A composite microbial agent and its preparation method and application

Abstract

The present invention discloses a composite microbial agent and its preparation method and application. The composite microbial agent comprises Pseudomonas alcaligenes, Citrobacter rodinii and Micrococcus. The preparation method comprises the following steps: culturing Pseudomonas alcaligenes, Citrobacter rodinii and Micrococcus respectively; collecting the bacterial bodies respectively and drying them to prepare Pseudomonas alcaligenes powder, Citrobacter rodinii powder and Micrococcus powder; counting the Pseudomonas alcaligenes powder, Citrobacter rodinii powder and Micrococcus powder respectively, and mixing the Pseudomonas alcaligenes powder, Citrobacter rodinii powder and Micrococcus powder according to the ratio of live bacteria to obtain the composite microbial agent. When the composite microbial agent in the present invention is used to repair the acidified soil in the coal mining area, the pH of the acidified soil can be quickly increased in a short period of time, and the repair effect can be maintained, and the pH of the acidified soil can be continuously increased to neutral, so as to achieve a good soil repair effect.

Description

Technical Field

[0001] The present invention relates to a soil remediation microbial preparation and a preparation method thereof, and in particular to a composite microbial agent for remediating acidic soil in coal mining areas and a preparation method and application thereof. Background Art

[0002] Coal mining causes many ecological problems, one of which is the acidification of surrounding water bodies and soil. The reason for this phenomenon is that a large amount of acidic wastewater containing aluminum, iron and sulfur is discharged during coal mining. At the same time, the solid waste generated during coal mining is rich in acidic components such as quartz, clay, iron-rich dolomite, silicide and iron sulfide. Therefore, the soil pH value in the coal mining area and the spoil dump is low. Compared with before mining, coal mining activities will reduce the soil pH value. Soil acidification refers to the process in which the soil absorption complex accepts a certain amount of exchangeable hydrogen ions or aluminum ions, causing the alkaline (base) ions in the soil to be leached, while the pH continues to decrease and the exchangeable acid in the soil continues to increase. It is a natural process that accompanies the occurrence and development of the soil, a manifestation of soil degradation, and a long natural process. The acidification of the surrounding soil caused by coal mining is not conducive to future vegetation restoration and ecological improvement.

[0003] The remediation measures for the above-mentioned soil acidification generally include: biological improvement and application of soil conditioners. Among them, the application of soil conditioners is generally accepted due to its relatively simple operation, quick effect and low cost. Commonly used soil conditioners mainly include alkaline inorganic conditioners such as lime, microbial agent conditioners and organic conditioners such as biochar. Microbial agent is also a commonly used acidified soil conditioner. The application of microbial agent can significantly improve soil fertility, increase soil pH, promote plant growth, and effectively improve soil acidification.

[0004] The screening and compounding of microbial strains are extremely important for the soil remediation effect of microbial agent improvers. Although some existing microorganisms have been proven in the laboratory to be able to secrete alkaline substances to neutralize acidity, these microorganisms do not show excellent neutralization and remediation performance in the acidic soil of coal mining areas, making these microbial agents unable to meet the remediation needs of acidic soil in coal mining areas. The possible reason is that these strains cannot stably colonize in the acidic soil environment of coal mining areas, and their growth is inhibited, and thus they cannot secrete sufficient alkaline substances. Therefore, how to make full use of existing microbial strains to develop a composite microbial strain suitable for the remediation of acidic soil in coal mining areas has become a technical problem that needs to be solved urgently. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a composite microbial agent to solve the problem that existing microbial strains cannot meet the needs of remediation of acidic soil in coal mining areas. Another purpose of the present invention is to propose a preparation method of a composite microbial agent to solve the problem of how to obtain the composite microbial agent. The third purpose of the present invention is to provide an application of a composite microbial agent in the remediation of acidified soil in coal mining areas to solve the problem of how to use the composite microbial agent to remediate acidified soil in coal mining areas.

[0006] Technical solution: The composite microbial agent described in the present invention comprises Pseudomonas alcaligenes, Citrobacter rodentium, and Micrococcus.

