Application of halotolerant amylase producing strain and complex microbial inoculant thereof in efficient anaerobic fermentation hydrogen production of kitchen wastewater

Abstract

The invention discloses a salt-tolerant amylase producing strain and application of a complex microbial inoculant thereof in efficient anaerobic fermentation hydrogen production of kitchen wastewater. According to the invention, a salt-tolerant amylase producing strain, namely bacillus paralicheniformis Q4 (CCTCC NO: M 20241043), is obtained through separation and screening, and the bacillus paralicheniformis Q4 can be used for producing the salt-tolerant amylase. According to the present invention, with the complex microbial inoculum prepared by compatibility of the complex microbial inoculum with activation solutions of three strains such as a salt-tolerant fermentation hydrogen production bacterium-bacillus altitudinis K3 (CCTCC NO: M 20241042) and a salt-tolerant protease production bacterium-arthrobacter protophila A1 (CCTCC NO: M 20241041) according to a volume ratio of (1-2): (1-2): 1, the fermentation hydrogen production efficiency of the kitchen wastewater can be synergistically enhanced, and the hydrogen yield and the production intensity can respectively achieve more than 100 mL / g-COD and more than 720 L / m < 3 > / d;

Description

Technical Field

[0001] The present invention belongs to the technical field of environmental microorganisms, and specifically relates to the application of a salt-tolerant amylase-producing bacterium and a composite bacterial agent thereof in the efficient anaerobic fermentation of kitchen wastewater to produce hydrogen. Background Art

[0002] In 2022, my country's food waste generation exceeded 140 million tons, accounting for approximately 30% of the nation's total urban domestic waste. The total amount of food waste is projected to exceed 170 million tons by 2025. With the launch of 100 pilot cities nationwide for food waste management, food waste management projects across the country have entered a period of rapid growth. Pre-treatment of food waste generates a large amount of organic wastewater, characterized by high COD, organic nitrogen, and salinity.

[0003] Anaerobic fermentation hydrogen production technology, a biological treatment method that can both degrade macromolecular organic matter and generate hydrogen energy, offers advantages such as low energy consumption, low operating costs, and simple processes, making it considered a highly promising hydrogen production method. However, when treating high-salinity organic wastewater, such as food wastewater, high salinity can inhibit the growth and metabolism of anaerobic microorganisms, thereby affecting the efficiency of organic matter conversion and hydrogen production.

[0004] Based on the concept of bioaugmentation, targeted screening of salt-tolerant functional strains and their introduction into a food wastewater fermentation hydrogen production system to enhance hydrogen production under high salt stress is one viable solution. Bioaugmentation technology requires no changes to the treatment process and is low-cost. It has demonstrated promising application potential in areas such as bioenergy production, pollutant biodegradation, and ecological restoration.

[0005] Patent CN 118703371 A discloses a composite bacterial agent for efficiently treating high-salt pharmaceutical and chemical wastewater, as well as its preparation method and application. The agent involves strain BWBX2402, with a deposit number of GDMCC No: 64264 and a classification name of Bacillus paralicheniformis. In this invention, Bacillus paralicheniformis BWBX2402 is used for denitrification of high-salt pharmaceutical and chemical wastewater. According to the principle of biological denitrification of wastewater, the general reaction process is: under aerobic conditions, ammonia nitrogen is converted into nitrate nitrogen through nitrification, and then the nitrate nitrogen is further converted into nitrogen gas through denitrification. This is completely inconsistent with the mechanism of action of Bacillus paralicheniformis Q4 disclosed in the present invention. In the present invention, Bacillus paralicheniformis Q4 has the function of secreting amylase under high-salt anaerobic conditions, and then further anaerobically degrades the starch hydrolysis products to produce hydrogen through coupling with hydrogen-producing bacteria. Summary of the Invention

[0006] This patent discloses the application of a salt-tolerant amylase-producing bacterium and its composite bacterial agent in the efficient anaerobic fermentation of kitchen wastewater to produce hydrogen, which significantly improves the anaerobic hydrogen production efficiency using kitchen wastewater as a substrate.

