A method for combining plant-microbial agent-biochar to repair high-molybdenum contaminated soil

Abstract

A method for the combined restoration of high-molybdenum contaminated soil by plant-microorganism-biochar belongs to the technical field of soil restoration, and specifically relates to a method for the combined restoration of high-molybdenum contaminated soil by plant-microorganism-biochar. The purpose of the present invention is to solve the problem that the reports on existing molybdenum-contaminated soil treatment methods are extremely limited, and there is a lack of a bioremediation method suitable for heavy metal molybdenum in cold areas. Method: Disperse biochar evenly into the high-molybdenum contaminated soil, then sprinkle ryegrass seeds into the high-molybdenum contaminated soil, cover the top with soil, water the soil regularly to keep it moist, and then pour the liquid microorganism into the roots of the ryegrass, cultivate at room temperature or low temperature, and the restoration of the high-molybdenum contaminated soil is completed. Using the method of the present invention, under low temperature (10°C), the content of readily usable molybdenum is reduced by 32.2%. The present invention can obtain a method for the combined restoration of high-molybdenum contaminated soil by plant-microorganism-biochar.

Description

Technical Field

[0001] The present invention belongs to the technical field of soil remediation, and in particular relates to a method for combining plants, bacterial agents and biochar to remediate high-molybdenum contaminated soil. Background Art

[0002] As a rare heavy metal, molybdenum has a high melting point, making it an important raw material for various industries. Its large-scale mining and use will lead to molybdenum pollution. Molybdenum that enters the soil will continue to be transmitted and accumulated along the food chain. When the content of forage is higher than 2 mg / kg, it will pose a threat to animals. Therefore, the control of soil molybdenum pollution has gradually attracted widespread attention from researchers. However, since the harm of molybdenum to humans is far less than its harm to ruminants, and its frequency of occurrence is lower than that of other heavy metal pollution, reports on molybdenum-contaminated soil treatment methods are extremely limited. In addition, the current research on molybdenum mainly focuses on its impact on crop yields as a trace element, and there are fewer studies on molybdenum as a pollutant. Most of the research focuses on the reduction effect of microorganisms on it. There are only a few studies on molybdenum removal, so it is urgent to propose a remediation plan for molybdenum-contaminated soil in cold regions. Summary of the invention

[0003] The purpose of the present invention is to solve the problem that the reports on existing molybdenum-contaminated soil treatment methods are extremely limited and there is a lack of a bioremediation method suitable for heavy metal molybdenum in cold regions, and to provide a method for the combined restoration of high-molybdenum-contaminated soil by plants, bacterial agents and biochar.

[0004] A method for combining plant-microbial agent-biochar to repair high-molybdenum contaminated soil is completed by the following steps:

[0005] Disperse biochar evenly into high-molybdenum contaminated soil, then sprinkle ryegrass seeds into the high-molybdenum contaminated soil, cover the top with soil, water regularly to keep the soil moist, then pour liquid bacterial agent into the roots of ryegrass, and cultivate at room temperature or low temperature to complete the restoration of high-molybdenum contaminated soil;

[0006] The strain in the liquid bacterial agent is Raoultella ornithinolytica A1, which is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is May 26, 2022, and the deposit number is CGMCC No. 24973.

[0007] Morphological characteristics of the ornithine-degrading Raoult strain: The strain was streaked on beef extract peptone medium and cultured at 30°C for 24 hours. The colonies were regular circles with a diameter of about 0.5 mm, white in color, with a smooth, convex, moist, opaque and viscous surface.

[0008] The present invention has the following beneficial effects:

[0009] 1. The present invention significantly increases the biomass of plants, effectively reduces the content of molybdenum in the soil, and is conducive to the transformation of molybdenum in the soil into a stable form, with a good restoration effect;

[0010] 2. Under low temperature (10℃), although plant growth is inhibited and the total removal rate of soil molybdenum is reduced, the molybdenum in the soil is more likely to transform into a stable state;

[0011] 3. By adopting the method of the present invention, under low temperature (10° C.) conditions, it is relatively easy to reduce the content of active molybdenum by 32.2%.

[0012] The present invention can obtain a method for jointly repairing high-molybdenum contaminated soil by using plants, bacterial agents and biochar.

