Sulfate reducing bacteria nutritional agent as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of acid wastewater treatment, and particularly relates to a sulfate reducing bacteria nutritional agent as well as a preparation method and application thereof. According to the invention, fermentation liquor of non-edible plants which is rich in amino acids (greater than or equal to 20 g / L) and has soluble chemical oxygen demand greater than or equal to 2200 mg / L is used as a basic carrier, exogenous nutrient substances and electron mediators are compounded, and the fermentation liquor and the nutrient substances of the non-edible plants provide efficient electron donors and nutritional ingredients for sulfate reducing bacteria; the electron mediator improves the electron transfer efficiency of the SRB in an anoxic environment. According to the method, the acidic mine wastewater and the sulfate reducing bacteria are subjected to adaptive culture, so that the SRB adapts to the acidic and high-sulfate environment of AMD, the metabolic efficiency of the sulfate reducing bacteria can be remarkably improved through the synergistic effect of the two modes, the growth rate of the sulfate reducing bacteria and the sulfate reduction rate are increased, and the method has the advantages of low cost, high recycling degree, easiness in large-scale production and the like.

Description

Technical Field

[0001] This invention belongs to the field of acidic wastewater treatment technology, specifically relating to a sulfate-reducing bacteria nutrient agent, its preparation method, and its application. Background Technology

[0002] Acidic leaching water from abandoned mines (also known as "acid mine wastewater," AMD) is widely considered a major source of heavy metal pollution from the mining industry. Acidic mine wastewater also leads to surface water acidification, groundwater acidification, soil acidification, and damage to aquatic ecosystems. Current methods for treating acidic mine wastewater primarily involve adding alkaline neutralizing agents such as lime and sodium hydroxide to adjust the pH value, followed by the addition of flocculants to rapidly precipitate metal ions. The supernatant is then discharged after meeting standards, and the sludge is treated by filtration and landfill. Because the generation of mine wastewater is a continuous process, it requires long-term, ongoing treatment, resulting in high costs. Low-cost, nature-based microbial treatment technologies have become a research hotspot.

[0003] Sulfate-reducing bacteria (SRB) are a class of anaerobic microorganisms that use sulfate as an electron acceptor and organic matter as an electron donor. They have significant application value in areas such as sulfate-containing wastewater treatment, soil heavy metal remediation, and oil and gas pipeline corrosion control. However, traditional SRB nutrient solutions mainly rely on organic carbon sources (such as low-molecular-weight organic acids, alcohols, and sugars), with inorganic carbon sources as a secondary source. However, existing carbon sources all have significant drawbacks: chemically synthesized organic carbon sources (such as glucose and sodium acetate) rely on sophisticated industrial production methods, resulting in high costs, and some compounds (such as artificially synthesized organic acids) are prone to residues, causing secondary pollution and failing to meet green environmental protection requirements; fermented organic carbon sources using corn, wheat, soybeans, straw, and forage grown in fertile fields as raw materials pose a problem of competition with humans and livestock for food. Traditional SRB nutrient solutions, relying on chemically synthesized carbon sources or fertile crops, suffer from problems such as competing with food crops for land, high costs, and the potential for secondary pollution. Therefore, developing a low-cost, resource-efficient, and highly effective SRB nutrient solution has become an urgent need for industry development. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a sulfate-reducing bacteria nutrient agent, its preparation method and application, to treat acidic mine wastewater at low cost and improve the sulfate reduction rate.

[0005] This invention provides a method for preparing a sulfate-reducing bacteria nutrient solution, comprising the following steps: Fermentation broth from non-edible plants, nutrients, and electronic mediators are mixed to obtain a fermentation broth-based nutrient solution. The fermentation liquid-based nutrient solution and acidic mine wastewater were mixed to obtain a fermentation substrate, which was then inoculated with sulfate-reducing bacteria and cultured to obtain a sulfate-reducing bacteria nutrient solution. The fermentation broth of the non-edible plants has an amino acid content ≥20 g / L and a dissolved chemical oxygen demand ≥2200 mg / L; The nutrients include nitrogen, phosphorus, and trace elements; the trace elements include iron, magnesium, and zinc. The electronic mediator includes one or more of phenazine, phenazine-1-carboxylic acid, 2-hydroxyphenazine, sodium anthraquinone-2-sulfonate, neutral red, vitamin B2, and vitamin B2 derivatives; The adaptive culture was conducted at a pH of 4.0–5.0, with a dissolved oxygen content of <0.5 mg / L, a temperature of 25–35°C, and a time of 48–96 h.

[0006] Preferably, the fermentation broth-based nutrient solution comprises 0.2-0.3 g / L nitrogen, 0.1-0.15 g / L phosphorus, 0.01-0.02 g / L iron, 0.05-0.07 mg / L magnesium, and 0.04-0.05 mg / L zinc; the mass of nitrogen, phosphorus, iron, magnesium, and zinc is calculated in terms of the mass of urea, potassium dihydrogen phosphate, ferrous sulfate, magnesium sulfate, and zinc sulfate, respectively. The concentration of electron mediators in the fermentation liquid-based nutrient is 0.05~0.06 mg / L.

[0007] Preferably, the volume ratio of the fermentation liquid-based nutrient to the acidic mine wastewater is 8-8.5:1; and the inoculation amount of the sulfate-reducing bacteria is 10-15% of the mass of the fermentation substrate.

