Preparation method and application of complex microbial inoculant for kitchen waste

Abstract

The invention discloses a preparation method and application of a complex microbial inoculant for kitchen waste, and belongs to the technical field of waste treatment. The compound bacterial agent disclosed by the invention comprises bacillus amyloliquefaciens CY-3, bacillus amyloliquefaciens CY-12, bacillus tequilensis HB-10 and bacillus licheniformis B13. According to the invention, four bacilli which can be used for fermentation and acid production of the kitchen waste are screened out and are further prepared into the complex microbial inoculant, so that the yield of organic acid in the fermentation process of the kitchen waste can be increased, a new microbial resource is developed for efficient acid production of the kitchen waste, and a more efficient method is provided for kitchen waste treatment and sewage treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of waste treatment, and particularly to a preparation method and application of a composite bacterium agent for kitchen waste. Background Art

[0002] Kitchen waste is a kind of solid waste formed in people's life consumption and is also a main component of urban domestic waste. Compared with other wastes, it has complex components and rich nutrients. Its main components are starch, protein, fat and cellulose, and it has the characteristic of being easy to pollute the environment. According to data, the annual compound growth rate of kitchen waste in China will continue to increase at an increment of 12%-15%. Therefore, the reasonable resource utilization of kitchen waste is of great significance to environmental protection and the rational utilization of resources.

[0003] A carbon source is a carbon-containing compound that can provide nutrients for the growth and metabolism of microorganisms in a sewage (wastewater) biochemical treatment system. Its effective components are organic compounds with a single molecular formula and molecular structure and are easily utilized by microorganisms, including small molecule alcohols such as methanol, ethanol, propanol, butanol, ethylene glycol, glycerol, butanol, pentanol, etc., small molecule organic acids and organic acid salts such as formic acid, acetic acid, propionic acid, lactic acid, butyric acid, acetate, citric acid, citrate, etc., and sugar substances such as glucose, fructose, sucrose. Among them, organic acids are high-quality carbon sources produced by microorganisms decomposing large substances, and have the characteristics of low cost, high utilization rate and environmental friendliness.

[0004] The nutritional components of kitchen waste are first hydrolyzed by microorganisms: large molecules are first hydrolyzed into small molecules, and then oxidized to produce organic acids under the action of acidifying bacteria. That is, the process of microbial acid production is a process of first hydrolysis and then acidification. Therefore, the kitchen waste raw material becomes a good source of organic acid carbon source. However, at present, lactic acid bacteria are mostly used when preparing organic acids from kitchen waste. The ability of lactic acid bacteria to decompose macromolecular organic matter is limited, and the lactic acid content in the fermentation broth of kitchen waste fermented by lactic acid bacteria is high. Research shows that the phosphorus removal effect is extremely low when using lactic acid as a carbon source for enhanced biological phosphorus removal (EBPR) of wastewater. The high proportion of lactic acid in organic acids has a certain impact on the application of kitchen waste fermentation broth in sewage treatment, restricting the subsequent application of the fermentation products of kitchen waste. Summary of the Invention

[0005] The purpose of the present invention is to provide a preparation method and application of a composite bacterium agent for kitchen waste to solve the problems existing in the above-mentioned prior art. The present invention has screened out four types of Bacillus that can be used for the fermentation of kitchen waste to produce acid, and further formulated them into a composite bacterium agent, which can improve the production of organic acids during the fermentation of kitchen waste, develop new microbial resources for the efficient acid production of kitchen waste, and provide a more efficient method for the treatment of kitchen waste and sewage treatment.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a Bacillus for fermenting kitchen waste to produce acid, including Bacillus amyloliquefaciens CY-3, Bacillus amyloliquefaciens CY-12, and Bacillus tequilensis HB-10;

[0008] The preservation number of Bacillus amyloliquefaciens CY-3 is CGMCC No. 33561; the preservation number of Bacillus amyloliquefaciens CY-12 is CGMCC No. 33562; the preservation number of Bacillus tequilensis HB-10 is CGMCC No. 33563.

