Microbial liquid bacterial fertilizer prepared from high-salt mine gushing water, preparation method of microbial liquid bacterial fertilizer and method for treating polluted soil in coal mine area

Abstract

The invention provides a microbial liquid bacterial fertilizer prepared from high-salt mine gushing water, a preparation method of the microbial liquid bacterial fertilizer and application of the microbial liquid bacterial fertilizer in treatment of polluted soil in a coal mine area. The microbial liquid bacterial fertilizer consists of a first group of liquid bacterial fertilizer and a second group of liquid bacterial fertilizer. The preparation method comprises the following steps: 1) pretreating mine gushing water; 2) preparing a fermentation substrate; 3) preparing a first group of liquid bacterial fertilizer; and 4) preparing a second group of liquid bacterial fertilizer. The first group of liquid bacterial manure is used for solidifying heavy metals in soil and reducing the mobility of the heavy metals; the second group of liquid bacterial fertilizer is applied after 30 days, the content of phosphorus, potassium and nitrogen in the soil is increased, and the soil fertility is improved. According to the method, the high-salt mine gushing water is effectively utilized, resource reutilization is achieved, meanwhile, the dual effects of heavy metal stabilization and soil remediation are achieved through staged bacterial fertilizer application, and an efficient solution is provided for ecological remediation of a coal mine area.

Description

Technical Field

[0001] The invention belongs to the technical field of environmental protection and mining ecological restoration, and relates to a method for preparing a microbial liquid fertilizer by utilizing high-salt mine water, and applying the microbial liquid fertilizer to the treatment and restoration of contaminated soil in coal mining areas. Background Art

[0002] Mine water is one of the main by-products produced during coal mining, especially high-salt mine water. If it is discharged directly 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 they are usually accompanied by high treatment costs and waste of resources. In addition, the soil in coal mining areas is often contaminated by coal gangue and other mineral wastes for a long time, and often faces problems such as excessive heavy metals, soil acidification or salinization, 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-fixing bacteria such as sulfate-reducing bacteria can effectively fix 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 circulation of important nutrients such as phosphorus, potassium and nitrogen in the soil, but also improves soil fertility, improves the soil environment and promotes the recovery of the ecosystem.

[0004] However, there are still many problems in the use of high-salinity mine water and the treatment of contaminated soil in coal mining areas using microbial technology: 1) Poor environmental adaptability: Traditional microbial treatment performs poorly under the conditions of high-salinity mine water. High-salinity environments often inhibit the activity of microorganisms, resulting in a significant decrease in the treatment effect, and there is a lack of effective microbial strain combinations in existing technologies to adapt to such environments. 2) Limited effect of heavy metal solidification: Many microbial treatment technologies are difficult to effectively solidify heavy metals in the soil. Even in a low-salinity environment, some microorganisms are still unable to stably adsorb or deposit heavy metals for a long time, resulting in the continued existence of heavy metal migration and biological toxicity problems. 3) Insufficient nutrient recycling: Traditional microbial treatment focuses more on removing pollutants, while paying less attention to the circulation and supplementation of soil nutrients. Treatment technologies that lack a combination of multifunctional strains make it difficult to increase the nitrogen, phosphorus, and potassium content of the soil while stabilizing heavy metals, and cannot meet the comprehensive needs of soil ecological restoration. 4) Low resource utilization: Existing microbial treatment schemes generally do not consider the reuse of high-salinity mine water, but instead remove salt through high-cost physical or chemical treatments, resulting in a waste of resources.

[0005] Therefore, it is urgent to provide a microbial technology that can effectively treat contaminated soil in coal mining areas, achieve good heavy metal solidification effects, modify contaminated soil, and realize the technical solution of reusing high-salt mine water. Summary of the invention

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

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

[0008] The third object of the present invention is to provide a method for the treatment of contaminated soil in coal mining areas, which achieves the compliant disposal of mine water, while promoting the stabilization of heavy metals in the soil and the circulation of nutrients such as phosphorus, potassium, and nitrogen, thereby improving soil fertility and providing effective technical support for the ecological restoration of coal mining areas.