[0007] The present invention can give full play to the acid neutralization characteristics of the three microorganisms by rationally compounding the three microorganisms, using Citrobacter and Micrococcus to quickly neutralize acidic substances and fix metal ions in the early stage, creating an excellent colonization environment for Pseudomonas alcaligenes, and Pseudomonas alcaligenes continuously secretes alkaline substances after colonization in the middle and late stages, continuously neutralizes and repairs acidic soil, and finally achieves excellent repair effect. The three microorganisms have a good synergistic effect, solving the problem of poor repair effect of a single microorganism.

[0008] Preferably, the ratio of the viable counts of Pseudomonas alcaligenes, Citrobacter rodentium and Micrococcus is 1-3:1-3:1-3. The ratio is preferably 1:3:1.

[0009] Preferably, the Pseudomonas alcaligenes is a strain with a preservation number of CICC 20696, the Citrobacter rodentium is a strain with a preservation number of CICC 21596, and the Micrococcus is a strain with a preservation number of CICC 10641.

[0010] Preferably, the total number of viable bacteria in the bacterial agent is ≥ 10 9 cfu / g.

[0011] In order to obtain the above-mentioned composite microbial agent, the present invention discloses a preparation method of the composite microbial agent, comprising the following steps:

[0012] (1) Cultivating Pseudomonas alcaligenes, Citrobacter rodentium and Micrococcus respectively;

[0013] (2) collecting the bacterial cells and drying them to prepare Pseudomonas alcaligenes powder, Citrobacter powder and Micrococcus powder;

[0014] (3) After counting the number of Pseudomonas alcaligenes powder, Citrobacter powder and Micrococcus powder respectively, the Pseudomonas alcaligenes powder, Citrobacter powder and Micrococcus powder are mixed according to the ratio of viable bacteria count to obtain a composite microbial agent.

[0015] The invention further discloses a method for repairing acidified soil in coal mining areas by using the composite microbial agent.

[0016] Preferably, the above method comprises the following steps:

[0017] (1) dissolving the composite microbial agent in water to prepare a bacterial solution;

[0018] (2) Regularly spray the bacterial solution onto the surface of the acidified soil.

[0019] Preferably, in step (1), the mass ratio of the composite microbial agent to water is 1-100:1000.

[0020] Preferably, in step (2), the bacterial solution is sprayed at 5-15 mL / m 2 Spray the bacterial solution evenly on the surface of the acidified soil in a ratio of .

[0021] Preferably, in step (2), the periodicity is every 5-15 days.

[0022] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: when the composite microbial agent in the present invention is used to repair acidified soil in coal mining areas, the pH of the acidified soil can be quickly increased in a short period of time, and the repair effect can be maintained, and the pH of the acidified soil can be continuously increased to neutral, thereby achieving a good soil repair effect. DETAILED DESCRIPTION

[0023] The technical solution of the present invention is further described below.

[0024] The test materials used in the examples of the present invention are all conventional test materials in the art and can be purchased through normal commercial channels.

[0025] Example 1: A composite microbial agent, comprising Pseudomonas alcaligenes, Citrobacter sp. and Micrococcus in a ratio of 1:3:1. Pseudomonas alcaligenes, Citrobacter sp. and Micrococcus used in this example were purchased from the China Industrial Microbiological Culture Collection Center. The collection number of Pseudomonas alcaligenes is CICC 20696, the collection number of Citrobacter sp. is CICC 21596, and the collection number of Micrococcus is CICC 10641. The total number of live bacteria in the agent is 10 9 cfu / g.

[0026] The preparation method of the composite microbial agent comprises the following steps:

[0027] (1) Prepare culture media for Pseudomonas alcaligenes, Citrobacter rodentium and Micrococcus according to the following formulas:

[0028] Pseudomonas alcaligenes culture medium: yeast extract 5.0 g, sodium lactate 5.0 g, magnesium sulfate heptahydrate 0.975 g, dipotassium hydrogen phosphate 0.02 g, sodium sulfate 0.994 g, calcium chloride 0.051 g, agar 15.0 g, deionized water 1000 mL, pH 7.0.

[0029] Citrobacter culture medium: 3 g tryptone, 10 g yeast extract powder, 5 g sodium chloride, 1000 mL deionized water, 15.0 g agar powder, pH 7.0.

[0030] Micrococcus culture medium: 3 g tryptone, 10 g yeast extract powder, 5 g sodium chloride, 1000 mL deionized water, 15.0 g agar powder, pH 7.0.