[0009] The present invention also provides a method for preparing a liquid compound bacterial agent for fermenting kitchen waste to produce acid, including the following steps:

[0010] (1) Inoculate single colonies of Bacillus amyloliquefaciens CY-3, Bacillus amyloliquefaciens CY-12, Bacillus tequilensis HB-10, and Bacillus licheniformis B13 into a beef extract peptone liquid medium for culture respectively to prepare a primary seed liquid;

[0011] The preservation number of Bacillus amyloliquefaciens CY-3 is CGMCC No. 33561; the preservation number of Bacillus amyloliquefaciens CY-12 is CGMCC No. 33562; the preservation number of Bacillus tequilensis HB-10 is CGMCC No. 33563; the preservation number of Bacillus licheniformis B13 is CGMCC 1.16278;

[0012] (2) After mixing the primary seed liquids of Bacillus amyloliquefaciens CY-3, Bacillus amyloliquefaciens CY-12, Bacillus tequilensis HB-10, and Bacillus licheniformis B13, inoculate them into a beef extract peptone liquid medium for culture to obtain a secondary seed liquid;

[0013] (3) Inoculate the secondary seed liquid into a beef extract peptone liquid medium for culture to obtain the liquid compound bacterial agent.

[0014] Optionally, in step (2), when mixing the first-stage seed solutions of Bacillus amyloliquefaciens CY-3, Bacillus amyloliquefaciens CY-12, Bacillus tequilensis HB-10, and Bacillus licheniformis B13, the volume ratio is (3-5):(1-3):(1-3):1.

[0015] Optionally, in steps (1)-(3), the temperature of the cultivation is 30°C, and the time of the cultivation is 40-50 h; in steps (2) and (3), the inoculation amount of the inoculation is 4%-8%.

[0016] The present invention also provides a preparation method of a solid composite bacterial agent for fermenting kitchen waste to produce acid, comprising the following steps:

[0017] (1) Respectively mix the bacterial cells of Bacillus amyloliquefaciens CY-3, Bacillus amyloliquefaciens CY-12, Bacillus tequilensis HB-10, and Bacillus licheniformis B13 with skim milk powder, and obtain single-strain bacterial powders through freeze-drying;

[0018] The preservation number of Bacillus amyloliquefaciens CY-3 is CGMCC No. 33561; the preservation number of Bacillus amyloliquefaciens CY-12 is CGMCC No. 33562; the preservation number of Bacillus tequilensis HB-10 is CGMCC No. 33563; the preservation number of Bacillus licheniformis B13 is CGMCC 1.16278;

[0019] (2) After mixing the single-strain bacterial powders of Bacillus amyloliquefaciens CY-3, Bacillus amyloliquefaciens CY-12, Bacillus tequilensis HB-10, and Bacillus licheniformis B13, obtain a composite bacterial powder;

[0020] (3) Mix the composite bacterial powder with peanut shells, wheat bran, wood chips, and beef bone powder to obtain the solid composite bacterial agent.

[0021] Optionally, in step (1), the mass ratio of the bacterial cells to the skim milk powder during mixing is 1:1;

[0022] In step (2), the mass ratio of the single-strain bacterial powders of Bacillus amyloliquefaciens CY-3, Bacillus amyloliquefaciens CY-12, Bacillus tequilensis HB-10, and Bacillus licheniformis B13 during mixing is 4:2:2:1;

[0023] In step (3), the mass ratio of the composite bacterial powder to peanut hulls, wheat bran, wood chips, and bovine bone meal during mixing is 1:2:1:1:1.

[0024] The present invention also provides a liquid composite bacterial agent prepared by the above preparation method.

[0025] The present invention also provides a solid composite bacterial agent prepared by the above preparation method.

[0026] The present invention also provides the application of the above-mentioned Bacillus, the above-mentioned liquid composite bacterial agent, or the above-mentioned solid composite bacterial agent in the fermentation of food waste to produce acid.

