Preparation and application method of biochar-urease-producing bacterium gel beads for synchronously improving fertility and strength of ecological slope protection base material

Abstract

The invention provides a preparation and application method of biochar-urease-producing bacterium coagulated beads capable of synchronously improving fertility and strength of an ecological slope protection base material, and the method comprises the following steps: mixing urease-producing bacteria and biochar for culturing, then adding sodium alginate, and dropwise adding the mixture into a calcium chloride solution for curing to prepare the biochar-urease-producing bacterium coagulated beads. The prepared gel beads can be stored at low temperature after being mixed with glycerol, and the method is low in cost, simple in process and convenient to apply. The construction method for ecological restoration of the side slope comprises the following steps that the slope surface is cleaned; embedding ground anchors and laying meshes; adding gel beads to prepare and spray a base material; adding coagulation beads to prepare and spray a surface layer material; watering and maintaining. The application process of the biochar-mediated urease-producing bacteria can be optimized, and uniform precipitation of calcium carbonate in a base material can be promoted, so that a firmer stress skeleton is formed in the base material, and the overall stability and sustainability of a side slope are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of slope protection, and specifically to a preparation and application method of biochar-urease-producing bacteria beads for synchronously improving the fertility and strength of ecological slope protection substrates. Background Technique

[0002] In recent years, while vigorously promoting the construction of large-scale water conservancy and hydropower projects in China, a large amount of earthwork excavation has led to the formation of many high-steep and bare slopes. These slopes not only damage the original landform and landscape, but also bring serious ecological problems over time, such as ecosystem degradation, loss of species diversity, landslides, and soil erosion. These problems seriously affect the stability of the ecological environment and also restrict the sustainable development of human society.

[0003] The research on ecological slope protection substrates requires them to have two core characteristics: one is strong mechanical strength, and the other is certain fertility to support vegetation growth and ecological restoration. The microbial-induced calcium carbonate precipitation (MICP) technology can effectively reinforce the soil, while biochar has the effect of increasing fertility. When biochar-mediated urease-producing bacteria are applied to ecological slope protection substrates, the synergistic effect between the two can significantly improve the performance of the substrates. However, when applying the MICP technology to ecological slope protection through a sprinkler irrigation system, some challenges still exist. Due to the filtering effect during the injection of the bacterial solution, the absorption of the bacterial solution by the soil results in uneven distribution of calcium carbonate precipitation along the substrate, leading to insufficient overall mechanical strength of the repaired slope, and the construction process is complex, reducing the construction efficiency.

[0004] Therefore, in order to more effectively promote the application of the MICP ecological slope protection technology to engineering practice, a new method for slope ecological restoration and reinforcement is needed. Summary of the Invention

[0005] To solve the current technical problems, the main purpose of the present invention is to provide a preparation and application method of biochar-urease-producing bacteria beads for synchronously improving the fertility and strength of ecological slope protection substrates. This method can not only optimize the application process of biochar-mediated urease-producing bacteria, but also promote the uniform precipitation of calcium carbonate in the substrate, thereby forming a more solid stress skeleton in the substrate, improving the overall stability and sustainability of the slope; in addition, this method can significantly improve the construction efficiency, effectively reinforce the slope to maintain long-term stability, and at the same time take into account the ecological restoration of the slope and improve the vegetation coverage rate of the slope.

[0006] In order to achieve the above technical features, the object of the present invention is realized as follows: A preparation method of biochar-urease-producing bacteria beads for synchronously improving the fertility and strength of ecological slope protection substrates, comprising the following steps: Step 1: Inoculate the urease-producing bacteria into the sterilized liquid medium, and at the same time add the sterilized biochar, mix evenly to ensure that the biochar fully immobilizes the urease-producing bacteria, and culture for 1 to 2 days; Step 2: Prepare a 0.2 - 0.4 mol / L calcium chloride solution and sterilize it for later use; Step 3: Add sodium alginate to the biochar-urease-producing bacteria mixed solution and stir evenly; Step 4: Slowly and evenly drip the mixed biochar / urease-producing bacteria / sodium alginate solution into the calcium chloride solution, and keep stirring the calcium chloride solution while dripping. After dripping, solidify for 0.5 to 1 hour, take out the solid, and freeze-dry to obtain biochar-urease-producing bacteria beads; Step 5: Mix the obtained biochar-urease-producing bacteria beads with the sterilized glycerol in a volume ratio of 1:1, and store them in a refrigerator at -20 to -28 °C.