[0008] Preferably, the method for preparing the fermentation broth from the non-edible plant includes the following steps: Non-edible plant powder is mixed with a medium and soaked to obtain fermentation raw material; the medium includes effluent from the primary sedimentation tank. Lactobacillus and cellulase are inoculated into the fermentation raw materials to carry out the first fermentation and obtain the first fermentation product. Bacillus subtilis and yeast are inoculated into the first fermentation product to carry out a second fermentation, thereby obtaining a second fermentation product; during the second fermentation process, materials are added; the materials include starch and fish meal. The second fermentation product is subjected to solid-liquid separation, and the collected liquid is sterilized to obtain the fermentation broth of the non-edible plant.

[0009] Preferably, the particle size of the non-edible plant powder is ≤1 mm; the mass-to-volume ratio of the non-edible plant powder to the medium is 1 g: 5 mL; the chemical oxygen demand of the effluent from the primary sedimentation tank is 300~500 mg / L, and the concentration of suspended solids is <10 mg / L. The non-edible plants include chicory and / or leafy grasses; The soaking temperature is 20~30℃, and the soaking time is 12~36 h.

[0010] Preferably, the first fermentation temperature is 35-42℃, the pH is 5.0-6.0, the dissolved oxygen content is ≤0.5 mg / L, and the time is 24-48 h; the inoculum size of the lactobacillus is 10. 8 ~10 9 CFU / mL, wherein the inoculum amount of cellulase is ≥5000 U / g; The second fermentation temperature is 30-35℃, pH is 6.0-7.0, and dissolved oxygen content is ≤0.5 mg / L; the inoculum size of Bacillus subtilis is 10... 5 ~10 6 CFU / mL, the inoculum size of the yeast was 10. 5 ~10 6 CFU / mL.

[0011] Preferably, the mass ratio of starch to fishmeal in the material is 0.5~2:1, and the total amount of the material added is 5~10% of the mass of the plant raw material; The feeding is carried out twice during the second fermentation, at 24-48 h and 48-72 h, with each feeding amount being 50% of the total material.

[0012] This invention provides a sulfate-reducing bacteria nutrient agent prepared by the method described in the above technical solution.

[0013] This invention provides the application of the sulfate-reducing bacteria nutrient agent described in the above technical solution for treating acidic mine wastewater.

[0014] This invention provides a method for treating acidic mine wastewater, comprising the following steps: The acidic mine wastewater to be treated is mixed with the sulfate-reducing bacteria nutrient solution described in the above technical solution, and sulfate-reducing bacteria are inoculated and anaerobic fermentation is carried out at 32°C.

[0015] Beneficial effects: This invention utilizes the fermentation broth of non-edible plants rich in amino acids (≥20 g / L) and with a dissolved chemical oxygen demand (COD) ≥2200 mg / L. Exogenous nutrients (urea, potassium dihydrogen phosphate, and trace elements, including ferrous sulfate, magnesium sulfate, and zinc sulfate) and electron mediators are added. The fermentation broth and nutrients from the non-edible plants provide efficient electron donors and nutrients for sulfate-reducing bacteria (SRBs), while the electron mediators enhance electron transfer efficiency in anaerobic environments. This invention also involves adaptively culturing acidic mine wastewater and sulfate-reducing bacteria to simulate the chemical niche of the treatment site. This effectively improves the resistance of SRBs to acidic, high-sulfate environments associated with adverse mineral discharge (AMD), while simultaneously increasing the subsequent growth rate and sulfate reduction efficiency of SRBs. This achieves in-situ integration of the treatment agent and nutrients, eliminating the application delay period and offering advantages such as low cost, high resource utilization, and ease of scalability.

[0016] Furthermore, the non-edible plants selected in this invention, such as chicory and / or leafy grasses, are sourced from contaminated soil in mining areas, eliminating the need for arable land and offering low prices. Rural domestic sewage is used as a dilution medium, and a "enzymatic hydrolysis-microbial co-fermentation" process is employed to directionally convert macromolecular organic matter into small-molecule nutrients (such as amino acids and small-molecule organic acids) for the adaptive cultivation of SRB, further reducing the treatment cost of AMD. This invention promotes lactic acid fermentation (an anaerobic process) throughout the entire fermentation process by maintaining low dissolved oxygen levels (≤0.5 mg / L). In the first fermentation stage, this promotes lactic acid fermentation, shifting Lactobacillus from aerobic respiration to more efficient lactic acid fermentation (anaerobic metabolism). In the second fermentation stage, limited oxygen is utilized to activate the metabolic potential of aerobic and facultative anaerobic microorganisms. Under these microaerobic conditions, the growth of Bacillus subtilis is restricted, but the secretory activity of its proteolytic enzymes and amylases is significantly enhanced. This successfully guides the metabolic flow of Bacillus subtilis, focusing it on hydrolysis rather than its own growth, thereby co-producing with yeast to achieve deep degradation and nutrient conversion of the substrate. Detailed Implementation

[0017] This invention provides a method for preparing a sulfate-reducing bacteria nutrient solution, comprising the following steps: Fermentation broth from non-edible plants, nutrients, and electronic mediators are mixed to obtain a fermentation broth-based nutrient solution. The fermentation liquid-based nutrient solution and acidic mine wastewater were mixed to obtain a fermentation substrate, which was then inoculated with sulfate-reducing bacteria and cultured to obtain a sulfate-reducing bacteria nutrient solution. The fermentation broth of the non-edible plants has an amino acid content ≥20 g / L and a dissolved chemical oxygen demand ≥2200 mg / L; The nutrients include nitrogen, phosphorus, and trace elements; the trace elements include iron, magnesium, and zinc. The adaptation culture was conducted at a pH of 4.0–5.0, with a dissolved oxygen content of <0.5 mg / L, a temperature of 30°C, and a duration of 72 h.