[0027] The present invention also provides a fermentation method for the efficient production of acid from food waste, which includes the step of inoculating the above-mentioned liquid composite bacterial agent into the slurry of food waste and performing fermentation culture to produce organic acids.

[0028] Optionally, the inoculation amount is not less than 0.3% of the mass of the food waste slurry; the temperature of the fermentation culture is 37°C.

[0029] The present invention discloses the following technical effects:

[0030] Four Bacillus strains with acid-producing characteristics were screened from the food waste fermentation broth of the present invention, including Bacillus amyloliquefaciens CY-3, Bacillus amyloliquefaciens CY-12, Bacillus tequilensis HB-10, and Bacillus licheniformis CGMCC1.16278. Among them, CY-12 has a protein hydrolysis function, CY-3 has a starch hydrolysis function, HB-10 has a fat hydrolysis function, and B13 has a cellulose hydrolysis function. The present invention uses these four Bacillus strains to prepare a composite bacterial agent for the fermentation of food waste, which greatly improves the fermentation process of food waste and increases the yield of organic acids, develops new microbial resources for the efficient production of acid from food waste, and provides a more efficient method for the treatment of food waste and sewage treatment. Description of the Drawings

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0032] Figure 1Results of the nutrient degradation experiments for strains CY-12, CY-3, HB-10, and B13; CY-12 shows the degradation effect of strain CY-12 on the protein medium; CY-3 shows the degradation effect of strain CY-3 on the starch medium; HB-10 shows the degradation effect of strain HB-10 on the Tween 80 medium; B13 shows the degradation effect of strain B13 on the cellulose medium;

[0033] Figure 2 Phylogenetic tree of 16S rDNA for strains CY-12, CY-3, HB-10, and B13;

[0034] Figure 3 Schematic diagram of a semi-continuous flow anaerobic digestion reactor device;

[0035] Figure 4 Culture results of the plate confrontation experiments for strains CY-12, CY-3, HB-10, and B13;

[0036] Figure 5 Is 4m 3 Physical diagram of a 4m aerobic fermentation tank;

[0037] Figure 6 Growth of the diluted suspension of the solid composite microbial agent on the plate; among them, A, B, and C are the results of three parallel experiments;

[0038] Figure 7 Organic acid production results of the solid composite microbial agent under the semi-continuous flow anaerobic digestion reactor. Detailed implementation mode

[0039] Now, various exemplary embodiments of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0040] It should be understood that the terms described in the present invention are only for describing specific implementation modes and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0041] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to those documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0042] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the description of the present invention, which are obvious to those skilled in the art. Other embodiments obtained from the description of the present invention are obvious to those skilled in the art. The description and examples of the present invention are merely illustrative.

[0043] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0044] The culture medium formulations used in the following examples of the present invention are as follows:

[0045] TSB medium: Tryptone 17.0 g / L, soybean papain digest 3.0 g / L, sodium chloride 5.0 g / L, dipotassium hydrogen phosphate 2.5 g / L, glucose 2.5 g / L, pH 7.3 ± 0.2.

[0046] TSA medium: Tryptone 15.0 g / L, soybean peptone 5.0 g / L, sodium chloride 5.0 g / L, agar 15.0 g / L, pH 7.3 ± 0.2.

[0047] Beef extract peptone medium: Beef extract 3.0 g, peptone 10.0 g, sodium chloride 5.0 g, water 1000 ml, pH 7.0 - 7.5.

[0048] Isolation and identification of the strain in Example 1

[0049] 1. High-throughput isolation and culture of the strain: Add 5 mL of kitchen waste fermentation broth (collected from a kitchen waste treatment center in Beijing, which is the waste liquid discharged after anaerobic fermentation of kitchen waste after crushing, that is, kitchen waste fermentation broth) to 45 mL of TSB medium, and dilute it in five gradients from 10 -3 to 10 -7 Each dilution of each gradient was separately transferred to 3 96-well cell culture plates, and 160 μL was added to each well. Another blank TSB medium was used as a negative control. After 1 week, observe the growth of bacteria in the 96-well plates, and select the dilution concentration with about 30% of the wells being turbid as the optimal concentration. Take the optimal dilution gradient (10-7 ) Perform high-throughput separation and cultivation. Transfer the dilution to 30 96-well plates, 160 μL per well, and use blank TSB medium as the negative control. Incubate in the dark at 30 °C for 2 weeks. Select the 96-well plates with bacterial growth in approximately 30% of the wells for subsequent streak isolation and purification.