[0009] In order to achieve the above object, the present invention provides a microbial liquid fertilizer group prepared by using high-salt mine water, the microbial liquid fertilizer group consists of a first group of liquid fertilizer and a second group of liquid fertilizer; wherein the first group of liquid fertilizer contains anaerobic sulfate-reducing bacteria, the concentration of the anaerobic sulfate-reducing bacteria is ≥10 6 CFU / g; The second group of liquid fertilizer contains aerobic phosphate-solubilizing bacteria, potassium-solubilizing bacteria and nitrogen-fixing bacteria, and the total concentration of aerobic phosphate-solubilizing bacteria, potassium-solubilizing bacteria and nitrogen-fixing bacteria is ≥10 6 CFU / g.

[0010] Furthermore, the anaerobic sulfate-reducing bacteria is classified and named as sulfate-reducing bacteria (Desulfovibriovulgaris) EM2, the depository is China Center for Type Culture Collection, Wuhan University, Wuhan, China, the deposit date is November 1, 2017, and the deposit number is CCTCC M 2017645;

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

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

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

[0014] The present invention also provides a method for preparing liquid microbial fertilizer using high-salt mine water, comprising the following steps:

[0015] 1) Pretreatment of mine water

[0016] Use physical precipitation to remove suspended solids from mine water; then if the salt concentration of mine water is greater than 3000 mg / L, use fresh water to dilute the mine water to a salt concentration of 2500-3000 mg / L, and then use chemical treatment to reduce the sulfate ion concentration to 500-1000 mg / L, so that the mine water is suitable as a fermentation substrate;

[0017] 2) Preparation of fermentation substrate

[0018] Adding carbon source, nitrogen source and phosphorus source to the pretreated mine water to prepare a fermentation matrix suitable for microbial growth;

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

[0020] The anaerobic sulfate-reducing bacteria are inoculated into the fermentation matrix prepared in step 2), and the fermentation is performed under anoxic conditions at a constant temperature. After the fermentation is completed, solid-liquid separation is performed, and a medium-pore filter cloth filter with a pore size of 0.5-1 mm is used to remove the completely fermented residue to obtain a first group of liquid bacterial fertilizer;

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

[0022] Aerobic phosphate-solubilizing bacteria, potassium-solubilizing bacteria and nitrogen-fixing bacteria are inoculated into the fermentation matrix prepared in step b), and constant temperature fermentation is carried out under an oxygen-permeable environment to obtain a second group of liquid bacterial fertilizers.

[0023] Furthermore, the physical precipitation method in step 1) is: firstly, the mine water is allowed to stand for 12 hours, and the precipitate is removed by filtering using a sand filter;

[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 and preventing high-concentration sulfate from interfering with the subsequent fermentation process.

[0025] Furthermore, the calcium-based material is lime or calcium carbonate.

[0026] Furthermore, in 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.

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

[0028] Furthermore, in step 3), the fermentation culture conditions of the anaerobic sulfate-reducing bacteria in the first group of liquid fertilizer are: temperature of 25-35°C, pH of 6.5-7.5, fermentation time of 5-7 days, and the concentration of live bacteria in the bacterial liquid is ≥10 6 CFU / g.

[0029] Furthermore, in step 4), the fermentation culture conditions of aerobic phosphate-solubilizing bacteria, potassium-solubilizing bacteria and nitrogen-fixing bacteria in the second group of liquid fertilizer are: 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 live bacteria in the bacterial liquid is ≥10 6 CFU / g.

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

[0031] 1) Dilute the first group of liquid bacterial fertilizers prepared from the above-mentioned microbial liquid bacterial fertilizers using high-salt mine water to 1×10 6 CFU / g, applied to contaminated soil in coal mining areas by spraying;

[0032] 2) After 30 days of spraying the first group of liquid fertilizer, the second group of liquid fertilizer freshly prepared as described above using high-salt mine water was diluted to 1×10 6 CFU / g, applied to contaminated soil in the same coal mining area by spraying or irrigation.

[0033] Furthermore, in step 1), the spraying amount of the spraying method is 2-5 liters / square meter of soil, the spraying time is 5-10 minutes, the spraying is done once every 3 days, and the spraying is done 10 times; in step 2), the spraying amount of the spraying method is 2-5 liters / square meter of soil, the spraying time is 5-10 minutes, the spraying is done once every 7 days, and the spraying is done 10 times.