[0007] To achieve the above object, the technical solution of the present invention is:

[0008] The first object of the present invention is to provide a salt-tolerant amylase-producing bacterium, which was deposited in the China Center for Type Culture Collection on May 23, 2024, with a preservation address of Wuhan, China, a preservation number of CCTCC NO: M 20241043, and a classification name of Bacillus paralicheniformis Q4.

[0009] In some embodiments of the present invention, the 16S rDNA sequence of the Bacillus paralicheniformis Q4 is shown as SEQ ID NO.1.

[0010] In some embodiments of the present invention, the salt-tolerant amylase-producing bacteria can tolerate 6.0% salinity growth under anaerobic conditions, and the starch degradation rate of kitchen wastewater is higher than 80%.

[0011] In some embodiments of the present invention, the salt-tolerant amylase-producing bacteria can tolerate 6.0% salinity growth under anaerobic conditions, and the starch degradation rate of kitchen wastewater is higher than 90%.

[0012] The second object of the present invention is to provide use of any of the above salt-tolerant amylase-producing bacteria in high-salt anaerobic fermentation to produce amylase.

[0013] In some embodiments of the present invention, in the high-salt anaerobic fermentation reaction for producing amylase, high salt means a salinity of ≥3%.

[0014] The third object of the present invention is to provide a composite bacterial agent, comprising:

[0015] Salt-tolerant amylase-producing bacteria: Bacillus paralicheniformis Q4, deposit number: CCTCC NO: M 20241043;

[0016] Salt-tolerant fermentative hydrogen-producing bacteria: Bacillus altitudinis K3, deposit number: CCTCCNO: M 20241042; and

[0017] Salt-tolerant protease-producing bacteria: Arthrobacter protophormiae A1, with a deposit number of CCTCC NO: M 20241041.

[0018] In some embodiments of the present invention, in the composite bacterial agent, the volume ratio of the bacterial liquid of Bacillus paralicheniformis Q4, Bacillus highland K3 and Arthrobacter protovitaminosis A1 is (1-2):(1-2):1.

[0019] In some embodiments of the present invention, in the composite bacterial agent, the volume ratio of the bacterial liquid of Bacillus paralicheniformis Q4, Bacillus altaica K3 and Arthrobacter protocisnerii A1 is 2:2:1.

[0020] The fourth object of the present invention is to provide a method for preparing any of the above composite bacterial agents, comprising the following steps: activating and culturing the paralicheniformis Q4, the high ground Bacillus K3 and the original glass fly arthrobacter A1 in LB liquid culture medium to obtain the corresponding three bacterial liquids; 600 After reaching 1.2-1.6, the three bacterial solutions are combined to prepare a composite bacterial agent.

[0021] The fifth object of the present invention is to provide the use of any of the above-mentioned composite bacterial agents in the efficient anaerobic fermentation of kitchen wastewater to produce hydrogen.

[0022] In some embodiments of the present invention, in the high-efficiency anaerobic fermentation hydrogen production reaction of kitchen wastewater, the inoculation amount of the composite bacterial agent is 5%-10%.

[0023] In some embodiments of the present invention, in the efficient anaerobic fermentation hydrogen production reaction of food wastewater, the fermentation pH is 6.5-7.0.

[0024] In some embodiments of the present invention, during the efficient anaerobic fermentation of kitchen wastewater to produce hydrogen, the temperature is 30-40°C.

[0025] In some embodiments of the present invention, in the high-efficiency anaerobic fermentation hydrogen production reaction of kitchen wastewater, the organic load is 3.0-7.2 kg-COD / m 3 / d.

[0026] In some embodiments of the present invention, in the high-efficiency anaerobic fermentation hydrogen production reaction of kitchen wastewater, the organic load is 5.0-7.2 kg-COD / m 3 / d. Furthermore, the organic load is 7.2kg-COD / m 3 / d.

[0027] In some embodiments of the present invention, in the high-efficiency anaerobic fermentation hydrogen production reaction of food wastewater, the hydrogen yield reaches more than 100 mL / g-COD.

[0028] In some embodiments of the present invention, the production intensity reaches 720L / m in the high-efficiency anaerobic fermentation hydrogen production reaction of kitchen wastewater.3 / d or more.

