Complex microbial inoculant and application thereof in deodorization

Abstract

The invention relates to the technical field of deodorization, in particular to a complex microbial inoculant and application in deodorization, the complex microbial inoculant comprises bacillus subtilis, bacillus licheniformis and aspergillus oryzae, and the deodorization effect of microorganisms is further improved through cooperation of various microorganisms, the immobilized microorganism technology and optimization of a carrier structure. And a new thought is provided for popularization and use of the microbial deodorant and landfill infiltration treatment. The prepared complex microbial inoculant has a good removal effect on the odor of the landfill leachate, and can be applied to the deodorization process of the landfill leachate.

Description

Technical Field

[0001] The present invention relates to the technical field of deodorization, in particular to a composite bacterial agent and its application in deodorization. Background Art

[0002] Malodor, a major atmospheric pollution nuisance, has received widespread attention. Landfill odor treatment is currently a challenging and hot topic. Landfill leachate, a complex and diverse product, contains high concentrations of ammonia, hydrogen sulfide, methane, and other malodorous gases, significantly polluting the surrounding environment and severely impacting people's daily lives.

[0003] Existing research has found that many microbial metabolites can eliminate odor molecules or degrade organic pollutants, thereby achieving a deodorizing effect. By combining multiple microorganisms, immobilizing microorganisms, or optimizing carrier structures, the deodorizing effect of microorganisms can be further improved. This provides new ideas for the promotion and use of microbial deodorants and the treatment of landfill leachate. Summary of the Invention

[0004] Purpose of the invention: In order to solve the above technical problems, the present invention proposes a composite bacterial agent and its application in deodorization.

[0005] The technical solutions adopted are as follows: A composite bacterial agent comprises Bacillus subtilis, Bacillus licheniformis and Aspergillus oryzae.

[0006] Furthermore, a carrier is also included.

[0007] Furthermore, the carrier is an amino acid-metal ion complex intercalated clay mineral.

[0008] Furthermore, the amino acid is any one or a combination of two or more of glycine, alanine, valine, leucine, methionine, proline, tyrosine, cysteine, phenylalanine, threonine, aspartic acid, glutamic acid, arginine, and histidine.

[0009] Furthermore, the metal ions are transition metal ions.

[0010] Furthermore, the transition metal ion is Mn 2+ 、Zn 2+ 、Fe 2+ Any one or a combination of two or more.

[0011] Furthermore, the clay mineral is kaolin.

[0012] Furthermore, the preparation method of the amino acid-metal ion complex is as follows: After dissolving the amino acid in water, add a water-soluble metal salt, stir in a water bath at 60-80°C for 1-10 hours, then let stand at room temperature, filter out the precipitate and dry it.

[0013] Furthermore, the preparation method of the amino acid-metal ion complex intercalated clay mineral is as follows: First, clay mineral, DMSO and water are mixed to react to obtain a clay mineral / DMSO intercalation complex, the clay mineral / DMSO intercalation complex is then mixed with methanol to react to obtain a clay mineral / methanol intercalation complex, and finally, an amino acid-metal ion complex is mixed with the clay mineral / methanol intercalation complex to react to obtain an amino acid-metal ion complex intercalated clay mineral.

[0014] Application of the above composite bacterial agent in deodorization.

