Microbial liquid bacterial fertilizer prepared from high-salt mine water inflow, its preparation method, and method for treating polluted soil in coal mining areas

Abstract

The present invention provides a microbial liquid fertilizer prepared by using high-salt mine drainage, a preparation method thereof, and an application thereof in the treatment of polluted soil in coal mining areas. The microbial liquid fertilizer is composed of a first group of liquid fertilizer and a second group of liquid fertilizer. The preparation method includes: 1) pretreatment of mine drainage; 2) preparation of a fermentation substrate; 3) preparation of the first group of liquid fertilizer; 4) preparation of the second group of liquid fertilizer. The first group of liquid fertilizer is used to solidify heavy metals in the soil and reduce their mobility; the second group of liquid fertilizer is applied after 30 days to increase the contents of phosphorus, potassium, and nitrogen in the soil and improve soil fertility. The present invention effectively utilizes high-salt mine drainage, realizes resource recycling, and at the same time, through staged application of the fertilizer, achieves the dual effects of heavy metal stabilization and soil remediation, providing an efficient solution for ecological restoration in coal mining areas.

Description

Technical Field

[0001] The present invention belongs to the technical fields of environmental protection and mine ecological restoration, and relates to a method for preparing a microbial liquid bacterial fertilizer by using high-salt mine water inflow and its preparation method, and applying the microbial liquid bacterial fertilizer to the treatment and restoration of polluted soil in coal mine areas. Background Art

[0002] Mine water inflow is one of the main by-products generated during coal mining, especially high-salt mine water inflow. If directly discharged without treatment, it will cause serious pollution to the surrounding environment. Traditional mine water treatment methods such as chemical precipitation and physical filtration can reduce the content of pollutants, but usually involve high treatment costs and resource waste problems. In addition, the soil in coal mine areas often faces problems such as heavy metal over-standard, soil acidification or salinization due to long-term pollution by coal gangue and other mineral waste, and effective treatment measures are urgently needed.

[0003] In recent years, the application of microbial technology in environmental governance has gradually received attention. Highly efficient heavy metal immobilizing bacterial communities such as sulfate-reducing bacteria can effectively immobilize heavy metals in the soil, reducing their mobility and bioavailability. At the same time, the presence of phosphate-solubilizing bacteria, potassium-solubilizing bacteria and nitrogen-fixing bacteria not only promotes the cycling of important nutrients such as phosphorus, potassium and nitrogen in the soil, but also improves soil fertility, improves the soil environment and promotes the restoration of the ecosystem.

[0004] However, there are still many problems in the application of microbial technology in the treatment of high-salt mine water inflow and the treatment of polluted soil in coal mine areas: 1) Poor environmental adaptability: Traditional microbial treatment performs poorly under the conditions of high-salt mine water inflow. The high-salt environment often inhibits the activity of microorganisms, resulting in a significant decline in treatment effects, and there is a lack of effective microbial strain combinations in the existing technology to adapt to such environments. 2) Limited heavy metal immobilization effect: Many microbial treatment technologies are difficult to effectively immobilize heavy metals in the soil. Even in a low-salt environment, some microorganisms still cannot stably adsorb or deposit heavy metals for a long time, resulting in the problems of heavy metal migration and biological toxicity still existing. 3) Insufficient nutrient cycling: Traditional microbial treatment focuses more on removing pollutants and pays less attention to the cycling and replenishment of soil nutrients. Treatment technologies lacking multifunctional strain combinations are difficult to enhance the nitrogen, phosphorus and potassium content of the soil while stabilizing heavy metals, and cannot meet the comprehensive requirements of soil ecological restoration. 4) Low resource utilization rate: Existing microbial treatment schemes usually do not consider the reuse of high-salt mine water inflow, but remove salts through high-cost physical or chemical treatments, resulting in resource waste.

[0005] Therefore, it is an urgent problem to be solved to provide a microbial technology that can effectively treat polluted soil in coal mine areas, achieve good heavy metal immobilization effect, achieve the improvement of polluted soil and achieve the reuse of high-salt mine water inflow. Summary of the Invention

[0006] To solve the above problems, the object of the present invention is to provide a microbial liquid fertilizer prepared by using high-salt mine water inflow, which can effectively utilize high-salt mine water inflow to achieve the treatment of polluted soil in coal mining areas.

[0007] Another object of the present invention is to provide a preparation method of a microbial liquid fertilizer prepared by using high-salt mine water inflow.

[0008] The third object of the present invention is to provide a method for treating polluted soil in coal mining areas. This method realizes the compliant consumption of mine water inflow, promotes the stabilization of heavy metals and the cycling of nutrient elements such as phosphorus, potassium, and nitrogen in the soil, improves soil fertility, and provides effective technical support for the ecological restoration of coal mining areas.