[0050] 2. Isolation and purification of strains: Use a microplate reader to measure the OD 600 value of the 96-well plate. The OD 600 value of the blank control is 0.103 ± 0.002. Therefore, when the OD 600 value is close to 0.1, it is considered aseptic growth. Inoculate the bacterial liquid in the wells with an OD 600 value greater than 0.1 onto TSA medium and continuously streak and culture three times using the three-zone streaking method. When the morphology is consistent under microscopic examination, confirm that the grown single colonies are pure strains.

[0051] 3. Screening of nutrient-degrading bacteria

[0052] Spot the above pure strains onto the differential media for starch, protein, cellulose, and fat. Use a vernier caliper to measure the diameters of the hydrolysis zone and the colony, and select the strain with a larger ratio (D / d) of the diameter of the clear zone (D) to the diameter of the colony (d) as the target strain.

[0053] Protein medium (g / L): Skim milk powder 50.0, soluble starch 10.0, yeast extract 5.0, KH2PO4 1, MgSO4·7H2O 0.2, agar 20.0, pH 7.0 - 7.2. Spot the bacteria onto the protein medium and incubate in an incubator at 37 °C. After 72 h, perform screening. After the skim milk is degraded by protease, a colorless and transparent degradation zone will be produced around the colony. Measure the diameter of the hydrolysis zone (D) and the diameter of the colony (d) respectively, and calculate their ratio (D / d). Judge the protease-producing ability of the strain according to the size of the ratio.

[0054] Starch medium (g / L): Soluble starch 5.0, peptone 5.0, beef extract 5.0, NaCl 5.0, agar 20.0, pH 7.0 - 7.2. Spot the bacteria onto the starch medium and incubate in an incubator at 37 °C. After 72 h, add iodine indicator for screening. The unhydrolyzed starch around turns blue when encountering iodine, and a colorless and transparent hydrolysis zone is formed in the starch hydrolysis area. Measure the diameter of the hydrolysis zone (D) and the diameter of the colony (d) respectively, and calculate their ratio (D / d). Judge the amylase-producing ability of the strain according to the size of the ratio.

[0055] Cellulose medium (g / L): Sodium carboxymethylcellulose 20.0, Na2HPO4 2.5, KH2PO4 1.5, peptone 2.5, agar 20, pH 7.0 - 7.2. The bacteria were spotted onto the cellulose medium and cultured in an incubator at 37°C. After 72 h, it was stained with 1 mg·mL -1 Congo red staining solution for 30 min. Then, the used Congo red staining solution was discarded, and 1 mol·L -1 NaCl solution was used for decolorization for 30 min. A distinct transparent circle appeared around the colonies producing cellulase. The diameters of the hydrolysis circle (D) and the colony (d) were measured respectively, and the ratio (D / d) was calculated. The ability of the strain to produce cellulase was judged according to the size of this ratio.

[0056] Tween 80 medium (g / L): Peptone 10, yeast extract powder 5, NaCl 5, CaCl2·2H2O 0.1, Tween-80 10 mL, agar 20, pH 7.0 - 7.2. The bacteria were spotted onto the fat medium and cultured in an incubator at 37°C. After 72 h, it was stained with Sudan Black B staining solution (0.08% w / v, 95% ethanol). Sudan Black B can specifically bind to hydrophobic lipids and present a dark blue color, and a colorless and transparent hydrolysis circle was formed in the fat degradation area. The diameters of the hydrolysis circle (D) and the colony (d) were measured respectively, and the ratio (D / d) was calculated. The ability of the strain to produce lipase was judged according to the size of this ratio.

