Microbial composite microbial inoculant and preparation method thereof

Abstract

The application discloses a kind of microbial compound microbial inoculant and preparation method thereof, the microbial compound microbial inoculant includes microbial inoculum and its fermentation product and carrier;The microbial inoculum includes bacillus subtilis and bacillus licheniformis;The carrier is nitrogen-rich grafted biochar;The nitrogen-rich grafted biochar is prepared after grafting acrylic acid and modified monomer after being modified by nitrogen-rich biochar.The microbial compound microbial inoculant provided in the application can significantly improve the adsorption capacity and flocculation capacity of the microbial inoculant for particles in wastewater by compounding microbial inoculum with excellent flocculation capacity with charge-modified and adsorbed carrier to make microbial inoculant, thereby effectively reducing the content of COD components, ammonia nitrogen and total phosphorus in wastewater, and can be widely applied in the field of wastewater treatment.

Description

Technical Field

[0001] This invention relates to the field of microbial inoculants, specifically to a microbial compound inoculant and its preparation method. Background Technology

[0002] Water resources, especially freshwater resources, are essential for sustaining life and are irreplaceable for social operation and industrial production. However, freshwater resources are scarce on Earth, making the protection and rational use of freshwater resources an issue that cannot be ignored.

[0003] People's current lifestyles are very different from those of the past, and their water usage habits have also changed. Increased water consumption in various aspects such as bathing and washing has led to higher nitrogen and phosphorus content in domestic sewage. Secondly, since most cities did not separate rainwater and sewage during construction, rainwater dilutes sewage, resulting in sewage with a low carbon-to-nitrogen ratio. In addition, the low winter temperature affects the activity of microorganisms, further reducing the efficiency of nitrogen and phosphorus removal.

[0004] Currently, the main methods for treating industrial wastewater include chemical oxidation, extraction, adsorption, electrodialysis, and flocculation. The first four methods are limited by factors such as large infrastructure investment, complex processes, and high energy consumption, making large-scale promotion difficult. Flocculation, on the other hand, is very common due to its low cost and relatively simple process.

[0005] Currently, flocculants are mainly classified into three categories: organic synthetic polymer flocculants, inorganic flocculants, and bioflocculants. Bioflocculants primarily refer to those produced by microorganisms such as bacteria and fungi, and their metabolic products, through interaction with particles in wastewater to achieve a flocculation effect. Compared with conventional flocculants, bioflocculants have advantages such as high flocculation efficiency, no secondary pollution, naturally biodegradable products, and sustainable regeneration.

[0006] However, existing bioflocculants have insufficient adsorption capacity for particles in wastewater and cannot fully treat water pollution. At the same time, their flocculation capacity still needs to be improved, as they cannot effectively aggregate pollutant particles of all sizes, and their treatment capacity still needs to be improved.

[0007] Therefore, there is an urgent need for a microbial compound agent with excellent adsorption and flocculation capabilities for wastewater treatment. Summary of the Invention

[0008] Purpose of the invention: In view of the deficiencies of the prior art, the purpose of this invention is to provide a microbial composite agent for wastewater treatment with excellent adsorption and flocculation capabilities, and a method for preparing the same.

[0009] Technical solution: In one aspect, the present invention provides a microbial compound inoculant, comprising microbial inoculum, its fermentation products, and a carrier; The microbial inoculum includes Bacillus subtilis and Bacillus licheniformis; The carrier is nitrogen-rich grafted biochar. The nitrogen-enriched grafted biochar is prepared by grafting acrylic acid and modified monomers onto biochar after it has been modified to be nitrogen-enriched.

[0010] This invention uses nitrogen-rich grafted biochar that has undergone charge modification and adsorption modification as a carrier, which can not only significantly improve the flocculation ability of the bacterial agent for particulate pollutants in wastewater, but also expand the influence range of the bacterial agent in wastewater through its excellent adsorption capacity, thereby improving the wastewater treatment capacity of the bacterial agent.