[0009] To achieve the above object, the present invention provides a group of microbial liquid fertilizers prepared by using high-salt mine water inflow. This group of microbial liquid fertilizers is composed of a first group of liquid fertilizers and a second group of liquid fertilizers. Among them, the first group of liquid fertilizers contains anaerobic sulfate-reducing bacteria, and the concentration of the anaerobic sulfate-reducing bacteria is ≥10 6 CFU / g; the second group of liquid fertilizers contains aerobic phosphorus-solubilizing bacteria, potassium-solubilizing bacteria, and nitrogen-fixing bacteria, and the total concentration of the aerobic phosphorus-solubilizing bacteria, potassium-solubilizing bacteria, and nitrogen-fixing bacteria is ≥10 6 CFU / g.

[0010] Furthermore, the anaerobic sulfate-reducing bacteria are classified and named as Desulfovibrio vulgaris EM2, the preservation unit is the China Center for Type Culture Collection, the address is Wuhan University, Wuhan, China, the preservation date is November 1, 2017, and the preservation number is CCTCC M 2017645;

[0011] The phosphorus-solubilizing bacteria are classified and named as Ochrobactrum intermedium, and its preservation number is CCTCC AB 2015151;

[0012] The potassium-solubilizing bacteria are classified and named as Bacillus circulans, and its preservation number is CCTCC NB 20083559;

[0013] The nitrogen-fixing bacteria are classified and named as Ochrobactrum pseudintermedium, and its preservation number is CCTCC AB 2015139.

[0014] The present invention also provides a preparation method of a microbial liquid fertilizer prepared by using high-salt mine water inflow, including the following steps:

[0015] 1) Pretreatment of mine water inflow

[0016] Use physical sedimentation to remove suspended solids in mine water inflow; then, if the salt concentration of mine water inflow is greater than 3000 mg / L, dilute the mine water inflow with fresh water to a salt concentration of 2500 - 3000 mg / L, and then reduce the sulfate ion concentration to 500 - 1000 mg / L through chemical treatment to make the mine water inflow suitable as a fermentation substrate;

[0017] 2) Preparation of fermentation substrate

[0018] Add carbon source, nitrogen source and phosphorus source to the pretreated mine water inflow to prepare a fermentation substrate suitable for the growth of microorganisms;

[0019] 3) Preparation of the first group of liquid biofertilizer

[0020] Inoculate anaerobic sulfate-reducing bacteria into the fermentation substrate prepared in step 2), carry out static thermostatic fermentation in an anoxic environment, after fermentation is completed, carry out solid-liquid separation, and use a mesoporous filter cloth filter with a pore size of 0.5 - 1 mm to remove the completely fermented residue to obtain the first group of liquid biofertilizer;

[0021] 4) Preparation of the second group of liquid biofertilizer

[0022] Inoculate aerobic phosphate-solubilizing bacteria, potassium-solubilizing bacteria and nitrogen-fixing bacteria into the fermentation substrate prepared in step b), and carry out thermostatic fermentation in an oxygen-permeable environment to obtain the second group of liquid biofertilizer.

[0023] Furthermore, the physical sedimentation method in step 1) is: first, let the mine water inflow stand for 12 hours, and use a sand and gravel filter to filter and remove the sediment;

[0024] The chemical treatment in step 1) is to add calcium-based materials to precipitate sulfate ions, thereby reducing the sulfate concentration in the mine water inflow and preventing the interference of high-concentration sulfates on the subsequent fermentation process.

[0025] Furthermore, the calcium-based materials are lime or calcium carbonate.

[0026] Furthermore, the carbon source in step 2) is acetic acid, glucose, lactic acid, propionic acid or starch; the nitrogen source is ammonium nitrate, urea, ammonium chloride, sodium nitrate or ammonia water; the phosphorus source is potassium dihydrogen phosphate, calcium phosphate, tricalcium phosphate or sodium phosphate.

[0027] The addition of the carbon source can provide a suitable energy source for microorganisms; the addition of the nitrogen source can effectively promote the growth and reproduction of microorganisms; the addition of the phosphorus source is used to meet the phosphorus requirements of microorganisms.

[0028] Further, the fermentation and cultivation conditions of the anaerobic sulfate-reducing bacteria in the first group of liquid bacterial fertilizers in step 3) are as follows: the temperature is 25 - 35 °C, the pH value is 6.5 - 7.5, and the fermentation time is 5 - 7 days, so that the viable bacteria concentration in the bacterial liquid ≥ 10 6 CFU / g.

[0029] Further, the fermentation and cultivation conditions of the aerobic phosphorus-solubilizing bacteria, potassium-solubilizing bacteria and nitrogen-fixing bacteria in the second group of liquid bacterial fertilizers in step 4) are as follows: the temperature is 25 - 35 °C, the pH value is 6.5 - 7.5, and they are fermented by shaking or continuously stirred in an oxygen-permeable environment for 3 - 5 days until the total viable bacteria concentration in the bacterial liquid ≥ 10 6 CFU / g.