[0018] This invention mixes fermentation broth from non-edible plants, nutrients, and electronic mediators to obtain a fermentation broth-based nutrient solution.

[0019] In one embodiment, the arginine content in the fermentation broth of the non-edible plants of the present invention is 30-50% by mass; in another embodiment, the arginine content in the fermentation broth of the non-edible plants of the present invention is 40% by mass. In one embodiment, the dissolved chemical oxygen demand (COD) of the fermentation broth of the non-edible plants of the present invention is 3000-4000 mg / L.

[0020] In one embodiment, the nutrients described in this invention include urea, potassium dihydrogen phosphate, and trace elements, wherein the trace elements include ferrous sulfate, magnesium sulfate, and zinc sulfate.

[0021] In one embodiment, the fermentation broth-based nutrient solution of the present invention comprises 0.2-0.3 g / L nitrogen, 0.1-0.15 g / L phosphorus, 0.01-0.02 g / L iron, 0.05-0.07 mg / L magnesium, and 0.04-0.05 mg / L zinc; the mass of nitrogen, phosphorus, iron, magnesium, and zinc is calculated in terms of the mass of urea, potassium dihydrogen phosphate, ferrous sulfate, magnesium sulfate, and zinc sulfate, respectively. In another embodiment, the fermentation broth-based nutrient solution of the present invention comprises 0.2 g / L nitrogen, 0.12 g / L phosphorus, 0.01 g / L iron, 0.05 mg / L magnesium, and 0.04 mg / L zinc. The present invention limits the concentrations of nitrogen, phosphorus, and trace elements to meet the requirement of a C:N:P ratio of 100:5:1 during SRB growth. The urea described in this invention serves as a nitrogen source to promote cell synthesis; the potassium dihydrogen phosphate serves as a phosphorus source to participate in energy metabolism; the ferrous sulfate serves as a coenzyme for the enzyme system, magnesium sulfate maintains osmotic pressure, and zinc sulfate promotes enzyme activity and supplements the metal ions necessary for the SRB enzyme system.

[0022] In one embodiment, the concentration of the electron mediator in the fermentation broth-based nutrient solution of the present invention is 0.05~0.06 mg / L. In another embodiment, the electron mediator of the present invention includes one or more of phenazine, phenazine-1-carboxylic acid, 2-hydroxyphenazine, sodium anthraquinone-2-sulfonate, neutral red, vitamin B2, and vitamin B2 derivatives. In the present invention, the electron mediator solves the problem of low electron transfer efficiency of SRB under hypoxic conditions, significantly improving its growth rate and metabolic activity.

[0023] As one embodiment, the method for preparing the fermentation broth of non-edible plants according to the present invention includes the following steps: Non-edible plant powder is mixed with a medium and soaked to obtain fermentation raw material; the medium includes effluent from the primary sedimentation tank. Lactobacillus and cellulase are inoculated into the fermentation raw materials to carry out the first fermentation and obtain the first fermentation product. Bacillus subtilis and yeast are inoculated into the first fermentation product to carry out a second fermentation, thereby obtaining a second fermentation product; during the second fermentation process, materials are added; the materials include starch and fish meal. The second fermentation product is subjected to solid-liquid separation, and the collected liquid is sterilized to obtain the fermentation broth of the non-edible plant.

[0024] In one embodiment, the present invention mixes non-edible plant powder with a medium, soaks the mixture, and obtains fermentation raw material. In one embodiment, the particle size of the non-edible plant powder is ≤1 mm; in another embodiment, the particle size is 0.8 mm. This limitation on the particle size of the non-edible plant powder increases the specific surface area by 3-5 times, promoting enzymatic hydrolysis. In one embodiment, the mass-to-volume ratio of the non-edible plant powder to the medium is 0.5-1.5 g: 4-6 mL; in another embodiment, the mass-to-volume ratio is 1 g: 5 mL. In one embodiment, the non-edible plant includes chicory and / or leafy greens. In one embodiment, the mass ratio of chicory to leafy greens is 0.5-1.5:1; in another embodiment, the mass ratio is 1:1. This invention uses non-edible plants as raw materials, specifically non-edible plants that can be safely utilized from polluted soil around mining areas. These plants are characterized by high yield, high nutritional value (rich in cellulose, protein, etc.), and renewability. They solve the problems of traditional SRB nutrients relying on fossil raw materials or expensive organic substrates, as well as the problem of utilizing polluted soil. They do not require the use of arable land, are inexpensive, and realize the treatment and resource utilization of polluted soil.

[0025] In one embodiment, the effluent from the primary sedimentation tank of this invention is the effluent from a primary sedimentation tank for rural domestic sewage. In one embodiment, the chemical oxygen demand (COD) of the effluent from the primary sedimentation tank of this invention is 300-500 mg / L; in another embodiment, the COD is 350-450 mg / L; in yet another embodiment, the COD is 300 mg / L. In one embodiment, the suspended solids (SS) concentration of the effluent from the primary sedimentation tank of this invention is <10 mg / L; in yet another embodiment, the SS concentration is 8 mg / L. In one embodiment, the soaking temperature is 20-30℃; in yet another embodiment, the soaking temperature is 25℃. In one embodiment, the soaking time is 12-36 h; in yet another embodiment, the soaking time is 24 h. This invention utilizes the effluent from the primary sedimentation tank for soaking, enabling non-edible plant materials to absorb water and swell, with a cellulose structure destruction rate ≥60%. Furthermore, by using rural domestic sewage (effluent from the primary sedimentation tank) instead of clean water, there is no need for processes such as boiling or rinsing. This not only reduces water consumption but also enables the resource utilization of organic matter (such as small amounts of carbohydrates and nitrogen) in the sewage. This reduces preparation costs while achieving the synergistic treatment of domestic sewage.

