standing and separating the layers, the aqueous phase was collected to obtain the germanium-rich solution. The germanium leaching rate was determined to be 96.5%.

[0034] Example 2 This embodiment provides a method for the microbial recovery of germanium from fly ash, specifically including the following steps: S1: Fly ash pretreatment: Grind the fly ash through a 200-mesh sieve and dry it at 60℃ to constant weight; S2: Selective culture of functional strains: Selective cultures of *Thiobacillus acidophilus*, *Pseudomonas putida*, and *Saccharomyces cerevisiae* were performed, specifically as follows: (a) Thiobacillus acidophilus was inoculated into 9K liquid medium and cultured for 48 h at pH 2.0, temperature 28℃ and shaking speed 200 rpm to obtain Thiobacillus culture medium; (b) Inoculate *Pseudomonas putida* into LB liquid medium and culture for 36 h at pH 6.5, temperature 30 °C and shaking speed 200 rpm. Collect the bacterial cells by centrifugation and resuspend them in sterile deionized water to obtain a *Pseudomonas putida* bacterial suspension. (c) Inoculate the Saccharomyces cerevisiae into YPD liquid medium and culture it for 24 h at pH 5.0, temperature 28℃ and shaking speed 180 rpm. Collect the cells by centrifugation and resuspend them in sterile deionized water to obtain a Saccharomyces cerevisiae suspension.

[0035] S3: Osmotic gradient acclimatization: The Thiobacillus culture obtained in step S2 was sequentially transferred to 9K medium with sodium chloride concentrations of 5 g / L, 10 g / L, 15 g / L and 20 g / L for osmotic gradient acclimatization. At each concentration, 10% of the culture was inoculated, and the culture was passaged three times for 48 hours each time to obtain osmotically acclimatized Thiobacillus culture. S4: Spatial stepwise inoculation and leaching: 15g of pretreated fly ash obtained in step S1 was mixed with the basic leaching medium (sterile deionized water) at a ratio of 1:10 (g / mL) to obtain a mixture, and then three stages of spatial stepwise inoculation and leaching were performed sequentially: First stage: Inoculate the mixture with the osmotically acclimated Thiobacillus culture obtained in step S3, the inoculation amount is 5% of the total volume of the mixture, and add sulfur powder, the amount of which is 0.5% of the mass of fly ash. The leaching reaction is carried out under the conditions of pH 2.5, temperature of 30℃, and shaking speed of 180 rpm for 3 days. Second stage: Inoculate the Pseudomonas suspension obtained in step S2 into the reaction system after leaching in the first stage. The inoculation amount is 5% of the total volume of the reaction system after leaching in the first stage. Add glucose, which is 0.5% of the mass of fly ash. The leaching reaction is carried out under the conditions of pH 4.0, temperature 30℃, and shaking speed 180 rpm for 5 days. The third stage: The Saccharomyces cerevisiae suspension obtained in step S2 was inoculated into the reaction system after the second stage leaching. The inoculation amount was 5% of the total volume of the reaction system after the second stage leaching. Sucrose was added at a rate of 0.3% of the fly ash mass. The leaching reaction was carried out under the conditions of pH 5.5, temperature 28℃, and shaking speed 180 rpm for 5 days. S5: Time-gradient replenishment: In the above three stages, the corresponding functional strain bacterial solution is replenished 24 hours after the start of each stage, and the replenishment amount is 50% of the initial inoculum amount for that stage; for stages with a leaching time of 4 days or more, a second replenishment is made at 48 hours, and the replenishment amount is 30% of the initial inoculum amount for that stage; in addition, half the amount of sulfur powder is added before the end of the first stage, half the amount of glucose is added before the end of the second stage, and half the amount of sucrose is added before the end of the third stage. S6: Separation and Recovery: After the leaching reaction was completed, the reaction mixture was centrifuged at 4000 rpm for 20 min, and the supernatant was collected as the germanium-containing leachate. Germanium was separated and recovered from the germanium-containing leachate using solvent extraction (same as in Example 1). The results showed that the germanium leaching rate was 92.1%.