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

[0035] The present invention provides a method for preparing a microbial liquid fertilizer group by utilizing high-salt mine water and a method for treating contaminated soil in a coal mining area. By using two groups of mixed bacterial 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 the heavy metals in the soil are effectively fixed and their mobility is reduced, but also the circulation of nutrients such as phosphorus, potassium and nitrogen in the soil is promoted, soil fertility is improved, and the ecological environment of the coal mining area is further improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a statistical graph of changes in the suspended matter content of mine water before and after the physical treatment in the preparation method provided by the present invention.

[0037] Figure 2A comparison chart of plant growth conditions after applying the microbial liquid fertilizer prepared using high-salt mine water provided by the present invention. DETAILED DESCRIPTION

[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 method for preparing a microbial liquid fertilizer by utilizing high-salt mine water. The microbial liquid fertilizer consists of a first group of liquid fertilizers and a second group of liquid fertilizers.

[0040] 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 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.

[0042] The anaerobic sulfate-reducing bacteria were classified and named as Desulfovibrio vulgaris EM2. The depository was China Center for Type Culture Collection, Wuhan University, Wuhan, China. The deposit date was November 1, 2017, and the deposit number was CCTCC M 2017645.

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

[0044] The potassium-solubilizing bacteria was classified as Bacillus circulans, with a collection number of CCTCC NB20083559, and was purchased from China Center for Type Culture Collection.

[0045] The nitrogen-fixing bacteria was classified as Ochrobactrum pseudintermedium, with a collection number of CCTCC AB 2015139, and was purchased from China Center for Type Culture Collection.

[0046] Embodiment 1:

[0047] The high-salinity mine water samples were collected from a coal mining area in Inner Mongolia Autonomous Region. The initial salt concentration was close to 3500 mg / L. The specific composition is shown in Table 1.

[0048] Table 1 Analysis of components of high-salt mine water samples in a coal mining area in Inner Mongolia Autonomous Region

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

[0050] 1. Pretreatment of mine water

[0051] (1) The mine water was allowed to settle for 12 hours to allow large particles of impurities to settle naturally. After sedimentation, the suspended matter was further removed by gravity filtration using a sand filter. The filtration process was carried out at room temperature, and the filtered water sample was used for the subsequent preparation of the fermentation matrix.

[0052] Since the high-salt mine water collected from coal mines contains suspended matter and large-particle impurities, it is left to stand for physical precipitation to allow the large-particle impurities to settle naturally, and then a sand and gravel filter is used to filter out the sediment, thereby reducing the suspended matter load in the water.

[0053] The sand and gravel filter is a commercial device, which contains several layers of sand and gravel with different particle sizes to ensure the removal of suspended particles layer by layer. The particle sizes of the sand and gravel are: 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] Changes in suspended matter content in mine water before and after physical treatment Figure 1 As shown. Figure 1 It can be seen that by gradually treating the mine water, the initial suspended matter content was greatly reduced from 606 mg / L to 141 mg / L, and the treatment effect was significant. After 12 hours of static sedimentation, large particles of impurities settled and the suspended matter content was reduced by 42% to 352 mg / L. This step effectively removed larger suspended particles and significantly reduced the suspended matter load in the water. Then, further filtration was performed through a sand and gravel filter to reduce the suspended matter content to 141 mg / L again, a reduction rate of 60%. The entire treatment process achieved a total reduction rate of 77% in suspended matter, 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 in the fermentation process, thereby ensuring the smooth progress of fermentation and the quality of the final product.

[0055] (2) Dilution treatment: After completing the physical treatment, the mine water with a salt concentration higher than 3000 mg / L is diluted to control the salt concentration below 3000 mg / L. This step reduces the salt concentration by adding fresh water to meet the requirements of the fermentation matrix and ensure that the growth of the bacterial flora during the fermentation process is not inhibited by high salt concentrations. In order 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 concentrations 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 colonies growing; “+” indicates very few colonies growing; “-” indicates no colony growing.