[0029] The present invention uses salt-tolerant amylase-producing bacteria - Bacillus paralicheniformis Q4, salt-tolerant fermentation hydrogen-producing bacteria - Bacillus altitudinis K3 and salt-tolerant protease-producing bacteria - Arthrobacter protophormiae A1 to construct a composite bacterial agent to enhance the fermentation and hydrogen production of kitchen wastewater. The three functional bacterial strains were deposited in the China Center for Type Culture Collection on May 23, 2024, and the deposit numbers are CCTCC NO: M 20241043, CCTCC NO: M 20241042 and CCTCC NO: M 20241041, respectively.

[0030] In the present invention, the 16S rDNA sequence of the Bacillus paralicheniformis Q4 is shown as SEQ ID NO.1.

[0031] In the present invention, the 16S rDNA sequence of the Bacillus subtilis K3 is shown as SEQ ID NO.2.

[0032] In the present invention, the 16S rDNA sequence of the original Arthrobacterium vitae A1 is shown as SEQ ID NO.3.

[0033] In the present invention, the Bacillus paralicheniformis Q4 has a strong amylase production ability (starch degradation rate is higher than 85%, or higher than 90%), and can tolerate 6.0% salinity growth under anaerobic conditions.

[0034] In the present invention, the Geobacillus sp. K3 has strong fermentation hydrogen production ability and salt tolerance, and can tolerate 6.0% salinity growth under anaerobic conditions.

[0035] In the present invention, the original Arthrobacterium vitae A1 has strong protease production ability and salt tolerance, and can tolerate 6.0% salinity growth under anaerobic conditions.

[0036] Beneficial effects:

[0037] Compared with the prior art, the application of a salt-tolerant amylase-producing bacterium and its composite bacterial agent disclosed in the present invention in efficient anaerobic fermentation of kitchen wastewater to produce hydrogen has the following beneficial effects:

[0038] The salt-tolerant amylase-producing strain, Bacillus paralicheniformis Q4 (CCTCC NO: M 20241043), can tolerate 6.0% salinity under anaerobic conditions and achieves a starch degradation rate exceeding 85% in food wastewater. It can be used in high-salt anaerobic fermentation to produce amylase (salinity ≥ 3%).

[0039] 2. The salt-tolerant amylase-producing bacteria provided by the present invention, Bacillus paralicheniformis Q4 (CCTCC NO: M 20241043), salt-tolerant fermentation hydrogen-producing bacteria, Bacillus altitudinis K3, and salt-tolerant protease-producing bacteria, Arthrobacter protophormiae A1, are combined to form a composite bacterial agent. This composite bacterial agent can effectively improve the yield and production intensity of anaerobic fermentation hydrogen production from food wastewater. The organic load is 7.2 kg-COD / m 3 / d, the hydrogen production rate reaches more than 100mL / g-COD, and the hydrogen production intensity can reach 720L / m 3 / d or more.

[0040] 3. The salt-tolerant amylase-producing bacteria and the composite bacterial agent provided by the present invention can provide an effective bacterial resource for enhancing the anaerobic fermentation hydrogen production of high-salt organic wastewater such as kitchen wastewater, thereby significantly increasing hydrogen production. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 : Screening results of salt-tolerant amylase-producing bacteria;

[0042] Figure 2 : Screening results of salt-tolerant hydrogen-producing bacteria;

[0043] Figure 3 : Colony morphology of Bacillus paralicheniformis Q4, Bacillus altaica K3 and Arthrobacter protocis A1;

[0044] Figure 4 :Effects of different bacterial agent ratios on anaerobic fermentation of kitchen wastewater for hydrogen production;

[0045] Figure 5 : Effect of mixed bacterial agents on hydrogen production from food wastewater under different organic loads. DETAILED DESCRIPTION

[0046] Restaurant wastewater has a high organic matter content, making it a very suitable feedstock for anaerobic fermentation to produce hydrogen. However, the high salt concentration limits the efficiency of organic matter bioconversion and the intensity of hydrogen production. To address this technical problem, the present invention discloses a salt-tolerant amylase-producing bacterium and its composite bacterial agent for efficient anaerobic fermentation of restaurant wastewater for hydrogen production, belonging to the field of environmental microbiology. The present invention provides three salt-tolerant functional bacterial strains, including:

[0047] ① A salt-tolerant amylase-producing bacterium, Bacillus paralicheniformis Q4, with a deposit number of CCTCC NO: M 20241043, deposited on May 23, 2024, at the China Center for Type Culture Collection, Wuhan, China, with a deposit number of CCTCC NO: M 20241043 and a taxonomic name of Bacillus paralicheniformis Q4;

[0048] ② A salt-tolerant, fermentative, hydrogen-producing bacterium, Bacillus altitudinis K3, with a deposition number of CCTCC NO: M 20241042, deposited on May 23, 2024, at the China Center for Type Culture Collection, Wuhan, China, with a deposition number of CCTCC NO: M 20241042 and a taxonomic name of Bacillus saltitudinis K3;

[0049] ③ A salt-tolerant protease-producing bacterium - Arthrobacter protophormiae A1, with a deposit number of CCTCC NO: M 20241041, was deposited in the China Center for Type Culture Collection on May 23, 2024, with a deposit address of Wuhan, China, with a deposit number of CCTCC NO: M 20241041, and a classification name of Arthrobacter protophormiae A1.

[0050] The present invention's research shows that a composite bacterial agent prepared by combining the activated solutions of three strains in a volume ratio of (1-2):(1-2):1 can synergistically enhance the efficiency of hydrogen production from food wastewater fermentation. When the inoculum size is 5%-10%, the fermentation pH and temperature are controlled at 6.5-7.0 and 30-40°C, respectively, and the organic load is 3.0-7.2 kg-COD / m 3 / d, the hydrogen production rate reaches above 100mL / g-COD.

[0051] The present invention discloses a composite bacterial agent for efficient anaerobic fermentation hydrogen production of restaurant wastewater. The composite bacterial agent is prepared by directional screening of salt-tolerant amylase-producing bacteria, salt-tolerant fermentation hydrogen-producing bacteria and salt-tolerant protease-producing bacteria to improve the efficiency of anaerobic fermentation hydrogen production of restaurant wastewater.

[0052] The present invention screened out a salt-tolerant amylase-producing bacterium - Bacillus paralicheniformis Q4, with a preservation number of CCTCCNO: M 20241043, a preservation time of May 23, 2024, a preservation location of China Center for Type Culture Collection, and a classification name of Bacillus paralicheniformis Q4.

[0053] The present invention adopts a salt-tolerant fermentation hydrogen-producing bacterium - Bacillus altitudinis K3, with a preservation number of CCTCCNO: M20241042, a preservation time of May 23, 2024, a preservation location of the China Center for Type Culture Collection, and a classification name of Bacillus altitudinis K3.

[0054] The present invention adopts a salt-tolerant protease-producing bacterium - Arthrobacter protophormiae A1, with a preservation number of CCTCC NO: M20241041, a preservation time of May 23, 2024, a preservation location of the China Center for Type Culture Collection, and a classification name of Arthrobacter protophormiae A1.

[0055] The Bacillus paralicheniformis Q4 strain has a strong amylase production capacity, can tolerate 6.0% salinity under anaerobic conditions, and has a starch degradation rate exceeding 85% in food wastewater. The screening process involves using anaerobic activated sludge pretreated at 105°C for one hour as the strain source, acclimating and culturing it at 3.0% salinity using starch as the sole carbon source; isolating and purifying it on a 6.0% salinity Lugol's iodine starch clearing zone medium; and further rescreening the target strain for starch hydrolysis ability.

[0056] The Bacillus subtilis K3 has strong fermentation hydrogen production capacity and salt tolerance, and can tolerate growth at 6.0% salinity under anaerobic conditions. The screening steps are as follows: anaerobic activated sludge pretreated with 105°C wet heat for 1 hour is used as the strain source, glucose is used as the carbon source, and the strain is cultivated under the combined selective pressure of 3.0% salinity, 8mmol / L 4-methylpyrazole (alcohol dehydrogenase inhibitor) and 15mmol / L sodium oxamate (lactate dehydrogenase inhibitor); the strain is then isolated and purified on a solid culture medium at 6.0% salinity; and the target strain is further screened for hydrogen production capacity to obtain the target strain.

