Complex microbial inoculant for producing hydrogen through fermentation and application of complex microbial inoculant

Abstract

The invention provides a complex microbial inoculant for producing hydrogen through fermentation and application thereof. The complex microbial inoculant disclosed by the invention is prepared from the following components: Pristia aryabhattai YLS2, Niallia nealsonii YLS4 and Lysinibacillus boroni S5, and the complex microbial inoculant has the advantages that the complex microbial inoculant is prepared from the following components: the P.aryabhattai YLS2, the Niallia nealsonii YLS4 and the Lysinibacillus boroni S5, and the complex microbial inoculant is prepared from the following components: the P.aryabhattai YLS2, the Niallia nealsonii YLS4 and the B.boroni S5; wherein the preservation number of the prelisteria aryabhattai YLS2 is CGMCC (China General Microbiological Culture Collection Center) NO.32138, the preservation number of the bacillus nissei YLS4 is CGMCC NO.32137, and the preservation number of the boron-resistant lysine bacillus S5 is CGMCC NO.32139. The invention further discloses a preparation method of the feed additive. The hydrogen production process is optimized through the synergistic effect of multiple microorganisms, the hydrogen yield and the stability in the gas production process can be improved through the composite microbial agent for producing hydrogen through fermentation, and remarkable environmental benefits and economic potential are achieved.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a composite bacterial agent for hydrogen production by fermentation and application thereof. Background Art

[0003] Existing hydrogen production methods are mainly divided into hydrogen production by reforming fossil energy, hydrogen production by industrial byproducts, and hydrogen production by combining renewable energy with water electrolysis. The implementation of these technologies has solved the demand for hydrogen energy to a certain extent, but it will also consume a lot of energy. Biohydrogen production is a technology that uses biological resources such as biomass and microorganisms to produce hydrogen. As one of the production methods of hydrogen energy, it has broad development prospects. The biohydrogen production process produces almost no greenhouse gases and harmful substances, which helps to mitigate climate change, and can produce hydrogen by utilizing waste, which is conducive to promoting the recycling of resources and reducing the environmental burden. Hydrogen production by microbial fermentation is an important form of biohydrogen production, so selecting and constructing suitable and efficient microbial populations is an important research direction for technicians in this field.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The object of the present invention is to provide a composite bacterial agent for hydrogen production by fermentation and application thereof, wherein the composite bacterial agent can improve hydrogen production and stability during the gas production process.

[0006] The invention provides a composite bacterial agent for hydrogen production by fermentation, comprising Priestia aryabhattai YLS2, Niallia nealsonii YLS4 and Lysinibacillus boronitolerans S5; wherein the preservation number of Priestia aryabhattai YLS2 is CGMCC NO.32138, the preservation number of Niallia nealsonii YLS4 is CGMCC NO.32137, and the preservation number of Lysinibacillus boronitolerans S5 is CGMCC NO.32139.

[0007] The classification name of Priestia aryabhattai YLS2 is Priestia aryabhattai. It was screened from the water sample of Yanqi Lake in Yanqi Lake Scenic Area, Huairou District, Beijing. It was deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC for short, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101) on September 30, 2024, and the deposit number is CGMCC No.32138.

[0008] The classification name of Bacillus nealsonii YLS4 is Niallia nealsonii. It was screened from the water sample of Yanqi Lake in Yanqi Lake Scenic Area, Huairou District, Beijing. It was deposited in the General Microbiology Center of China Culture Collection Administration, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing on September 30, 2024, with the deposit number CGMCC No. 32137.

[0009] Boron-resistant lysine Bacillus S5 is classified and named Lysinibacillus boronitolerans, which was screened from the anaerobic activated sludge of the sewage treatment plant in Huairou District, Beijing, and was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing on September 30, 2024. The deposit number is CGMCC No. 32139.