[0030] The present invention also provides a method for treating polluted soil in coal mining areas, including the following steps:

[0031] 1) Dilute the first group of liquid bacterial fertilizers in the above-mentioned microbial liquid bacterial fertilizers prepared from high-salt mine water to 1×10 6 CFU / g, and apply it to the polluted soil in the coal mining area by spraying.

[0032] 2) After 30 days of spraying the first group of liquid bacterial fertilizers, dilute the second group of liquid bacterial fertilizers in the above-mentioned microbial liquid bacterial fertilizers prepared from high-salt mine water to 1×10 6 CFU / g, and apply it to the same polluted soil in the coal mining area by spraying or irrigation.

[0033] Further, in step 1), the spraying amount of the spraying method is 2 - 5 L / m² of soil, the spraying time is 5 - 10 minutes, spraying is carried out once every 3 days, and spraying is carried out 10 times; in step 2), the spraying amount of the spraying method is 2 - 5 L / m² of soil, the spraying time is 5 - 10 minutes, spraying is carried out once every 7 days, and spraying is carried out 10 times.

[0034] The beneficial effects of the present invention are as follows:

[0035] The present invention provides a method for preparing a group of microbial liquid bacterial fertilizers using high-salt mine water and a method for treating polluted soil in coal mining areas. By using two groups of mixed bacterial populations, including anaerobic sulfate-reducing bacteria and aerobic phosphorus-solubilizing bacteria, potassium-solubilizing bacteria and nitrogen-fixing bacteria, and applying them in stages, it not only effectively solidifies heavy metals in the soil and reduces their mobility, but also promotes the cycling of nutrient elements such as phosphorus, potassium and nitrogen in the soil, improves soil fertility, and further improves the ecological environment of coal mining areas. Description of the Drawings

[0036] Figure 1 It is a statistical chart of the change in the suspended solid content of mine water before and after physical treatment in the preparation method provided by the present invention.

[0037] Figure 2Comparison chart of plant growth status after applying the microbial liquid fertilizer prepared from high-salt mine water provided by the present invention. Detailed implementation mode

[0038] The embodiments of the present invention will be described in detail and comprehensively below, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.

[0039] The present invention provides a microbial liquid fertilizer prepared from high-salt mine water, and the microbial liquid fertilizer is composed of a first group of liquid fertilizer and a second group of liquid fertilizer;

[0040] Among them, the first group of liquid fertilizer contains anaerobic sulfate-reducing bacteria, and the concentration of the anaerobic sulfate-reducing bacteria is ≥ 10 6 CFU / g;

[0041] The second group of liquid fertilizer contains aerobic phosphate-solubilizing bacteria, potassium-solubilizing bacteria and nitrogen-fixing bacteria, and the total concentration of the aerobic phosphate-solubilizing bacteria, potassium-solubilizing bacteria and nitrogen-fixing bacteria is ≥ 10 6 CFU / g.

[0042] The anaerobic sulfate-reducing bacteria are classified and named as Desulfovibrio vulgaris EM2, the preservation unit is the China Center for Type Culture Collection, the address is Wuhan University, Wuhan, China, the preservation date is November 1, 2017, and the preservation number is CCTCC M 2017645.

[0043] The phosphate-solubilizing bacteria are classified and named as Ochrobactrum intermedium, and its preservation number is CCTCC AB 2015151, purchased from the China Center for Type Culture Collection.

[0044] The potassium-solubilizing bacteria are classified and named as Bacillus circulans, and its preservation number is CCTCC NB20083559, purchased from the China Center for Type Culture Collection.

[0045] The nitrogen-fixing bacteria are classified and named as Ochrobactrum pseudintermedium, and its preservation number is CCTCC AB 2015139, purchased from the China Center for Type Culture Collection.

[0046] Example 1:

[0047] A high-salt mine water sample collected from a coal mining area in Inner Mongolia Autonomous Region has an initial salt concentration close to 3500 mg / L, and the specific components are shown in Table 1.

[0048] Table 1 Composition Analysis of High-Salt Mine Water Samples in a Coal Mine Area of Inner Mongolia Autonomous Region

[0049] Component Concentration (mg / L) Remarks <![CDATA[Sodium (Na + )]]> 758.4 Main cation, accounting for the main proportion <![CDATA[Calcium (Ca 2+ )]]> 262.3 Secondary cation, usually with a relatively high content <![CDATA[Magnesium (Mg 2+ )]]> 219.7 One of the cations, second only to sodium and calcium <![CDATA[Potassium (K + )]]> 81.2 Cation with a relatively low content <![CDATA[Sulfate (SO4 2- )]]> 1563.5 One of the main anions, with a relatively high concentration <![CDATA[Chlorine (Cl - )]]> 451.6 Main anion, with a concentration second only to sulfate <![CDATA[Bicarbonate radical (HCO3 - )]]> 125.8 The content fluctuates greatly, depending on the specific characteristics of the mine water inflow

[0050] 1. Pretreatment of Mine Water

[0051] (1) Let the mine water stand and precipitate for 12 hours to allow large particle impurities to settle naturally. After precipitation, use a gravity filtration method and a sand filter to further remove suspended solids. The filtration process is carried out at room temperature, and the filtered water sample is used for subsequent fermentation substrate preparation.