[0015] Beneficial effects of the present invention: The present invention provides a composite bacterial agent. Bacillus subtilis can secrete multiple extracellular enzymes (such as proteases, lipases, and amylases). The multiple enzymes secreted by the Bacillus subtilis can effectively decompose sulfur- and nitrogen-containing organic matter in landfill leachate, converting them into odorless or low-odor small molecules. The antibacterial substances (such as subtilisin and organic acids) produced by the Bacillus subtilis can directly inhibit the activity of odor-producing pathogens (such as Escherichia coli). Bacillus licheniformis focuses on macromolecular degradation and ecological regulation. It decomposes macromolecular organic matter (such as plant residues and humus) in landfill leachate by secreting cellulase and pectinase, reducing malodorous substances such as hydrogen sulfide and ammonia produced by the decay of organic matter. It also inhibits the growth of anaerobic odor-producing bacteria by rapidly consuming oxygen in the environment. At the same time, it secretes antimicrobial proteins to destroy the cell walls of pathogens, reducing their metabolic odor-producing ability. It can also regulate the microbial community structure in the leachate, inhibit the dominance of odor-producing bacteria, and promote the proliferation of beneficial bacteria. Cellulase and ligninase secreted by Aspergillus oryzae can degrade difficult-to-decompose substances such as cellulose and lignin in the leachate, reducing odor precursors such as volatile organic acids produced during the humification process. Furthermore, through fermentation, macromolecular organic matter is converted into carbon dioxide and water, and stable substances such as humic acid are generated, thereby reducing the release of malodor. Amino acid-metal ion complex intercalated clay minerals as microbial carriers can provide microorganisms with attachment sites with high specific surface area, protecting them from extreme pH or toxic substances, and making it easy to remove the composite bacterial agent from the leachate. It can be used continuously and repeatedly. The metal ions in the amino acid-metal ion complex activate microorganisms and promote the secretion of their extracellular enzymes, thereby greatly improving the deodorization effect on landfill leachate.

[0016] This invention further improves the deodorizing effect of microorganisms by combining multiple microorganisms, immobilizing microorganisms, and optimizing carrier structures. This provides new insights into the widespread use of microbial deodorants and the management of landfill leachate. The prepared composite bacterial agent exhibits excellent odor removal effectiveness in landfill leachate and can be applied to landfill leachate deodorization processes. DETAILED DESCRIPTION

[0017] Unless otherwise specified, the following examples and comparative examples were conducted in parallel, using the same processing steps and parameters.

[0018] The invention provides a composite bacterial agent consisting of Bacillus subtilis, Bacillus licheniformis and Aspergillus oryzae.

[0019] Bacillus subtilis can secrete a variety of extracellular enzymes (such as proteases, lipases, and amylases). The various enzymes it secretes can effectively decompose sulfur- and nitrogen-containing organic matter in landfill leachate, converting them into odorless or low-odor small molecules. The antibacterial substances it produces (such as subtilisin and organic acids) can directly inhibit the activity of odor-producing pathogens (such as Escherichia coli).

[0020] Bacillus licheniformis focuses on macromolecular degradation and ecological regulation. It decomposes macromolecular organic matter (such as plant residues and humus) in landfill leachate by secreting cellulase, pectinase, etc., reducing malodorous substances such as hydrogen sulfide and ammonia produced by the corruption of organic matter, and inhibiting the growth of anaerobic odor-producing bacteria by rapidly consuming oxygen in the environment. At the same time, it secretes antimicrobial proteins to destroy the cell walls of pathogens, reduce their metabolic odor-producing ability, and regulate the microbial community structure in the leachate, inhibit the dominance of odor-producing bacteria, and promote the proliferation of beneficial bacteria.

[0021] The cellulase and ligninase secreted by Aspergillus oryzae can degrade difficult-to-decompose substances such as cellulose and lignin in the leachate, reduce the odor precursors such as volatile organic acids produced during the humification process, and convert large molecular organic matter into carbon dioxide and water through fermentation, and generate stable substances such as humic acid, thereby reducing the release of odor.

[0022] The invention deodorizes garbage leachate by synergistically using Bacillus subtilis, Bacillus licheniformis and Aspergillus oryzae.

[0023] The composite bacterial agent of the present invention further comprises a carrier.

[0024] The role of carriers in microbial encapsulation is primarily reflected in physical protection, functional enhancement, and environmental adaptability regulation. Carriers form physical barriers through adsorption or encapsulation, reducing damage to microorganisms from external environments (such as ultraviolet light, extreme pH, and water loss). Carriers provide microorganisms with high-surface-area attachment sites, immobilizing them and preventing their rapid loss in dynamic environments (such as water flow). Carriers can also promote microbial proliferation, stimulate bacterial metabolic activity, and promote the secretion of active substances.