[0007] Preferably, each liter of the liquid medium in Step 1 includes 20 g - 25 g of yeast extract, 10 g - 12.5 g of (NH4)2SO4, and 0.13 - 0.2 mol / L Tris hydrochloride buffer solution, and use 0.1 mol / L hydrochloric acid to adjust the solution pH to 9.0.

[0008] Preferably, the biochar selected in Step 1 is 100 - 400 mesh activated carbon, and 1 - 4 g of biochar is added to every 100 mL of the culture solution.

[0009] Preferably, in Step 1, the culture ensures that the OD600 value of the urease-producing bacteria solution is greater than 1.5.

[0010] Preferably, during the addition of sodium alginate in Step 3, 1 - 2 g of sodium alginate is added to every 100 mL of the biochar-urease-producing bacteria mixed solution.

[0011] Preferably, in the stirring process of Step 4, a centrifuge is used, and the set parameters are 2800 - 3200 r / min, and centrifuge for 3 - 4 minutes.

[0012] Preferably, in Steps 1, 2, and 5, sterilization is carried out using an autoclave, the temperature is set at 121 °C - 125 °C, and the time is set at 30 min - 35 min.

[0013] Preferably, on the other hand, the present invention provides an application method of applying biochar-urease-producing bacteria beads in slope ecological restoration, including the following steps: Step 1: Pretreat the slope surface, remove the floating soil, loose stones, and loose floating roots on the slope surface to keep the slope surface flat; Step 2: Set up the reinforcement system, install anchor rods on the slope at a certain interval, and then lay the mesh, and tie the mesh and anchor rods firmly; Step 3: After fully mixing 95-105 parts of sand, 23-26 parts of organic materials, 1-2 parts of organic fertilizer, biochar-urease-producing bacteria beads and cementing liquid, use a wet sprayer to spray the base layer onto the slope surface with a spraying thickness of 50-100 mm; Step 4: After fully mixing 95-105 parts of sand, 30-33 parts of organic materials, 3-5 parts of organic fertilizer, biochar-urease-producing bacteria beads, cementing liquid and plant seeds, use a wet sprayer to spray the surface layer onto the slope surface with a spraying thickness of 20-50 mm; Step 5: Cover the surface with non-woven fabric for insulation, sprinkle water every day for maintenance, and remove the non-woven fabric after the grass seeds germinate to complete the construction.

[0014] Preferably, the biochar-urease-producing bacteria beads in step 3 and step 4 need to be activated by incubation in a water bath shaker at a speed of 150-200 r / min and a constant temperature of 25-35°C for 1-3 days before use, and 0.15-0.2 times the pore volume of the sand and soil used for the base layer, and 0.1-0.15 times the pore volume of the sand and soil used for the surface layer; the binder is prepared by dissolving 1 mol / L urea and anhydrous calcium chloride in an equimolar ratio in deionized water, and stirring to form a uniform mixture.

[0015] Preferably, 0.2 to 0.3 times the pore volume of the sand and soil used is added to the base layer, and 0.15 to 0.2 times the pore volume of the sand and soil used is added to the surface layer.

[0016] The present invention has the following beneficial effects: 1. The present invention designs a urease-producing bacteria gel bead with sodium alginate and biochar as a composite carrier, wherein the urease-producing bacteria is loaded on the composite carrier. Since biochar has abundant micropores and surface functional groups, it can interact with the cell wall of microorganisms through electrostatic attraction, covalent bond formation, hydrogen bonding, etc., and its large surface area provides a stable attachment and living space for urease-producing bacteria. Compared with the addition of biochar and urease-producing bacteria alone, the present invention effectively improves the survival rate and biological activity of urease-producing bacteria in the soil, thereby promoting the formation of calcium carbonate and enhancing the soil reinforcement effect. In addition, since the urease-producing bacteria are attached to the biochar in advance, after the binder is added, the generated calcium carbonate can wrap the biochar, delaying its aging and breakage. The gel beads are low in preparation cost, simple in process, easy to transport, and easy to achieve industrial promotion.