[0011] Furthermore, the number of viable bacteria in the microbial culture solution is not less than 3 × 10⁻⁶. 9 CFU / g; The effective viable count ratio of Bacillus subtilis to Bacillus licheniformis is (2-3):(1-2).

[0012] This invention prepares a microbial inoculum by mixing Bacillus subtilis and Bacillus licheniformis. On the one hand, the extracellular polysaccharides produced by Bacillus licheniformis fermentation can act as a bioflocculant to effectively flocculate particulate pollutants in wastewater. On the other hand, Bacillus subtilis can effectively inhibit the growth of various bacteria, such as Gram-positive bacteria, in wastewater. At the same time, its fermentation products can improve the aggregation stability of flocs, facilitating subsequent filtration and separation, and further enhancing the flocculation ability of the inoculum.

[0013] Furthermore, the mass ratio of the microbial culture to the carrier is (6-8):(15-20).

[0014] Furthermore, the modified monomer is prepared by the following steps: in a reactor, itaconic acid and tetrahydrofuran are added, stirred and dissolved, then 4-hydroxycyclohexanone and concentrated sulfuric acid are added, the temperature is raised to 60-70°C, and after reacting for 4-6 hours, the modified monomer is obtained by vacuum distillation.

[0015] Furthermore, the molar ratio of itaconic acid to 4-hydroxycyclohexanone is 1.2-1.3:1; and the mass concentration of the concentrated sulfuric acid is 90-99%.

[0016] Furthermore, the nitrogen-rich grafted biochar is prepared through the following steps: (1) Nitrogen-rich biochar is obtained by dry distillation of a mixture of biological straw and urea; (2) The nitrogen-rich grafted biochar is prepared by reacting nitrogen-rich biochar with acrylic acid and modified monomers.

[0017] Further, step (1) specifically involves: crushing the biological straw and mixing it with urea, then dry distilling it at 400-600℃ for 1-2 hours to obtain the nitrogen-rich biochar; The biological straw is selected from at least one of wheat straw, rice straw, or corn straw; the average particle size of the crushed biological straw is 30-80 mm; and the mass ratio of the biological straw to urea is 25-35:1.

[0018] After being modified with nitrogen, the biochar of the present invention forms an electron-deficient region inside. On the one hand, it can form hydrogen bonds with the polysaccharides of the microbial cell wall, thereby stabilizing the loading of microorganisms and their fermentation products inside the biochar. On the other hand, it can increase the positive charge density inside the biochar, thereby enhancing its adsorption capacity for particulate pollutants through electrostatic adsorption and improving the flocculation effect of the microbial agent on particulate pollutants.

[0019] Further, step (2) specifically involves: adding nitrogen-rich biochar, dilute sulfuric acid and deionized water to a reactor, stirring evenly, then adding acrylic acid, modified monomer and catalyst, heating to 60-80℃ and reacting for 6-8 hours, filtering, washing and drying to obtain the nitrogen-rich grafted biochar. The mass concentration of the dilute sulfuric acid is 30-50%; the mass ratio of the nitrogen-rich biochar, acrylic acid, and modified monomer is (15-20):(3-5):(2-3); the catalyst is selected from sodium persulfate, potassium persulfate, or ammonium persulfate.

[0020] The nitrogen-rich biochar of the present invention can be further grafted with acrylic acid and modified monomers on its surface, which can significantly increase the carboxyl content on the surface of the biochar and expand its steric hindrance, thereby expanding the adsorption capacity of the bacterial agent for ammonia nitrogen and phosphorus pollutants through ion exchange, while further expanding the adsorption range and enhancing the sewage treatment capacity of the bacterial agent.

[0021] Another aspect of the present invention provides a method for preparing any one of the above-mentioned microbial compound agents, comprising the following steps: adding microbial liquid and carrier in proportion in a fermenter, controlling dissolved oxygen >1 mg / L during fermentation, stopping fermentation after 36-48 hours, and obtaining the microbial compound agent.