[0013] The present invention also provides a method for screening and identifying the above-mentioned Raoultella ornithinesolytica, comprising the following steps: enriching and culturing the collected soil in sequence, and screening step by step to obtain the Raoultella ornithinesolytica;

[0014] The soil was collected from the topsoil of a garbage recycling station in Harbin. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 A statistical diagram of the average molybdenum content in the soil after restoration using the embodiment;

[0016] Figure 2 A statistical chart showing the proportion of each form of molybdenum in the soil after restoration using the embodiment;

[0017] Figure 3 A statistical diagram of pH in the rhizosphere soil of plants after restoration using the embodiment;

[0018] Figure 4 A statistical diagram of the length of the aboveground part of ryegrass after restoration using the embodiment. DETAILED DESCRIPTION

[0019] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the essence of the present invention, modifications and substitutions made to the methods, steps or conditions of the present invention all fall within the scope of the present invention.

[0020] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the essence of the present invention, modifications and substitutions made to the methods, steps or conditions of the present invention all fall within the scope of the present invention.

[0021] Specific implementation method 1: This implementation method is a screening and identification of ornithine-degrading Raoult strains, comprising the following steps:

[0022] (I) Enrichment and screening: Surface soil was collected from a garbage station in Harbin to obtain soil samples. 5 g of soil sample was placed in 45 mL of beef extract peptone medium and cultured in a shaker at 30 °C and 200 r / min for 24 h to enrich the strains. 2 mL of the enriched culture medium was added to a MoO4-containing 2- The concentration of beef extract peptone medium is 100 mg / L. When the enrichment bottle becomes turbid, take 1 mL of enrichment culture medium and add it to the MoO4 2- The concentration of beef extract peptone medium is 200 mg / L, and MoO4 is gradually increased. 2- The concentration reached 3000 mg / L, and the enriched culture solution was added to sterile saline for 10 -1 ~10 -9 9 gradient dilutions were prepared, and 20 μL of the dilution solution was applied to the MoO4 2- The solid plate was inverted and incubated at 30°C for 3 days. The white single colony on the plate was selected and grown on the plate containing MoO4 2- The solid beef extract peptone plates were streaked three times to obtain pure bacteria, which were numbered and preserved in glycerol.

[0023] The beef extract peptone culture medium is formulated as follows: sodium molybdate, 3.0 g beef extract, 10.0 g protein, 5.0 g NaCl, 1000 mL water, pH 7.4-7.6, sterilized at 120° C. for 30 min;

[0024] The MoO4 2- The solid beef extract peptone formula is: 3000 mg sodium molybdate, 15.0 g agar, 3.0 g beef extract, 10.0 g protein, 5.0 g NaCl, 1000 mL water, pH 7.4-7.6, sterilized at 120°C for 30 min.

[0025] (II) Identification: The white strain obtained by screening was sent to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. for sequencing. The nucleotide sequence is as follows:

[0026]

[0027] The sequencing results were compared by BLAST on the NCBI website and confirmed to be Raoultella ornithinolytica, as shown in Table 1;

[0028] Table 1

[0029]

[0030]

[0031] Specific implementation method 2: This implementation method is a method for combining plant-microbial agent-biochar to repair high-molybdenum contaminated soil, which is completed by the following steps:

[0032] Disperse biochar evenly into high-molybdenum contaminated soil, then sprinkle ryegrass seeds into the high-molybdenum contaminated soil, cover the top with soil, water regularly to keep the soil moist, then pour liquid bacterial agent into the roots of ryegrass, and cultivate at room temperature or low temperature to complete the restoration of high-molybdenum contaminated soil;

[0033] The strain in the liquid bacterial agent is Raoultella ornithinolytica A1, which is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, with the deposit address being No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit date being May 26, 2022, and the deposit number being CGMCC No. 24973. The other steps are the same as those in the first embodiment.

[0034] Specific implementation method 3: This implementation method is different from specific implementation method 1 or 2 in that the particle size of the biochar is 200-250 meshes. The other steps are the same as those of specific implementation method 1 or 2.