[0025] Wherein, the carrier is an amino acid-metal ion complex intercalated clay mineral.

[0026] Furthermore, the amino acid is any one or a combination of two or more of glycine, alanine, valine, leucine, methionine, proline, tyrosine, cysteine, phenylalanine, threonine, aspartic acid, glutamic acid, arginine, and histidine.

[0027] Wherein, the metal ions are transition metal ions.

[0028] The above-mentioned transition metal ions refer to charged particles formed by the loss of electrons by transition metal elements (located in the d zone of the periodic table, including elements from groups 3 to 12). Their core characteristic is that they have incompletely filled d-orbital electrons (or ions that can form such an electronic configuration).

[0029] Wherein, the transition metal ion is Mn 2+ 、Zn 2+ 、Fe 2+ Any one or a combination of two or more.

[0030] Wherein, the clay mineral is kaolin.

[0031] Kaolin is a layered silicate mineral with a rich microporous and mesoporous structure (pore size 0.1-10 μm), resulting in a specific surface area of 10-30 m² / g³. This structure provides numerous attachment sites for microorganisms, promoting efficient colonization and proliferation. Kaolin immobilizes microorganisms through van der Waals forces, hydrogen bonds, and electrostatic interactions, making it particularly suitable for adsorption to prevent bacterial loss. Kaolin, due to its high adsorption capacity, chemical stability, low cost, and environmental friendliness, is an ideal carrier for immobilizing microorganisms.

[0032] Wherein, the preparation method of the amino acid-metal ion complex is as follows: After dissolving the amino acid in water, add a water-soluble metal salt, stir in a water bath at 60-80°C for 1-10 hours, then let stand at room temperature, filter out the precipitate and dry it.

[0033] The preparation method of the amino acid-metal ion complex intercalated clay mineral is as follows: First, clay mineral, DMSO and water are mixed to react to obtain a clay mineral / DMSO intercalation complex, the clay mineral / DMSO intercalation complex is then mixed with methanol to react to obtain a clay mineral / methanol intercalation complex, and finally, an amino acid-metal ion complex is mixed with the clay mineral / methanol intercalation complex to react to obtain an amino acid-metal ion complex intercalated clay mineral.

[0034] The three-step intercalation method addresses the problem of tight interlayer bonding in clay minerals, hindering the direct insertion of amino acid-metal ion complexes, by gradually expanding the interlayer spacing and strengthening the hydrogen-bonding network. A small polar molecule (dimethyl sulfoxide (DMSO)) is first used as an initial intercalation agent. These molecules, through their functional groups, form hydrogen bonds with the hydroxyl (-OH) groups of the aluminum oxide layer of kaolin, disrupting the existing interlayer hydrogen-bonding network and increasing the interlayer spacing from 0.72 nm to 1.07-1.13 nm. The clay mineral / DMSO intercalation complex is then mixed with methanol, where the polar hydroxyl groups of methanol displace the DMSO molecules. Methanol molecules further expand the interlayer spacing through OHO-type hydrogen bonds and provide a more relaxed interlayer environment for the subsequent amino acid-metal ion complex. Finally, the amino acid-metal ion complex forms multiple hydrogen bonds with the oxygen atoms of the clay mineral's silicon oxide layer or the aluminum oxide layer's hydroxyl groups via NH or OH groups, achieving intercalation.

[0035] Application of the above composite bacterial agent in deodorization.

[0036] Furthermore, the above composite bacterial agent is used in the deodorization of landfill leachate.