[0036] Example 3 This embodiment provides a method for the microbial recovery of germanium from fly ash, specifically including the following steps: S1: Fly ash pretreatment: Grind the fly ash through a 400-mesh sieve and dry it at 80℃ to constant weight; S2: Selective culture of functional strains: Selective cultures of *Thiobacillus acidophilus*, *Pseudomonas putida*, and *Saccharomyces cerevisiae* were performed, specifically as follows: (a) Thiobacillus acidophilus was inoculated into 9K liquid medium and cultured for 72 h at pH 3.0, temperature 32℃ and shaking speed 150 rpm to obtain Thiobacillus culture medium; (b) Inoculate *Pseudomonas putida* into LB liquid medium and culture for 48 h at pH 7.2, temperature 35 °C and shaking speed 150 rpm. Collect the bacterial cells by centrifugation and resuspend them in sterile deionized water to obtain a *Pseudomonas putida* bacterial suspension. (c) Inoculate the Saccharomyces cerevisiae into YPD liquid medium and culture it for 36 h at pH 6.0, temperature 30℃ and shaking speed 120 rpm. Collect the cells by centrifugation and resuspend them in sterile deionized water to obtain a Saccharomyces cerevisiae suspension.

[0037] S3: Osmotic gradient acclimatization: The Thiobacillus culture obtained in step S2 was sequentially transferred to 9K medium with sodium chloride concentrations of 5 g / L, 10 g / L, 15 g / L and 20 g / L for osmotic gradient acclimatization. At each concentration, 10% inoculum was used, and the culture was passaged twice for 72 h each time to obtain osmotically acclimatized Thiobacillus culture. S4: Spatial stepwise inoculation and leaching: 5g of pretreated fly ash obtained in step S1 was mixed with the basic leaching medium (sterile deionized water) at a ratio of 1:20 (g / mL) to obtain a mixture, and then three stages of spatial stepwise inoculation and leaching were performed sequentially: First stage: Inoculate the mixture with the osmotically acclimated Thiobacillus culture obtained in step S3, the inoculation amount is 15% of the total volume of the mixture, and add sulfur powder, the amount of which is 1.5% of the mass of fly ash. The leaching reaction is carried out under the conditions of pH 3.5, temperature 35℃, and shaking speed 120 rpm for 5 days. Second stage: Inoculate the Pseudomonas suspension obtained in step S2 into the reaction system after leaching in the first stage. The inoculation amount is 10% of the total volume of the reaction system after leaching in the first stage. Add glucose at a rate of 2.0% of the fly ash mass. The leaching reaction is carried out under the conditions of pH 5.0, temperature 35℃, and shaking speed 120 rpm for 4 days. The third stage: The Saccharomyces cerevisiae suspension obtained in step S2 was inoculated into the reaction system after the second stage leaching. The inoculation amount was 10% of the total volume of the reaction system after the second stage leaching. Sucrose was added at a rate of 1.0% of the mass of fly ash. The leaching reaction was carried out under the conditions of pH 6.5, temperature 32℃, and shaking speed 120 rpm for 4 days. S5: Time-gradient replenishment: In the above three stages, the corresponding functional strain bacterial solution is replenished 24 hours after the start of each stage, and the replenishment amount is 50% of the initial inoculum amount for that stage; for stages with a leaching time of 4 days or more, a second replenishment is made at 48 hours, and the replenishment amount is 30% of the initial inoculum amount for that stage; in addition, half the amount of sulfur powder is added before the end of the first stage, half the amount of glucose is added before the end of the second stage, and half the amount of sucrose is added before the end of the third stage. S6: Separation and Recovery: After the leaching reaction, the reaction mixture was centrifuged at 8000 rpm for 10 min. The supernatant was collected as the germanium-containing leachate. Germanium was separated and recovered from the germanium-containing leachate using ion exchange. Specifically, the germanium-containing leachate was passed through an exchange column packed with a 201×7 type strong base anion exchange resin at a flow rate of 3 times the bed volume / hour. The resin was then washed with deionized water until neutral, and eluted with 0.8 mol / L sodium hydroxide solution at a flow rate of 1 times the bed volume / hour. The eluent was collected to obtain the germanium-rich solution. The determination result was: the germanium leaching rate was 93.7%.