[0052] Since the high-salt mine water collected from the coal mine contains suspended solids and large particle impurities, let it stand for physical precipitation to allow large particle impurities to settle naturally, and then filter out the sediment with a sand filter to reduce the suspended solid load in the water.

[0053] The sand filter is a commercial device, which contains several layers of sand with different particle sizes inside to ensure the removal of suspended particles layer by layer. The particle size of the sand is as follows: the top layer is coarse sand with a particle size of about 2 - 5 mm; the middle layer is medium sand with a particle size of about 1 - 2 mm; the bottom layer is fine sand with a particle size of about 0.5 - 1 mm.

[0054] The change in the suspended solid content of mine water before and after physical treatment is as Figure 1 shown. As Figure 1 can be seen, by gradually treating the mine water, the initial suspended solid content is significantly reduced from 606 mg / L to 141 mg / L, and the treatment effect is remarkable. After 12 hours of standing precipitation, large particle impurities settle, and the suspended solid content decreases by 42% to 352 mg / L. This step effectively removes larger suspended particles and significantly reduces the suspended solid load in the water. Then, through further filtration with a sand filtration device, the suspended solid content is reduced to 141 mg / L again, with a reduction rate of 60%. The total reduction rate of suspended solids in the whole treatment process reaches 77%, greatly improving the purity of the mine water. This efficient physical treatment provides more ideal water quality conditions for the subsequent microbial fermentation process, helps to avoid the interference of sediments on the fermentation process, and thus ensures the smooth progress of fermentation and the quality of the final product.

[0055] (2) Dilution treatment: After the physical treatment, the mine water with a salt concentration higher than 3000 mg / L is diluted, and the salt concentration needs to be controlled below 3000 mg / L. This step reduces the salt concentration by adding fresh water to meet the requirements of the fermentation substrate and ensure that the growth of the microbial community during the fermentation process is not inhibited by the high salt concentration. To facilitate the comparison of the growth of microorganisms with a salt concentration exceeding 3000 mg / L, the salt concentration of some mine water is also adjusted to 3500 mg / L. The growth of microorganisms under different salt concentration conditions is compared, as shown in Table 2.

[0056] Table 2 Growth of functional microorganisms under different salt concentration conditions

[0057]

[0058] Note: "++" indicates a large number of colony growth; "+" indicates very few colony growth; "-" indicates no colony growth.

[0059] As can be seen from Table 2, when the salt concentration exceeds 3000 mg / L, the growth rate of microorganisms significantly decreases. Therefore, adjusting the salt concentration below 3000 mg / L is crucial for the fermentation substrate, which helps maintain the activity and stability of the microbial community and promotes an effective fermentation process. At the same time, the dilution treatment with the salt concentration controlled within the range of 2500 - 3000 mg / L can ensure that the subsequent chemical treatment can reduce the sulfate concentration below 800 mg / L, thus meeting the growth requirements of microorganisms.

[0060] In this embodiment, the salt concentration of the mine water after physical treatment is adjusted to 3500 mg / L. Through dilution treatment, the mine water is mixed with fresh water at a ratio of 5:1, and the diluted salt concentration is reduced to about 2900 mg / L, a reduction of about 17%; the mine water is mixed with fresh water at a ratio of 4:1, and the diluted salt concentration is reduced to about 2800 mg / L, a reduction of about 20%; the mine water is mixed with fresh water at a ratio of 3:1, and the diluted salt concentration is reduced to about 2600 mg / L, a reduction of about 26%; the mine water is mixed with fresh water at a ratio of 2:1, and the diluted salt concentration is reduced to about 2300 mg / L, a reduction of about 34%. The comparison of the salt concentration before and after treatment is shown in Table 3. This step effectively reduces the salt concentration of the mine water and provides a suitable environment for the growth of microorganisms in the subsequent fermentation process.

[0061] Table 3 Comparison table of salt concentration of mine water before and after dilution treatment

[0062]

[0063] Since the fresh water used for dilution is the local fresh water resource in the coal mining area (such as tap water), which is in short supply, the use of fresh water should be minimized in practical applications. The set lower limit of the target concentration (2500 mg / L) has ensured the growth of functional microorganisms, while also minimizing the risk of land salinization as much as possible to meet the needs of ecological restoration.

[0064] (3) Chemical treatment: The diluted mine water inflow enters the chemical treatment stage. By adding calcium-based material lime (calcium oxide), precipitation reaction occurs to separate the sulfate in it in the form of precipitate, thereby reducing the concentration of sulfate ions in the water. This step can prevent the potential inhibitory effect of high-concentration sulfate ions on the microbial fermentation process and provide a suitable environment for the subsequent process.