[0037] Example 1: A composite bacterial agent, composed of Bacillus subtilis, Bacillus licheniformis, Aspergillus oryzae and a carrier, is prepared as follows: Bacillus subtilis and Bacillus licheniformis were inoculated into LB liquid medium respectively, and cultured in a shaking incubator at 37°C for 24 h (speed 180 rpm) to obtain their respective activated bacterial solutions. The activated bacterial solutions were transferred to fermentation tanks at a 5% inoculation rate. The fermentation medium contained 20 g / L glucose, 15 g / L soybean meal, and 5 g / L ammonium sulfate. The pH was controlled at 6.8-7.2. The fermentation was carried out at 37°C for 36 h to obtain bacterial fermentation liquid. The bacterial fermentation liquid was centrifuged (8000 rpm, 15 min) to collect bacterial mud, and resuspended to 10 10CFU / mL was used to obtain a bacterial suspension. Aspergillus oryzae was inoculated into PDA slant medium and cultured at 28°C for 7 days to form mature spores. The spore suspension was inoculated into solid medium (wheat bran: rice husk = 7:3) with a humidity of 60% and cultured at 30°C for 72 h. The Aspergillus oryzae culture was dried with hot air at 40°C and then crushed through an 80-mesh sieve to obtain Aspergillus oryzae spore powder with a spore concentration of ≥1×10 9 CFU / g; After dissolving glycine in an appropriate amount of deionized water, ferrous chloride was added, and the molar ratio of glycine to ferrous chloride was 2:1. The reaction solution was stirred in a water bath at 70°C for 10 hours, then the water bath was removed, and the mixture was allowed to stand at room temperature for 24 hours, then filtered. The resulting precipitate was dried to obtain a glycine ferrous complex. First, 2 g of kaolin, 30 mL of DMSO, and 3 mL of deionized water were mixed, stirred at room temperature for 72 hours, and then filtered and dried to obtain a kaolin / DMSO intercalation complex. 1 g of kaolin / DMSO intercalation complex was then mixed with 20 mL of methanol, stirred at room temperature for 6 days, and then filtered and dried to obtain a kaolin / methanol intercalation complex. Finally, 1 g of glycine ferrous complex, 1 g of kaolin / methanol intercalation complex, and 10 mL of deionized water were mixed, stirred in a water bath at 50°C for 10 hours, and then filtered and dried to obtain a carrier. The Bacillus subtilis suspension, the Bacillus licheniformis suspension, the Aspergillus oryzae spore powder and the carrier were mixed in a mass ratio of 3:3:2:2, stirred at a low speed (40 rpm) for 2 h in a sterile mixer and then dried in a fluidized bed at 40°C.

[0038] Example 2: The process is basically the same as Example 1, except that the ferrous glycine complex is replaced by the manganese glycine complex.

[0039] A composite bacterial agent, composed of Bacillus subtilis, Bacillus licheniformis, Aspergillus oryzae and a carrier, is prepared as follows: Bacillus subtilis and Bacillus licheniformis were inoculated into LB liquid medium respectively, and cultured in a shaking incubator at 37°C for 24 h (speed 180 rpm) to obtain their respective activated bacterial solutions. The activated bacterial solutions were transferred to fermentation tanks at a 5% inoculation rate. The fermentation medium contained 20 g / L glucose, 15 g / L soybean meal, and 5 g / L ammonium sulfate. The pH was controlled at 6.8-7.2. The fermentation was carried out at 37°C for 36 h to obtain bacterial fermentation liquid. The bacterial fermentation liquid was centrifuged (8000 rpm, 15 min) to collect bacterial mud, and resuspended to 10 10CFU / mL was used to obtain a bacterial suspension. Aspergillus oryzae was inoculated into PDA slant medium and cultured at 28°C for 7 days to form mature spores. The spore suspension was inoculated into solid medium (wheat bran: rice husk = 7:3) with a humidity of 60% and cultured at 30°C for 72 h. The Aspergillus oryzae culture was dried with hot air at 40°C and then crushed through an 80-mesh sieve to obtain Aspergillus oryzae spore powder with a spore concentration of ≥1×10 9 CFU / g; After dissolving glycine in an appropriate amount of deionized water, manganese sulfate was added, and the molar ratio of glycine to manganese sulfate was 2:1. The reaction solution was stirred in a water bath at 70°C for 10 hours, then the water bath was removed, and the mixture was allowed to stand at room temperature for 24 hours before filtering. The resulting precipitate was dried to obtain a glycine manganese complex. First, 2 g of kaolin, 30 mL of DMSO, and 3 mL of deionized water were mixed, stirred at room temperature for 72 hours, filtered, and dried to obtain a kaolin / DMSO intercalation complex. 1 g of the kaolin / DMSO intercalation complex was then mixed with 20 mL of methanol, stirred at room temperature for 6 days, filtered, and dried to obtain a kaolin / methanol intercalation complex. Finally, 1 g of the glycine manganese complex, 1 g of the kaolin / methanol intercalation complex, and 10 mL of deionized water were mixed, stirred in a water bath at 50°C for 10 hours, filtered, and dried to obtain a carrier. The Bacillus subtilis suspension, the Bacillus licheniformis suspension, the Aspergillus oryzae spore powder and the carrier were mixed in a mass ratio of 3:3:2:2, stirred at a low speed (40 rpm) for 2 h in a sterile mixer and then dried in a fluidized bed at 40°C.