[0038] Comparative Example 1: Blank Control The difference from Example 1 is that no bacterial strain was added; only sterile deionized water was mixed with fly ash at a solid-liquid ratio of 1:15, and the mixture was shaken for 13 days at pH 3.5, 32°C, and 150 rpm. The result was that the germanium leaching rate was 2.5%.

[0039] Comparative Example 2: Thiobacillus acidophilus alone The difference from Example 1 is that step S2 only involves culturing *Thiobacillus acidophilus*, and step S4 only involves the first stage, leaching for 13 days at pH 3.0, 32°C, and 150 rpm. Step S5 only involves adding *Thiobacillus* culture medium and sulfur powder, with other conditions the same as in Example 1. The measured result was: germanium leaching rate was 33.0%.

[0040] Comparative Example 3: Only *Pseudomonas putida* The difference from Example 1 is that step S2 only involves culturing *Pseudomonas putida*, and step S4 only involves the second stage, leaching for 13 days at pH 4.5, 32°C, and 150 rpm. Step S5 only involves adding *Pseudomonas putida* suspension and glucose, with other conditions the same as in Example 1. The measured result was: germanium leaching rate was 38.7%.

[0041] Comparative Example 4: Saccharomyces cerevisiae only The difference from Example 1 is that step S2 only involves culturing *Saccharomyces cerevisiae*, and step S4 only involves the third stage, leaching for 13 days at pH 6.0, 30°C, and 150 rpm. Step S5 only involves adding *Saccharomyces cerevisiae* suspension and sucrose, with other conditions the same as in Example 1. The measured result was: germanium leaching rate was 21.2%.

[0042] Comparative Example 5: Spatially staggered inoculation without temporal booster. The difference from Example 1 is that step S5 does not involve any supplementation (i.e., the bacterial strain is only inoculated once at the beginning of each stage, and no additional bacterial solution or carbon / energy source is added in between). Other conditions are the same as in Example 1. The germanium leaching rate was determined to be 79.3%.

[0043] Comparative Example 6: Temporal gradient supplementation without spatial stepwise inoculation. The difference from Example 1 is that spatial stepwise inoculation was not performed. Instead, the three bacterial strains were mixed at the final inoculation amount of Example 1 and added all at once. Leaching was carried out continuously for 13 days under a single pH of 4.5, with time-gradient replenishment (the mixed bacterial solution was added every 24 hours, and the replenishment amount was calculated as the sum of the replenishment amounts at each stage in Example 1). Other conditions were the same as in Example 1. The germanium leaching rate was determined to be 65.6%.

[0044] Comparative Example 7: The three-strain compound was added once without any supplementation (as a baseline). The difference from Example 1 is that Thiobacillus, Pseudomonas, and Saccharomyces cerevisiae were mixed and added all at once according to the final inoculum amount in Example 1, and leached for 13 days under a single pH 4.5 condition without any supplementation. The germanium leaching rate was determined to be 55.0%.

[0045] Comparative Example 8: Acclimation without Osmotic Gradient The difference from Example 1 is that step S3 does not involve osmotic gradient acclimation (unacclimated Thiobacillus culture medium is used directly). Everything else is the same as in Example 1. The germanium leaching rate was determined to be 81.0%.

[0046] Comparative Example 9: pH Regulation Without Stages The difference from Example 1 is that all three stages were conducted at pH 4.5 (without pH adjustment). Everything else was the same as in Example 1. The germanium leaching rate was determined to be 78.0%.

[0047] The experimental results of Examples 1-3 and Comparative Examples 1-9 were analyzed: (1) Compared with Comparative Example 1 (blank control group), the net values ​​of Examples 1-3 (i.e., germanium leaching rate of Examples 1-3 - germanium leaching rate of Comparative Example 1, which were 94.0%, 89.6%, and 91.2%, respectively) were greater than the sum of the net values ​​of Comparative Examples 1, 2, and 3 (30.5% + 36.2% + 18.7% = 85.4%). This indicates that there is a significant synergistic effect among the three strains: Thiobacillus acidophilus, Pseudomonas putida, and Saccharomyces cerevisiae. The three strains are not simply superimposed, but rather produce an effect of 1+1+1>3 through functional complementarity (structural destruction - complexation dissolution - adsorption and desorption).