[0022] Beneficial effects: (1) The microbial compound agent provided by the present invention is made by combining a compound bacterial liquid with excellent flocculation ability with a carrier that has undergone charge modification and adsorption modification. It can significantly improve the adsorption and flocculation ability of the agent for particles in sewage, thereby effectively reducing the content of COD, ammonia nitrogen and total phosphorus in sewage. It can be widely used in the field of sewage treatment.

[0023] (2) The microbial compound agent provided by the present invention is prepared by mixing Bacillus subtilis and Bacillus licheniformis to form a compound bacterial solution. On the one hand, the extracellular polysaccharide produced by Bacillus licheniformis after fermentation can be used as a bioflocculant to effectively flocculate particulate pollutants in sewage. On the other hand, Bacillus subtilis can effectively inhibit the growth of various bacteria such as Gram-positive bacteria in sewage. At the same time, its fermentation products can improve the aggregation stability of flocs, which is convenient for subsequent filtration and separation, and further improve the flocculation ability of the agent.

[0024] (3) The microbial composite agent provided by the present invention uses nitrogen-rich grafted biochar that has been modified by charge and adsorption as a carrier. It can not only significantly improve the flocculation ability of the agent for particulate pollutants in sewage, but also expand the influence range of the agent in sewage through its excellent adsorption capacity, thereby improving the sewage treatment capacity of the agent.

[0025] (4) The microbial compound agent provided by the present invention has an electron-deficient region formed inside the biochar after nitrogen enrichment modification. On the one hand, it can form hydrogen bonds with the polysaccharides of the microbial cell wall, thereby stabilizing the loading of microorganisms and their fermentation products inside the biochar; on the other hand, it can increase the positive charge density inside the biochar, thereby enhancing its adsorption capacity for particulate pollutants through electrostatic adsorption and improving the flocculation effect of the agent on particulate pollutants.

[0026] (5) The microbial composite agent provided by the present invention can further graft acrylic acid and modified monomers on the surface of nitrogen-rich biochar, which can significantly increase the carboxyl content on the surface of biochar and expand its steric hindrance, thereby expanding the adsorption capacity of the agent for ammonia nitrogen and phosphorus pollutants through ion exchange, and further expanding the adsorption range and enhancing the wastewater treatment capacity of the agent. Detailed Implementation

[0027] The present invention will be described below with reference to specific embodiments. It should be noted that the following embodiments are examples of the present invention and are used only to illustrate the invention, not to limit it. Other combinations and various modifications within the scope of the present invention can be made without departing from its spirit or scope.

[0028] In this invention, Bacillus subtilis MES 810 was selected. This strain was deposited by the applicant at the China General Microbiological Culture Collection Center (CGMCC) on August 10, 2017, with accession number CGMCC 14514. It should be noted that the Bacillus subtilis MES 810 strain of the present invention has been patented by our company, application number 201710862006.3; In this invention, the Bacillus licheniformis strain selected is Bacillus licheniformis MES816, which was deposited by the applicant at the China General Microbiological Culture Collection Center (CGMCC) on May 8, 2018, with accession number CGMCC 15744. It should be noted that the Bacillus licheniformis MES816 of this invention has been patented by our company, application number 202111399261.1; The remaining reagents and equipment are conventional reagents and equipment in this technical field.

[0029] Preparation of modified monomers The modified monomer was prepared by the following steps: In a reactor, 0.12 mol itaconic acid and 100 mL tetrahydrofuran were added and stirred to dissolve. Then, 0.1 mol 4-hydroxycyclohexanone and 3 mL concentrated sulfuric acid with a mass concentration of 98% were added. The temperature was raised to 70 °C and the reaction was carried out for 6 hours. The modified monomer was obtained by vacuum distillation. Mass spectrometry data of the modified monomers: The products were analyzed by LC-MS, and the m / z of the products were 226.08 (100.0%) and 227.17 (13.1%).