[0059] It can be seen from Table 2 that when the salt concentration exceeds 3000 mg / L, the growth rate of microorganisms is significantly reduced. Therefore, it is crucial to adjust the salt concentration to below 3000 mg / L for the fermentation matrix, which helps to maintain the activity and stability of the bacterial flora and promote 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 to below 800 mg / L, thereby 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 gushing water is mixed with fresh water in a ratio of 5:1, and the salt concentration after dilution is reduced to about 2900 mg / L, which is reduced by about 17%; the gushing water is mixed with fresh water in a ratio of 4:1, and the salt concentration after dilution is reduced to about 2800 mg / L, which is reduced by about 20%; the gushing water is mixed with fresh water in a ratio of 3:1, and the salt concentration after dilution is reduced to about 2600 mg / L, which is reduced by about 26%; the gushing water is mixed with fresh water in a ratio of 2:1, and the salt concentration after dilution is reduced to about 2300 mg / L, which is reduced by about 34%. The changes in salt concentration before and after treatment are 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 of salt concentration before and after dilution treatment of mine water

[0062]

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

[0064] (3) Chemical treatment: The diluted mine water enters the chemical treatment stage, where the calcium-based material lime (calcium oxide) is added to perform a precipitation reaction, so that the sulfate in it is separated in the form of precipitate, thereby reducing the sulfate ion concentration 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 subsequent processes.

[0065] The sulfate concentration in mine water often exceeds 1000mg / L. When the sulfate concentration exceeds 1000mg / L, it will inhibit the fermentation process of many microorganisms, especially in fermentation systems containing sensitive bacteria. For example, when the sulfate concentration is higher than 1000-1500mg / L, the activity of microorganisms such as anaerobic sulfate-reducing bacteria (SRB) will be significantly affected. Therefore, chemical treatment is used to reduce the sulfate ion concentration to below 1000mg / L to keep the anaerobic sulfate-reducing bacteria (SRB) active.

[0066] The reason why sulfate concentration interferes with microorganisms is that high concentration of sulfate in the fermentation environment will affect the normal metabolic activities of microorganisms, mainly through the following two aspects:

[0067] The first is the increase in osmotic pressure: sulfate is a strong electrolyte. At high concentrations, it will significantly increase the osmotic pressure of the fermentation matrix, forcing the water in the microbial cells to flow out, resulting in increased dehydration pressure, thereby inhibiting the activity and growth of the microorganisms.

[0068] The second is the production of inhibitory metabolites: Under anaerobic conditions, sulfate-reducing bacteria convert sulfate into hydrogen sulfide (H2S), which is a highly toxic substance that inhibits the growth of other microorganisms and may even have an adverse effect on its own bacteria. Therefore, under high sulfate concentrations, the microbial diversity and balance of the fermentation environment will be disrupted.

[0069] In this embodiment, 7 g / L of lime is added to the diluted mine water, and the mine water sample is stirred for 30 minutes and then left to stand for 24 hours. In the experiment, mine water with different salt concentrations is used for chemical treatment, and the concentration change of sulfate ions is recorded. The specific changes of sulfate ion concentration before and after treatment are shown in Table 4.

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

[0071]

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

[0073] In addition, for cost considerations, further reduction of sulfate will use more lime, and retaining 500-1000 mg / L of sulfate is beneficial for the fermentation of anaerobic sulfate-reducing bacteria EM2. Anaerobic sulfate-reducing bacteria 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 matrix

[0075] In the pretreated mine water, add 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) and phosphorus sources (monopotassium phosphate, calcium phosphate, tricalcium phosphate or sodium phosphate). These nutrients will provide the necessary growth conditions for the microbial flora. The specific amount of nutrients added should be adjusted according to the initial nutrient composition of the mine water to ensure that the nutrients in the fermentation matrix are balanced and suitable for the growth of the flora.

[0076] In this embodiment, in order to rationally prepare the fermentation matrix according to the nutritional characteristics of different mine water, 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, namely sulfate-reducing bacteria, phosphate-solubilizing bacteria, potassium-solubilizing bacteria and nitrogen-fixing bacteria, are selected to establish a mixed culture system. Under aerobic conditions, since phosphate-solubilizing bacteria, potassium-solubilizing bacteria and nitrogen-fixing bacteria are aerobic bacteria, their growth will be dominant; sulfate-reducing bacteria are facultative anaerobic bacteria and can maintain a certain growth activity under this condition. Therefore, the nutrient addition scheme adopted is based on a mixed system of four bacteria, and its overall growth performance under different nutritional levels is evaluated. The specific nutritional scheme is graded from "special low nutritional requirements" to "super high nutritional requirements" according to the nutritional status of mine water. Using OD 600The value was used as an indicator of the overall biomass of the mixed flora, and the pH value was recorded to evaluate the effects of different nutritional schemes on the flora growth environment. The specific nutritional addition schemes are shown in Table 5. The experimental conditions of each scheme were laboratory-scale fermentation with a reaction cycle of 72 hours.