[0057] The results showed that a total of 39 protein-degrading bacteria, 39 starch-degrading bacteria, 11 fat-degrading bacteria, and 7 cellulose-degrading bacteria were screened, and there were differences in the hydrolysis effects of different strains. One strain with the best effect was selected from each function for the statistics of the D / d ratio, and the results are shown in Table 1 and Figure 1 .

[0058] Table 1 Degradation effects of 4 degrading strains

[0059] Strain number Degradation function D / d CY-12 Protein 2.7±0.5 CY-3 Starch 4.3±0.4 HB-10 Fat 1.5±0.1 B13 Cellulose 3.1±0.4

[0060] 4. Identification of strains: The genomic DNA of the isolated strains was extracted according to the operating method of the bacterial genomic DNA extraction kit. PCR amplification was performed using the 16S rRNA gene primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO.1) and 1492R (5'-TACGACTTAACCCCAATCGC-3', SEQ ID NO.2). The PCR reaction system was 50 μL, including 25 μL of 2×Taqmix, 2 μL of each upstream and downstream primer, 2 μL of template DNA, and 19 μL of ddH2O. The PCR program was as follows: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 35 s, annealing at 50°C for 30 s, extension at 72°C for 1 min, for 30 cycles, and final extension at 72°C for 10 min. After the PCR products were detected by 1% agarose gel electrophoresis, they were sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for sequencing. The sequencing results were analyzed by alignment using the BLAST online tool, and a phylogenetic tree was constructed using the MEGAX software.

[0061] The sequencing result of strain CY-3 was:

[0062]

[0063] The sequencing result of strain CY-12 is as follows:

[0064]

[0065] The sequencing result of strain HB-10 is as follows:

[0066]

[0067] The sequencing result of strain B13 is as follows:

[0068]

[0069] The phylogenetic trees of each strain are as Figure 2 shown. It can be seen that strains CY-3 and CY-12 are closely related to Bacillus amyloliquefaciens. CY-3 and CY-12 are classified as Bacillus amyloliquefaciens and were deposited in the China General Microbiological Culture Collection Center on February 18, 2025, with the deposit numbers CGMCC No. 33561 and CGMCC No. 33562, respectively. Strain HB-10 is closely related to Bacillus tequilensis. HB-10 is classified as Bacillus tequilensis and was deposited in the China General Microbiological Culture Collection Center on February 18, 2025, with the deposit number CGMCC No. 33563. Strain B13 is closely related to Bacillus licheniformis. After further identification, this strain is Bacillus licheniformis B13, which has been deposited in the China General Microbiological Culture Collection Center, with the deposit number: CGMCC 1.16278.

[0070] Example 2 Application of single strain in fermenting kitchen waste to produce acid

[0071] 1. Strains CY-3, CY-12, HB-10, and B13 were respectively inoculated into 250 mL conical flasks containing 150 mL of nutrient broth liquid medium and cultured in a constant temperature shaker at 30 °C and 200 r / min for 48 h to complete single-strain fermentation and prepare the primary seed liquid; after culturing the primary seed liquid in the nutrient broth medium for 48 h at an inoculation ratio of 5% (v / v), the secondary seed liquid was prepared; the secondary seed liquid was inoculated into a 20 L small fermenter at the same inoculation ratio of 5% (v / v) for 48 h to obtain the single-strain fermentation inoculant. After identification, the bacterial concentrations of the CY-3, CY-12, HB-10, and B13 fermentation inoculants were 4.2×10 8 cfu / mL, 4.8×10 8 cfu / mL, 3.5×10 8 cfu / mL, and 6.3×10 8 cfu / mL, respectively.

[0072] 2. A semi-continuous anaerobic digestion reactor was built with blue-capped bottles with a total volume of 1000 mL (as Figure 3 shown). In the startup stage, 850 mL of kitchen waste slurry was inoculated into the reactor, purged with nitrogen for 10 min, and placed in a constant temperature shaking water bath at 37 °C and shaken at 110 rpm for 24 h to discharge the nitrogen above the reactor. After 24 h, the fermentation inoculant could be inoculated.