[0026] In one embodiment, after obtaining the fermentation raw material, the present invention inoculates lactobacillus and cellulase into the fermentation raw material to carry out a first fermentation and obtain a first fermentation product.

[0027] In one embodiment, the temperature of the first fermentation in this invention is 35~42℃; in another embodiment, the temperature of the first fermentation in this invention is 37℃. In one embodiment, the pH of the first fermentation in this invention is 5.0~6.0; in another embodiment, the pH of the first fermentation in this invention is 5.5. In one embodiment, the dissolved oxygen content of the first fermentation in this invention is ≤0.5 mg / L. In one embodiment, the fermentation time of the first fermentation in this invention is 24~48 h; in another embodiment, the fermentation time of the first fermentation in this invention is 36 h. In one embodiment, the inoculum size of the lactobacillus in this invention is 10... 8 ~10 9 CFU / mL; as another embodiment, the inoculum quantity of Lactobacillus described in this invention is 10. 8CFU / mL. As one embodiment, the inoculum amount of cellulase described in this invention is ≥5000 U / g. This invention utilizes lactobacillus and cellulase for the first fermentation. The cellulase decomposes cellulose and hemicellulose into fermentable sugars, while the lactobacillus converts the sugars into small molecules such as lactic acid and amino acids. Simultaneously, it lowers the pH to inhibit contaminating bacteria (contaminating bacteria survival rate ≤10%).

[0028] In one embodiment, after obtaining the first fermentation product, the present invention inoculates Bacillus subtilis and yeast into the first fermentation product to carry out a second fermentation, thereby obtaining a fermentation broth of non-edible plants. In one embodiment, the temperature of the second fermentation is 30-35°C; in another embodiment, the temperature of the second fermentation is 32°C. In one embodiment, the pH of the second fermentation is 6.0-7.0; in another embodiment, the pH of the second fermentation is 6.5. In one embodiment, the dissolved oxygen content of the second fermentation is ≤0.5 mg / L. In one embodiment, the inoculum size of Bacillus subtilis is 10... 5 ~10 6 CFU / mL. As one embodiment, the inoculum size of the yeast described in this invention is 10... 5 ~10 6 CFU / mL. This invention utilizes Bacillus subtilis and yeast for secondary fermentation. Bacillus subtilis systematically degrades residual and additional proteins (fish meal) and starches in the primary fermentation products through enzymatic hydrolysis, converting them into easily absorbed small-molecule peptides, amino acids, and organic acids, providing SRB with more direct and diversified nitrogen and carbon sources. Yeast utilizes various fermentable sugars and small-molecule organic acids to produce volatile metabolites such as ethanol and esters through its own fermentation metabolic pathway, optimizing the carbon source structure and maintaining the anaerobic environment, creating more suitable conditions for SRB.

[0029] In one embodiment, materials are added during the second fermentation process of the present invention; the materials include starch and fish meal. In one embodiment, the mass ratio of starch to fish meal in the materials is (0.5~2):1; in another embodiment, the mass ratio of starch to fish meal is 1:1. In one embodiment, the total amount of materials added is 5~10% of the mass of the plant raw material; in another embodiment, the total amount of materials added is 8% of the mass of the plant raw material. In one embodiment, the feeding is carried out twice during the second fermentation, at 24~48 h and 48~72 h, with each feeding amount being 50% of the total materials; in another embodiment, the feeding is carried out twice during the second fermentation, at 36 h and 60 h, with each feeding amount being 50% of the total materials. This invention uses batch feeding to avoid excessively high substrate concentrations at once.

[0030] In one embodiment, after obtaining the second fermentation product, the present invention performs solid-liquid separation on the second fermentation product, collects the liquid and sterilizes it to obtain the fermentation broth of the non-edible plant.

[0031] In one embodiment, the solid-liquid separation method of the present invention uses double-layer gauze or a 100-mesh sieve to remove undecomposed plant residues (residue rate ≤5%). In another embodiment, the sterilization method of the present invention includes sequential centrifugation and ultraviolet sterilization. Centrifugation removes minute suspended matter, reducing turbidity to ≤10 NTU, while ultraviolet sterilization thoroughly removes contaminants (such as aerobic bacteria and molds), achieving a contaminant removal rate ≥99.9%. The fermentation broth of non-edible plants obtained by the present invention contains ≥20 g / L of amino acids (≥40% arginine) and is rich in lactic acid, vitamins, and trace elements.

[0032] After obtaining the fermentation liquid-based nutrient, the present invention mixes the fermentation liquid-based nutrient with acidic mine wastewater to obtain a fermentation substrate, inoculates sulfate-reducing bacteria, and performs adaptive culture to obtain a sulfate-reducing bacteria nutrient.