[0017] 2. The present invention pre - colonizes urease - producing bacteria on biochar, which can effectively improve the survival rate and biological activity of urease - producing bacteria in soil, thereby enhancing the soil reinforcement effect. At the same time, the calcium carbonate generated by urease - producing bacteria during the later stage of repair can further wrap the biochar, delaying the aging and fragmentation of the biochar, and solving the problems of cumbersome processes, poor reinforcement effects, and aging and fragmentation of biochar in the engineering application of urease - producing bacteria.

[0018] 3. The construction method of the biochar - urease - producing bacteria beads in slope ecological restoration. First, the biochar - urease - producing bacteria beads are premixed with the binder liquid and the base material. Compared with the existing single - injection methods such as drip irrigation or perfusion, since the bacterial liquid is evenly distributed, the cured body obtained after the reaction is relatively uniform, more calcium carbonate is generated, and the construction method is simple, greatly improving the construction efficiency.

[0019] 4. The method of the present invention can not only optimize the application process of biochar - mediated urease - producing bacteria, but also promote the uniform precipitation of calcium carbonate in the base material, thereby forming a more solid stress skeleton in the base material and improving the overall stability and sustainability of the slope.

[0020] 5. The method of the present invention can significantly improve the construction efficiency, effectively reinforce the slope to maintain long - term stability, and at the same time take into account the ecological restoration of the slope, improve the vegetation coverage rate of the slope, and provide a construction method applicable to the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The present invention will be further described below in conjunction with the drawings and embodiments.

[0022] Figure 1 It is a flow chart for the preparation of biochar - urease - producing bacteria beads. Figure 2 It is a flow chart for the application of biochar - urease - producing bacteria beads in slope ecological restoration. Figure 3 It is the calcium carbonate production rate curves of each group.

[0023] Figure 4 It is the calcium carbonate production rate distribution curves of each layer in each group. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0024] The embodiments of the present invention will be further described below in conjunction with the drawings.

[0025] Example 1: A method for preparing biochar - urease - producing bacteria beads for synchronously improving the fertility and strength of ecological slope base materials, comprising the following steps: Step 1: Inoculate urease - producing bacteria into the sterilized liquid medium, and at the same time add the sterilized biochar, mix evenly to ensure that the biochar fully fixes the urease - producing bacteria, and culture for 1 - 2 days; Step 2: Prepare a 0.2 - 0.4 mol / L calcium chloride solution and sterilize it for later use; Step 3: Add sodium alginate to the biochar - urease - producing bacteria mixed solution and stir evenly; Step 4: Slowly and evenly drip the mixed biochar / urease - producing bacteria / sodium alginate solution into the calcium chloride solution, while continuously stirring the calcium chloride solution during the dripping process. After the dripping is completed, solidify for 0.5 - 1 hour, take out the solid, and freeze - dry it to obtain biochar - urease - producing bacteria beads. After the biochar / urease - producing bacteria / sodium alginate solution is dripped into the calcium chloride solution, the calcium ions in calcium chloride react with the carboxyl groups in sodium alginate to form an ion - crosslinked calcium alginate film with a certain elasticity and mechanical strength, which wraps the biochar - urease - producing bacteria solution, facilitating storage and later use; Step 5: Mix the obtained biochar - urease - producing bacteria beads with sterilized glycerol in a volume ratio of 1:1 and store them in a refrigerator at - 20~ - 28 °C.

[0026] Further, each liter of the liquid medium in Step 1 includes 20 g of yeast extract, 10 g of (NH 4 ) 2 SO 4 and 0.13 mol / L Tris - hydrochloride buffer solution, and use 0.1 mol / L hydrochloric acid to adjust the solution pH to 9.0.

[0027] Further, the biochar selected in Step 1 is 100 - 400 - mesh activated carbon, and 1 - 4 g of biochar is added to every 100 mL of the culture solution. The biochar selected in this example is woody activated carbon, which is processed and produced by the zinc chloride method using agricultural waste as raw materials according to national standards. The biochar should not be too fine. The size of urease - producing bacteria is generally 1 - 5 μm, and too fine biochar may cause its pores to be unable to load urease - producing bacteria.