[0077] Table 5 Carbon, nitrogen and phosphorus addition scheme for mine water fermentation matrix

[0078]

[0079] As can be seen from Table 5, under the conditions of "low nutritional demand" (Scheme C) and "medium nutritional demand" (Scheme D), the OD 600 The values ​​reached 0.72 and 0.85, respectively, showing the best growth conditions. These schemes provide a suitable nutritional environment for sulfate-reducing bacteria, phosphate-solubilizing bacteria, potassium-solubilizing bacteria and nitrogen-fixing bacteria, meeting their growth and metabolic needs. In addition, under these two conditions, the pH of the configured mine water was 6.5 and 6.8, respectively, both within the pH range (6.0 to 7.4) for the most suitable growth of microorganisms. In the undernourished schemes (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, undernourished nutrition could not meet the growth needs of microorganisms. On the other hand, the OD600 value of the ultra-high nutritional demand scheme (F) was 0.54. Although its pH was 7.4, excessive nutrition may cause an increase in osmotic pressure and accumulation of metabolites, resulting in inhibition of bacterial growth.

[0080] Therefore, moderate nutrient concentration is crucial for the successful preparation of fermentation substrate. The conclusion shows that the "low nutrient requirement" to "medium nutrient requirement" schemes (C and D) not only meet the nutrient requirements of the flora, 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. Combined with the initial conditions of mine water, a low nutrient requirement scheme (Scheme C) or a medium nutrient requirement scheme (Scheme D) is selected in practical applications to ensure optimal growth and metabolic activity of the flora.

[0081] 3. Inoculation and cultivation of efficient bacterial flora

[0082] (1) Inoculation and cultivation of the first group of mixed bacteria: anaerobic sulfate-reducing bacteria are inoculated into the prepared fermentation matrix. The culture conditions must be strictly controlled to ensure the growth and metabolic activity 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 anaerobic environment is created. The fermentation culture time is generally 5-7 days, and the concentration of live bacteria in the bacterial solution is ≥10 6 CFU / g, the specific time can be adjusted according to the progress of fermentation. The obtained liquid fertilizer is mainly used for the first soil treatment to promote the solidification 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 bacterial fertilizer, aerobic phosphate-solubilizing bacteria, potassium-solubilizing bacteria and nitrogen-fixing bacteria are inoculated into the new fermentation matrix. The culture conditions must be maintained in a suitable oxygen-permeable environment to ensure the effective growth of such aerobic bacteria. 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 culture time is 3-5 days until the total concentration of live bacteria in the bacterial solution is ≥10 6 CFU / g, and the obtained liquid fertilizer is used for the second soil treatment to increase the phosphorus, potassium and nitrogen content in the soil and promote the improvement of soil fertility.

[0084] The three bacteria selected by the present invention are all aerobic bacteria, and the composition of the culture medium has been optimized to support their common growth in the same environment. On this basis, suitable culture conditions are adopted to ensure that they can cooperate with each other in an aerobic environment without unfavorable competition. Because those microorganisms that cannot coexist in the environment have been eliminated through screening and optimization, what remains are strains that can grow stably together in a system. Therefore, under the culture conditions defined by the present invention, there will be no obvious dominant bacteria phenomenon, and the three bacteria can coexist and achieve the desired effect.

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

[0086] (1) First application: After fermentation, the anaerobic sulfate-reducing bacteria fermentation liquid is separated into solid and liquid, and a medium-pore filter cloth filter with a pore size of 0.5-1 mm is used to remove the incompletely fermented residue to obtain a purified liquid fertilizer. The pore size of the filter is about 0.5-1 mm, which can effectively remove the incompletely fermented residue, but will not trap the bacteria, ensuring that the active microorganisms in the fertilizer are not affected.

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

[0088] (2) Second application: 30 days after the first application of bacterial fertilizer, liquid bacterial fertilizer prepared from fermentation liquid 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 effective phosphorus, potassium and nitrogen content of the soil, improve soil fertility, and promote plant growth and ecosystem recovery.