[0057] The present invention will be further explained below with reference to specific examples. However, it will be readily understood by those skilled in the art that the specific process conditions and results described in the examples are merely illustrative of the present invention and should not and will not limit the present invention described in detail in the claims.

[0058] Example 1 Screening of Bacillus paralicheniformis Q4

[0059] (1) Isolation and screening of culture medium components for salt-tolerant amylase-producing bacteria

[0060] Starch 10 g / L, peptone 3.0 g / L, NaCl 60 g / L, K2HPO4 1.5 g / L, NH4Cl 1.0 g / L, MgCl2 0.1 g / L, FeSO4·7H2O 0.1 g / L, L-cysteine ​​0.5 g / L, vitamin solution 1.0 mL / L, trace element solution 1.0 mL / L, pH 6.5. Add 20 g / L agar to the solid medium.

[0061] (2) Isolation and purification of salt-tolerant amylase-producing bacteria

[0062] Anaerobic activated sludge pretreated at 105°C for 1 hour was used as the bacterial strain source. The strain was first cultured for 15 days at 3.0% salinity (36°C, pH 6.5) using starch as the sole carbon source. The bacterial suspension was then placed in an anaerobic tube and glass beads added. After vortexing and dispersion, it was diluted in a gradient using saline. The diluted bacterial suspension was spread onto a solid screening medium containing Lugol's iodine at 6.0% salinity and incubated inverted in a 36°C anaerobic incubator for 48 hours. Colonies with larger clearing zones were selected and repeatedly streaked onto the 6.0% salinity solid medium, resulting in the identification of 11 salt-tolerant amylase-producing strains. After activation and culture in LB liquid medium for 36 hours, the strains were transferred to a 5% inoculum in 3.0% salinity liquid screening medium and cultured anaerobically for 36 hours. Starch hydrolysis ability was rescreened by measuring the reducing sugar and starch concentrations in the fermentation broth.

[0063] like Figure 1 As shown, strain Q4 had the strongest starch hydrolysis ability, with a starch hydrolysis rate of 59.5% and a reducing sugar concentration of 3.8 g / L in the fermentation broth.

[0064] (3) Identification of bacterial species

[0065] The 16S rDNA sequence of the salt-tolerant amylase-producing strain Q4 was amplified using bacterial universal primers (27F: 5'-AGAGTTTGATCATGGCTCAG-3'; 1492R: 5'-GGTTACCTTGTTACGACTT-3') and then sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The 16S rDNA sequence was compared with GenBank.

[0066] The results showed that the strain's 16S rDNA sequence was 1486 bp long, and the nucleotide sequence, shown in SEQ ID NO. 1, shared 99.87% similarity with the 16S rDNA sequence of Bacillus paralicheniformis. The strain was identified and named Bacillus paralicheniformis Q4 and deposited with the China Center for Type Culture Collection (CCTCC NO: M 20241043).

[0067] Example 2 Screening of Bacillus subtilis K3

[0068] (1) Culture medium components for the isolation and screening of salt-tolerant hydrogen-producing bacteria

[0069] Glucose 15 g / L, peptone 3 g / L, yeast extract 1 g / L, NaCl 60 g / L, K2HPO4 1.5 g / L, NH4Cl 1 g / L, MgCl2 0.1 g / L, FeSO4·7H2O 0.1 g / L, L-cysteine ​​0.5 g / L, vitamin solution 1 mL / L, trace element solution 1 mL / L, 4-methylpyrazole 8 mM, oxamate 15 mM, pH 6.5. For solid medium, add 20 g / L agar.