[0030] Preparation of nitrogen-rich grafted biochar-1 Nitrogen-rich grafted biochar-1 was prepared using the following steps: (1) 500g of wheat straw was crushed to an average particle size of 50mm and mixed with 20g of urea. The nitrogen-rich biochar was obtained by dry distillation at 500℃ for 2 hours. (2) In the reactor, add 100g of nitrogen-rich biochar, 10mL of 35% dilute sulfuric acid and 300mL of deionized water, stir evenly, add 25g of acrylic acid, 15g of modified monomer and 2g of ammonium persulfate, heat to 70℃ and react for 8 hours, filter, wash and dry to obtain the nitrogen-rich grafted biochar-1.

[0031] Preparation of nitrogen-rich grafted biochar-2 The preparation method is basically the same as that of nitrogen-rich grafted biochar-1, except that the modified monomer in step (2) is replaced with an equal amount of acrylic acid.

[0032] Example 1 Microbial compound inoculants are prepared by the following steps: By weight, 8 parts of compound bacterial solution and 20 parts of nitrogen-rich grafted biochar-1 were added to the fermenter. Dissolved oxygen was controlled to be >1 mg / L during fermentation. Fermentation was stopped after 48 hours to obtain the microbial compound inoculant. The effective viable bacteria count in the compound microbial agent is 5 × 10⁶. 9 CFU / g; The effective viable count ratio of Bacillus subtilis to Bacillus licheniformis is 2:1.

[0033] Example 2 The process is basically the same as in Example 1, except that the number of effective viable bacteria in the compound microbial agent is 3 × 10⁻⁶. 9 CFU / g; the effective viable count ratio of Bacillus subtilis and Bacillus licheniformis is 1:1.

[0034] Example 3 The process is basically the same as in Example 1, except that the nitrogen-rich grafted biochar-1 is changed to 15 parts.

[0035] Comparative Example 1 The method is basically the same as in Example 1, except that nitrogen-rich grafted biochar-1 is replaced with an equal amount of wheat straw biochar.

[0036] Comparative Example 2 The process is basically the same as in Example 1, except that nitrogen-rich grafted biochar-1 is replaced with nitrogen-rich biochar prepared by an equal amount of nitrogen-rich grafted biochar-1.

[0037] Comparative Example 3 The method is basically the same as in Example 1, except that nitrogen-rich grafted biochar-1 is replaced with an equal amount of nitrogen-rich grafted biochar-2.

[0038] Comparative Example 4 Basically the same as Example 1, except that the compound microbial agent is changed to have an effective viable count of 5 × 10⁻⁶. 9 Bacillus subtilis CFU / g.

[0039] Comparative Example 5 Basically the same as Example 1, except that the compound microbial agent is changed to have an effective viable count of 5 × 10⁻⁶. 9 Bacillus licheniformis CFU / g.

[0040] Performance testing 1 kg of the products from Examples 1-3 and Comparative Examples 1-5 were uniformly added to 1 ml of water. 3 The wastewater was stirred for 1 hour and then allowed to stand for 24 hours. The COD, ammonia nitrogen, and total chlorine content in the wastewater were then measured. The results are shown in the table below: COD (mg / L) Ammonia nitrogen (mg / L) Total chlorine (mg / L) Blank control group 580 380 36 Example 1 53 0.24 0.29 Example 2 55 0.25 0.28 Example 3 55 0.24 0.28 Comparative Example 1 96 0.62 0.87 Comparative Example 2 80 0.57 0.79 Comparative Example 3 65 0.39 0.44 Comparative Example 4 244 1.32 1.58 Comparative Example 5 127 0.87 0.98 According to the comparison of the test results of Examples 1-3 and Comparative Examples 1-3, it can be seen that the microbial compound agent provided by the present invention, by using overcharge-modified and adsorption-modified biochar as a carrier, can significantly improve the treatment effect of pollutants such as COD components, ammonia nitrogen and total chlorine in sewage, and thus can be widely used in the field of sewage treatment.