[0065] Since the sulfate concentration in mine water inflow mostly exceeds 1000 mg / L, and when the sulfate concentration exceeds 1000 mg / L, it will inhibit the fermentation process of many microorganisms, especially in the fermentation system containing sensitive strains. For example, when the sulfate concentration is higher than 1000 - 1500 mg / L, the activity of microorganisms such as anaerobic sulfate-reducing bacteria (SRB) will be significantly affected. Therefore, the sulfate ion concentration is reduced to below 1000 mg / L through chemical treatment to keep the anaerobic sulfate-reducing bacteria (SRB) with normal activity.

[0066] The reason for the interference of sulfate concentration on microorganisms is that high-concentration sulfate will affect the normal metabolic activities of microorganisms in the fermentation environment, mainly through the following two aspects to cause interference:

[0067] The first is the increase in osmotic pressure: Sulfate belongs to strong electrolyte, and at high concentration, it will significantly increase the osmotic pressure of the fermentation substrate, forcing the water in the microbial cells to flow out, resulting in an increase in dehydration pressure, and then inhibiting the activity and growth of microorganisms.

[0068] The second is the generation of inhibitory metabolites: Under anaerobic conditions, sulfate-reducing bacteria convert sulfate into hydrogen sulfide (H2S), and hydrogen sulfide is a substance with relatively high toxicity, which will inhibit the growth of other microorganisms and may even have an adverse effect on its own strain. Therefore, under high sulfate concentration, the microbial diversity and flora balance in the fermentation environment will be damaged.

[0069] In this embodiment, 7 grams per liter of lime is added to the diluted mine water inflow, and the mine water sample reacts for 30 minutes under stirring conditions and then stands for 24 hours. In the experiment, mine water inflow with different salt concentrations is used for chemical treatment, and the concentration changes of sulfate ions are recorded. The specific changes in sulfate ion concentration before and after treatment are shown in Table 4.

[0070] Table 4 Comparison table of sulfate ion concentration in mine water before and after chemical treatment

[0071]

[0072] As can be seen from Table 4, when the initial salt concentration was 2900 mg / L, the sulfate ion concentration decreased by approximately 32%. When the initial salt concentration was 2800 mg / L, the sulfate ion concentration decreased by approximately 35%. When the initial salt concentration was 2600 mg / L, the sulfate ion concentration decreased by approximately 37%. When the initial salt concentration was 2300 mg / L, the sulfate ion concentration decreased by approximately 40%. The experimental results show that as the salt concentration decreases, the removal effect of sulfate ions shows a gradually increasing trend, that is, the effectiveness of chemical treatment is higher at lower salt concentrations. In addition, after chemical treatment, the sulfate ion concentration in the diluted mine water decreased significantly, thereby reducing the adverse effects on the growth of microorganisms and making it suitable for the fermentation process.

[0073] In addition, considering cost, further reduction of sulfate will require more lime, and retaining 500 - 1000 mg / L of sulfate is beneficial for the fermentation of anaerobic sulfate-reducing bacterium EM2. Anaerobic sulfate-reducing bacterium EM2 requires sulfate as an electron acceptor and reduces it to sulfide during anaerobic fermentation, thereby promoting the solidification and stabilization of heavy metals.

[0074] 2. Preparation of Fermentation Substrate

[0075] In the pretreated mine water, appropriate amounts of carbon sources (acetic acid, glucose, lactic acid, propionic acid or starch), nitrogen sources (ammonium nitrate, urea, ammonium chloride, sodium nitrate or ammonia water) and phosphorus sources (potassium dihydrogen phosphate, calcium phosphate, tricalcium phosphate or sodium phosphate) are added. These nutrients will provide the necessary growth conditions for the microbial flora. The specific addition amounts of the nutrients should be adjusted according to the initial nutrient composition of the mine water to ensure a balanced nutrient content in the fermentation substrate and suitable for the growth of the flora.

[0076] In this embodiment, in order to rationally prepare the fermentation substrate according to the nutrient composition characteristics of different mine waters, the pretreated mine water is supplemented with carbon source (acetic acid), nitrogen source (ammonium nitrate) and phosphorus source (potassium dihydrogen phosphate) according to six different schemes. Four functional bacteria, sulfate-reducing bacteria, phosphorus-solubilizing bacteria, potassium-solubilizing bacteria and nitrogen-fixing bacteria, are selected to establish a mixed culture system. Under aerobic conditions, since phosphorus-solubilizing bacteria, potassium-solubilizing bacteria and nitrogen-fixing bacteria are aerobic bacteria, their growth will be dominant; sulfate-reducing bacteria are facultative anaerobes and can also maintain a certain growth activity under this condition. Therefore, the adopted nutrient addition scheme is based on the mixed system of the four bacteria to evaluate their overall growth performance at different nutrient levels. The specific nutrient schemes are classified from "special low nutrient requirements" to "ultra-high nutrient requirements" according to the nutrient status of the mine water. Using OD 600The value is used as an indicator of the overall biomass of the mixed microbial community, and the pH value is recorded to evaluate the impact of different nutrient regimens on the growth environment of the microbial community. The specific nutrient addition regimens are shown in Table 5, and the experimental conditions for each regimen are fermentation at the laboratory scale with a reaction cycle of 72 hours.