[0010] The present invention does not impose strict restrictions on the number of live bacteria of Priesteria azadirachta YLS2, Bacillus nissoni YLS4 and boron-resistant lysine Bacillus S5 in the composite bacterial agent. The ratio of the number of live bacteria of Priesteria azadirachta YLS2, Bacillus nissoni YLS4 and boron-resistant lysine Bacillus S5 in the composite bacterial agent can be, for example, 1:(0.8-1.2):(0.8-1.2).

[0011] The present invention does not impose strict restrictions on the composite method of the composite bacterial agent. Apricotibacterium axetii YLS2, Bacillus niger YLS4 and boron-resistant lysine Bacillus S5 can exist in the form of a single bacterial agent or in the form of a mixed bacterial agent.

[0012] The present invention also provides a fermentation hydrogen production method, which is carried out using the above-mentioned composite bacterial agent. The fermentation hydrogen production method comprises: inoculating Apricot Bacillus YLS2, Bacillus nissori YLS4 and boron-resistant lysine Bacillus S5 into a hydrogen production medium, anaerobically treating the inoculated hydrogen production medium, and then fermenting and producing hydrogen under anaerobic and light-free conditions.

[0013] Furthermore, before inoculation, the method further comprises: activating and culturing Priesteria axytokoux YLS2, Bacillus niger YLS4 and boron-resistant lysine Bacillus S5 respectively.

[0014] Specifically, LB culture medium is used for activation; the composition of LB culture medium includes: 8-12 g / L tryptone, 4-6 g / L yeast extract, 8-12 g / L sodium chloride; the temperature of activation culture is 28-37°C, and the time of activation culture is 24-48 hours.

[0015] The anaerobic treatment includes: introducing nitrogen to purge until the oxygen in the hydrogen production medium is completely eliminated and then sealing; there is no strict limit on the nitrogen purge time, as long as the oxygen in the hydrogen production medium can be completely eliminated, and the purge time is, for example, 5-8 minutes.

[0016] The composition of the hydrogen production medium includes: 8-12 g / L anhydrous glucose, 4-6 g / L beef extract powder, 8-12 g / L peptone, 4-6 g / L sodium chloride, 3-4 g / L potassium dihydrogen phosphate, 5-6 g / L dipotassium hydrogen phosphate, 0.4-0.6 g / L magnesium sulfate heptahydrate, and a pH value of 7±0.2; the fermentation temperature is 28-37°C, preferably 36-37°C; the fermentation time is 12-240 h, preferably 48-96 h.

[0017] In the above-mentioned fermentation hydrogen production method of the present invention, the hydrogen production capacity of each 100mL hydrogen production culture medium during fermentation hydrogen production is 7-35mL, preferably 20-30mL; the mixed gas output is 11-62mL, preferably 33-51mL; the volume percentage of hydrogen in the mixed gas is 52-61%.

[0018] The composite bacterial agent of the present invention is composed of different types of hydrogen-producing strains, and the composite bacterial agent can produce hydrogen through a fermentation process using organic matter as a substrate under a specific nutritional environment and temperature; by adjusting the culture conditions of different strains during the fermentation process, such as pH value, temperature, culture time, substrate concentration and other parameters, the hydrogen production is significantly increased, and the gas production process is ensured to be stable. The composite bacterial agent of the present invention and its application can not only improve the production efficiency of hydrogen, but also reduce the emission of organic waste, have significant environmental benefits and economic potential, and are suitable for large-scale industrial hydrogen production, wastewater treatment and comprehensive utilization of agricultural waste. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 The growth diagram of the strains on plates; (a) is Priesteria axyti YLS2, (b) is Bacillus niger YLS4, and (c) is boron-resistant Bacillus lysine S5;

[0021] Figure 2 It is the trend of cumulative mixed gas production, cumulative hydrogen production, and hydrogen percentage content changing over time;

[0022] Figure 3It is the incremental diagram of mixed gas production at different times;

[0023] Figure 4 This is a graph showing the increase in hydrogen production at different times. DETAILED DESCRIPTION

[0024] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0025] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form also includes the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this description, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0026] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] Example 1

[0028] 1. Compound bacterial agent

[0029] The composite bacterial agent of this embodiment is composed of Priestia aryabhattai YLS2, Niallia nealsonii YLS4 and Lysinibacillus boronitolans S5; the strain information is shown in Table 1.