[0033] As one embodiment, the acidic mine wastewater described in this invention has a pH of 2.0~6.0 and an SO4 content of [missing information]. 2- The concentration is 500~15000 mg / L; as another embodiment, the acidic mine wastewater of the present invention has a pH of 3.5 and an SO42- content of 0.5%. 2- The concentration is 3200 mg / L. As one embodiment, the volume ratio of the fermentation broth-based nutrient to the acidic mine wastewater is 8-8.5:1. As one embodiment, the inoculum amount of sulfate-reducing bacteria is 10-15% of the fermentation substrate mass. As one embodiment, the bacterial concentration of sulfate-reducing bacteria is ≥10.7 CFU / mL. In one embodiment, the pH of the adaptive culture in this invention is 4.5. The dissolved oxygen content of the adaptive culture in this invention is <0.5 mg / L. The temperature of the adaptive culture in this invention is 25~35℃; in one embodiment, the temperature of the adaptive culture in this invention is 30℃. The culture time of the adaptive culture in this invention is 48~96 h; in one embodiment, the culture time of the adaptive culture in this invention is 72 h. This invention directionally adapts the fermentation broth-based nutrient to SRB, simulating the chemical niche of the treatment site, which can effectively improve the stress resistance of SRB to the acidic and high sulfate environment of AMD, while improving the subsequent growth rate and sulfate reduction efficiency of SRB, realizing the in-situ integration of the treatment agent and the nutrient, and eliminating the application delay period.

[0034] This invention provides a sulfate-reducing bacteria nutrient agent prepared by the method described in the above technical solution. This invention utilizes a first-stage enzymatic hydrolysis ("Lactobacillus + cellulase") to produce acid, followed by a second-stage enhanced degradation process ("Bacillus subtilis + yeast"), combined with batch feeding to avoid substrate inhibition, thereby improving the degradation efficiency of macromolecular organic matter (such as cellulose) and increasing the content of small molecule nutrients (such as amino acids); amino acids ≥20 g / L (arginine ≥40%), SCOD ≥2200 mg / L (small molecule carbon source ≥80%). After obtaining a fermentation broth from non-plant raw materials, nutrients and electron mediators are compounded, resulting in a fermentation broth-based nutrient agent that fully meets the electron donor requirements of SRB. Furthermore, this invention directionally adapts the fermentation broth-based nutrient agent to SRB, simulating the chemical niche of the treatment site, effectively enhancing the SRB's resistance to the acidic, high-sulfate environment of AMD. The resulting sulfate-reducing bacteria nutrient agent can improve the subsequent growth rate and sulfate reduction efficiency of SRB, achieving in-situ integration of the treatment agent and nutrient agent, eliminating the application delay period.

[0035] Given the effectiveness of the sulfate-reducing bacteria nutrient agent provided by this invention, its application in treating acidic mine wastewater also falls within the scope of protection of this invention. As one embodiment, the acidic mine wastewater described in this invention has a pH of 2.0~6.0 and contains SO42-22-32-4 ... 2- The concentration is 500~15000 mg / L; as another embodiment, the acidic mine wastewater of the present invention has a pH of 3.5 and an SO42- content of 0.5%. 2- The concentration is 3200 mg / L.

[0036] This invention provides a method for treating acidic mine wastewater, comprising the following steps: The acidic mine wastewater to be treated is mixed with the sulfate-reducing bacteria nutrient solution described in the above technical solution, and sulfate-reducing bacteria are inoculated and anaerobic fermentation is carried out at 32°C.

[0037] In one embodiment, the volume ratio of the acidic mine wastewater to be treated to the sulfate-reducing bacteria nutrient agent is (5~15):1; in another embodiment, the volume ratio is 10:1. In one embodiment, the sulfate-reducing bacteria concentration is ≥10. 7 CFU / mL, the inoculum amount is 5% of the total volume of sulfate-reducing bacteria nutrient solution and AMD.

[0038] In one embodiment, the anaerobic fermentation time of the present invention is 72-120 h; in another embodiment, the anaerobic fermentation time of the present invention is 72 h. In one embodiment, the dissolved oxygen content of the anaerobic fermentation of the present invention is <0.5 mg / L. Using the method of the present invention to treat acidic mine wastewater, the sulfate reduction rate reaches 82.06%-92.00%, and the treatment cost is as low as 650-720 yuan / ton. For a 10,000-ton-scale AMD treatment project, the annual carbon source cost can be reduced from 1.8-1.95 million yuan to 650,000-720,000 yuan, saving 1.15-1.3 million yuan.

[0039] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of a sulfate-reducing bacteria nutrient agent, its preparation method, and its application, is provided by the present invention, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0040] Example 1 A method for preparing a sulfate-reducing bacteria nutrient solution comprises the following steps: 1. Raw material pretreatment Fresh chicory, grown in non-edible soil in the vicinity of a mining area in Shaoguan, was safely utilized. The chicory was crushed to 0.8 mm. The effluent from the primary sedimentation tank of a nearby rural centralized domestic sewage treatment plant (COD 400 mg / L, SS 8 mg / L) was used as the soaking medium. The mixture was prepared at a material-to-liquid ratio of 1 g: 5 mL and soaked at 25 °C for 24 h to obtain the fermentation raw material.

[0041] 2. Staged fermentation (1) First stage fermentation: Lactobacillus and cellulase were inoculated into the raw materials soaked in step 1, and fermentation was carried out for 48 h at 37℃, pH 5.5, and dissolved oxygen (DO) ≤ 0.5 mg / L; wherein, the inoculation amount of Lactobacillus was 10 8 CFU / mL, cellulase inoculum ≥5000 U / g (based on the mass of non-edible plant dry powder).

[0042] (2) Second stage fermentation: Bacillus subtilis and yeast were inoculated into the fermentation product of step (1), stirred evenly, and fermented at 32℃ and pH 6.5. Feeding was carried out once after 36 h and 60 h of fermentation, respectively. The total amount of feeding was 8% of the weight of fresh chicory, and the amount of feeding each time was 50% of the total amount of feeding. Stirring was carried out for 3 h after each feeding. The inoculation amount of Bacillus subtilis and yeast was 10. 6 CFU / mL, with starch and fish meal added each time, at a mass ratio of 1:1.