[0077] Table 5 Addition regimens of carbon, nitrogen, and phosphorus in the fermentation substrate of mine water

[0078]

[0079] As can be seen from Table 5, under the conditions of "low nutrient demand" (Regimen C) and "medium nutrient demand" (Regimen D), the OD 600 values of the microbial community reached 0.72 and 0.85 respectively, showing the best growth conditions. These regimens provided a suitable nutrient environment for sulfate-reducing bacteria, phosphorus-solubilizing bacteria, potassium-solubilizing bacteria, and nitrogen-fixing bacteria, meeting their growth and metabolic requirements. In addition, under these two conditions, the pH values of the configured mine water were 6.5 and 6.8 respectively, both within the pH range (6.0 to 7.4) most suitable for microbial growth. In the nutrient-deficient regimens (A and B), although the pH was between 6.0 and 6.2, the OD600 values were only 0.22 and 0.35, indicating that even if the pH was suitable, nutrient deficiency could not meet the growth requirements of microorganisms. On the other hand, the OD600 value of the ultra-high nutrient demand regimen (F) was 0.54. Although its pH value was 7.4, excessive nutrients might cause an increase in osmotic pressure and accumulation of metabolites, resulting in inhibited growth of the microbial community.

[0080] Therefore, moderate nutrient concentration is crucial for the successful preparation of the fermentation substrate. The conclusion shows that the regimens of "low nutrient demand" to "medium nutrient demand" (C and D) not only meet the nutrient requirements of the microbial community but also provide a suitable pH environment. This combination maximizes the growth efficiency of microorganisms and avoids the inhibitory effects caused by nutrient imbalance or pH fluctuations. Considering the initial conditions of mine water, in practical applications, choose the low nutrient demand regimen (Regimen C) or the medium nutrient demand regimen (Regimen D) to ensure the best growth and metabolic activities of the microbial community.

[0081] 3. Inoculation and cultivation of highly efficient microbial communities

[0082] (1) Inoculation and cultivation of the first group of mixed microbial communities: Inoculate anaerobic sulfate-reducing bacteria into the prepared fermentation substrate. The cultivation conditions need to be strictly controlled to ensure the growth and metabolic activities of sulfate-reducing bacteria. The specific conditions are as follows: The temperature is maintained at 25 - 35 °C, the pH value is adjusted within the range of 6.5 - 7.5, and an anoxic environment is created. The fermentation and cultivation time is generally 5 - 7 days to make the viable cell concentration in the bacterial liquid ≥ 10 6 CFU / g, and the specific time can be adjusted according to the progress of fermentation. The obtained liquid biofertilizer is mainly used for the first soil treatment to promote the immobilization of heavy metals in the soil.

[0083] (2) Inoculation and cultivation of the second group of mixed bacteria: 30 days after the first application of the bacterial fertilizer, aerobic phosphate-solubilizing bacteria, potassium-solubilizing bacteria, and nitrogen-fixing bacteria are inoculated into a new fermentation substrate. The cultivation conditions need to be maintained in a suitable aerobic environment to ensure the effective growth of such aerobic bacterial communities. The specific conditions are as follows: the temperature is controlled at 25 - 35 °C, and the pH value is 6.5 - 7.5. The fermentation and cultivation time is 3 - 5 days until the total viable concentration in the bacterial liquid ≥ 10 6 CFU / g, and the obtained liquid bacterial fertilizer is used for the second soil treatment to increase the phosphorus, potassium, and nitrogen contents in the soil and promote the improvement of soil fertility.

[0084] The three types of bacteria selected in this invention are all aerobic bacteria, and the composition of the culture medium has been optimized to support their co-growth in the same environment. On this basis, appropriate cultivation conditions are adopted to ensure that they can cooperate with each other in an aerobic environment without adverse competition. Since those microorganisms that cannot coexist in this environment have been screened out and optimized, the remaining are the bacterial strains that can stably grow together in a system. Therefore, under the cultivation conditions defined in this invention, there will be no obvious dominant bacteria phenomenon, and the three types of bacteria can coexist synergistically and achieve the expected effect.