[0028] Further, in Step 1, the culture ensures that the OD 600 value of the urease - producing bacteria solution is greater than 1.5.

[0029] Further, for the sodium alginate in Step 2, 1 - 2 g of sodium alginate is added to every 100 mL of the biochar - urease - producing bacteria mixed solution.

[0030] Further, in Step 4, the centrifuge is set with parameters of 2800 - 3200 r / min and centrifuged for 3 - 4 minutes.

[0031] Further, in Steps 1, 2, and 5, sterilization is carried out using an autoclave, with the temperature set at 121 °C and the time set at 30 min.

[0032] Embodiment 2: In addition, the present invention also discloses a method for applying biochar-urease-producing bacteria beads in slope ecological restoration, which comprises the following steps: Step 1: Pre-treat the slope surface, remove loose soil, loose stones and loose roots on the slope surface, and keep the slope surface flat; Step 2: Set up the reinforcement system, install anchor rods on the slope at a certain interval, and then lay the mesh, and tie the mesh and anchor rods firmly; Step 3: After fully mixing 95-105 parts of sand, 23-26 parts of organic materials, 1-2 parts of organic fertilizer, biochar-urease-producing bacteria beads and the binder, a wet sprayer is used to spray the base layer onto the slope surface with a spraying thickness of 50-100 mm. In this construction step, the mixing method is used to treat the bacterial solution and the binder to avoid the linear decrease of bacterial concentration along the injection path due to soil filtration after direct injection of the bacterial solution, and the uneven distribution of calcium carbonate, resulting in the instability of the soil structure after reinforcement.

[0033] Step 4: After fully mixing 95-105 parts of sand, 30-33 parts of organic materials, 3-5 parts of organic fertilizer, biochar-urease-producing bacteria beads, cementing liquid and plant seeds, use a wet sprayer to spray the surface layer onto the slope surface with a spraying thickness of 20-50 mm; Step 5: Cover the surface with non-woven fabric for insulation, sprinkle water every day for maintenance, and remove the non-woven fabric after the grass seeds germinate to complete the construction.

[0034] The biochar-urease-producing bacteria beads in step 3 and step 4 need to be activated by incubating in a water bath shaker at a speed of 150-200 r / min and a constant temperature of 25-35°C for 1-3 days before use. The base layer is added with 0.15-0.2 times the pore volume of the sand and soil used, and the surface layer is added with 0.1-0.15 times the pore volume of the sand and soil used; the cementing liquid is prepared by dissolving 1 mol / L urea and anhydrous calcium chloride in deionized water in an equal molar ratio and stirring and mixing them evenly. The base layer is added with 0.2-0.3 times the pore volume of the sand and soil used, and the surface layer is added with 0.15-0.2 times the pore volume of the sand and soil used. By designing the bacterial liquid content in the surface and bottom layers of the restoration layer to increase successively, the strength of the substrate can be improved layer by layer with increasing depth, while the organic fertilizer content decreases successively, thereby achieving an increase in the soil's demand for mechanical properties with increasing depth and a decrease in the plant's demand for nutrients, thus achieving a balance between the substrate's strength performance and the plant's growth environment.

[0035] Embodiment 3: According to Example 1, biochar-Bacillus pasteurianus beads are prepared, comprising the following steps: Step 1: Inoculate *Bacillus pasteurii* into the sterilized liquid medium, and at the same time add the sterilized biochar. Mix them evenly to ensure that the biochar fully immobilizes *Bacillus pasteurii*, and culture for 1 day; Step 2: Prepare 0.2 mol / L calcium chloride solution and sterilize it for later use; Step 3: Add 1.5 g of sodium alginate to every 100 mL of the biochar-*Bacillus pasteurii* mixed solution and stir evenly; Step 4: Slowly and evenly drip the mixed biochar / *Bacillus pasteurii* / sodium alginate solution into the calcium chloride solution, and continuously stir the calcium chloride solution while dripping. After dripping, solidify for 1 hour, take out the solid, and freeze-dry to obtain the biochar-*Bacillus pasteurii* beads. After the biochar / *Bacillus pasteurii* / sodium alginate solution is dripped into the calcium chloride solution, the calcium ions in the calcium chloride react with the carboxyl groups in the sodium alginate to form an ion cross-linking, quickly forming a calcium alginate film with a certain elasticity and mechanical strength, which wraps the biochar-*Bacillus pasteurii* solution, facilitating storage and later use; Step 5: Prepare 1 mol / L cementing liquid of urea and anhydrous calcium chloride in an equimolar ratio and sterilize it for later use.