[0043] 3. Post-treatment of fermentation broth (1) Filtration: Filter the fermentation liquid from step 2 with double-layer gauze (or 100-mesh sieve) to remove undecomposed plant residue (residue rate ≤5%). (2) Sterilization: After filtration, the fermentation broth is centrifuged at 3000 rpm for 10 min to remove small suspended matter. The turbidity is ≤10 NTU. Then, it is sterilized with ultraviolet light to remove ≥99.9% of miscellaneous bacteria (such as aerobic bacteria and mold). If the volume of the fermentation broth is too large, it is concentrated to 1 / 2 to 1 / 3 of the original volume by vacuum concentration (50~60℃, vacuum degree 0.08 MPa) (SCOD is increased to 3000~4000 mg / L to reduce the amount of subsequent compounding).

[0044] 4. Exogenous nutritional regulation After sterilization in step 3, add 0.20 g / L urea, 0.12 g / L potassium dihydrogen phosphate, 0.01 g / L ferrous sulfate, 0.05 mg / L magnesium sulfate, 0.04 mg / L zinc sulfate, and 0.05 mg / L phenazine to the fermentation broth in sequence. Stir with an electric stirrer at 300 rpm for 30 min to achieve a homogeneity of ≥95% to obtain the fermentation broth-based nutrient solution.

[0045] 5. Adaptation culture The fermentation broth-based nutrient solution obtained in step 4 was mixed with acidic mine wastewater (AMD) at a volume ratio of 8:1. Sulfate-reducing bacteria (SRB) were inoculated, the pH was adjusted to approximately 5.0, and the dissolved oxygen (DO) was controlled to be <0.5 mg / L. Adaptation culture (anaerobic culture) was then carried out at 30℃. OD was monitored during the culture process. 600 When OD 600 Stop heating when the temperature reaches ≥1.5℃ to obtain sulfate-reducing bacteria nutrient solution. Store in a sealed container at room temperature in a well-ventilated and dry place for one month. The bacterial concentration of sulfate-reducing bacteria should be ≥10. 7 The inoculum concentration was CFU / mL, and the inoculum size was 5% of the total volume of the fermentation broth-based nutrient solution and AMD. The AMD was obtained from acidic wastewater leached from a waste rock dump in a mining area in Shaoguan, with a pH of 3.5 and SO42-. 2- The concentration was 3600 mg / L. In this example, a sulfate-reducing bacterial nutrient solution was obtained after 72 h of adaptive culture.

[0046] Example 2 A sulfate-reducing bacteria nutrient solution similar to that of Example 1 was developed, the only difference being the raw materials used. In Example 1, fresh chicory was replaced with fresh leafy grass grown in the soil surrounding a mining area in Shaoguan. This example yielded the sulfate-reducing bacteria nutrient solution after 72 hours of adaptability culture.

[0047] Example 3 A sulfate-reducing bacteria nutrient solution similar to that of Example 1 was developed, the only difference being the raw materials used. In Example 1, fresh chicory was replaced with fresh leafy grass grown in the soil surrounding a mining area in Shaoguan. This example yielded the sulfate-reducing bacteria nutrient solution after 72 hours of adaptability culture.

[0048] Comparative Example 1 A sulfate-reducing bacteria nutrient solution similar to that of Example 1 was obtained, the only difference being the absence of phenazine. This comparative example was cultured for 72 h to obtain the sulfate-reducing bacteria nutrient solution.

[0049] Comparative Example 2 A sulfate-reducing bacteria nutrient solution similar to that in Example 1, the only difference being that no adaptation culture is performed, and the resulting fermentation broth-based nutrient solution is used as the sulfate-reducing bacteria nutrient solution.

[0050] Comparative Example 3 A sulfate-reducing bacteria nutrient solution similar to that in Example 1, the only difference being that no phenazine is added and no adaptation culture is performed, and the resulting fermentation broth-based nutrient solution is used as a sulfate-reducing bacteria nutrient solution.

[0051] Comparative Example 4 A sulfate-reducing bacteria nutrient solution similar to that of Example 2 was obtained, the only difference being the absence of phenazine. This comparative example was cultured for 72 h to obtain the sulfate-reducing bacteria nutrient solution.

[0052] Comparative Example 5 A sulfate-reducing bacteria nutrient solution similar to that in Example 2, the only difference being that no adaptation culture is performed, and the resulting fermentation broth-based nutrient solution is used as the sulfate-reducing bacteria nutrient solution.

[0053] Comparative Example 6 A sulfate-reducing bacteria nutrient solution similar to that in Example 2, the only difference being that no phenazine is added and no adaptation culture is performed, and the resulting fermentation broth-based nutrient solution is used as a sulfate-reducing bacteria nutrient solution.

[0054] Comparative Example 7 A sulfate-reducing bacteria nutrient solution similar to that in Example 3 was obtained, the only difference being the absence of phenazine. This comparative example was cultured for 72 h to obtain the sulfate-reducing bacteria nutrient solution.

[0055] Comparative Example 8 A sulfate-reducing bacteria nutrient solution similar to that in Example 3, the only difference being that no adaptation culture is performed, and the resulting fermentation broth-based nutrient solution is used as the sulfate-reducing bacteria nutrient solution.

[0056] Comparative Example 9 A sulfate-reducing bacteria nutrient solution similar to that in Example 3, the only difference being that no phenazine is added and no adaptation culture is performed, and the resulting fermentation broth-based nutrient solution is used as a sulfate-reducing bacteria nutrient solution.