[0070] (2) Isolation and purification of salt-tolerant hydrogen-producing bacteria

[0071] Anaerobic activated sludge pretreated at 105°C for 1 hour was used as the bacterial strain source, with glucose as the carbon source. The strains were cultured for 15 days (36°C, pH 6.5) under the combined selective pressure of 3.0% salinity, 8 mmol / L 4-methylpyrazole (an alcohol dehydrogenase inhibitor), and 15 mmol / L sodium oxamate (a lactate dehydrogenase inhibitor). Hydrogen-producing strains were isolated using the flat-plate sandwich culture method. The bacterial suspension was placed in an anaerobic tube and glass beads were added. After oscillation and dispersion, it was diluted in a gradient using physiological saline. The diluted bacterial suspension was spread on the surface of a solid culture medium with a salinity of 6.0%. After 5 minutes, agar was poured again for sandwich culture. The suspension was inverted and cultured in an anaerobic constant-temperature incubator at 36°C. When colonies grew in the solid sandwich, single colonies were picked and repeatedly streaked to isolate them. A total of 17 salt-tolerant hydrogen-producing bacteria were initially screened. After the initial screening strains were activated and cultured in LB liquid medium for 36 hours, they were transferred to 3.0% salinity liquid screening medium with a 5% inoculum and cultured anaerobically for 48 hours. The strains were rescreened by measuring hydrogen production. Figure 2 As shown, strain K3 had the strongest hydrogen production ability, with glucose degradation rate and hydrogen production reaching 81.7% and 1280 mL, respectively.

[0072] (3) Identification of bacterial species

[0073] The 16S rDNA sequence of the salt-tolerant hydrogen-producing bacterium K3 was amplified and screened using bacterial universal primers, and then sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing, and the 16S rDNA sequence was compared with GenBank.

[0074] The results showed that the strain's 16S rDNA sequence was 1489 bp long and shared 100% similarity with the 16S rDNA sequence of Bacillus altitudinis. The strain was identified and named Bacillus altitudinis K3 and deposited with the China Center for Type Culture Collection (CCTCC NO: M 20241042).

[0075] Figure 3 The colony morphology of salt-tolerant hydrogen-producing bacteria K3 is shown.

[0076] Example 3: Efficacy of Composite Bacterial Agent in Enhancing Fermentation of Kitchen Wastewater to Produce Hydrogen

[0077] Existing organic wastewater anaerobic fermentation hydrogen production mostly uses heat-pretreated anaerobic sludge as inoculum. Therefore, the present invention uses anaerobic sludge pretreated with 105°C heat for 1 hour and activated and cultured as the control inoculum (inoculum size is 30g / L).

[0078] The Bacillus paralicheniformis Q4, Bacillus thuringiensis K3 and Arthrobacter spp. A1 obtained in the above examples were activated and cultured in LB liquid medium. 600 After reaching a pH of 1.2-1.6, we first analyzed the effects of different inoculant ratios on anaerobic hydrogen production from food wastewater in a batch fermentation mode. Furthermore, we conducted experiments on hydrogen production from food wastewater in batch fermentation, comprehensively analyzing the fermentation system's hydrogen yield, starch degradation, and protein degradation rates to identify inoculant ratios that improved hydrogen yield. The fermentation temperature and pH were controlled at 35°C and 6.5, respectively, with a total inoculum level of 5%.

[0079] (1) Effect test of a single bacterial agent on anaerobic fermentation of food wastewater to produce hydrogen

[0080] Bacillus paralicheniformis Q4, Bacillus altaica K3 and Arthrobacter protocis A1 were used separately as inoculation strains for the anaerobic fermentation hydrogen production system of food wastewater, and their hydrogen yields, starch degradation rates and protein degradation rates were determined.

[0081] The fermentation results of the anaerobic fermentation hydrogen production system of food wastewater are as follows Figure 4 As shown, the hydrogen production of the control inoculum was 76.3 mL / g-COD, the starch degradation rate in the kitchen wastewater was 68.9%, and the protein degradation rate was 52.4%.

[0082] In the fermentation system with only Bacillus paralicheniformis Q4, although the starch degradation rate increased to 90.1%, the hydrogen production rate was only 29.3 mL / g-COD, and the protein degradation rate was only 25.7%.

[0083] In the fermentation system with only Geobacillus K3, the hydrogen production, starch degradation rate and protein degradation rate were only 49.4 mL / g-COD, 32.5% and 28.2%, respectively.

[0084] In the fermentation system with only Arthrobacter citrinum A1, although the protein degradation rate reached 90.6%, the hydrogen production rate was only 16.4 mL / g-COD, and the starch degradation rate was only 29.5%.