[0030] Table 1 Strain information

[0031]

[0032]

[0033] 2. Preparation of culture medium

[0034] LB agar medium was prepared according to the composition in Table 2, and hydrogen production medium (pH value was 7±0.2) was prepared according to the composition in Table 3.

[0035] Table 2 LB agar medium composition

[0036] composition concentration Tryptone 10g / L Yeast Extract 5g / L Sodium chloride 10g / L Agar 15-20g / L

[0037] Table 3 Hydrogen production culture medium

[0038] composition concentration Anhydrous glucose 10g / L Beef dip powder 5g / L Peptone 10g / L Sodium chloride 5g / L Potassium dihydrogen phosphate 3.63g / L Dipotassium hydrogen phosphate 5.7g / L Magnesium sulfate heptahydrate 0.5g / L

[0039] 3. Fermentation to produce hydrogen

[0040] Priesteria afaik YLS2, Bacillus nissori YLS4 and boron-resistant lysine Bacillus S5 were inoculated into LB agar medium for activation culture respectively. The activation culture temperature was 28-37° C. and the activation culture time was 24-48 hours.

[0041] When healthy, clear, single and uniformly growing colonies (such as Figure 1 As shown), use a sterile inoculation loop to gently pick a single pure colony and inoculate it into the hydrogen production medium. The culture system is 100mL. The reaction system is treated anaerobically, 99.9% nitrogen is passed, and it is purged for 5-8 minutes to ensure that the oxygen in the bottle is completely excluded. After the purging is completed, it is immediately sealed with a rubber stopper. Use tin foil to strictly wrap the anaerobic bottle for light-shielding treatment to ensure that the composite bacterial agent reacts under anaerobic and lightless conditions, and use an incubator to adjust the culture temperature to 37°C. During the reaction period, the gas sampling is carried out at 12h, 24h, 36h, 48h, 60h, 72h, 96h, 120h, 144h, 168h, 192h, 216h, and 240h, and the gas production and proportion are analyzed using a gas chromatograph.

[0042] 4. Testing

[0043] The gas was quantitatively analyzed, and the gas composition was analyzed by a gas chromatograph equipped with a thermal conductivity detector (TCD) column, model GC-7860D, manufactured by Shanghai Youke Instrument Co., Ltd. Before starting the test, the test baseline must be leveled to ensure the accuracy of the test results. Use a 1mL syringe to extract 1mL of mixed gas and inject it into the TCD detection port of the gas chromatograph to start the test. When the detection peak line is completely completed and the baseline remains horizontal, the test is ended and analyzed by gas chromatography; the test results are shown in Table 4 and Figure 2-Figure 4 .

[0044] Table 4 Cumulative mixed gas production, cumulative hydrogen production and hydrogen percentage

[0045]

[0046]

[0047] The data analysis is as follows:

[0048] 1) Analyze the cumulative mixed gas production:

[0049] The increment is the largest in the early stage of the reaction, indicating that the mixed bacterial community can quickly degrade organic matter to react and produce gas; then it gradually decreases and tends to stabilize, indicating that the system's production increase rate has reached a relatively stable state.

[0050] 2) Analysis of cumulative hydrogen production:

[0051] The production of hydrogen continued to increase over time, with a growth trend of rapid growth in the early stage and gradually stabilizing in the later stage. The increment was the largest (8.81 mL) in 12-24 hours, and then gradually decreased and became constant (about 0.7 mL). The reason for this was that the concentration of organic matter and nutrients in the system was high in the early stage, the reaction rate was driven by maximum kinetics, and the temperature, pressure and other parameters were in the optimal state of reaction, which promoted the rapid formation of hydrogen. The main reactants in the middle stage of the system were gradually consumed, causing the reaction rate to decrease. The consumption of the reactants in the later stage was close to completion, and the system entered the final stage of the reaction. The pressure, temperature and other conditions in the system no longer changed, and the generation of hydrogen was close to the saturation level.