[0031] (2) Cultivating Pseudomonas alcaligenes, Citrobacter rodentium and Micrococcus and preparing microbial powders:

[0032] After being activated, Pseudomonas alcaligenes is inoculated into a Pseudomonas alcaligenes culture medium at a volume ratio of 5%, and fermented and cultured at 30°C for 48 hours. The bacteria are collected by centrifugation, a protective agent is added, and the bacteria are freeze-dried to prepare Pseudomonas alcaligenes powder.

[0033] After activating Citrobacter, 5% (volume ratio) was inoculated into Citrobacter culture medium, fermented and cultured at 30°C for 48 hours, the bacteria were collected by centrifugation, a protective agent was added, and freeze-dried to prepare Citrobacter powder.

[0034] The activated Micrococcus is inoculated into a Micrococcus culture medium at 5% (volume ratio), fermented and cultured at 30° C. for 48 hours, the bacteria are collected by centrifugation, a protective agent is added, and freeze-dried to prepare a Micrococcus powder.

[0035] (3) After counting the number of Pseudomonas alcaligenes powder, Citrobacter powder and Micrococcus powder respectively, the Pseudomonas alcaligenes powder, Citrobacter powder and Micrococcus powder are mixed according to the ratio of viable bacteria count to obtain a composite microbial agent.

[0036] Embodiment 2: The rest is the same as Embodiment 1, except that:

[0037] The ratio of the number of live bacteria of Pseudomonas alcaligenes, Citrobacter and Micrococcus in the composite microbial agent is 3:1:3. The total number of live bacteria in the agent is 10 10 cfu / g.

[0038] Embodiment 3: The rest is the same as Embodiment 1, except that:

[0039] The ratio of the live counts of Pseudomonas alcaligenes, Citrobacter rodentium and Micrococcus in the composite microbial agent is 2:2:1.

[0040] Example 4: The composite microbial agent prepared in Example 1 is used to repair the acidified soil in the coal mining area, and the method is as follows:

[0041] (1) dissolving the composite microbial agent in water to prepare a bacterial solution at a mass ratio of the composite microbial agent to water of 50:1000;

[0042] (2) 10 mL / m 2 Spray the bacterial solution evenly on the surface of the acidified soil in a ratio of. Spray the bacterial solution evenly on the surface of the acidified soil.

[0043] The pH value and main pollutant content of the acidified soil in the coal mining area in this example are as follows:

[0044] Table 1 pH values ​​and main pollutant contents of acidified soil in coal mining areas

[0045]

[0046] Example 5: The composite microbial agent prepared in Example 2 is used to repair the acidified soil in the coal mining area, and the method is as follows:

[0047] (1) dissolving the composite microbial agent in water at a mass ratio of composite microbial agent to water of 1:1000 to prepare a bacterial solution;

[0048] (2) 15 mL / m 2 Spray the bacterial solution evenly on the surface of the acidified soil at a ratio of . Repeat the spraying every 5 days.

[0049] The acidified soil in the coal mining area in this embodiment is the same as that in Example 4.

[0050] Example 6: The composite microbial agent prepared in Example 3 is used to repair the acidified soil in the coal mining area, and the method is as follows:

[0051] (1) dissolving the composite microbial agent in water at a mass ratio of composite microbial agent to water of 100:1000 to prepare a bacterial solution;

[0052] (2) 5 mL / m 2 Spray the bacterial solution evenly on the surface of the acidified soil at a ratio of . Repeat the spraying every 15 days.

[0053] The acidified soil in the coal mining area in this embodiment is the same as that in Example 4.

[0054] Comparative Example 1: The rest is the same as Example 4, except that:

[0055] Replace the composite microbial agent with Pseudomonas alcaligenes powder, with a total viable count of 109 cfu / g.

[0056] Comparative Example 2: The rest is the same as Example 4, except that:

[0057] Replace the composite microbial agent with Citrobacter acidophilus powder, with a total viable count of 10 9 cfu / g.

[0058] Comparative Example 3: The rest is the same as Example 4, except that:

[0059] Replace the composite microbial agent with micrococcal powder, with a total viable count of 10 9 cfu / g.