[0040] Example 3: The method is basically the same as Example 1, except that the ferrous glycine complex is replaced by the zinc glycine complex.

[0041] A composite bacterial agent, composed of Bacillus subtilis, Bacillus licheniformis, Aspergillus oryzae and a carrier, is prepared as follows: Bacillus subtilis and Bacillus licheniformis were inoculated into LB liquid medium respectively, and cultured in a shaking incubator at 37°C for 24 h (speed 180 rpm) to obtain their respective activated bacterial solutions. The activated bacterial solutions were transferred to fermentation tanks at a 5% inoculation rate. The fermentation medium contained 20 g / L glucose, 15 g / L soybean meal, and 5 g / L ammonium sulfate. The pH was controlled at 6.8-7.2. The fermentation was carried out at 37°C for 36 h to obtain bacterial fermentation liquid. The bacterial fermentation liquid was centrifuged (8000 rpm, 15 min) to collect bacterial mud, and resuspended to 10 10CFU / mL was used to obtain a bacterial suspension. Aspergillus oryzae was inoculated into PDA slant medium and cultured at 28°C for 7 days to form mature spores. The spore suspension was inoculated into solid medium (wheat bran: rice husk = 7:3) with a humidity of 60% and cultured at 30°C for 72 h. The Aspergillus oryzae culture was dried with hot air at 40°C and then crushed through an 80-mesh sieve to obtain Aspergillus oryzae spore powder with a spore concentration of ≥1×10 9 CFU / g; After dissolving glycine in an appropriate amount of deionized water, zinc chloride was added, and the molar ratio of glycine to zinc chloride was 2:1. The reaction solution was stirred in a water bath at 70°C for 10 hours, then the water bath was removed, and the mixture was allowed to stand at room temperature for 24 hours, then filtered, and the resulting precipitate was dried to obtain a glycine zinc complex. First, 2 g of kaolin, 30 mL of DMSO, and 3 mL of deionized water were mixed, stirred at room temperature for 72 hours, and then filtered and dried to obtain a kaolin / DMSO intercalation complex. 1 g of kaolin / DMSO intercalation complex was then mixed with 20 mL of methanol, stirred at room temperature for 6 days, and then filtered and dried to obtain a kaolin / methanol intercalation complex. Finally, 1 g of glycine zinc complex, 1 g of kaolin / methanol intercalation complex, and 10 mL of deionized water were mixed, stirred in a water bath at 50°C for 10 hours, and then filtered and dried to obtain a carrier. The Bacillus subtilis suspension, the Bacillus licheniformis suspension, the Aspergillus oryzae spore powder and the carrier were mixed in a mass ratio of 3:3:2:2, stirred at a low speed (40 rpm) for 2 h in a sterile mixer and then dried in a fluidized bed at 40°C.

[0042] Comparative Example 1: basically the same as Example 1, except that the composite bacterial agent does not contain a carrier.

[0043] Comparative Example 2: basically the same as Example 1, except that kaolin is directly used as the carrier.

[0044] Comparative Example 3: basically the same as Example 1, except that the composite bacterial agent does not contain Bacillus subtilis.

[0045] Comparative Example 4: basically the same as Example 1, except that the composite bacterial agent does not contain Bacillus licheniformis.