[0041] According to the comparison of the test results of Examples 1-3 and Comparative Examples 4-5, the microbial compound agent provided by the present invention, by mixing Bacillus subtilis and Bacillus licheniformis to prepare a compound bacterial solution, can effectively flocculate particulate pollutants in sewage through the synergistic effect of the two strains and their fermentation products, thereby effectively reducing the content of COD components, ammonia nitrogen and total chlorine in sewage.

[0042] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it. They should not be used to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A microbial compound inoculant, characterized in that, This includes microbial inoculum, its fermentation products, and carriers; The microbial inoculum includes Bacillus subtilis and Bacillus licheniformis; The carrier is nitrogen-rich grafted biochar. The nitrogen-enriched grafted biochar is prepared by grafting acrylic acid and modified monomers onto biochar after it has been modified to be nitrogen-enriched.

2. The microbial compound inoculant according to claim 1, characterized in that, The number of viable bacteria in the microbial culture solution is not less than 3 × 10⁻⁶. 9 CFU / g; The effective viable count ratio of Bacillus subtilis to Bacillus licheniformis is (2-3):(1-2).

3. The microbial compound inoculant according to claim 1, characterized in that, The mass ratio of the microbial culture to the carrier is (6-8):(15-20).

4. The microbial compound inoculant according to claim 1, characterized in that, The modified monomer is prepared by the following steps: in a reactor, itaconic acid and tetrahydrofuran are added, stirred and dissolved, then 4-hydroxycyclohexanone and concentrated sulfuric acid are added, the temperature is raised to 60-70°C, and after reacting for 4-6 hours, the modified monomer is obtained by vacuum distillation.

5. The microbial compound inoculant according to claim 4, characterized in that, The molar ratio of itaconic acid to 4-hydroxycyclohexanone is 1.2-1.3:1; the mass concentration of the concentrated sulfuric acid is 90-99%.

6. The microbial compound inoculant according to claim 1, characterized in that, The nitrogen-rich grafted biochar is prepared through the following steps: (1) Nitrogen-rich biochar is obtained by dry distillation of a mixture of biological straw and urea; (2) The nitrogen-rich grafted biochar is prepared by reacting nitrogen-rich biochar with acrylic acid and modified monomers.

7. The microbial compound inoculant according to claim 6, characterized in that, The specific steps (1) are as follows: after crushing the biological straw, it is mixed with urea and then dry distilled at 400-600℃ for 1-2 hours to obtain the nitrogen-rich biochar. The biological straw is selected from at least one of wheat straw, rice straw, or corn straw; the average particle size of the crushed biological straw is 30-80 mm; and the mass ratio of the biological straw to urea is 25-35:

1.

8. The microbial compound inoculant according to claim 6, characterized in that, The specific steps (2) are as follows: In the reactor, nitrogen-rich biochar, dilute sulfuric acid and deionized water are added, and after stirring evenly, acrylic acid, modified monomer and catalyst are added. After heating to 60-80℃, the reaction is carried out for 6-8 hours. After filtration, washing and drying, the nitrogen-rich grafted biochar is obtained. The mass concentration of the dilute sulfuric acid is 30-50%; the mass ratio of the nitrogen-rich biochar, acrylic acid, and modified monomer is (15-20):(3-5):(2-3); the catalyst is selected from sodium persulfate, potassium persulfate, or ammonium persulfate.

9. The method for preparing the microbial compound inoculant according to any one of claims 1-8, characterized in that, The process includes the following steps: adding microbial inoculum and carrier to a fermenter in proportion, controlling dissolved oxygen >1mg / L during fermentation, stopping fermentation after 36-48 hours, and obtaining the microbial compound inoculum.