[0035] Specific embodiment 4: This embodiment differs from specific embodiments 1 to 3 in that the preparation method of the liquid bacterial agent is:

[0036] 1. Inoculate the culture medium with glycerol-preserved Raoultella ornithinolytica A1, the inoculation amount of Raoultella ornithinolytica A1 being 1%;

[0037] The culture medium formula described in step 1 is: 3.0 g beef extract, 10.0 g peptone, 5.0 g NaCl, 1000 mL water, pH 7.4-7.6, sterilized at 120°C for 30 min;

[0038] 2. Place the culture medium inoculated with Raoultella ornithinolytica A1 in a shaker and shake at 30° C. for 24 hours to obtain a liquid bacterial agent. The other steps are the same as those in the first to third embodiments.

[0039] Specific implementation method 5: This implementation method is different from specific implementation methods 1 to 4 in that the mass ratio of the biochar to the high-molybdenum contaminated soil is (6-8): 100. The other steps are the same as those of specific implementation methods 1 to 4.

[0040] Specific implementation method 6: The difference between this implementation method and specific implementation methods 1 to 5 is that the sowing rate of ryegrass seeds is 20g / m 2 ~25g / m 2 The other steps are the same as those in Specific Embodiments 1 to 5.

[0041] Specific embodiment 7: The difference between this embodiment and specific embodiments 1 to 6 is that the mass ratio of the bacterial content in the liquid bacterial agent to the high-molybdenum contaminated soil is (1×10 7 CFU~1.2×10 7 CFU): 1g. The other steps are the same as those in the first to sixth embodiments.

[0042] Specific embodiment 8: This embodiment differs from specific embodiments 1 to 7 in that the number of days for room temperature culture is 40 to 50 days. The other steps are the same as those of specific embodiments 1 to 7.

[0043] Specific embodiment 9: This embodiment differs from specific embodiments 1 to 8 in that the low-temperature culture temperature is 10° C. The other steps are the same as those of specific embodiments 1 to 8.

[0044] The following examples are used to verify the beneficial effects of the present invention:

[0045] Example 1: A method for combining plant-microbial agent-biochar to repair high-molybdenum contaminated soil at room temperature (H-A1-B) is completed by the following steps:

[0046] Evenly disperse the biochar into the high-molybdenum contaminated soil, then sprinkle ryegrass seeds into the high-molybdenum contaminated soil, cover the top with soil, water the soil regularly to keep it moist, and then pour the liquid bacterial agent into the roots of the ryegrass. Cultivate at room temperature for 40 days to complete the restoration of the high-molybdenum contaminated soil.

[0047] The strain in the liquid bacterial agent is Raoultella ornithinolytica A1, which is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is May 26, 2022, and the deposit number is CGMCC No. 24973.

[0048] The high-molybdenum contaminated soil was purchased from Harbin Hongli Flower Base, where the total molybdenum concentration was 800 mg / kg;

[0049] The biochar was purchased from Beishankou Industrial Park, Gongyi City, Zhengzhou City, Henan Province, and the particle size of the biochar was 200-250 meshes;

[0050] The preparation method of the liquid bacterial agent is as follows:

[0051] 1. Inoculate the culture medium with glycerol-preserved Raoultella ornithinolytica A1, the inoculation amount of Raoultella ornithinolytica A1 being 1%;

[0052] The culture medium formula described in step 1 is: 3.0 g beef extract, 10.0 g peptone, 5.0 g NaCl, 1000 mL water, pH 7.4-7.6, sterilized at 120°C for 30 min;

[0053] 2. Place the culture medium inoculated with Raoultella ornithinolytica A1 in a shaker and shake at 30° C. for 24 hours to obtain a liquid bacterial agent;

[0054] The mass ratio of the biochar to the high-molybdenum contaminated soil is 6g:100g;

[0055] The sowing rate of the ryegrass seeds is 20g / m 2 ;

[0056] The mass ratio of the bacterial content in the liquid bacterial agent to the high-molybdenum contaminated soil is 1×10 7 CFU:1g.