[0060] Comparative Example 4: The rest is the same as Example 4, except that:

[0061] The alkaline Pseudomonas powder was replaced with the alkaline Pseudomonas powder with the deposit number of CICC 23069 (hereinafter referred to as bacteria A), and the total viable count of the composite microbial agent was 10 9 The preparation method of the bacteria A powder is the same as that of the Pseudomonas alcaligenes powder in Example 1.

[0062] Comparative Example 5: The rest is the same as Example 4, except that:

[0063] The Citrobacter rodi powder was replaced with Citrobacter rodi powder with a preservation number of CICC10296 (hereinafter referred to as bacteria B), and the total viable count of the composite microbial agent was 10 9 The preparation method of Bacteria B powder is the same as that of Citrobacter acidophilus powder in Example 1.

[0064] Comparative Example 6: The rest is the same as Example 4, except that:

[0065] The micrococcal powder was replaced with micrococcal powder with a preservation number of CICC 24055 (hereinafter referred to as micrococcal C), and the total viable count of the composite microbial agent was 10 9 The preparation method of the micrococcal powder of bacteria C is the same as that of the micrococcal powder of Example 1.

[0066] Comparative Example 7: The rest is the same as Example 4, except that:

[0067] No alkaline-producing Pseudomonas powder is added to the composite microbial agent, and the total number of live bacteria in the composite microbial agent is 10 9 cfu / g, the ratio of the live counts of Citrobacter and Micrococcus in the composite microbial agent is 3:1.

[0068] Comparative Example 8: The rest is the same as Example 4, except that:

[0069] No Citrobacter acidophilus powder is added to the composite microbial agent, and the total number of live bacteria in the composite microbial agent is 10 9 cfu / g, the ratio of the number of live bacteria of Pseudomonas alcaligenes and Micrococcus in the composite microbial agent is 1:1.

[0070] Comparative Example 9: The rest is the same as Example 4, except that:

[0071] No micrococcal powder is added to the composite microbial agent, and the total number of live bacteria in the composite microbial agent is 10 9 cfu / g, the ratio of the number of live bacteria of Pseudomonas alcaligenes and Citrobacter rodentium in the composite microbial agent is 1:3.

[0072] Comparative Example 10: The rest is the same as Example 4, except that:

[0073] The ratio of the live counts of Pseudomonas alcaligenes, Citrobacter rodentium and Micrococcus in the composite microbial agent is 1:15:5.

[0074] Comparative Example 11: The rest is the same as Example 4, except that:

[0075] The ratio of the live counts of Pseudomonas alcaligenes, Citrobacter rodentium and Micrococcus in the composite microbial agent is 20:1:1.

[0076] In order to test the soil improvement effect of the composite microbial agent, the physical and chemical indicators of the acidified soil in the coal mining area treated with Examples 4-6 and Comparative Examples 1-11 were tested every other day after spraying the composite microbial solution. The results are as follows:

[0077] Table 2 Effects of different microbial agents on pH value of acidified soil in coal mining areas

[0078]

[0079]

[0080] The values ​​in Table 2 are the average values ​​of three different sampling points in the bacterial liquid spraying area.

[0081] Table 3 Retention rate of different microorganisms in acidified soil in coal mining area during improvement process

[0082]

[0083]

[0084] The calculation formula for the retention rate of microorganisms in Table 3 is:

[0085] Microbial retention rate = (actual number of live bacteria / initial total number of live bacteria of microorganisms) * 100%.

[0086] Among them, the measured number of live bacteria is the measured number of live microorganisms in the soil after spraying the bacterial solution, and the initial total number of live microorganisms is the number of live microorganisms in the composite microbial agent used to prepare the bacterial solution.