[0085] 4. Preparation and application of the fermented liquid bacterial fertilizer

[0086] (1) First application: After the fermentation is completed, the anaerobic sulfate-reducing bacteria fermentation broth is subjected to solid-liquid separation, and a medium-hole filter cloth filter with a pore size of 0.5 - 1 mm is used to remove the incompletely fermented residues, obtaining a purified liquid bacterial fertilizer. The pore size of the filter is about between 0.5 - 1 mm, which can effectively remove the incompletely fermented residues but will not intercept the bacterial cells, ensuring that the active microorganisms in the bacterial fertilizer are not affected.

[0087] This bacterial fertilizer is directly applied to the polluted soil in the coal mining area by surface spraying, mainly for the solidification of heavy metals and the preliminary soil improvement.

[0088] (2) Second application: 30 days after the first application of the bacterial fertilizer, the liquid bacterial fertilizer prepared from the fermentation broth of phosphate-solubilizing bacteria, potassium-solubilizing bacteria, and nitrogen-fixing bacteria is applied to the same soil area by surface spraying to further increase the available phosphorus, potassium, and nitrogen contents in the soil, improve soil fertility, and promote plant growth and ecosystem restoration.

[0089] In this embodiment, sulfate-reducing bacteria were inoculated into the previously prepared fermentation substrate. It was cultured under anaerobic conditions at 30 °C and a pH value of 6.8 for 5 - 7 days. The obtained liquid bacterial fertilizer was directly applied to the polluted soil in a coal mining area in Inner Mongolia Autonomous Region by surface spraying to promote the immobilization of heavy metals in the soil. The spraying cycle was 3 days and the spraying time was 30 days. 30 days after the first application of the bacterial fertilizer, phosphate-solubilizing bacteria, potassium-solubilizing bacteria, and nitrogen-fixing bacteria were respectively inoculated into a new fermentation substrate. The obtained liquid bacterial fertilizer was used for the second soil treatment. The spraying cycle was 7 days and the spraying time was 70 days. After two applications of the liquid bacterial fertilizer, the plant growth conditions were compared as Figure 2 shown, and the changes in the comprehensive soil quality indicators are shown in Table 6.

[0090] Table 6 Comprehensive soil quality indicators before and after applying two groups of liquid bacterial fertilizers

[0091]

[0092] From Figure 2 and Table 6, it can be seen that after two applications of the liquid bacterial fertilizer, significant improvements have been achieved in both the heavy metal content and nutrient content of the soil in this area. First, in terms of heavy metal removal, the total Cd and total Pb contents in the soil have decreased significantly. The content of Cd has dropped from 0.10 mg / kg to below the detection limit, and the content of Pb has decreased from 18.9 mg / kg to 12.0 mg / kg. This indicates that sulfate-reducing bacteria have a significant effect on the immobilization of toxic heavy metals in the soil, helping to reduce their mobility and bioavailability. Although the As content has increased slightly, the overall change is small, which may be related to the soil environmental conditions. In addition, the nutrient content in the soil has also been significantly improved. The total nitrogen content has increased from 703 mg / kg to 858 mg / kg, the available potassium has risen from 0.15 g / kg to 0.17 g / kg, and the available phosphorus has increased significantly from 9.55 mg / kg to 21.2 mg / kg. The application of phosphate-solubilizing bacteria, potassium-solubilizing bacteria, and nitrogen-fixing bacteria has significantly promoted the cycling of key nutrient elements in the soil and improved soil fertility. The increase in soil organic matter is also very significant, increasing from 5.7 g / kg to 14.6 g / kg, indicating that the application of bacterial fertilizer has an obvious effect on improving soil quality. The total number of bacteria in the soil has increased from 3.9×10 5 CFU / g to 1.8×10 6 CFU / g, showing a significant enhancement of soil microbial activity, which will contribute to the stability and sustainable health of the soil ecosystem.

[0093] As can be seen from the above embodiments, the present invention provides a method for using high-salt mine water inflow as a fermentation substrate for microbial bacterial liquid to prepare a microbial liquid fertilizer suitable for the remediation of polluted soil in coal mining areas. By using two groups of mixed microbial communities, including anaerobic sulfate-reducing bacteria and aerobic phosphate-solubilizing bacteria, potassium-solubilizing bacteria, and nitrogen-fixing bacteria, and applying them in stages, not only effectively solidifies heavy metals in the soil and reduces their mobility, but also promotes the cycling of nutrient elements such as phosphorus, potassium, and nitrogen in the soil, improves soil fertility, and further improves the ecological environment of coal mining areas.

[0094] The above-described embodiments only express the implementation modes of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the appended claims.