[0046] Comparative Example 5: is basically the same as Example 1, except that the composite bacterial agent does not contain Aspergillus oryzae.

[0047] Performance testing: Leachate from a landfill was collected in a sealed container. The composite bacterial agents from Examples 1-3 and Comparative Examples 1-5 were added at a rate of 10% by mass of the leachate. The mixture was incubated at 28°C, with a constant shaking speed of 180 rpm for 3 days, followed by a constant static incubation for 2 days. The ammoniacal nitrogen content of the leachate was determined using the distillation-neutralization titration method described in HJ-537-2009, Water Quality - Determination of Ammoniacal Nitrogen. Odor thresholds were also determined using the odor threshold method.

[0048] The odor threshold of landfill leachate is determined using the "Odor Threshold Determination Method in Water Quality Testing Methods" (2000) issued by the United States, that is, the water sample is diluted with odorless water until the lowest concentration of discernible odor is detected (odor threshold concentration), which is used to represent the odor threshold. The dilution multiple at which the water sample is diluted to the point where the odor is just detected is called the odor threshold.

[0049] Odor threshold = (water sample volume + odorless water sample volume) / water sample volume; The test results are shown in Table 1 below:

[0050] As can be seen from Table 1 above, the composite bacterial agent prepared by the present invention has a good effect in removing the odor of landfill leachate.

[0051] From the comparison of Examples 1-3, it can be seen that the composite bacterial agents prepared by using different amino acid-metal ion complexes have a good effect on removing the odor of landfill leachate.

[0052] By comparing Example 1 with Comparative Example 1, it can be seen that when no carrier is included, the odor removal effect of the composite bacterial agent on landfill leachate is reduced.

[0053] By comparing Example 1 and Comparative Example 2, it can be seen that when kaolin intercalated with ferrous glycine complex is used as a carrier, the removal effect of the composite bacterial agent on the odor of landfill leachate is greatly improved.

[0054] By comparing Example 1 with Comparative Examples 1-3, it can be seen that Bacillus subtilis, Bacillus licheniformis and Aspergillus oryzae in the composite bacterial agent all play an important role in removing odor from landfill leachate.

[0055] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A composite bacterial agent, characterized in that: Its composition includes Bacillus subtilis, Bacillus licheniformis and Aspergillus oryzae.

2. The composite bacterial agent according to claim 1, wherein Also included are carriers.

3. The composite bacterial agent according to claim 2, wherein The carrier is an amino acid-metal ion complex intercalated clay mineral.

4. The composite bacterial agent according to claim 3, wherein The amino acid is any one or a combination of two or more of glycine, alanine, valine, leucine, methionine, proline, tyrosine, cysteine, phenylalanine, threonine, aspartic acid, glutamic acid, arginine, and histidine.

5. The composite bacterial agent according to claim 3, characterized in that The metal ions are transition metal ions.

6. The composite bacterial agent according to claim 5, wherein The transition metal ion is Mn 2+ 、Zn 2+ 、Fe 2+ Any one or a combination of two or more.

7. The composite bacterial agent according to claim 3, characterized in that The clay mineral is kaolin.

8. The composite bacterial agent according to claim 3, wherein The preparation method of the amino acid-metal ion complex is as follows: After dissolving the amino acid in water, add a water-soluble metal salt, stir in a water bath at 60-80°C for 1-10 hours, then let stand at room temperature, filter out the precipitate and dry it.

9. The composite bacterial agent according to claim 3, wherein The preparation method of the amino acid-metal ion complex intercalated clay mineral is as follows: First, clay mineral, DMSO and water are mixed to react to obtain a clay mineral / DMSO intercalation complex, the clay mineral / DMSO intercalation complex is then mixed with methanol to react to obtain a clay mineral / methanol intercalation complex, and finally, an amino acid-metal ion complex is mixed with the clay mineral / methanol intercalation complex to react to obtain an amino acid-metal ion complex intercalated clay mineral.

10. Use of the composite bacterial agent according to any one of claims 2 to 9 in deodorization.

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