[0073] The kitchen waste slurry is obtained by sieving out large substances and crushing and separating the kitchen waste (collected from the kitchen waste harmless treatment center in Jingzhou City, Hubei Province), and then adjusting the pH of the slurry to 7.0 with sodium hydroxide.

[0074] 3. Respectively inoculate CY-3, CY-12, HB-10, and B13 fermentation inoculants into a semi-continuous flow anaerobic digestion reactor containing kitchen waste slurry at a ratio of 1% (w / w). At the same time, set an experimental group without adding inoculants as a blank control (CK), and place it in a constant temperature shaking incubator at 37 °C and 110 rpm. According to the set organic load 3, calculate the feeding amount of the hydrolyzed kitchen waste slurry added every 24 h according to the following formula, and take out an equal amount of digestate from the device, and use a FOS / TAC 2000 titrator (PRONOVA, Germany) to measure the content of organic acids.

[0075]

[0076] In the formula, M is the feeding amount, in g; OLR is the organic loading rate, in g VS / L·d; L is the effective volume of the reactor, in L; TS is the total solid content (%) of the hydrolysis raw material; VS is the volatile solid content (%) of the hydrolysis raw material.

[0077] The acid production amounts of each strain are shown in Table 2.

[0078] Table 2 Acid production amounts of each strain (g / L)

[0079] Strain CY-3 CY-12 HB-10 B13 CK 0d 5.0 5.0 5.0 5.0 5.0 1d 8.4 7.0 9.9 12.6 8.1 2d 15.1 13.2 10.0 12.6 6.9 3d 16.5 12.6 10.1 13.0 8.2 4d 18.3 14.5 11.0 13.5 10.6 5d 18.3 14.5 12.5 15.0 12.3 6d 20.6 15.0 12.6 15.0 12.3 7d 21.0 15.2 14.0 15.5 13.0 8d 21.5 15.5 14.2 16.0 13.0

[0080] Example 3 Compound of inoculants

[0081] Adopt the plate confrontation culture method to evaluate the interaction between strains CY-3, CY-12, HB-10, and B13, and verify whether the four strains can be used to prepare a compound inoculant. The results are as Figure 4 shown, and the results show that there is no antagonism between the four strains, and they can be used to prepare a compound inoculant.

[0082] Example 4 Liquid compound inoculant 4m 3 Acid production test in a fermenter

[0083] 1. Use an inoculation loop to inoculate the four strains after preservation and activation into 250 ml conical flasks containing 150 ml of beef extract peptone liquid medium respectively. Cultivate them in a constant temperature shaker at 30 °C and 200 r / min for 48 h to complete the preparation of the first-stage seed liquid by single-strain fermentation. Mix the first-stage seed liquids of strains CY-3, CY-12, HB-10, and B13 according to a volume ratio of 4:2:2:1, and inoculate them into the beef extract peptone medium according to an inoculation ratio of 5% (v / v). After culturing for 48 h, complete the preparation of the second-stage seed liquid; inoculate the second-stage seed liquid into a 20 L small fermenter containing beef extract peptone medium according to the same inoculation ratio of 5% (v / v). After culturing for 48 h, a compound microbial agent is prepared. After identification, the bacterial concentration of this compound microbial agent is 6.03×10 8 cfu / mL.

[0084] 2. After screening out large substances and crushing and separating the kitchen waste (collected from the kitchen waste harmless treatment center in Jingzhou City, Hubei Province), a slurry is obtained and put into a 4 m 3 fermenter (as Figure 5 shown). Use sodium hydroxide to adjust the pH to 6.5 - 7.5 to complete the pretreatment of the slurry raw material.

[0085] 3. Inoculate the compound microbial agent in step 1 into a 4 m 3 fermenter containing kitchen waste slurry at a ratio of 0.3% (w / w), and ferment at 37 °C. Take samples every 1 d to measure the organic acid content.