[0052] 3) Analysis of hydrogen content:

[0053] In the initial stage, the reactants are consumed intensively, and the rate of hydrogen generation is high, resulting in a short-term increase in the percentage of hydrogen. The proportion of hydrogen in the mixed gas is high, and there is a significant fluctuation in the percentage in the early stage; as time goes by, other gases (such as carbon dioxide or methane) begin to increase, diluting the relative proportion of hydrogen. The system gradually approaches saturation, the rate of hydrogen generation decreases, but the total amount of hydrogen is still increasing. In the later stage (144-240h), the decline in the percentage of hydrogen tends to stabilize, indicating that the composition of the mixed gas in the system is close to dynamic equilibrium.

[0054] In summary, the composite bacterial agent for hydrogen production by fermentation of the present invention and its application optimize the hydrogen production process through the synergistic action of multiple microorganisms. By rationally designing and optimizing the composition of the bacterial agent, fermentation conditions and related production processes, the hydrogen production can be effectively improved and widely used in the fields of energy, environmental protection, etc., and has broad market prospects and application value.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. 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 replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composite bacterial agent for hydrogen production by fermentation, characterized in that: Including Priestia aryabhattai YLS2, Niallia nealsonii YLS4 and Lysinibacillus boronitolerans S5; among them, the preservation number of Priestia aryabhattai YLS2 is CGMCC NO.32138, the preservation number of Niallia nealsonii YLS4 is CGMCC NO.32137, and the preservation number of Lysinibacillus boronitolerans S5 is CGMCC NO.32139.

2. The composite bacterial agent according to claim 1, characterized in that The ratio of the number of live bacteria of Bacillus axetii YLS2, Bacillus niger YLS4, and boron-resistant Bacillus lysine S5 in the composite bacterial agent is 1:(0.8-1.2):(0.8-1.2).

3. A method for producing hydrogen by fermentation, characterized in that: The fermentation hydrogen production method using the composite bacterial agent according to claim 1 or 2 comprises: inoculating Apricot Bacillus YLS2, Bacillus niger YLS4 and boron-resistant lysine Bacillus S5 into a hydrogen production medium, anaerobically treating the inoculated hydrogen production medium, and then fermenting and producing hydrogen under anaerobic and light-free conditions.

4. The method for producing hydrogen through fermentation according to claim 3, characterized in that: Before inoculation, the method also includes: activating and culturing Priesteria axytokoux YLS2, Bacillus niger YLS4 and boron-resistant lysine Bacillus S5 respectively.

5. The method for producing hydrogen through fermentation according to claim 4, characterized in that: LB medium was used for activation; the composition of LB medium included: 8-12 g / L tryptone, 4-6 g / L yeast extract, and 8-12 g / L sodium chloride.

6. The method for producing hydrogen through fermentation according to claim 4, characterized in that: The temperature of activation culture is 28-37°C, and the time of activation culture is 24-48h.

7. The method for producing hydrogen through fermentation according to claim 3, characterized in that: The anaerobic treatment includes: introducing nitrogen gas to purge until the oxygen in the hydrogen-producing culture medium is completely eliminated and then sealing.

8. The method for producing hydrogen through fermentation according to claim 3, characterized in that: The composition of the hydrogen-producing culture medium includes: 8-12 g / L anhydrous glucose, 4-6 g / L beef extract powder, 8-12 g / L peptone, 4-6 g / L sodium chloride, 3-4 g / L potassium dihydrogen phosphate, 5-6 g / L dipotassium hydrogen phosphate, and 0.4-0.6 g / L magnesium sulfate heptahydrate.

9. The method for producing hydrogen through fermentation according to claim 3, characterized in that: The fermentation temperature is 28-37°C and the fermentation time is 12-240h.

10. The method for producing hydrogen through fermentation according to claim 3, characterized in that: The hydrogen production during fermentation is 7-35 mL per 100 mL of hydrogen production culture medium.

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