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

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

[0091]

[0092] Depend on Figure 2 As can be seen from Table 6, after two applications of liquid bacterial fertilizer, the soil in this area has achieved significant improvements in heavy metal content and nutrient content. First, in terms of heavy metal removal, the total Cd and total Pb content of heavy metals in the soil decreased significantly, the Cd content dropped from 0.10 mg / kg to below the detection limit, and the Pb content decreased from 18.9 mg / kg to 12.0 mg / kg. This shows that sulfate-reducing bacteria have a significant effect on the fixation of toxic heavy metals in the soil, which helps to reduce their mobility and bioavailability. Although the As content increased slightly, the overall change was 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 increased from 703 mg / kg to 858 mg / kg, the available potassium increased from 0.15 g / kg to 0.17 g / kg, and the available phosphorus 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 significantly promoted the circulation of key nutrients in the soil and improved soil fertility. The increase in soil organic matter was also very significant, from 5.7 g / kg to 14.6 g / kg, indicating that the application of bacterial fertilizer has a significant effect in improving soil quality. The total number of bacteria in the soil increased from 3.9×10 5 CFU / g increased to 1.8×10 6 CFU / g, showing a significant enhancement in soil microbial activity, which will contribute to the stability and continued health of the soil ecosystem.

[0093] It can be seen from the above examples that the present invention provides a method for preparing a microbial liquid fertilizer suitable for the remediation of contaminated soil in coal mining areas by using high-salt mine water as a microbial fermentation matrix. By using two groups of mixed bacterial 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 the heavy metals in the soil are effectively fixed and their mobility is reduced, but also the circulation of nutrients such as phosphorus, potassium and nitrogen in the soil is promoted, soil fertility is improved, and the ecological environment of the coal mining area is further improved.

[0094] The above-mentioned embodiments only express the implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached 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.

2. The microbial liquid fertilizer prepared by using high-salt mine water according to claim 1, characterized in that: The anaerobic sulfate-reducing bacteria is classified and named as Desulfovibrio vulgaris EM2, and the depository is China Center for Type Culture Collection, located at Wuhan University, Wuhan, China. The deposit date is November 1, 2017, and the deposit number is CCTCC M 2017645; The phosphate-solubilizing bacteria is classified and named as Ochrobactrum intermedium, and its deposit number is CCTCC AB 2015151; The potassium-dissolving bacteria is classified and named as Bacillus circulans, and its deposit number is CCTCC NB20083559; The nitrogen-fixing bacteria is classified and named as Ochrobactrum pseudintermedium, and its preservation number is CCTCC AB 2015139.

3. A method for preparing microbial liquid fertilizer using high-salt mine water according to claim 1 or 2, 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 mine water is greater than 3000 mg / L, use fresh water to dilute the mine water to a salt concentration of 2500-3000 mg / L, and then use chemical treatment to reduce the sulfate ion concentration to 500-1000 mg / L; 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 The anaerobic sulfate-reducing bacteria are inoculated into the fermentation matrix prepared in step 2), and the fermentation is performed under anoxic conditions at a constant temperature. After the fermentation is completed, solid-liquid separation is performed, and a medium-pore filter cloth filter with a pore size of 0.5-1 mm is used to remove the completely fermented residue 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 constant temperature fermentation is carried out under an oxygen-permeable environment to obtain a second group of liquid bacterial fertilizers.

4. The preparation method according to claim 3, 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 using a sand filter to remove the precipitate; Step 1) The chemical treatment is to add calcium-based materials to precipitate sulfate ions.

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

6. The preparation method according to claim 3, 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; the phosphorus source is potassium dihydrogen phosphate, calcium phosphate, tricalcium phosphate or sodium phosphate.

7. The preparation method according to claim 3, 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 of 25-35°C, pH of 6.5-7.5, fermentation time of 5-7 days, and the concentration of live bacteria in the bacterial liquid is ≥10 6 CFU / g.

8. The preparation method according to claim 3, characterized in that: Step 4) The fermentation culture 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 live bacteria in the bacterial liquid is ≥10 6 CFU / g.

9. A method for treating contaminated soil in coal mining areas, characterized in that: The steps include: 1) diluting the first group of liquid bacterial fertilizers prepared by using high-salt mine water as claimed in claim 1 or 2 to 1×10 6 CFU / g, applied to contaminated soil in coal mining areas by spraying; 2) 30 days after the first group of liquid fertilizer was sprayed, 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.

10. The method for treating contaminated soil in coal mining areas according to claim 9, 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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