[0048] (2) Based on Comparative Example 7 (three bacteria added at once, without supplementation), the contribution of spatial stepwise inoculation (net value of Comparative Example 5 - net value of Comparative Example 7) was 24.3%, and the contribution of time-gradient supplementation (net value of Comparative Example 6 - net value of Comparative Example 7) was 10.6%, with the sum of the two contributions being 34.9%. In contrast, the contribution of Example 1 (net value of Example 1 - net value of Comparative Example 7) was 41.5%, and the contributions of Examples 2 and 3 were 37.1% and 38.7% respectively, all greater than 34.9%, demonstrating that the combined use of spatial stepwise inoculation and time-gradient supplementation produced a synergistic effect. In addition, the leaching rate of Example 1 (96.5%) was significantly higher than that of Comparative Example 5 (79.3%) and Comparative Example 6 (65.6%), which also intuitively illustrates the superiority of the synergistic effect of the two strategies.

[0049] (3) The germanium leaching rates of Examples 1-3 (92.1%-96.5%) were higher than those of Comparative Example 8 (81.0%), indicating that the osmotic pressure gradient acclimatization effectively improved the adaptability and metabolic activity of Thiobacillus in the high-salt environment of fly ash, which played an important role in improving the germanium leaching rate.

[0050] (4) The germanium leaching rate of Examples 1-3 (92.1%-96.5%) was higher than that of Comparative Example 9 (78.0%), indicating that adjusting the pH according to the functional requirements of microorganisms at different stages, so that each strain can play its role under the optimal conditions, is an indispensable key link in the technical solution of this invention.

[0051] In summary, this invention achieves highly efficient microbial recovery of germanium from fly ash by constructing a three-strain complex system composed of *Thiobacillus acidophilus*, *Pseudomonas putida*, and *Saccharomyces cerevisiae*, and employing a spatial stepwise inoculation strategy and a temporal gradient supplementation strategy, combined with osmotic pressure gradient acclimation and staged pH control. Experimental results show that the three-strain complex produces a significant synergistic effect, and the combined use of spatial stepwise inoculation and temporal gradient supplementation also produces a synergistic effect. Under optimal conditions, the germanium leaching rate of this invention can reach 96.5%, which is significantly better than the existing technology. Furthermore, the process is mild, simple to operate, and environmentally friendly, providing a novel technical approach for the efficient and green recovery of germanium resources from fly ash, and has broad prospects for industrial application.

[0052] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for microbial recovery of germanium from fly ash, characterized in that: Specifically, it includes the following steps: S1: Fly ash pretreatment: Grind the fly ash, sieve it, and dry it to constant weight; S2: Selective culture of functional strains: culture of *Thiobacillus acidophilus* (… Acidithiobacillus thiooxidans ), Pseudomonas putida ( Pseudomonas putida ) and brewer's yeast ( Saccharomyces cerevisiae Selective culture was performed to obtain Thiobacillus culture medium, Pseudomonas suspension and Saccharomyces cerevisiae suspension, respectively; S3: Osmotic gradient acclimatization: The Thiobacillus culture medium obtained in step S2 is subjected to osmotic gradient acclimatization in a culture medium containing a sodium chloride gradient to obtain an osmotically acclimatized Thiobacillus culture medium. S4: Spatial stepwise inoculation and leaching: The pretreated fly ash obtained in step S1 is mixed with the basic leaching medium to obtain a mixture, and then three stages of spatial stepwise inoculation and leaching are performed sequentially: First stage: Inoculate the mixture with the osmotically acclimatized Thiobacillus culture obtained in step S3, add sulfur powder, and carry out the leaching reaction under pH 2.5~3.5 conditions; Second stage: Inoculate the Pseudomonas suspension obtained in step S2 into the reaction system after leaching in the first stage, add glucose, and carry out the leaching reaction under pH 4.0~5.0 conditions; Third stage: Inoculate the Saccharomyces cerevisiae suspension obtained in step S2 into the reaction system after leaching in the second stage, add sucrose, and carry out the leaching reaction under pH 5.5~6.5 conditions; S5: Time-gradient replenishment: In the above three stages, after the start of each stage, the corresponding functional strain of bacterial solution is replenished at regular intervals, and half of the corresponding carbon source or energy substance is replenished before the end of each stage. S6: Separation and Recovery: After the leaching reaction is completed, the reaction mixture is centrifuged and the supernatant is collected as germanium-containing leachate. Germanium element is separated and recovered from germanium-containing leachate.