Claims

1. A microbial liquid fertilizer prepared by using high-salt mine water, characterized in that: The microbial liquid fertilizer consists of a first group of liquid fertilizers and a second group of liquid fertilizers; The first group of liquid fertilizer contains anaerobic sulfate-reducing bacteria, and the concentration of the anaerobic sulfate-reducing bacteria is ≥10 6 CFU / g; The second group of liquid bacterial fertilizer contains aerobic phosphate-solubilizing bacteria, potassium-solubilizing bacteria and nitrogen-fixing bacteria, and the total concentration of the aerobic phosphate-solubilizing bacteria, potassium-solubilizing bacteria and nitrogen-fixing bacteria is ≥10 6 CFU / g; The anaerobic sulfate-reducing bacteria is classified as Desulfovibrio vulgaris EM2, deposited with China Center for Type Culture Collection, Wuhan University, Wuhan, China, on November 1, 2017, with a deposit number of CCTCC M 2017645. The phosphate-solubilizing bacteria is classified as Ochrobactrum intermedium, and its deposit number is CCTCC AB 2015151; The potassium-dissolving bacteria is classified as Bacillus circulans, and its deposit number is CCTCC NB20083559; The nitrogen-fixing bacteria is classified as Ochrobactrum pseudintermedium, and its preservation number is CCTCC AB 2015139.

2. A method for preparing microbial liquid fertilizer using high-salt mine water according to claim 1, characterized in that: The steps include: 1) Pretreatment of mine water Use physical precipitation to remove suspended solids from mine water; if the salt concentration of the mine water is greater than 3000mg / L, dilute the mine water with fresh water to a salt concentration of 2500-3000mg / L, and then reduce the sulfate ion concentration to 500-1000mg / L through chemical treatment; 2) Preparation of fermentation substrate Adding 4-5 g / L of carbon source, 0.5-1 g / L of nitrogen source, and 0.3-0.5 g / L of phosphorus source to the pretreated mine water to prepare a fermentation matrix suitable for microbial growth; 3) Preparation of the first group of liquid fertilizer inoculating anaerobic sulfate-reducing bacteria into the fermentation matrix prepared in step 2), performing static constant-temperature fermentation in an anoxic environment, performing solid-liquid separation after the fermentation is completed, and removing the completely fermented residue using a medium-pore filter cloth filter with a pore size of 0.5-1 mm to obtain a first group of liquid bacterial fertilizer; 4) Preparation of the second group of liquid fertilizer Aerobic phosphate-solubilizing bacteria, potassium-solubilizing bacteria and nitrogen-fixing bacteria are inoculated into the fermentation matrix prepared in step b), and fermented at a constant temperature under an oxygen-permeable environment to obtain a second group of liquid bacterial fertilizer.

3. The preparation method according to claim 2, characterized in that Step 1) The physical precipitation method is as follows: first, the mine water is allowed to stand for 12 hours, and then filtered through a sand filter to remove the precipitate; The chemical treatment in step 1) is to add calcium-based materials to precipitate sulfate ions.

4. The preparation method according to claim 3, characterized in that The calcium-based material is lime or calcium carbonate.

5. The preparation method according to claim 2, characterized in that Step 2) The carbon source is acetic acid, glucose, lactic acid, propionic acid or starch; the nitrogen source is ammonium nitrate, urea, ammonium chloride, sodium nitrate or ammonia water; and the phosphorus source is potassium dihydrogen phosphate, calcium phosphate, tricalcium phosphate or sodium phosphate.

6. The preparation method according to claim 2, characterized in that Step 3) The fermentation conditions of the anaerobic sulfate-reducing bacteria in the first group of liquid fertilizer are as follows: temperature 25-35°C, pH 6.5-7.5, fermentation time 5-7 days, and the concentration of live bacteria in the bacterial solution is ≥10 6 CFU / g.

7. The preparation method according to claim 2, characterized in that Step 4) The fermentation conditions of aerobic phosphate-solubilizing bacteria, potassium-solubilizing bacteria and nitrogen-fixing bacteria in the second group of liquid fertilizer are as follows: temperature 25-35°C, pH 6.5-7.5, and shaking fermentation or continuous stirring fermentation in an aerobic environment for 3-5 days until the total concentration of viable bacteria in the bacterial liquid is ≥10 6 CFU / g.

8. A method for treating contaminated soil in coal mining areas, characterized in that: The steps include: 1) Dilute the first group of liquid microbial fertilizers prepared by using high-salt mine water as claimed in claim 1 to 1×10 6 CFU / g, applied to contaminated soil in coal mining areas by spraying; 2) After 30 days of spraying the first group of liquid fertilizer, the second group of liquid fertilizer in the freshly prepared microbial liquid fertilizer prepared by using high-salt mine water as claimed in claim 1 or 2 was diluted to 1×10 6 CFU / g were applied to the contaminated soil in the same coal mining area by spraying or irrigation.

9. The method for treating contaminated soil in coal mining areas according to claim 8, characterized in that: Step 1) The spraying method has a spraying volume of 2-5 liters / square meter of soil, a spraying time of 5-10 minutes, spraying once every 3 days, and spraying 10 times; Step 2) The spraying method has a spraying volume of 2-5 liters per square meter of soil, a spraying time of 5-10 minutes, spraying once every 7 days, and spraying 10 times.

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