[0036] Further, each liter of the liquid medium in Step 1 includes 20 g of yeast extract, 10 g of (NH 4 ) 2 SO 4 and 0.13 mol / L Tris hydrochloride buffer solution, and use 0.1 mol / L hydrochloric acid to adjust the pH of the solution to 9.0.

[0037] Further, the biochar selected in Step 1 is 200-mesh activated carbon, and 4 g of biochar is added to every 100 mL of the culture solution.

[0038] Further, the culture in Step 1 ensures that the OD 600 value of the *Bacillus pasteurii* bacterial solution is greater than 1.5.

[0039] Further, in Step 4, a centrifuge is used with the parameters set at 3000 r / min and centrifuged for 3 - 4 minutes.

[0040] Further, in Steps 1, 2, and 5, autoclaving is used for sterilization, with the temperature set at 121 °C and the time set at 30 min.

[0041] Use the prepared biochar-*Bacillus pasteurii* beads for the soil column test. The test selects sandy soil with a pore volume of 33 cm³, which is divided into 4 groups, and 3 replicate experiments are set for each group: Group 1: Mix 0.2 g of biochar, 0.15 times the pore volume of the bacterial solution, and 0.2 times the pore volume of the cementing solution directly with the sandy soil. Group 2: First, mix 0.2 g of biochar into the sandy soil in advance, and then use a peristaltic pump to drip 0.15 times the pore volume of the bacterial solution and 0.2 times the pore volume of the cementing solution into the soil column. Group 3: Mix 0.15 times the pore volume of biochar - Bacillus pasteurii beads directly with 0.2 times the pore volume of the cementing solution into the sandy soil. Group 4: First, mix 0.15 times the pore volume of biochar - Bacillus pasteurii beads into the sandy soil in advance, and then use a peristaltic pump to drip 0.2 times the pore volume of the cementing solution into the soil column.

[0042] After the test, divide the soil column into upper, middle, and lower parts to explore the distribution law of CaCO 3 production in the soil column under different treatment conditions.

[0043] According to Figure 3 and Figure 4 shown, the influence results of different treatment methods on the CaCO 3 production distribution are as follows: From Figure 3 it can be seen that the CaCO 3 production rate of the group directly mixed with biochar - Bacillus pasteurii beads is better than the other three groups. From Figure 4 it can be seen that the CaCO 3 production rate of the specimens treated by dripping is higher in the upper layer of the soil column than that of the specimens directly mixed; overall, the CaCO 3 production rate distribution of the mixing treatment is more uniform, meeting the requirements of actual engineering. From Figure 3 and Figure 4 it can be obtained that under the same additive method, the CaCO 3 production rate of the specimens directly mixed is higher; under the same mixing method, the CaCO 3 production rate of the specimens added with biochar - Bacillus pasteurii beads is higher. The results show that this method significantly improves the survival rate and biological activity of Bacillus pasteurii in the soil, thereby promoting the CaCO 3 generation, which proves indirectly that this method can enhance the reinforcement effect of urease - producing bacteria on the soil.

Claims

1. A method for preparing biochar-urease-producing bacteria beads for simultaneously improving the fertility and strength of ecological slope protection substrates, characterized in that: The following steps are involved: Step 1: Inoculate urease-producing bacteria into sterilized liquid culture medium, add sterilized biochar, mix well, ensure that biochar fully fixes urease-producing bacteria, and culture for 1 to 2 days; Step 2: Prepare 0.2~0.4 mol / L calcium chloride solution and sterilize it for later use; Step 3: Add sodium alginate to the biochar-urease-producing bacteria mixed solution and stir evenly; Step 4: The mixed biochar / urease-producing bacteria / sodium alginate solution is slowly and evenly added to the calcium chloride solution while stirring the calcium chloride solution continuously. After the addition is completed, the solution is solidified for 0.5 to 1 hour, and the solid is taken out and freeze-dried to obtain biochar-urease-producing bacteria beads; Step 5: Mix the obtained biochar-urease-producing bacteria beads with sterilized glycerol in a volume ratio of 1:1 and store them in a -20~-28℃ refrigerator.