[0085] The fermentation results of single bacterial agents showed that the three strains were not conducive to improving hydrogen production when used independently.

[0086] (2) Test on the effect of the composite bacterial agent of Bacillus paralicheniformis Q4 and Bacillus subtilis K3 on the anaerobic fermentation of kitchen wastewater to produce hydrogen

[0087] To test the effect of a composite inoculant formed by combining Bacillus paralicheniformis Q4 with other strains on hydrogen yield, this example first combined Bacillus paralicheniformis Q4 with the salt-tolerant Geobacillus K3 to create a dual-strain composite inoculant. This composite inoculant was then used as an inoculum in an anaerobic fermentation hydrogen production system using food wastewater. The inoculant's hydrogen yield, starch degradation rate, and protein degradation rate were measured.

[0088] The fermentation results of the anaerobic fermentation hydrogen production system of food wastewater are as follows Figure 4 As shown in the figure, in the fermentation system with an inoculation ratio of Bacillus paralicheniformis Q4 and Bacillus subtilis K3 of 1:1, the hydrogen yield and starch degradation rate were significantly improved, reaching 92.1 mL / g-COD and 91.7%, respectively, but the protein degradation rate was only 33.8%.

[0089] This shows that with the addition of Bacillus subtilis K3, the hydrogen production rate of the fermentation system was improved, but the protein degradation rate was not effectively improved.

[0090] (3) Test on the effect of different bacterial agent ratios on anaerobic fermentation of kitchen wastewater to produce hydrogen

[0091] Based on the results of part (2) above, in order to improve the protein degradation rate of the fermentation system, this example further introduced the original glass fly Arthrobacter A1 with strong protease production ability and salt tolerance, and combined it with Bacillus paralicheniformis Q4 and Bacillus high K3 to prepare a three-strain composite bacterial agent, which was used as the inoculation strain for the anaerobic fermentation hydrogen production system of food wastewater, and its hydrogen production rate, starch degradation rate and protein degradation rate were measured.

[0092] The fermentation results of the anaerobic fermentation hydrogen production system of food wastewater are as follows Figure 4 As shown, in the fermentation system with an inoculation ratio of 1:1:1 of Bacillus paralicheniformis Q4, Bacillus highland K3 and Arthrobacter protothecoides A1, the hydrogen yield, starch degradation rate and protein degradation rate were significantly improved, reaching 102.3 mL / g-COD, 87.9% and 84.5%, respectively.

[0093] In the fermentation system with an inoculation ratio of 2:2:1 of Bacillus paralicheniformis Q4, Bacillus highi K3 and Arthrobacter protothecoides A1, the hydrogen yield, starch degradation rate and protein degradation rate further increased to 116.7 mL / g-COD, 93.1% and 81.9% respectively.

[0094] These results demonstrate that efficient anaerobic fermentation of food wastewater for hydrogen production is only possible with the synergistic action of the three bacterial strains. A volume ratio of (1-2):(1-2):1 among the three bacterial cultures effectively increased hydrogen production, starch degradation, and protein degradation. The 2:2:1 group achieved a hydrogen production rate of 116.7 mL / g-COD, a 52.9% increase compared to the conventional hydrogen production inoculum (control group). The starch degradation rate reached 93.1% in the 2:2:1 group, a 35.1% increase compared to the conventional hydrogen production inoculum (control group). The protein degradation rate reached 81.9% in the 2:2:1 group, a 56.3% increase compared to the conventional hydrogen production inoculum (control group).

[0095] Example 4 Effect of the composite bacterial agent on hydrogen production from kitchen wastewater under different organic loads

[0096] To test the effect of organic load on the fermentation of hydrogen production from food wastewater using a composite bacterial agent, this example, based on the results of Example 2, used a fermentation system with an inoculation ratio of 2:2:1 of Bacillus paralicheniformis Q4, Bacillus thunbergii K3, and Arthrobacter protocis A1 as the research object. The composite bacterial agent with the optimal ratio was added at an inoculum rate of 5% to a continuous flow hydrogen production reactor using food wastewater to analyze the hydrogen production effect of the composite bacterial agent under different organic loads.