[0057] Comparative Example 10 A method for preparing a sulfate-reducing bacteria nutrient solution: A 200 g / L aqueous solution of a carbon source (glucose) is prepared and mixed with acidic mine wastewater (AMD) at a volume ratio of 1:8. Sulfate-reducing bacteria (SRB) are inoculated, the pH is adjusted to approximately 5.0, and the dissolved oxygen (DO) is controlled to <0.5 mg / L. The mixture is then anaerobically cultured at 30℃ for 72 h. OD is monitored during the anaerobic culture process. 600 When OD 600 Stop heating when the temperature reaches ≥1.5℃ to obtain sulfate-reducing bacteria nutrient solution. Store in a sealed container at room temperature in a well-ventilated and dry place for one month. The bacterial concentration of sulfate-reducing bacteria should be ≥10. 7 The inoculum concentration was CFU / mL, and the inoculum size was 5% of the total volume of glucose and AMD. The AMD was obtained from acidic wastewater leached from a waste rock dump in a mining area in Shaoguan, with a pH of 3.5 and SO42-. 2- The concentration is 3600 mg / L.

[0058] Comparative Example 11 A method for preparing a sulfate-reducing bacteria nutrient solution: A carbon source (sodium acetate) is prepared into a 200 g / L aqueous solution, which is then mixed with acidic mine wastewater (AMD) at a volume ratio of 1:8. Sulfate-reducing bacteria (SRB) are inoculated, the pH is adjusted to approximately 5.0, and the dissolved oxygen (DO) is controlled to <0.5 mg / L. The mixture is then anaerobically cultured at 30℃ for 72 h. OD is monitored during the anaerobic culture process. 600 When OD 600 Stop heating when the temperature reaches ≥1.5℃ to obtain sulfate-reducing bacteria nutrient solution. Store in a sealed container at room temperature in a well-ventilated and dry place for one month. The bacterial concentration of sulfate-reducing bacteria should be ≥10. 7 The inoculum concentration was CFU / mL, and the inoculum size was 5% of the total volume of sodium acetate and AMD. The AMD was obtained from acidic wastewater leached from a waste rock dump in a mining area in Shaoguan, with a pH of 3.5 and SO42-. 2- The concentration is 3600 mg / L.

[0059] Test Example 1 The sulfate-reducing bacteria nutrients obtained in Examples 1-3 and Comparative Examples 1-11 were tested for physicochemical and biological indicators. The specific indicators tested were pH, turbidity, chemical oxygen demand, proportion of small molecule carbon source, total amino acid and arginine proportion. The specific test methods and results are shown in Table 1.

[0060] Detection methods: pH value was measured on-site using a portable pH meter; turbidity value was measured using the turbidimeter method (scattering method, HJ1075-2019); chemical oxygen demand was measured using the dichromate method (HJ 828-2017); the proportion of small molecule carbon sources was measured using the BOD5 / COD ratio method; the total amount of amino acids was measured using high performance liquid chromatography; and the proportion of arginine was measured using the amino acid component separation-normalization method.

[0061] Table 1. Detection results of physicochemical and biological indicators of different sulfate-reducing bacteria nutrient solutions.

[0062] Application Example 1 Treatment of Acidic Mine Wastewater (AMD) 1. AMD was treated with sulfate-reducing bacteria nutrients obtained in Examples 1-3 and Comparative Examples 1-11, respectively. The specific steps were as follows: AMD was placed in an AMD reactor with an effective capacity of 1000L, inoculated with 10% (v / v) sulfate-reducing bacteria nutrients, and then inoculated with sulfate-reducing bacteria (SRB). The reactor temperature was controlled at 32℃, DO < 0.5 mg / L, and the reaction was carried out intermittently for 48 h. The AMD was obtained from acidic wastewater leached from a waste rock dump in a mining area in Shaoguan, with a pH of 3.5 and SO42-. 2- The concentration was 3600 mg / L; the bacterial concentration of sulfate-reducing bacteria was ≥10. 7 CFU / mL, the inoculum amount is 5% of the total volume of sulfate-reducing bacteria nutrient solution and AMD.

[0063] 2. After the reaction is complete, the pH of the reaction product, the number of viable SRB bacteria, the oxidation-reduction potential (ORP), and the SO4 levels before and after the reaction are measured. 2- Concentration, sulfate reduction rate, and reaction cost, along with specific detection methods and results, are shown in Table 2.

[0064] Detection methods: pH value was measured on-site using a portable pH meter; SRB viable count was determined using qPCR quantification (EPA Method 1631); ORP value was measured on-site using a portable ORP analyzer; SO4... 2- Concentration was determined using the turbidimetric method (HJ / T 342-2007).

[0065] Table 2. Application effects of different sulfate-reducing bacteria nutrient agents