[0086] 4. After 48 hours of reaction, the organic acid production is increased from the original 8.3 g / L to 28.5 g / L, and the organic acid production is increased by about 243%. Compared with the acid production effect of the single-strain microbial agent in Example 2, the acid production effect of the compound microbial agent is greatly improved, indicating that there is a synergistic effect among the four strains, and the acid production effect of the combined use is better.

[0087] Example 5 Preparation of solid compound microbial agent and verification of its acid production function

[0088] 1. Preparation of solid compound microbial agent:

[0089] Use an inoculation loop to enrich and expand the four strains after preservation and activation in beef extract peptone liquid medium. Mix the cells after centrifuging to remove the matrix with 20% skim milk powder according to a mass ratio of 1:1, and then freeze-dry them respectively to obtain single-strain bacterial powders. Referring to the previous research, prepare a compound bacterial powder according to the mass ratio of CY-12:CY-3:HB-10:B13 = 4:2:2:1. Then mix crushed peanut shells, wheat bran, wood chips, beef bone powder and the compound bacterial powder according to a mass ratio of 2:1:1:1:1 to prepare a solid compound microbial agent.

[0090] Prepare a suspension of this compound microbial agent and water according to a ratio of 1:9, and dilute it to 10-6 After determining the concentration, 200 μL of the diluted suspension was spread on the beef extract peptone medium and cultured at 37 °C for 48 h to observe the bacterial growth. Three parallels were set up for the experiment, and the results are as Figure 6 shown. It will be detected that the viable bacteria content of this composite bacterium agent is high, reaching 1.5×10 9 CFU / g.

[0091] 2. Verification of the function of the solid composite bacterium agent in fermenting kitchen waste to produce acid

[0092] A semi-continuous anaerobic digestion reactor was constructed with blue-cap bottles with a total volume of 1000 mL. In the startup stage, 850 mL of kitchen waste slurry was inoculated into the reactor, purged with nitrogen for 10 min, and placed in a constant temperature shaking incubator at 37 °C and shaken at 110 rpm for 24 h to discharge the nitrogen above the reactor. After 24 h, the solid composite bacterium agent was added to the reactor at a ratio of 1% (w / w). At the same time, an experiment without adding the bacterium agent was set as a blank control (CK), and it was placed in a constant temperature shaking incubator at 37 °C and 110 rpm. According to the set organic loading 3, the feeding amount of the kitchen waste hydrolysis slurry added every 24 h was calculated according to the following formula, and an equal amount of digested liquid was taken out from the device, and the content of organic acids was measured using a FOS / TAC 2000 titrator (PRONOVA, Germany).

[0093]

[0094] In the formula, M is the feeding amount, in g; OLR is the organic loading rate, in g VS / L·d; L is the effective volume of the reactor, in L; TS is the total solid content (%) of the hydrolysis raw material; VS is the volatile solid content (%) of the hydrolysis raw material.

[0095] The results are as Figure 7 shown. The organic acid content in the reactor with 1% solid composite bacterium agent added was significantly higher than that of CK, reaching 7.48 g / L, an increase of 27% compared with CK.

[0096] The above results indicate that strains CY-3, CY-12, HB-10, and B13 can also be used to prepare a solid composite bacterium agent for fermenting kitchen waste to produce acid. The bacterium agent carrier does not affect the ability of microorganisms to ferment and produce acid, and the solid composite bacterium agent has the advantages of being easy to store and transport.

[0097] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A Bacillus for acid production by fermentation of kitchen waste, characterized in that, It includes Bacillus amyloliquefaciens CY-3, Bacillus amyloliquefaciens CY-12, and Bacillus tequilensis HB-10; The preservation number of Bacillus amyloliquefaciens CY-3 is CGMCC No. 33561; the preservation number of Bacillus amyloliquefaciens CY-12 is CGMCC No. 33562; the preservation number of Bacillus tequilensis HB-10 is CGMCC No. 33563.