[0087] The results in Tables 2 and 3 show that the pH of Comparative Examples 1-3 changed little within 10 days after the bacterial liquid was sprayed, indicating that a single strain could hardly affect the pH value of the acidified soil in the coal mining area. This may be because the single strain lacked the auxiliary effect of other strains and could not colonize or adapt to the acidified soil in the coal mining area, resulting in very little secretion of alkaline substances and failure to improve the soil. The pH increase of the soil treated in Comparative Examples 4 and 7 in the first 4 days was similar to that in Example 4, mainly because Citrobacter and Micrococcus secreted a large amount of alkaline substances and improved the soil environment in the early stage of soil improvement, resulting in a rapid increase in the soil pH after the bacterial liquid was sprayed, but at 6-10 days, the pH value of the soil dropped rapidly, because the acidified soil in the coal mining area was not suitable for the continuous colonization of Citrobacter and Micrococcus, and after the death of Citrobacter and Micrococcus provided in the composite microbial agent, there was no subsequent microorganism to continuously improve the soil, which ultimately led to the failure of soil improvement. The improvement effect of Comparative Examples 5 and 6 is also significantly inferior to that of Example 4, because only when Citrobacter and Micrococcus are combined can they synergistically promote the secretion of alkaline substances, prompting the rapid increase of pH in the early stage of improvement. When one of the two is replaced by other strains, it is impossible to form an environment suitable for the colonization of Pseudomonas alcaligenes in a short time, that is, the pH increase is insufficient, which makes it difficult for Pseudomonas alcaligenes to colonize and continuously improve acidic soil, so it is manifested as a rapid decrease in pH in the later stage of improvement. Similarly, since only one of Citrobacter and Micrococcus is used in Comparative Examples 8 and 9, it is also impossible to form an environment suitable for the colonization of Pseudomonas alcaligenes in the early stage of improvement, which ultimately leads to improvement failure. In Comparative Example 10, since the number of Citrobacter and Micrococcus is much higher than that of Pseudomonas alcaligenes, even if an environment suitable for the colonization of Pseudomonas alcaligenes is formed in the early stage of the improvement, the initial number of Pseudomonas alcaligenes is too small, and Citrobacter and Micrococcus can only maintain an environment suitable for the colonization of Pseudomonas alcaligenes for a short time, which makes it difficult for Pseudomonas alcaligenes to colonize quickly and form a sufficient number of flora, which in turn leads to the lack of sufficient continuous production of alkaline substances. Therefore, after 6 days, the soil pH is greatly reduced. Although there is a sufficient amount of Pseudomonas alcaligenes in Comparative Example 11, there are too few Citrobacter and Micrococcus, which makes it difficult to quickly increase the pH and form an environment suitable for the colonization of Pseudomonas alcaligenes. Therefore, after 6 days, Pseudomonas alcaligenes, Citrobacter and Micrococcus cannot colonize or survive, so the improvement ultimately fails.

Claims

1. A composite microbial agent, characterized in that: Contains Pseudomonas alcaligenes, Citrobacter rodentium, Micrococcus; The ratio of the viable counts of Pseudomonas alcaligenes, Citrobacter rodentium, and Micrococcus is 1-3:1-3:1-3; The Pseudomonas alcaligenes is a strain with a preservation number of CICC 20696, the Citrobacter rodentium is a strain with a preservation number of CICC21596, and the Micrococcus is a strain with a preservation number of CICC 10641.

2. The composite microbial agent according to claim 1, characterized in that: The total number of viable bacteria in the inoculant is ≥10 9 cfu / g.

3. The method for preparing the composite microbial agent according to claim 1, characterized in that: The steps include: (1) Cultivate Pseudomonas alcaligenes, Citrobacter rodentium and Micrococcus respectively; (2) collecting the bacterial cells separately and drying them to prepare Pseudomonas alcaligenes powder, Citrobacter powder and Micrococcus powder; (3) After counting the number of Pseudomonas alcaligenes powder, Citrobacter powder and Micrococcus powder respectively, the Pseudomonas alcaligenes powder, Citrobacter powder and Micrococcus powder are mixed according to the ratio of live bacteria count to obtain a composite microbial agent.

4. Use of the composite microbial agent according to claim 1 in the remediation of acidified soil in coal mining areas.

5. The use according to claim 4, characterized in that: The steps include: (1) dissolving the composite microbial agent in water to prepare a bacterial solution; (2) Regularly spray the bacterial solution onto the surface of the acidified soil.

6. The use according to claim 5, characterized in that: In step (1), the mass ratio of the composite microbial agent to water is 1-100:1000.

7. The use according to claim 6, characterized in that: In step (2), the bacterial solution is sprayed at 5-15 mL / m 2 Spray the bacterial solution evenly on the surface of the acidified soil in a ratio of .

8. The use according to claim 6, characterized in that: In step (2), the regular period is every 5-15 days.

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