[0066] As shown in Table 2, when AMD was treated with the sulfate-reducing bacteria nutrient solutions prepared in Examples 1-3, the sulfate reduction rate was significantly higher than that of other treatment groups. Among them, Example 3 (chicory: edible leaves = 1:1) showed the best performance: the sulfate reduction rate reached 92.00%, and the viable SRB count reached 4.2 × 10⁻⁶. 9 CFU / mL. Comparative Examples 1, 4, and 7, due to the lack of phenazine utilization, showed sulfate reduction rates of 61.11–70.00%, indicating that phenazine, as an electron shuttle, can enhance SRB metabolic rate. Comparative Examples 2, 5, and 8, due to the lack of adaptation culture, showed sulfate reduction rates of 55.97–65.00%, highlighting the crucial role of adaptation culture pre-adaptation in SRB stress resistance. Comparative Examples 3, 6, and 9, without phenazine utilization or adaptation culture, showed sulfate reduction rates below 50%, with a minimum of 39.44%, demonstrating the synergistic effect of phenazine and adaptation culture in improving sulfate reduction. This invention uses chicory (6 yuan / kg) and / or leafy greens (16 yuan / kg) as raw materials to replace traditional carbon sources (glucose and sodium acetate), reducing costs to 650–720 yuan / ton, a reduction of 64–67% compared to glucose (1800 yuan / ton) and sodium acetate (1950 yuan / ton). For a 10,000-ton-scale AMD treatment project, the annual carbon source cost can be reduced from 1.8-1.95 million yuan to 650,000-720,000 yuan, saving 1.15-1.3 million yuan.

[0067] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing a sulfate-reducing bacteria nutrient solution, characterized in that, Includes the following steps: Fermentation broth from non-edible plants, nutrients, and electronic mediators are mixed to obtain a fermentation broth-based nutrient solution. The fermentation liquid-based nutrient solution and acidic mine wastewater were mixed to obtain a fermentation substrate, which was then inoculated with sulfate-reducing bacteria and cultured to obtain a sulfate-reducing bacteria nutrient solution. The fermentation broth of the non-edible plants has an amino acid content ≥20 g / L and a dissolved chemical oxygen demand ≥2200 mg / L; The nutrients include nitrogen, phosphorus, and trace elements; the trace elements include iron, magnesium, and zinc. The electronic mediator includes one or more of phenazine, phenazine-1-carboxylic acid, 2-hydroxyphenazine, sodium anthraquinone-2-sulfonate, neutral red, vitamin B2, and vitamin B2 derivatives; The adaptive culture was conducted at a pH of 4.0–5.0, with a dissolved oxygen content of <0.5 mg / L, a temperature of 25–35°C, and a time of 48–96 h.

2. The preparation method according to claim 1, characterized in that, The fermentation broth-based nutrient solution comprises nitrogen 0.2~0.3 g / L, phosphorus 0.1~0.15 g / L, iron 0.01~0.02 g / L, magnesium 0.05~0.07 mg / L, and zinc 0.04~0.05 mg / L; the mass of nitrogen, phosphorus, iron, magnesium, and zinc is calculated in terms of the mass of urea, potassium dihydrogen phosphate, ferrous sulfate, magnesium sulfate, and zinc sulfate, respectively. The concentration of electron mediators in the fermentation liquid-based nutrient is 0.05~0.06 mg / L.

3. The preparation method according to claim 1, characterized in that, The volume ratio of the fermentation liquid-based nutrient to the acidic mine wastewater is 8-8.5:1; the inoculation amount of the sulfate-reducing bacteria is 10-15% of the mass of the fermentation substrate.

4. The preparation method according to any one of claims 1 to 3, characterized in that, The preparation method of the fermentation broth from the non-edible plant includes the following steps: Non-edible plant powder is mixed with a medium and soaked to obtain fermentation raw material; the medium includes effluent from the primary sedimentation tank. Lactobacillus and cellulase are inoculated into the fermentation raw materials to carry out the first fermentation and obtain the first fermentation product. Bacillus subtilis and yeast are inoculated into the first fermentation product to carry out a second fermentation, thereby obtaining a second fermentation product; during the second fermentation process, materials are added; the materials include starch and fish meal. The second fermentation product is subjected to solid-liquid separation, and the collected liquid is sterilized to obtain the fermentation broth of the non-edible plant.

5. The preparation method according to claim 4, characterized in that, The particle size of the non-edible plant powder is ≤1mm; the mass-to-volume ratio of the non-edible plant powder to the medium is 0.5~1.5g:4~6mL; the chemical oxygen demand of the effluent from the primary sedimentation tank is 300~500 mg / L, and the concentration of suspended solids is <10 mg / L. The non-edible plants include chicory and / or leafy grasses; The soaking temperature is 20~30℃, and the soaking time is 12~36 h.

6. The preparation method according to claim 4, characterized in that, The first fermentation was carried out at a temperature of 35-42℃, a pH of 5.0-6.0, a dissolved oxygen content ≤0.5 mg / L, and a time of 24-48 h; the inoculum size of the lactobacillus was 10. 8 ~10 9 CFU / mL, wherein the inoculum amount of cellulase is ≥5000 U / g; The second fermentation temperature is 30-35℃, pH is 6.0-7.0, and dissolved oxygen content is ≤0.5 mg / L; the inoculum size of Bacillus subtilis is 10... 5 ~10 6 CFU / mL, the inoculum size of the yeast was 10. 5 ~10 6 CFU / mL.

7. The preparation method according to claim 4, characterized in that, The mass ratio of starch to fishmeal in the material is 0.5~2:1, and the total amount of the material added is 5~10% of the mass of the plant raw material. The feeding is carried out twice during the second fermentation, at 24-48 h and 48-72 h, with each feeding amount being 50% of the total material.

8. The sulfate-reducing bacteria nutrient agent obtained by the preparation method according to any one of claims 1 to 7.

9. The application of the sulfate-reducing bacteria nutrient agent according to claim 8 in the treatment of acidic mine wastewater.

10. A method for treating acidic mine wastewater, characterized in that, Includes the following steps: The acidic mine wastewater to be treated is mixed with the sulfate-reducing bacteria nutrient solution described in claim 8, and sulfate-reducing bacteria are inoculated. Anaerobic fermentation is carried out at 32°C.

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