2. A preparation method of a liquid compound bacterial agent for fermenting kitchen waste to produce acid, characterized in that It includes the following steps: (1) Inoculate single colonies of Bacillus amyloliquefaciens CY-3, Bacillus amyloliquefaciens CY-12, Bacillus tequilensis HB-10, and Bacillus licheniformis B13 into a beef extract peptone liquid medium for cultivation to prepare primary seed solutions; The preservation number of Bacillus amyloliquefaciens CY-3 is CGMCC No. 33561; the preservation number of Bacillus amyloliquefaciens CY-12 is CGMCC No. 33562; the preservation number of Bacillus tequilensis HB-10 is CGMCC No. 33563; the preservation number of Bacillus licheniformis B13 is CGMCC 1.16278; (2) After mixing the primary seed solutions of Bacillus amyloliquefaciens CY-3, Bacillus amyloliquefaciens CY-12, Bacillus tequilensis HB-10, and Bacillus licheniformis B13, inoculate them into a beef extract peptone liquid medium for cultivation to obtain secondary seed solutions; (3) Inoculate the secondary seed solutions into a beef extract peptone liquid medium for cultivation to obtain the liquid compound microbial agent.

3. The preparation method according to claim 2, characterized in that, In step (2), when mixing the primary seed solutions of Bacillus amyloliquefaciens CY-3, Bacillus amyloliquefaciens CY-12, Bacillus tequilensis HB-10, and Bacillus licheniformis B13, the volume ratio is (3 - 5):(1 - 3):(1 - 3):

1.

4. The preparation method according to claim 2, wherein In steps (1)-(3), the cultivation temperature is 30°C, and the cultivation time is 40 - 50 h; in steps (2) and (3), the inoculation amount for inoculation is 4% - 8%.

5. A preparation method of a solid composite microbial inoculum for fermenting kitchen waste to produce acid, characterized in that, It includes the following steps: (1) The cells of Bacillus amyloliquefaciens CY-3, Bacillus amyloliquefaciens CY-12, Bacillus tequilensis HB-10, and Bacillus licheniformis B13 were respectively mixed with skim milk powder and freeze-dried to obtain single-strain bacterial powders; The preservation number of Bacillus amyloliquefaciens CY-3 is CGMCC No. 33561; the preservation number of Bacillus amyloliquefaciens CY-12 is CGMCC No. 33562; the preservation number of Bacillus tequilensis HB-10 is CGMCC No. 33563; the preservation number of Bacillus licheniformis B13 is CGMCC 1.16278; (2) After mixing the single-strain bacterial powders of Bacillus amyloliquefaciens CY-3, Bacillus amyloliquefaciens CY-12, Bacillus tequilensis HB-10, and Bacillus licheniformis B13, a compound bacterial powder was obtained; (3) The compound bacterial powder was mixed with peanut hulls, wheat bran, wood chips, and bovine bone meal to obtain the solid compound bacterial agent.

6. The preparation method according to claim 5, characterized in that, In step (1), the mass ratio of the cells to the skim milk powder during mixing is 1:1; In step (2), the mass ratio of the single-strain bacterial powders of Bacillus amyloliquefaciens CY-3, Bacillus amyloliquefaciens CY-12, Bacillus tequilensis HB-10, and Bacillus licheniformis B13 during mixing is 4:2:2:1; In step (3), the mass ratio of the compound bacterial powder to peanut hulls, wheat bran, wood chips, and bovine bone meal during mixing is 1:2:1:1:

1.

7. A liquid compound bacterial agent prepared by the preparation method according to any one of claims 2-4.

8. A solid compound bacterial agent prepared by the preparation method according to claim 5 or 6.

9. The application of the Bacillus described in claim 1, the liquid compound bacterial agent described in claim 7, or the solid compound bacterial agent described in claim 8 in the fermentation of kitchen waste to produce acid.

10. A fermentation method for highly efficient acid production from kitchen waste, characterized in that, It includes the step of inoculating the liquid compound bacterial agent described in claim 7 into the slurry of kitchen waste and fermenting and culturing to produce organic acids.

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