[0097] Bacillus paralicheniformis Q4, Bacillus euclidinium K3, and Arthrobacter protocisinus A1 were activated and cultured in LB liquid medium. A composite inoculum was prepared at a volume ratio of 2:2:1. A 5% inoculum was then added to a continuous anaerobic fermentation reactor using food wastewater. The fermentation temperature and pH were controlled at 35°C and 6.5, respectively. The hydrogen production efficiency of the composite inoculum was evaluated under different influent organic loads.

[0098] The fermentation results of the anaerobic fermentation hydrogen production system of food wastewater are as follows Figure 5 As shown in Figure 2, the hydrogen yield is 3.0, 5.0 and 7.2 kg-COD / m3 / d can be maintained above 106mL / g-COD. When the organic load is 7.2kg-COD / m 3 / d, the hydrogen production intensity is the highest, reaching 765.4L / m 3 / d. However, when the organic load is further increased to 8.5kg-COD / m 3 / d, the hydrogen yield and production intensity decreased significantly to 71.5mL / g-COD and 607.8L / m 3 / d.

[0099] The results showed that organic load is an important factor affecting the fermentation effect of the composite bacterial agent in the anaerobic fermentation hydrogen production system of food wastewater. The optimal organic load range of the composite bacterial agent is 3.0-7.2 kg-COD / m 3 / d, and the hydrogen production rate reached above 100mL / g-COD.

[0100] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A salt-tolerant amylase-producing bacterium, characterized in that: The salt-tolerant amylase-producing bacteria was deposited in the China Center for Type Culture Collection on May 23, 2024, with the deposit address being Wuhan, China, the deposit number being CCTCC NO: M 20241043, and the classification name being Bacillus paralicheniformis Q4.

2. The salt-tolerant amylase-producing bacterium according to claim 1, characterized in that The salt-tolerant amylase-producing bacteria can tolerate 6.0% salinity and grow under anaerobic conditions, and the starch degradation rate of kitchen wastewater is higher than 85%.

3. Use of the salt-tolerant amylase-producing bacteria according to claim 1 or 2 in producing amylase by high-salt anaerobic fermentation.

4. A composite bacterial agent, characterized in that: The composite bacterial agent comprises: Salt-tolerant amylase-producing bacteria: Bacillus paralicheniformis Q4, deposit number: CCTCC NO: M 20241043; Salt-tolerant fermentative hydrogen-producing bacteria: Bacillus altitudinis K3, deposited with CCTCC NO: M20241042; Salt-tolerant protease-producing bacteria: Arthrobacter protophormiae A1, with a deposit number of CCTCC NO: M 20241041.

5. The composite bacterial agent according to claim 4, characterized in that In the composite bacterial agent, the volume ratio of the bacterial liquid of Bacillus paralicheniformis Q4, Bacillus highland K3 and Arthrobacter protovitilis A1 is (1-2): (1-2):

1.

6. The composite bacterial agent according to claim 5, characterized in that In the composite bacterial agent, the volume ratio of the bacterial liquid of Bacillus paralicheniformis Q4, Bacillus altaica K3 and Arthrobacter protovitilis A1 is 2:2:

1.

7. A method for preparing the composite bacterial agent according to any one of claims 4 to 6, characterized in that: The following steps are included: activating and culturing the Bacillus paralicheniformis Q4, Bacillus highland K3 and Arthrobacter protothecoides A1 in LB liquid culture medium to obtain three corresponding bacterial solutions; 600 After reaching 1.2-1.6, the three bacterial solutions are combined to prepare a composite bacterial agent.

8. Use of the composite bacterial agent according to any one of claims 4 to 6 in efficient anaerobic fermentation of kitchen wastewater to produce hydrogen.

9. The use according to claim 8, characterized in that In the high-efficiency anaerobic fermentation hydrogen production reaction of kitchen wastewater, the inoculation amount of the composite bacterial agent is 5%-10%, and / or Fermentation pH is 6.5-7.0, and / or Temperature of 30-40°C, and / or Organic load is 3.0-7.2 kg-COD / m 3 / d.

10. The use according to claim 8 or 9, characterized in that The hydrogen production rate reaches 100 mL / g-COD or above; and / or Production intensity reaches 720 L / m 3 / d or more.

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