2. A method for preparing biochar-urease-producing bacteria beads for simultaneously improving the fertility and strength of ecological slope protection substrates according to claim 1, characterized in that: The liquid culture medium in step 1 includes 20 g to 25 g yeast extract, 10 g to 12.5 g (NH4)2SO4 and 0.13 to 0.2 mol / L Tris hydrochloride buffer per liter of culture medium, and 0.1 mol / L hydrochloric acid is used to adjust the pH of the solution to 9.

0.

3. The method for preparing biochar-urease-producing bacteria beads for simultaneously improving the fertility and strength of ecological slope protection substrate according to claim 1, characterized in that: In the step 1, the biochar is selected from 100-400 mesh activated carbon, and 1-4 g of biochar is added to every 100 mL of culture solution.

4. The method for preparing biochar-urease-producing bacteria beads for simultaneously improving the fertility and strength of ecological slope protection substrate according to claim 1, characterized in that: In step 1, the OD of the urease-producing bacterial solution is guaranteed to be 600 Value greater than 1.

5.

5. The method for preparing biochar-urease-producing bacteria beads for simultaneously improving the fertility and strength of ecological slope protection substrate according to claim 1, characterized in that: During the addition of sodium alginate in step 3, 1-2 g of sodium alginate is added to every 100 mL of the biochar-urease-producing bacteria mixed solution.

6. The method for preparing biochar-urease-producing bacteria beads for simultaneously improving the fertility and strength of ecological slope protection substrate according to claim 1, characterized in that: In the step 4, a centrifuge is used during the stirring process, the parameters are set to 2800-3200 r / min, and the centrifugation is performed for 3-4 minutes.

7. The method for preparing biochar-urease-producing bacteria beads for simultaneously improving the fertility and strength of ecological slope protection substrate according to claim 1, characterized in that: In the step 1, step 2 and step 5, the sterilization is performed by a high pressure sterilizer, the temperature is set to 121° C. to 125° C., and the time is set to 30 min to 35 min.

8. A method for applying the biochar-urease-producing bacteria beads according to any one of claims 1 to 7 to slope ecological restoration, characterized in that: The steps include: Step 1: Pre-treat the slope surface, remove loose soil, loose stones and loose roots on the slope surface, and keep the slope surface flat; Step 2: Set up the reinforcement system, install anchor rods on the slope at a certain interval, and then lay the mesh, and tie the mesh and anchor rods firmly; Step 3: After fully mixing 95-105 parts of sand, 23-26 parts of organic materials, 1-2 parts of organic fertilizer, biochar-urease-producing bacteria beads and cementing liquid, use a wet sprayer to spray the base layer onto the slope surface with a spraying thickness of 50-100 mm; Step 4: After fully mixing 95-105 parts of sand, 30-33 parts of organic materials, 3-5 parts of organic fertilizer, biochar-urease-producing bacteria beads, cementing liquid and plant seeds, use a wet sprayer to spray the surface layer onto the slope surface with a spraying thickness of 20-50 mm; Step 5: Cover the surface with non-woven fabric for insulation, sprinkle water every day for maintenance, and remove the non-woven fabric after the grass seeds germinate to complete the construction.

9. The method for applying the biochar-urease-producing bacteria beads in slope ecological restoration according to claim 8, characterized in that: The biochar-urease-producing bacteria beads in step 3 and step 4 need to be activated by incubation in a water bath shaker at a speed of 150-200 r / min and a constant temperature of 25-35°C for 1-3 days before use. 0.15-0.2 times the pore volume of the sand and soil used are added to the base layer, and 0.1-0.15 times the pore volume of the sand and soil used are added to the surface layer. The binder is prepared by dissolving 1 mol / L urea and anhydrous calcium chloride in an equimolar ratio in deionized water and stirring to mix them evenly.

10. The method for applying the biochar-urease-producing bacteria beads in slope ecological restoration according to claim 8, characterized in that: Add 0.2~0.3 times the pore volume of the sand and soil used as the base layer, and add 0.15~0.2 times the pore volume of the sand and soil used as the surface layer.

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