[0057] Example 2: A method for combining plant-microbial agent-biochar to repair high-molybdenum contaminated soil under low temperature conditions (H-A1-B2) is completed by the following steps:

[0058] Evenly disperse the biochar into the high-molybdenum contaminated soil, then sprinkle ryegrass seeds into the high-molybdenum contaminated soil, cover the top with soil, water the soil regularly to keep it moist, and then pour the liquid bacterial agent into the roots of the ryegrass. Cultivate it at 10°C for 40 days to complete the restoration of the high-molybdenum contaminated soil.

[0059] The strain in the liquid bacterial agent is Raoultella ornithinolytica A1, which is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit address being No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit date being May 26, 2022, and the deposit number being CGMCC No. 24973;

[0060] The high-molybdenum contaminated soil was purchased from Harbin Hongli Flower Base, where the total molybdenum concentration was 800 mg / kg;

[0061] The biochar was purchased from Beishankou Industrial Park, Gongyi City, Zhengzhou City, Henan Province, and the particle size of the biochar was 200-250 meshes;

[0062] The preparation method of the liquid bacterial agent is as follows:

[0063] 1. Inoculate the culture medium with glycerol-preserved Raoultella ornithinolytica A1, the inoculation amount of Raoultella ornithinolytica A1 being 1%;

[0064] The culture medium formula described in step 1 is: 3.0 g beef extract, 10.0 g peptone, 5.0 g NaCl, 1000 mL water, pH 7.4-7.6, sterilized at 120°C for 30 min;

[0065] 2. Place the culture medium inoculated with Raoultella ornithinolytica A1 in a shaker and shake at 30° C. for 24 hours to obtain a liquid bacterial agent;

[0066] The mass ratio of the biochar to the high-molybdenum contaminated soil is 6g:100g;

[0067] The sowing rate of the ryegrass seeds is 20g / m 2 ;

[0068] The mass ratio of the bacterial content in the liquid bacterial agent to the high-molybdenum contaminated soil is 1×10 7 CFU:1g.

[0069] Example 3: A method (CK) for repairing high-molybdenum contaminated soil by planting ryegrass alone is accomplished by the following steps:

[0070] Sow ryegrass seeds in the high-molybdenum contaminated soil, cover the top with soil, water the soil regularly to keep it moist, and incubate it at room temperature for 40 days to complete the restoration of the high-molybdenum contaminated soil;

[0071] The high-molybdenum contaminated soil was purchased from Harbin Hongli Flower Base, where the total molybdenum concentration was 800 mg / kg;

[0072] The sowing rate of the ryegrass seeds is 20 g / m 2 .

[0073] Example 4: A method for remediating high-molybdenum contaminated soil by combining biochar and ryegrass (HB) is accomplished by the following steps:

[0074] The biochar was evenly dispersed in the high-molybdenum contaminated soil, and then ryegrass seeds were sprinkled in the high-molybdenum contaminated soil, the upper part was covered with soil, and the soil was watered regularly to keep it moist. After incubation at room temperature for 40 days, the high-molybdenum contaminated soil was repaired;

[0075] The high-molybdenum contaminated soil was purchased from Harbin Hongli Flower Base, where the total molybdenum concentration was 800 mg / kg;

[0076] The biochar was purchased from Beishankou Industrial Park, Gongyi City, Zhengzhou City, Henan Province, and the particle size of the biochar was 200-250 meshes;

[0077] The mass ratio of the biochar to the high-molybdenum contaminated soil is 6g:100g;

[0078] The sowing rate of the ryegrass seeds is 20 g / m 2 .

[0079] Example 5: A method for remediating high-molybdenum contaminated soil by combining a bacterial agent with ryegrass (H-A1) is accomplished by the following steps:

[0080] Sow ryegrass seeds in the high-molybdenum contaminated soil, cover the top with soil, water regularly to keep the soil moist, then pour liquid bacterial agent into the roots of ryegrass and cultivate at room temperature for 40 days to complete the restoration of high-molybdenum contaminated soil;

[0081] The strain in the liquid bacterial agent is Raoultella ornithinolytica A1, which is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is May 26, 2022, and the deposit number is CGMCC No. 24973.