2. The method according to claim 1, characterized in that: In step S1, the sieve size is 200-400 mesh, and the drying temperature is 60-80℃.

3. The method according to claim 1, characterized in that: In step S2, the selective culture specifically involves: (a) Inoculate Thiobacillus acidophilus into 9K liquid medium and culture for 48-72 h at pH 2.0-3.0, temperature 28-32℃ and shaking speed 150-200 rpm to obtain Thiobacillus culture medium; (b) Inoculate *Pseudomonas putida* into LB liquid medium and culture for 36-48 h at pH 6.5-7.2, temperature 30-35 °C and shaking speed 150-200 rpm. Collect the bacterial cells by centrifugation and resuspend them in sterile deionized water to obtain a *Pseudomonas putida* bacterial suspension. (c) Inoculate the Saccharomyces cerevisiae into YPD liquid medium and culture it for 24-36 h at pH 5.0-6.0, temperature 28-30℃ and shaking speed 120-180 rpm. Collect the cells by centrifugation and resuspend them in sterile deionized water to obtain a Saccharomyces cerevisiae suspension.

4. The method according to claim 3, characterized in that: The 9K liquid culture medium consists of: (NH4)2SO4 3.0 g / L, KCl 0.1 g / L, K2HPO4 0.5 g / L, MgSO4·7H2O 0.5 g / L, Ca(NO3)2 0.01 g / L, FeSO4·7H2O 4.2 g / L, with the pH adjusted to 2.0-3.0 using dilute sulfuric acid; the LB liquid culture medium consists of: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, with a pH of 7.0-7.2; the YPD liquid culture medium consists of: yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L, with a pH of 5.0-6.

0.

5. The method according to claim 1, characterized in that: In step S3, the osmotic gradient acclimatization specifically involves: transferring the Thiobacillus culture medium sequentially to 9K medium with sodium chloride concentrations of 5 g / L, 10 g / L, 15 g / L, and 20 g / L, respectively, inoculating at a 10% inoculum at each concentration, and subculturing 2-3 times consecutively, with each culture lasting 48-72 hours.

6. The method according to claim 1, characterized in that: In step S4, the basic leaching medium is sterilized deionized water, and the solid-liquid ratio of the pretreated fly ash to the basic leaching medium is 1:10~20 (g / mL). The leaching times for the first, second, and third stages are 3~5 days, 4~6 days, and 3~5 days, respectively. In the first stage, the inoculation amount of Thiobacillus culture medium is 5%~15% of the total volume of the mixture, and the amount of sulfur powder added is 0.5%~1.5% of the fly ash mass. In the second stage, the inoculation amount of Pseudomonas suspension is 5%~10% of the total volume of the reaction system after leaching in the first stage, and the amount of glucose added is 0.5%~2.0% of the fly ash mass. In the third stage, the inoculation amount of Saccharomyces cerevisiae suspension is 5%~10% of the total volume of the reaction system after leaching in the second stage, and the amount of sucrose added is 0.3%~1.0% of the fly ash mass. The reaction temperatures for the first, second, and third stages are 30~35℃, 30~35℃, and 28~32℃, respectively.

7. The method according to claim 1, characterized in that: In step S5, the specific operation of the timed replenishment is as follows: 24 hours after the start of each stage, the corresponding functional strain bacterial solution is replenished, and the replenishment amount is 50% of the initial inoculation amount for that stage; for stages with a leaching time of 4 days or more, it is replenished again at 48 hours, and the replenishment amount is 30% of the initial inoculation amount for that stage; the replenishment of half the amount of carbon source or energy substance corresponding to that stage before the end of each stage refers to: half the amount of sulfur powder added before the end of the first stage, half the amount of glucose added before the end of the second stage, and half the amount of sucrose added before the end of the third stage.

8. The method according to claim 1, characterized in that: In step S5, the centrifugation speed is 4000~8000 rpm and the centrifugation time is 10~20 min; the method for separating and recovering germanium from germanium-containing leachate is solvent extraction or ion exchange.