[0082] The high-molybdenum contaminated soil was purchased from Harbin Hongli Flower Base, where the total molybdenum concentration was 800 mg / kg;

[0083] The preparation method of the liquid bacterial agent is as follows:

[0084] 1. Inoculate the culture medium with glycerol-preserved Raoultella ornithinolytica A1, the inoculation amount of Raoultella ornithinolytica A1 being 1%;

[0085] The culture medium formula described in step 1 is: 3.0 g beef extract, 10.0 g peptone, 5.0 g NaCl, 1000 mL water, pH 7.4-7.6, sterilized at 120°C for 30 min;

[0086] 2. Place the culture medium inoculated with Raoultella ornithinolytica A1 in a shaker and shake at 30° C. for 24 hours to obtain a liquid bacterial agent;

[0087] The sowing rate of the ryegrass seeds is 20g / m 2 ;

[0088] The mass ratio of the bacterial content in the liquid bacterial agent to the high-molybdenum contaminated soil is 1×10 7 CFU:1g.

[0089] Figure 1 A statistical diagram of the average molybdenum content in the soil after restoration using the embodiment;

[0090] Figure 2 A statistical chart showing the proportion of each form of molybdenum in the soil after restoration using the embodiment;

[0091] Figure 3 A statistical diagram of pH in the rhizosphere soil of plants after restoration using the embodiment;

[0092] Figure 4 A statistical diagram of the length of the aboveground part of ryegrass after restoration using the embodiment.

[0093] Figure 1 to Figure 4 CK represents the control group with ryegrass planted alone; HB represents the experimental group with biochar and ryegrass added; H-A1 represents the experimental group with microbial agent and ryegrass added; H-A1-B represents the experimental group with microbial agent, biochar and ryegrass added at the same time under normal temperature; H-A1-B2 represents the experimental group with microbial agent, biochar and ryegrass added at the same time under low temperature. Each experimental group and control group were set up with 3 parallel groups.

[0094] from Figure 1 to Figure 4It can be seen that both the microbial agent and biochar will promote the increase of ryegrass biomass. Under the combined action of plant-microbial agent-biochar, after 40 days of restoration, the total molybdenum content in the soil under normal temperature conditions decreased by 24.78%, and the content of easily available molybdenum decreased by 23.03%; low temperature conditions will inhibit the growth of ryegrass and the removal of total molybdenum, but the content of easily available molybdenum will decrease by 32.2%.

Claims

1. A method for combining plant-microbial agent-biochar to repair high-molybdenum contaminated soil, characterized in that Under the low temperature condition of 10°C, the molybdenum in the soil is more easily converted to a stable state, and the content of the molybdenum in the more easily utilized state is reduced by 32.2%. The method is completed according to the following steps: The biochar was evenly dispersed in the high-molybdenum contaminated soil, and then ryegrass seeds were sprinkled in the high-molybdenum contaminated soil, the upper part was covered with soil, and the soil was watered regularly to keep it moist. Then the liquid bacterial agent was poured into the roots of the ryegrass, and the low-temperature culture temperature was 10°C for 40 days to complete the restoration of the high-molybdenum contaminated soil; The preparation method of the liquid bacterial agent is as follows:

1. Inoculate the culture medium with glycerol-preserved Raoultella ornithinolytica A1, the inoculation amount of Raoultella ornithinolytica A1 being 1%; The Raoultella ornithinolytica A1 described in step 1 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is May 26, 2022, and the deposit number is CGMCC No. 24973; The culture medium formula described in step 1 is: 3.0 g beef extract, 10.0 g peptone, 5.0 g NaCl, 1000 mL water, pH 7.4-7.6, sterilized at 120°C for 30 min; 2. Place the culture medium inoculated with Raoultella ornithinolytica A1 in a shaker and shake at 30° C. for 24 hours to obtain a liquid bacterial agent; The mass ratio of the biochar to the high-molybdenum contaminated soil is (6-8):100; The sowing rate of the ryegrass seeds is 20 g / m 2 ~25g / m 2 ; The mass ratio of the bacterial content in the liquid bacterial agent to the high-molybdenum contaminated soil is (1×10 7 CFU~1.2×10 7 CFU): 1g.

2. The method of claim 1 wherein the plant-microbial agent-biochar combined restoration method comprises the following steps: The particle size of the biochar is 200-250 meshes.

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